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This is to certify that the thesis entitled UNDERSTANDlNG THE STRUCTURE OF YSCF, THE TYPE III SECRETION PROTEIN FROM YERSINIA AND HOW IT FORMS PILI presented by Joel Sanya Lwande has been accepted towards fulfillment of the requirements for the PhD degree in Biochemistry and Molecular Biology Cob WW k; %QALWQI Major @fessor's Signature l3 flags“: 2930 Date MSU is an Affirmative Action/Equal Opportunity Employer LIBRARY Michigan State University — --.---.--- -.n.--a - PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProj/Acc&Pres/ClRC/Date0ue.indd UNDERSTANDING THE STRUCTURE OF YSCF, A TYPE III SECRETION PROTEIN FROM YERSINIA AND HOW IT FORMS PILI By Joel Sanya Lwande A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Biochemistry and Molecular Biology 2010 ABSTRACT UNDERSTANDING THE STRUCTURE OF YSCF, A TYPE III SECRETION PROTEIN FROM YERSINIA AND HOW IT FORMS PILI By Joel Sanya Lwande Pathogenic bacteria of the genus Yersinia inject effector proteins into mammalian host cells to interfere with the host immune response, thereby enabling the pathogens to thrive. The effector proteins are translocated through an extracellular, hollow needle structure that forms part of the Type III Secretion System (T388). The needle is made up of many copies of a single protein called YscF. The needle is anchored by interactions with the T388 base, which is embedded in the inner and outer bacterial membranes. In this thesis, we look for YscF residues that are likely to interact between the needle and the base in Y. pseudotuberculosis. We used multiple sequence alignment, secondary-structure prediction, homology modeling, and the crystal structures of YscF, in complex with YscE and Y306, and homologous proteins BsaL, MxiH, and Prgl. We identified the YscF residues Y65, N66, K76, D77, I82, 084 and F86 as potential residues interacting with the base in Y. pseudotuberculosis. We used site-directed mutagenesis, circular dichroism spectroscopy and transmission electron microscopy to analyze the role of these residues in YscF structure and needle assembly in vitro. This work also analyzed the role of the 19 N-terminal residues in YscF needle assembly. Finally, we used a GFP-YopQ hybrid protein to analyze the role of these residues in YopQ secretion, both in a YscF knockout strain and in wild-type Y. pseudotuberculosis. Our results show that the 19 N-terminal residues of YscF are not required for needle assembly. Circular dichroism spectroscopy shows that seven YscF variants (Y65A, N66A, K76A, D77A, l82A, 084A and F86A) are d-helical, whereas Y65F seems to be unstructured. Our results also show that the YscF mutations resulting in N66A and Y65F abolish needle assembly in vitro. whereas the mutations resulting in K76A, D77A, l82A, Q84A and F86A have little effect. The rate of needle assembly in Y65A is very slow compared to that of wild-type. Three YscF variants (Y65A, Y65F and l82A) did not reconstitute GFP—YopQ secretion in the YscF knockout strain; however, the remaining mutants reconstituted secretion in the knockout strain. Our secretion assays also show that mutations resulting in variants Y65A and Y65F have a dominant-negative phenotype for secretion; the Y65A and Y65F variant proteins inhibit secretion of GFP—YopQ in wild-type Y. pseudotuberculosis. Based on these results, residues Y65 and N66 are likely to be involved in the pilin-pilin interactions, although it seems likely that the pilin-pilin interactions are less disrupted by mutations of N66 than by mutations of Y65. The YscF residue I82 is likely to be nonessential in YscF monomer folding and pilus assembly (pilin-pilin interactions) because the l82A mutation has little effect on needle assembly. However, since the l82A variant inhibits GFP-YopQ53 secretion and I82 is strictly conserved, this residue likely makes a critical interaction for secretion and, in particular, may be involved in the needle-base interface. Cepyright by Joel Sanya Lwande 2010 ACKNOWLEDGEMENTS This work is a result of great effort and dedication that would not have been possible without support from people who surrounded me. I thank my wife Jane for helping me throughout the difficult moments I encountered in the course of my research. I express my heartfelt gratitude to my mentor Dr. William Wedemeyer who guided and supported me until completion of this project. His patience and dedication kept me focused even in times of immense pressure. It was a great privilege to work in your lab. I was lucky to work with great lab mates and friends: Terry, Kai-Chun, Amy, Kelly, and Jason. I will forever cherish the comfort and happiness you provided in that lab environment. I also thank my uncle Dr. Wilber Lwande for helping me follow this direction in life. Thank you for believing in me and providing me with the opportunity to pursue my dream. My sincere thanks go to my guidance committee members: Dr. Dean DellaPenna, Dr. Leslie Kuhn, Dr. Honggao Yan and Dr. Michael Bagdasarian for providing me with great support. I give special thanks to Dr. Michael Bagdasarian for helping me with research facilities and teaching me a lot of microbiology work. I thank Dr. Alicia Pastor-Lecha for teaching me Transmission Electron Microscopy. I appreciate the help I got from the department of Biochemistry at MSU and thank the graduate director, Dr. Jon Kaguni and chair, Dr. Tom Shakey for their support. Finally, I thank my special friends and family: Peter Sande, Betty Okwako, David Maine, and Salmon Aguko TABLE OF CONTENTS LIST OF TABLES ................................................................................................ viii LIST OF FIGURES ............................................................................................... ix ABBREVIATIONS ................................................................................................ xi Chapter 1: Introduction .......................................................................................... 1 INTRODUCTION TO YERSINIA PESTIS AND PLAGUE ..................................... 2 1. The type III secretion system (T3SS) ....................................................... 4 1.1 Yersinia type III secretion system ...................................................... 4 1.2 The translocon ................................................................................... 5 1.3 The base ............................................................................................ 6 1.4 The needle ......................................................................................... 8 2. Yersinia outer protein secretion (Yop secretion) ...................................... 9 2.1 T3SS chaperones .............................................................................. 9 3. T388 needle protein structures ............................................................... 9 3.1 MxiH and BsaL .................................................................................. 9 3.2 YscEFG complex ............................................................................. 19 3.3 YscF ................................................................................................ 21 CONCLUSIONS AND REMAINING QUESTIONS ............................................. 23 REFERENCES .................................................................................................... 25 Chapter 2: Structural Analysis of YscF and its in vitro Polymerization Examined by Secondary Structure Prediction, Homology Modeling, Electrophoresis, mutagenesis, Circular Dichroism, and Transmission Electron Microscopy ......... 33 ABSTRACT ........................................................................................................ 34 INTRODUCTION ................................................................................................. 35 EXPERIMENTAL PROCEDURES ...................................................................... 41 Multiple sequence alignment ................................................................. 41 YscF structure prediction ....................................................................... 41 Comparison of de novo protein structure predictions for YscF with experimental structures of its homologs and the YscEFG complex ....... 42 Cloning, protein expression and purification ..................................... 42 Site-directed mutagenesis ........................................................... 44 Analytical gel filtration chromatography ................................................. 45 Circular dichroism (CD) spectroscopy ................................................... 46 Needle formation in vitro and electron microscopy ................................ 46 RESULTS ........................................................................................................... 47 Multiple sequence alignment ................................................................. 47 YscF structure prediction ....................................................................... 47 Comparison of de novo protein structure predictions for YscF with experimental structures of its homologs and the YscEFG complex ....... 48 vi Site-directed mutagenesis ..................................................................... 51 Protein expression and purification ........................................................ 51 Analytical gel filtration chromatography ................................................. 52 Circular dichroism spectroscopy ............................................................ 52 Needle formation in vitro ........................................................................ 53 DISCUSSION ...................................................................................................... 72 REFERENCES .................................................................................................... 75 Chapter 3: In vivo Analysis of the Functions of YscF Variant Proteins ................ 79 ABSTRACT ........................................................................................................ 80 INTRODUCTION ................................................................................................. 81 EXPERIMENTAL PROCEDURES ...................................................................... 87 Site-directed mutagenesis ..................................................................... 87 Y. pseudotuberculosis competent cells and electroporation ................. 87 YscF knockout ....................................................................................... 88 Eliminating the helper plasmid pKD119 ................................................. 90 GFP-YopQ secretion studies ................................................................. 9O YscF reconstitution and the effect of 8 YscF mutants ............................ 93 RESULTS ........................................................................................................... 95 Site-directed mutagenesis ..................................................................... 95 Y. pseudotuberculosis competent cells and electroporation .................. 95 YscF knockout ....................................................................................... 96 Eliminating the helper plasmid pKD119 ................................................. 97 GFP-YopQ secretion studies ................................................................. 97 DISCUSSION .................................................................................................... 105 REFERENCES .................................................................................................. 108 Chapter 4: Conclusions and remaining questions ............................................. 113 CONCLUSIONS AND REMAINING QUESTIONS ........................................... 114 REFERENCES .................................................................................................. 119 Images in this dissertation are presented in color vii LIST OF TABLES Table 2.1: Oligonucleotides used to generate YscF mutants .............................. 70 Table 2.2: Comparing positions of the predicted YscF helices 01, 02, 03 and 04 in relation to the atomic structures of Prgl (18) (PDB ID: 2JOW), MxiH (10) (PDB ID: ZCA5), BsaL (11) (PDB ID: ZGOU) and the inactive form of YscF in the YscEFG complex (9) (PDB ID: 2P58) ................................................................. 70 Table 2.3: Deconvoluted far-UV CD spectra into various components of secondary structure in YscF, YscFA4 and its mutants ........................................ 71 Table 3.1: Oligonucleotides used to generate YscF mutants .............................. 94 viii LIST OF FIGURES Figure 1.1. A schematic diagram of a type III secretion apparatus ...................... 12 Figure 1.2. The crystal structure of the MxiH monomer ...................................... 13 Figure 1.3. The NMR structure of BsaL ............................................................... 14 Figure 1.4. The NMR structure of Prgl ................................................................ 15 Figure 1.5. The crystal structure of the YscEFG complex ................................... 16 Figure 1.6. Secondary structure predictions for YscF ......................................... 19 Figure 1.7. The YscF homology model based on the MxiH crystal structure ...... 22 Figure 2.1. YscF secondary-structure predictions for YscF ................................. 50 Figure 2.2. The YscF homology model ............................................................... 51 Figure 2.3. YscF structural families from de novo predictions ............................. 55 Figure 2.4. De novo YscF structure predictions .................................................. 58 Figure 2.5. T3SS secondary structure models .................................................... 60 Figure 2.6. 12% SDS-PAGE gel for purified YscFA4 .......................................... 61 Figure 2.7. 12% SDS-PAGE gel for Y65AYscFA4 .............................................. 61 Figure 2.8. Analysis of the Nickel column binding properties of YscFA4 and Y65AYscFA4 in absence of GnHCI ..................................................................... 62 Figure 2.9. Polymerization of A19YscFA4 ........................................................... 62 Figure 2.10. Analytical gel filtration of Y65AYscFA4 under two different pH Conditions ........................................................................................................... 63 Figure 2.11. CD spectra for YscF, YscFA4, and N66AYscFA4 ........................... 64 Figure 2.12. CD spectra comparing Y65AYscFA4 and Y65FYscFA4 ................. 66 Figure 2.13. In vitro needle formation for YscFA4, A19YscFA4, N66AYscFA4 and Y65FYscFA4 ....................................................................................................... 67 ix Figure 2.14. In vitro needle formation for Y65AYscFA4 in different buffers ......... 69 Figure 3.1. A schematic diagram of a simple gene disruption strategy and an agarose gel of the PCR product of H1-chloramphenicol-H2 gene ..................... 100 Figure 3.2. Verification of yscF knockout by PCR using primers complementary to genes flanking the knockout region (YscE and YscG). Results show the YscE- Chloramphenicol-YscG gene (1570 bp) from the knockout strain ..................... 101 Figure 3.3. Verification of yscF knockout by PCR using primers complementary to genes flanking the knockout region (YscE and YscG). Results show the YscEFG gene (810 bp) from wild-type Y. pseudotuberculosis .......................... 101 Figure 3.4. PCR amplification of the yopE gene from the yscF knockout strain to verify the presence of the virulence plasmid pW .............................................. 102 Figure 3.5. GFP-YopQ18 and GFP-Yop053 constructs for in vivo secretion Assays .............................................................................................................. 102 Figure 3.6. YopQ-GFP secretion assay results ................................................. 103 Figure 3.7. Western blot analysis of differential secretion ................................. 104 Figure 3.8. Western blot analysis of the knockout secretion assay ................... 104 ATP BHI BsaL CD A19YscFA4 EDTA EGTA F LP FRT GFP—YopQ1 8 GFP-YopQ53 GnHCI His5-tag IPTG MALDl-TOF MxiH NIAID PCR PDB Prgl stcFA4 ABBREVIATIONS adenosine triphosphate brain-heart infusion type III secretion system pilin from Burkholden'a Circular dichroism YscFA4 with 19 N-terminal residue truncation Ethylenediaminetetraacetic acid ethylene glycol tetraacetic acid Flippase recombinase FLP recognition target Green Fluorescent Protein-YopQ18 hybrid protein Green Fluorescent Protein-Yop053 hybrid protein guanidine hydrochloride Histidine tag isopropyl-B-D-thiogalactopyranoside Matrix-assisted laser desorption/ionization - time of flight type III secretion system pilin from Shigella National Institute of Allergy and Infectious Diseases Polymerase Chain Reaction Protein Data Bank type III secretion system pilin from Salmonella pET22b containing the YscFA4 gene xi SDS-PAGE T3SS TCA TEM TPR Yops Ysc YscF YscFA4 YscEFG sodium dodecyl sulfate polyacrylamide gel electrophoresis type III secretion system Trichloroacetic acid transmission electron microscopy tetratrico peptide repeat Yersinia outer proteins Yop secretion type III secretion system pilin from Yersinia YscF with 4 C-terminal residue truncation YscF in complex with YscE and YscG chaperones xii Chapter 1 Introduction INTRODUCTION TO YERSINIA PESTIS AND PLAGUE Y. pestis is a Gram-negative bacterium that causes bubonic, pneumonic and septicemic plague and is classified as a Category A priority pathogen by the National Institute of Allergy and Infectious Diseases (NIAID). During the Middle Ages, the plague was pandemic from Europe to China and killed approximately one-third of Europe’s population (~25 million) within a five-year span (1347-1351) (1). Y. pestis is still found in the developing world and is a potential weapon of bioterrorism and biowarfare (2). Although Y. pestis is generally susceptible to antibiotics such as streptomycin and chloramphenicol, partially resistant strains have been isolated from nature (3) and fully resistant strains may be developed by design. Hence, there is an urgent need to identify drug targets that are necessary for pathogenesis and against which the bacterium will be unlikely to develop resistance. Other bacterial species closely related to Yersinia within the family Enterobactenaceae include well-known pathogens such as Shigella dysenten’ae, Salmonella typhi and Eschericia coli. Yersinia is an eleven-species genus in the family Enterobacteriaceae. Only 3 of the 11 Yersinia species are human pathogens: Y. pestis, Y. pseudotuberculosis and Y. enterocolitica (4). Y. pestis, and Y. pseudotuberculosis are closely related to each other based on their chromosomal DNA sequence, but not as closely related to Y. enterocolitica (5, 6). Earlier suggestions were raised to classify Y. pestis as a subspecies of Y. pseudotuberculosis, but this has not been implemented because of public health safety and unique laboratory and historical considerations linked with plague (5, 7). Historically, the global pandemic caused by both bubonic and pneumonic plague is much greater than other infectious diseases (4). Throughout recorded history, almost 200 million deaths have been attributed to plague (8). The disease can occur in any one of the three primary forms: bubonic, septicemic or pneumonic (4). The classic form of plague is bubonic and is characterized by many symptoms including fever, headache, chills, swollen and extremely tender lymph nodes (buboes) within 2-6 days of infection (4). These infections can be treated successfully with antibiotics, especially streptomycin (9). There have been attempts to develop vaccines, but some of the existing vaccines cause an adverse reaction in significant percentage of vaccinees and sometimes the reactions can be severe (10). Experimental evidence has shown that the plague vaccine does not protect against pneumonic plague (11). The plague has a complex life cycle that makes it extremely difficult to eradicate (4). Three major pandemics have occurred, causing major calamities. The first, second and third pandemics occurred in AD. 541 — 544 (12, 8, 13), AD. 1347 — 1351 (1, 14) and 1855 - 1918 (15, 16, 8, 17) respectively. The mortality rates and outbreaks have since reduced because of good public-health measures and antibiotics (4). Plague affects rodents primarily, but the Y. pestis pathogen can be transmitted between rodents and humans by fleas that acquire it from an infected blood meal (4). Following ingestion of an infected blood meal, bacteria may grow into masses that block the gut of the flea after 3-9 days. This prevents ingested blood from reaching the stomach (18, 19, 20, 21, 22, 23). During feeding, the blood sucked from the host can mix with the pathogen and is then regurgitated into the host (19, 22). Although not all blocked fleas transmit the disease, blockage is central to successful transmission (24, 22, 25). The pathogen moves from the site of the bite to local lymph nodes, thereby multiplying and leading to formation of bubo (swollen lymph nodes) (4). The infection enters the bloodstream, at which point some pathogens are removed in the spleen and liver; however, the bacteria continue to grow and spread in the blood and other organs (4). Bubonic plague can then lead to secondary pneumonic plague, which becomes highly contagious due to respiratory droplets. This scenario can trigger an epidemic because of the human-human transmission (26, 17). 1. The type III secretion system (T 338) 1.1 Yersinia type III secretion system The type three secretion system (T3SS) (Figure 1.1) is a unique device known as the injectisome. The injectisome is used by many Gram-negative bacteria to translocate proteins across lipid membranes and also deliver effector proteins into the cytosol of the host cells (27). Following their delivery into host cells, these Yops affect signal transduction pathways that control phagocytosis, apoptosis, the actin cytoskeleton, and the inflammatory response, thereby helping the pathogen to survive (28). A properly assembled injectisome consists of approximately 25 proteins. Although the functions and positions of some of these proteins have been determined, most of them are yet to be defined (27). The genes encoding the secretion machinery are organized in three operons, virA (IchR genes), virB (yscNOPQRSTU genes) and WC (yscABCDEFGH/JKL genes) (29). The T3SS can be divided into three different regions: (i) the translocon, (ii) the needle, and (iii) the base. 1.2 The translocon The TBSS is used by many Gram-negative bacteria like Yersinia spp., Buntholden'a spp., and Shigella spp. to translocate effector proteins into host cells (30, 31, 27). The delivery of Yersinia outer proteins (Yops) into host cells involves two processes aimed at overcoming the membrane barriers: (a) secretion involving extracellular release of Yops through the bacterial membrane barrier and (b) translocation involving delivery of Yops into the host cell cytosol through the host cell membrane barrier (32, 33). The three structural parts of the T388 work in a well-coordinated fashion to accomplish the task (34). Effector proteins are released when the pathogen comes into contact with a host cell in a well-regulated way at the translocon (34). Secretion can also occur in vitro under conditions that resemble the host environment. In Yersinia, many proteins play a role in this kind of regulation by enabling the pathogen-host cell contact. Some of the Yersinia regulatory factors involved in this process include: SycN, YscB, Lch, YopN, and TyeA (35-39). It has been suggested that these regulation occurs in the bacterial cytosol because only two (YopN and Lch) of these regulatory proteins are secreted (40, 41). Due to the regulatory role played by these proteins, uncontrolled secretion occurs when regulatory proteins are mutated (42, 37). In Yersinia, a translocon is a pore that is formed by the pathogen in the host cell membrane to enable delivery of Yops to the host-cell cytoplasm. This pore is made by three proteins: Lch, YopB, and YopD under the control of the translocation regulatory proteins called the translocators (43-48). Lch is hydrophilic and located at the tip of the needle where it forms a multi-subunit complex (49) required for the translocon build-up. Unlike Lch, YopB, and YopD are hydrophobic and embedded in the host cell membrane where they form the translocon (50, 51). Translocation of Yops into host cell cytosol cannot occur without these three proteins (47, 52, 53), although extracellullar release of the effector proteins can still occur without them. In their work, Davis et al. (34) identified yscF mutants that were specifically altered in translocation but not in Yop secretion or needle assembly. Their results showed that YscF functions in Yop secretion and translocation can be genetically separated. 1.3 The base The Yersinia T3SS is made up of Ysc (Yop secretion) injectisome involved in Yop secretion and translocation (54). The genes encoding the Yops and the secretion system are located on a large virulence plasmid, known as pYV (55). A total of 27 Ysc proteins make up this complex of two pairs of rings. The first ring is embedded in the inner bacterial membrane while the other is in the outer bacterial membrane. This pair of rings forms the base of the T388 complex. The two rings are linked by a hollow needle of 10 nm diameter and 60 nm length that protrudes on the outside of the bacterial membrane (56-58). The function of some Ysc proteins in the base has been defined, although much is still unknown. The length of the Yersinia needle is determined by YscP (59), a protein that belongs to the base of the injectisome complex. It was proposed that YscP acts as a molecular ruler during the stepwise assembly of the injectisome (60). They hypothesized that one end of YscP is attached to the basal body while the other end is connected to the growing tip of the needle. The needle grows by addition of YscF at the tip. When the needle reaches its mature length, YscP would be fully stretched and signal, via its internal anchor, to the secretion apparatus, which would stop exporting YscF and switch to other substrates (59). As part of the base, the YscR/S/T group of proteins resembles flagellar proteins FliP/Q/R that form a membrane channeling structure (60). These three proteins are embedded in the inner bacterial membrane (61). Two other proteins, Lch and Ych in the Yersinia injectisome play a role in rod formation. Ych is in the inner bacterial membrane just like YscR/S/T. The rod is thought to act as a link between proteins in the two bacterial membranes just like the P rod in flagellum structure (61). Yst is a lipoprotein involved in the Yersinia TBSS-dependent secretion. It is also connected to other proteins in both the inner and outer bacterial membranes (61). Like YscP, Ych plays a role in regulation of effector and YscF secretion and is located in the inner bacterial membrane (61). The injectisome also has a cytoplasmic protein YscN with an ATP binding motif resembling the B—subunit of FoF1-ATPase (61). This is the energy source for the apparatus (27). Another protein required for effector secretion is Y500 and it is a mobile component of the T388 (61). Ych is a peripheral cytoplasmic protein that interacts with YscK and YscL (61 ). YscC is an outer membrane protein that exists as a stable oligomeric complex in the outer membrane of the bacterium (55). The YscC protein of Y. enterocolitica was isolated as a stable multimeric complex with an apparent molecular weight of 600 kDa in the outer membrane (55). They showed the YscC complex in a ring-shaped structure of ~ 20 nm with an apparent central pore. The study also showed that YscC plays a central role in the export of the Yop proteins. It is an outer membrane component of the Yop secretion machinery of Yersinia. chC seems to be in clear contact with the needle protein (YscF). The virG gene also results in products that improve the overall response and speed of the secretion process (62). It has been suggested that the VirG lipoprotein interacts with YscC in the bacterial outer membrane (62). 1.4 The needle The needle is a hollow tube protruding from the injectisome base in the bacterial membrane to the exterior. In Yersinia, the needle is a result of polymerization of a major subunit called YscF (63). The needle length is between 45 and 80 nm depending on the Yersinia species, but the interior diameter is 2.5 nm (27). Extension of the needle length in Yersinia is controlled by YscP, as described above (59). 2. Yersinia outer protein secretion (Yop secretion) 2.1 T3SS chaperones Some of the T388 proteins act as chaperones to prevent premature folding or the wrong protein-protein interaction. The hydrophilic translocator Lch acts as an extracellular chaperone that guides the two hydrophobic translocators, YopB and YopD, to integrate in the eukaryotic plasma membrane (64, 65). Secretion of some, but not all, T3S substrates requires the assistance in the, bacterial cytoplasm of a particular type of chaperones (65). These chaperones are mostly acidic proteins with low molecular mass ~ 15 kDa. Each chaperone enables proper secretion of its cognate substrate (65). Sch (66) is one such chaperone and it binds YopE. In absence of the chaperone, YopE is unstable and is rapidly degraded. Sch masks an aggregation-prone region of YopE (67) and was also shown to be necessary for YopE recognition by the secretion system (68, 69). As “chaperones”, YscG and YscE interact with YscF to prevent it from premature polymerization in the cytosol of the bacterium prior to assembly of the needle (70). 3. T3SS needle protein structures 3.1 MxiH and BsaL Structural determination of T3SS needle proteins has remained difficult because they tend to readily polymerize into much larger structures. Only three structures have been solved including MxiH (Figure 1.2), BsaL (Figure 1.3), and Prgl (Figure 1.4) (71, 72 and 73), although the YscF structure has also been solved in complex with the heterodimeric chaperone YscE/YscG (Figure 1.5). Determination of MxiH, BsaL and Prgl structures involved some modifications in form of truncation of the last five C-terminal residues and addition of a Hise-tag to the C-terminus. These modifications increased the solubility of those proteins while preventing their self-oligomerization. The reported crystal structure of the Shigella flexnen' T3SS needle subunit MxiH (Figure 1.2) (71) and the nuclear magnetic resonance structure of Burkholderia pseudomallei T3SS needle subunit BsaL (1.3) (72) have contributed important information towards further understanding of the T388. Both MxiH and BsaL are YscF homologs based on the multiple sequence alignment with each having a 30% sequence identity to YscF. This thesis predicts YscF secondary structure using four different programs (namely PsiPRED, SAM, PROFsec and SABLE2). The results suggest the possibility of four q-helices which we will denote here as 01, 02. (13 and 04 (Figure 1.6). Two of the four predicted YscF helices (02 and a3) align well with the two reported core helices in both MxiH (Figure 1.2) and BsaL (Figure 1.3). The stmcture of MxiH consists of two long anti-parallel helices (corresponding to our predicted G; as and a4 helices of YscF) connected by a short Pro-Ser-Asn-Pro (PSNP) turn. The structure of BsaL has a similar helix-tum-helix core domain with two well-defined a-helices that are joined by a PSDP linker. The two helices also correspond to our predicted 02 and 03 helices of YscF. The tetrapeptide P- (S/D)—(D/N)—P is conserved among four YscF homologs (71) namely, BsaL, MxiH, Prgl (S. typhimurium) and Eprl (E. 00/!) with YscF having Pro-Asp-Asn-Pro 10 (PDNP). Thus, there is a high probability that YscF forms an a-helical hairpin centered on this tetrapeptide. Although MxiH had been strongly predicted to form a helix (11 at the N- terminus (74), this prediction disagreed with the crystal structure in which the N- terminus is disordered. Two possible explanations (71) were suggested for this outcome: the truncated C-terminal residues might be essential for N-terminal helix formation; or this region becomes folded only within the intact needle, possibly in response to environmental factors. Based on these findings, Deane et al. (71) generated a model of the Shigella T3SS needle by docking the crystal structure for MxiH into the 16 A density map from their earlier three-dimensional EM reconstruction of the needle (75). This study shows that the C-terminal helix forms the outer shell of the needle core, whereas the PSNP loop directs the N- terminal helix to line the inner wall of the needle channel. Their model shows that the C terminus of MxiH is involved in extensive inter-subunit contacts. They also suggested that this interaction is important for needle stability because polymerization was blocked following truncation of the last 5 residues of MxiH. In their analysis of the interface between two subunits, the patch of the 02 residues L30, L34, A38, and Y50 on the head of one subunit contacts the patch of the 04 residues D73, D75, I78, I79, and Q80 on the tail of another subunit. 11 Host cell cytoplasm Host cell membrane 7nm ._,2nm Extracellular space Pilus . l Outer membrane I Periplasm I Inner membrane I Bacterial cytoplasm Figure 1.1. A schematic diagram of a type III secretion apparatus showing the width of the hollow needle and the membranes of the bacterium and the host cell. 12 Tail Figure 1.2. The crystal structure of the MxiH monomer. Two long anti-parallel helices connected by a short Pro-Ser-Asn-Pro (PSNP) turn. (Deane et al.) (71 ). 13 Figure 1.3. The NMR structure of BsaL showing the helix-tum-helix core domain with two well-defined a-helices that are joined by a PSDP linker (Zhang et al.) (72). 14 C Figure 1.4. PrglCA5 forms a two helix bundle stabilized by hydrophobic contacts at the helix 02- (:3 interface. (Wang et al.) (73). 15 Figure1.5. (a) Ribbon representation of the overall structure of the YscEFG complex. YscE, YscF and YscG are colored purple, lime and grey, respectively. (b) Ribbon representation of the overall structure of the YscEFG complex after a 90° rotation. (Sun et al.) (76). 16 YscE Figure 1.5 (cont’d) The tail region (residues 58 to 83 of 04) of MxiH has well conserved hydrophobic and polar residues namely, L59, Y60, A63, T67, V68, V74, I78, I79, F82 (hydrophobic) and N62, Q64, S65, K69, K72, D73, Q80, N81 (polar). This part of the C-terrninal helix does not participate in the 02-03 interhelix interactions, meaning that the hydrophobic residues listed above are exposed in monomeric MxiH. There is a high possibility that these residues are buried (shielded from the solvent) during needle formation. Only D73, I78, I79, and Q80 are reported to participate in MxiH-MxiH (tail to head) interaction. While some of these residues might be involved in lateral MxiH-MxiH interaction during needle formation, 17 others are presumably involved in interactions at the interface between MxiH and other proteins in the T388 base. Such residues might include K69 and K72, both of which are likely to be involved in formation of salt bridges needed to stabilize the needle. This explanation is also given for the BsaL structure and there is a possibility that this might also be true for YscF. The two-helix bundle structure of BsaL was reported (72) to be stabilized by interhelix hydrophobic contacts between the helices corresponding to our predicted C12 and (13 helices of YscF. In BsaL, these stabilizing residues at the interface between helices 02 and 03 are conserved among BsaL homologs including YscF. In the reported BsaL structure, the hydrophobic side of the amphipathic helix 02 directly contacts the hydrophobic side of helix 03. This interface involves interactions of Leu32, Leu36, Ala39, and Leu43 of helix <12 with Tyr63, Met60, lle59, Tyr56, Leu53, and Ala52 of helix (:3. In their paper, Zhang et al. reported that Asn 68 is conserved among needle proteins including YscF, but does not participate in contacts at the helix 02-03 interface. They suggested that this residue might be involved in functions other than stabilizing the core domain, such as the interaction between BsaL and other T3SS proteins of the base. This residue corresponds to N66 in YscF. There is a possibility that N66 of YscF participates in the interactions at the needle-base interface. Based on chemical shifts, the first seven residues of BsaL were reported to be unstructured while the regions flanking the well-defined core domain (Ala10 to Gly28 at the N terminus and Ser71 to lle84 at the C terminus) are in partial-helical conformations. 18 SABLE2 STRAND SAM PROFsec _ .-.—u— _. SABLE2 . fifimfi_ - LOOP PSIPREDE‘ ‘s s r“—:‘ ‘1:‘:-.‘.‘ "-1 my: SAM l A j r ‘ - l PROFsecIj 1 I," l ' , SABLE2 L53... .- - L‘ ._i_n_-.I_1__nti Figure 1.6. Secondary structure predictions for YscF by four different methods, PsiPRED, SAM, PROFsec and SABLE2. Four helices are predicted (structured regions of the sequence are highlighted in red). 3.2 YscEFG complex The recently determined crystal structure of the YscEFG protein complex (Figure 1.5) (76) shows that YscG binds tightly to the C-terrninal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure (76). The structure shows that YscE interacts with YscG, but makes very little direct contact with YscF. In the crystal structure, they could not observe electron density for the N-terminal 49 residues of YscF. This and additional evidence from their study suggest that the N-terminal region of YscF is disordered in the complex with YscE and Y306. 19 The structure shows that conserved residues in the C-terminal half of YscF mediate important intra- and inter-molecular interactions in the complex. The final YscEFG model consists of 204 amino acids, including residues 10—63 of YscE, 50—87 of YscF and 3—114 of YscG. YscF is bound within the hydrophobic groove of YscG generated by nonpolar residues. The crystal structure shows hydrophobic residues on the concave surface of YscG (Val7, Ala10, Leu14, Ala40, Leu43, Leu70, Pro72, Trp73, Leu76, Tyr79, Met109, and Phe105) interacting with hydrophobic residues on YscF (Asn66, lle71, Met75, Met78, Met79, lle82, Leu83, and Phe86). In the complex, YscF is reported to have two q-helices connected by a 5 residue loop (Ile64—Asn68). In addition to the numerous hydrophobic interactions between YscF and YscG described in their findings, hydrophilic interactions also seem to play an important role in maintaining the conformation of YscF within the complex. The loop in YscF is well-ordered and stabilized by a hydrogen bond with YscG. Additionally, the side chains of Tyr79 and Arg80 from the 3rd TPR (tetratrico peptide repeat) motif of YscG and that of G|n112 from the C-tenninal a-helix of YscG form hydrogen bonds with the main chain constituents of the loop residues in YscF. The side chain of YscF residue Asn68 forms a hydrogen bond with G|y108 of YscG. They also show that the hairpin loop between the two a- helices in YscF is further stabilized by intramolecular hydrogen bonds between Os of Gln55 and N; of Lys76, N8 of Gln55 and 06 of Asn59, and NC of Lys76 and 08 of Gln80. Based on the structure, they suggest that intramolecular hydrogen bond "bridge" between highly conserved Gln55 and Lys76 in YscF may play an 20 important role in maintaining the helical hairpin fold of YscF. They concluded that the a-helical hairpin conformation of YscF in the YscEFG complex may be an accurate representation of the biologically relevant structure. Several experiments in the report showed that the N-terminal 49 residues of YscF are present and disordered in the structure of the heterotrimer. 3.3 YscF In this work, we modeled YscF (Figure 1.7) based on the crystal structure of MxiH. We compared the YscF model to the three TBSS needle structures (MxiH, BsaL and Prgl) and the YscEFG complex. The following is a list of selected YscF residues located in the predicted tail region (04 helix in our model): V63, I64, Y65, N66, 869, R73, K76, D77, l82, L83, Q84 and F86. Conserved residues in YscF play different roles that together contribute to the proper functional mechanism of T3SS in Yersinia. These conserved residues are likely to fall under three different categories. The first category comprises residues that are likely to be important for the folding stability of YscF protein. These may include A30, 837 and L41 of helix oz and L50, L51, S57, l58 and W61 of helix (:3 likely to form the C12 — (:3 interaction interface of the predicted structure. The second category could be crucial for the self oligomerization of the protein leading to the formation of the needle. These may include V34, I38, K42, D53, L54, and L83. Although four out of the six residues are hydrophobic, the polar residues K42 and D53 may also be necessary for “indexing” the two subunits relative to one another. A specific polar interaction is often observed in protein- 21 protein interface, since purely hydrophobic interfaces are “greasy” and tend to slide upon each other. The third category of the conserved residues is likely to be important for interaction at the needle-base interface. This interaction is likely to be important for proper linkage between the needle and the base. In this category, we predict a mixture of hydrophobic and polar residues. The polar residues may help in “indexing” the interface (keeping the interacting hydrophobic residues in place). Figure 1.7. YscF homology model based on the MxiH crystal structure. Some of the selected conserved residues are shown on predicted oz and q3-a4 helices. Blue represents N terminus while red represents C terminus. Some of the residues are involved in G2 — o3 hydrophobic interactions to keep the two core helices close to each other. 22 CONCLUSIONS AND REMAINING QUESTIONS A previous study (77) examined the effect of individual amino acid substitutions on the regulation of type III secretion by YscF. They targeted conserved residues for replacement by alanine. Alanine substitutions at two positions (Asp77 and lle82) abolished the secretion of YscF. Based on the YscEFG structure (76), Sun et al. suggested that the highly conserved lle82 residue is located at the interface between YscG and YscF and the mutant may therefore destabilize the interaction between them. They also concluded that Asp77 is a solvent-accessible residue on the outer surface of YscF in the heterotrimeric complex. Therefore, the secretion defect exhibited by this mutant is not the result of the failure to properly interact with its chaperones YscE/G in cytosol. Instead, most likely, mutation in D77 will influence the architecture between needle monomers. In this work, we suggest that these two residues (Ile82 and D77) might be important for interactions at the interface between YscF and YscC. In the base of the Yersinia T3SS, YscC forms a ring in the outer membrane that seems to interact with the needle (78). This outer ring of the base is almost exclusively made of YscC. Indeed the Yersinia base has very few other proteins embedded in the outer membrane other than YscC. Almost all the other mentioned membrane proteins are embedded in the inner membrane of the base and two in the translocon. The presence of another structure called the rod linking the outer ring to the inner ring means that the needle originates from the outer ring (YscC) where it makes the needle-base interactions. 23 The aim of this work is to identify some conserved YscF residues that are likely to participate in the interaction at the needle-base interface. During needle formation, it is likely that YscF is released from the YscEFG complex enabling it to participate in new hydrophobic and hydrophilic interactions. The new interactions bury the interacting hydrophobic residues while maintaining the hydrogen bonding to form a stable needle structure. Some interactions are between YscF monomers (forming the needle) while others are between YscF and proteins in the base (linking the needle to the base). We have carried out a study to show that individual replacement of some of the interacting residues (both hydrophilic and hydrophobic) with alanine does not prevent in vitro needle formation. While previous studies have focused mainly on secretion analysis, our study goes a step further by looking at both the in vitro needle formation and in vivo YopQ secretion by the mutated YscF. If a given mutant is able to form needles in vitro but prevents Yop secretion and in vivo needle formation, then we propose that such a residue participates in linking the needle to the base. We have used the information from the four available T3SS structures (71, 72, 73, and 76) and our preliminary in vitro needle assembly results to identify 7 YscF residues that are likely to be important for the interactions between the needle and the base. 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Blocker, A., Jouihri, N., Larquet, E., Gounon, P., Ebel, F., Parsot, C., Sansonetti, P., and Allaoui, A. (2001) Structure and composition of the Shigella flexneri “needle complex” a part of its type III secreton. Mal Microbial 39, 652-663. Joumet, L., Agrain, C., Broz, P., and Cornelis, G. R. (2003) The Needle Length of Bacterial lnjectisomes ls Determined by a Molecular Ruler. Science 302, 1757-1760. Aizawa, S.-l. (2001) Bacterial flagella and type III secretion systems. FEMS Microbiol Lett 202, 157-164. Tampakaki, A. P., Fadouloglou, V. E., Gazi, A. D., Panopoulos, N. J., and Kokkinidis, M. (2004) Conserved features of type III secretion. Cellullar Microbiology 6 (9), 805-816. Allaoui, A., Sheen, R., Lambert de Rouvroit, C., and Cornelis, G. R. (1995) VirG, a Yersinia enterocolitica lipoprotein involved in the Ca2+ dependency is related to Est of pseudomonas aeruginosa. J Bacteriol 177, 4230- 4237. Hoiczyk, E., and Blobel, G. (2001) Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells. Prac Natl Acad Sci USA 98, 4669-4674. Goure, J., Broz, P., Attree, O., Cornelis, GR. and Attree, I. (2005) Protective anti-V antibodies inhibit Pseudamanas and Yersinia translocon assembly within host membranes. J Infect Dis 192, 218-225. Holmstrom, A., Olsson, J. and Cherepanov, P., Maier, E., Nordfelth, R., Pettersson, J., Benz, R., Wolf-Watz, H, and Forsberg, A. (2001) Lch is a channel size-determining component of the Yap effector translocon of Yersinia. Mol Microbiol 39, 620-632. Wattiau, P., and Cornelis, G. R. (1993) Sch, a chaperone-like protein of Yersinia enterocolitica involved in the secretion of YopE. Mol Microbiol 8, 123-131. Boyd, A. P., Lambemtont, I., and Cornelis, G. R. (2000) Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the Sch chaperone binding domain of YopE. 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Zhang, L., Wang, Y., Picking, W. L., Picking, W. D. and De Guzman, R. N. (2006) Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei. J Mol Biol 359, 322-330. Wang, Y., Ouellette, A. N., Egan, C. W., Rathinavelan, T., lm, W., and De Guzman, R. N. (2007) Differences in the electrostatic surfaces of the type III secretion needle proteins Prgl, BsaL, and MxiH. J Mol Biol 371(5), 1304-1314. Kenjale, R., Wilson, J., Zenk, S. F., Saurya, S., Picking, W. L., Picking, W. D., and Blocker, A. (2005) The Needle Component of the Type III Secreton of Shigella Regulates the Activity of the Secretion Apparatus. J Biol Chem 280, 42929-42937. Cordes, F. S., Komoriya, K., Larquet, E., Yang, S., Egelman, E. H., Blocker, A., and Lea, S. M. (2003) Helical Structure of the Needle of the Type III Secretion System of Shigella flexneri. J Biol Chem 278, 17103- 17107. Sun, P., Tropea, J. E, Austin, B. P., Cherry, 8., and Waugh, D. S. (2008) Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG. J Mol Biol 377, 819-830. Torruellas, J., Jackson, M. W., Pennock, J. W., and Plano, G. V. (2005) The Yersinia pestis type III secretion needle plays a role in the regulation of Yap secretion. Mal Microbial 57, 1719-1733. 31 78. Plano, G. V., Day, J. B., and Ferracci, F. (2001) Type III export: new uses for an old pathway. Mal Microbial 40, 1365-2958. 32 Chapter 2 Structural Analysis of YscF and its in vitro Polymerization Examined by Secondary Structure Prediction, Homology Modeling, Electrophoresis, Mutagenesis, Circular Dichroism, and Transmission Electron Microscopy 33 ABSTRACT Structural analysis of YscF and its in vitro needle assembly studies were performed by a combination of secondary-structure prediction, homology modeling, electrophoresis, mutagenesis, circular dichroism spectroscopy, and transmission electron microscopy. This analysis improves our understanding of YscF, whose structure has not been solved to date. Secondary-structure predictions of YscF show four q-helices while homology modeling results show that the N-terminal region of YscF is unstructured in the monomer. This model presents YscF in the form of an o-helical hairpin centered on the tetrapeptide PDNP. Deletion of the first 19 residues of YscF did not hinder in vitro polymerization. This analysis gave more evidence that the N-terminal region of YscF does not control its polymerization into the needle structure. Mutagenesis and circular dichroism spectroscopy show that 7 YscF mutants (Y65A, N66A, K76A, D77A, I82A, 084A and F86A) mentioned in chapter 1 of this work have a high helical content, but Y65F is a random coil. We use analytical gel filtration to elucidate the role of Y65 in YscF needle assembly. This work uses transmission electron microscopy to study in vitro polymerization (needle assembly) of YscF and its mutants listed above. Our results show that mutant N66A is able to prevent YscF needle assembly. 34 INTRODUCTION The Yersinia needle plays an important role in secretion and translocation of Yersinia outer proteins (Yops) into host cells (1-4). It also plays an important role in the cell-contact regulation of type III secretion (1). Cordes et al. (5) used X-ray fiber diffraction and EM to demonstrate that the Shigella flexneri needle shares an identical helical architecture (5.6 subunits per turn, 24-A helical pitch) and inner channel diameter (2 nm) with the flagellar rod, hook, and filament. This structural similarity is likely to exist among other TBSS needles that include Yersinia. The Yersinia needle structure is assembled through self-oligomerization of an 87-residue protein known as YscF (6, 7). During needle assembly, YscF is secreted, but YscG and YscE prevent it from premature polymerization in the cytosol of the bacterium prior to the assembly of the needle (8). The crystal structure of the YscEFG protein complex (9) shows that YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle stnicture (9). Sun et al. (9) showed that the N- terminal 49 residues of YscF are present and disordered in the structure of the heterotrimer YscEFG. The N terminal regions of both MxiH and BsaL (10, 11) are also shown to be disordered in the structures and may not be involved in needle assembly. Based on their study, Deane et al. (10) suggested that either the five C-terminal residues are essential for N-terminal helix formation or the N-terminal region becomes folded only within the intact needle, as it is thought to occur for flagellin (12-14). This suggestion was based on the fact that polymerization was 35 blocked following truncation of the last 5 residues of MxiH (10). The unfolded N- terminus is thought to facilitate easy movement through the central channel of the needle because the size of the channel in the center of the assembly is likely to allow secretion of only single helices and random-coil proteins, rather than fully folded structures (10). In their study, Torruellas et al. (15) examined the effect of individual amino acid substitutions on the regulation of type III secretion by YscF. By targeting conserved residues for replacement by alanine, they showed that substitution of a single amino acid can totally alter/interfere with the function of YscF. Deane et al. (10) generated a model of the Shigella T388 needle by docking the crystal stmcture for MxiH into the 16 A density map from their earlier three-dimensional EM reconstruction of the needle (5). The model shows that the C-terminal helix of MxiH forms the outer shell of the needle core, whereas the PSNP loop directs the N-tenninal helix to line the inner wall of the needle channel. Their model shows that the C terminal region of MxiH is involved in extensive inter-subunit contacts. They suggest that this interaction is important for needle stability because polymerization was blocked following truncation of the last 5 residues of MxiH. Mutational studies of MxiH (16) and YscF (15) have shown that these needle proteins determine induction of their secretion systems. Previous secondary structure predictions of the three YscF homologs, Prgl (Salmonella typhimurium), MxiH, and BsaL revealed q-helical structures (16) that were confirmed by circular dichroism spectroscopy (17). The three YscF homologs also show a big difference in thermal stabilities (17) in contrast with their 36 sequence conservation. Moreover, the electrostatic surfaces of Prgl were shown to differ radically from those of BsaL or MxiH (18). In all three needle proteins, deletion of the last five residues stopped self-polymerization resulting into respective monomers (17, 11, 18) whose structures were solved. Although the structures of three T3SS proteins (MxiH, BsaL and Prgl) have been solved, many questions remain concerning the structure and protein-protein interactions in type III secretion needle proteins. The sequences of these needle proteins suggest that electrostatic contacts are important in needle assembly (18). Wang et al. (18) suggested that needle packing interactions may be different among Shigella, Salmonella and Burkholderia. In the structure of Prgl (18), the surface of the two helix bundle is polar due to residues an: helix (:1 (N22, 021, Q24, T25, Q26, E29, K33, and D32), helix (:2 (Q48, S49, K50, S52, E53, N55, R58, N59, and S62), and the PxxP region (K27, $39, and D40). They also showed that most of these polar residues are pointed away from the two helix bundle and therefore are not needed in stabilizing the hydrophobic core of the two helix bundle. Their work shows that several polar residues are identical (Q48, N59, and S62) or conserved (Q24, E53, N55, and R58) among needle proteins, suggesting that they are important for function other than stabilizing the core domain. They also concluded that the polar surface of the needle protein is most likely important in pathogenesis. Kenjale et al. (16) performed point mutations on MxiH to analyze needle assembly and effector secretion. Their study shows that in S. flexneri MxiH, the L54A or Y57A mutation prevents needle polymerization and secretion of effector 37 proteins. These mutations disrupt the hydrophobic interactions at the helix 01-02 interface, thereby destabilizing the two helix bundle. A similar effect is reported in Prgl (18), another YscF homologue. The same effect is likely to exist in YscF due to the conservation of these residues among the needle proteins. These findings suggest that needle assembly depends on proper folding of the two-helix bundle. It has been shown that the last five residues of Prgl, BsaL, and MxiH are important in needle monomer-monomer interaction (16, 17). In addition, Wang et al. (18) demonstrated that electrostatic interaction is an important component of needle assembly. Their suggestion is based on the presence of distinct areas of positively and negatively charged surface potentials on Prgl, MxiH, and BsaL. They concluded that the most significant difference among these needle proteins is in the arrangement of their electrostatic surfaces, and that despite primary sequence conservation, protein-protein interaction maybe different among these needle proteins (18). Factors like ionic strength and electrostatic interaction affect needle assembly (18). By adjusting the pH from 8 to 10.5, Marlovits et al. (19) were able to dissociate the needle, but not the basal structure of the S. typhimurium needle apparatus. Their study showed the effect of pH on needle assembly. Wang et al. (18) have shown that the polar surface of the two-helix bundle is a common feature among needle proteins. They also demonstrated that conserved tyrosines contribute to surface polarity. In comparing Prgl to BsaL, and MxiH, their study confirmed that the three structures do not show a particular need for tyrosines in position Y47 and Y54 because phenylalanine would have maintained similar 38 hydrophobic contacts and the overall fold of the two-helix bundle. Owing to the fact that Y47 and Y54 are identical among Prgl, BsaL, and MxiH, they hypothesized that the tyrosyl hydroxyl groups of these conserved residues are important in protein-protein interactions with other needle monomers or with the tip complex (20). Based on our homology model of YscF, Y65 is likely to be involved in similar protein-protein interactions with other needle monomers or with the base proteins because it appears pointed away from the two-helix bundle and does not appear to contribute in stabilizing the core structure of the needle protein. Kenjale et al. (16) also developed a model to show that signaling of host-cell contact is relayed through the needle via intersubunit contacts. Here, we predict the YscF secondary structure (Figure 2.1) by submitting the sequence to four different programs, namely PsiPRED (21, 22), SAM (23), PROFsec and SABLE2 (24). Furthermore, we use homology modeling to build the YscF homology model (Figure 2.2) based on the existing structures of YscF homologs. We also show that cleavage of the first 19 residues of YscF does not hinder in vitro needle assembly. Using circular dichroism spectroscopy, we determine the secondary structure of YscF, YscFA4 (YscF with C-terminal truncation) and its mutants. We use analytical gel filtration to elucidate the role of Y65 in YscF polymerization. In MxiH, many single mutations (W10A, Y57A, K69A, K72A, and R83A) are capable of severely altering needle polymerization (16). We use single mutations and transmission electron microscopy to study in vitro YscF needle assembly and investigate the role of 7 YscF conserved 39 residues in needle assembly. We use this study to identify YscF conserved residues that don’t necessarily affect in vitro needle assembly. 40 EXPERIMENTAL PROCEDURES Multiple sequence alignment. The YscF sequence was submitted to the NCBI BLAST server (25, 15) to identify homologous sequences; using a cutoff E- value of 10's, forty-six sequences were identified. However, many of these homologs were redundant, e.g., several versions within the genus Yersinia or closely related genera. Therefore, the redundant sequences were eliminated to obtain a set of 21 sequences with no more than 75% sequence identity among them. These 21 species were submitted to the T-Coffee server (27) for multiple sequence alignment, the best-validated algorithm. The results were visualized by the publicly available server ESPript (28), and analyzed numerically to rank the residues by their degree of conservation (data not shown here). YscF structure prediction. The YscF secondary-structure prediction (Figure 2.1) was done by submitting the Sequence to four different programs, namely PsiPRED (21, 22), SAM (23), PROFsec and SABLE2 (24). The results were visualized using our web- serverzhttpzllproteinsmsu.edu/Servers/Secondary_Structure/visualize_secondary _structure_predictions.html. To get the homology model (Figure 2.2), the YscF sequence was submitted to http://bioinfo.pl/Meta/, a well-validated and publicly available server for consensus fold recognition. The server easily identified the two available structures of type III pilins (ZGOU and 2CA5) as the best parent structures. For each of these two structures, the best alignment to the YscF sequence was identified and converted into a backbone homology model for YscF using in-house laboratory software. The conformation of a side chain was 41 preserved if it was identical to its counterpart in the parent structures; athenrvise, the side chain was added using the publicly available SCWRL program. Due to their high sequence similarity, few if any insertions or deletions are needed to align the sequences; the YscF sequence and those of the parent structures match almost over their entire lengths. The resulting homology models were visualized using MOLMOL. Comparison of de nova protein structure predictions for YscF with experimental structures of its homologs and the YscEFG complex. The YscF sequence was subjected to the ROSETTA protocol for de novo folding (29), which produces predictions of protein conformations by gradually minimizing a statistical potential energy that measures how plausible a given conformation is for a given protein sequence. In the process, 30,000 such predictions were made for the YscF structure by my thesis advisor, Prof. William Wedemeyer. All predictions were independent of one another and were clustered structurally into families with similar conformations. As shown in Figure 2.3, the three largest clusters (denoted as clusters 0, 1 and 2) account for most of the predictions, meaning that the other clusters could be viewed as structurally similar to these three. Cloning, protein expression and purification. The YscFA4 construct involved C-terrninal modifications of yscF gene by removal of the three C- terminal amino acid residues (KFP), 084E single mutation and introduction of a C-terminal His-tag as indicated in the underlined sequence below: MSNFSGFTKGTDIADLDAVAQTLKKPADDANKAVNDSIAALKDKPDNPALLADL QHSINKWSVIYNINSTIVRSMKDLMQGILQKFP (YscF). 42 MSNFSGFTKGTDIADLDAVAQTLKKPADDANKAVNDSIAALKDKPDNPALLADL QHSINKWSVIYNINSTIVRSMKDLMQGILEHHHHHH (YscFA4). This modification was done based on results from other YscF homologs (10, 11) in which it prevented self-association of monomeric MxiH and BsaL. The YscF coding sequence was PCR-amplified (30 cycles of 40 s at 95 °C, 40 s at 55 °C and 1 min 30 s at 68 °C) from a plasmid (pMMB 867) containing the YscEFG construct obtained from Dr. Michael Bagdasarian (Michigan State University- Microbiology and Molecular Genetics) using primers 5'- GGA ATT CCA TAT §AG TAA ATT CTC TGG A‘l'l' TAC GAA AGG -3' (fonrvard) and 5'- CCG GQI _CQAQGA TGC CTT GCA 'ITA AGT CTT TCA TG -3' (reverse). We designed a second set of primers, 5’- GGA ATT CCA TAT GGCT CAA ACG CTC AAG AAG CCA GCA GA-3' (fonrvard) and 5'- CCG GCT CGA GGA TGC CTT GCA TTA AGT C'l'l’ TCA TG -3' (reverse) to truncate the first N-terminal 19 residues of YscFA4. This second construct was named A19YscFA4 and was used to study the role played by the N-terminal region in YscF needle formation. Both sets of fonrvard and reverse primers were designed with Ndel and Xhol restriction sites (underlined) respectively. The resulting PCR products were digested with Ndel and Xhol then ligated (30) into similarly digested pET22b vector. The correct sequences were verified by DNA sequencing then the expression plasmids containing YscFA4 and A19YscFA4 were transformed into E. coli DH50 for replication. The plasmids were isolated using the Qiagen plasmid isolation kit (Qiagen Inc.) then transformed into E.coli BL21(DE3) for protein production. 43 Cell cultures were grown in LB containing 100 pg/mL ampicilin. In each case a single colony was picked from the plate and used to inoculate 5 mL of liquid LB medium containing 100 ug/mL ampicilin. The culture was incubated at 37°C for 6 hours with shaking at 250 rpm. This culture was added to 1.0 L of LB medium as a starter culture. The 1.0 L culture was then incubated at 37°C with shaking to an 00600 of approximately 0.9. Protein expression was induced by addition of filter-sterilized isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM and grown for 4 more hours with shaking (250 rpm) at 37°C. The culture was pelleted by centrifugation (80009) at 4°C for 20 minutes then pellet resuspended in nickel-A buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM Imidazole, 5 mM B-Mercaptoethanol and EDTA-free Protease Inhibitor tablet) containing 6 M guanidine hydrochloride. Following sonication, the extract was incubated at room temperature with gentle shaking for 30 minutes. The extract was centrifuged at 20,000g for 20 min to remove insoluble debris. The protein was purified via the C-terminal His tag by nickel-chelation chromatography. The unbound protein was washed off by 3 column volumes of nickel-A buffer followed by 3 column volumes of wash buffer (nickel-A buffer plus 600 mM NaCl). The protein was eluted using nickel-B buffer (50 mM NaHzPO4, pH 8.0, 300 mM NaCl, 300 mM Imidazole, 5 mM B-Mercaptoethanol and EDTA- free Protease Inhibitor tablet) (31). The purified protein was verified by protein N- terminal sequencing, Western blot and MALDI-TOF mass spectrometry. Site—directed mutagenesis. The following six point mutations were each introduced on plasmid stcFA4 (pET22b containing the YscFA4 gene) using the 44 QuikChange XL Site-Directed Mutagenesis Kit (Stratagene): Y65A, Y65F, N66A, K76A, D77A and l82A. Each of the listed forward primers (Table 2.1) was used in a PCR reaction (18 cycles of 50 s at 95 °C, 50 S at 50 °C and 8 min at 72 °C) with its complement that is not listed (mutated codons are underlined). The PfuTurba® master mix (Stratagene) was used in the PCR reaction. After each PCR reaction, Dpnl was added then sample incubated at 37 °C for 1 h to digest the template plasmid. Each sample was used to transform E. coli DH5a cells for plasmid replication according to the mutagenesis kit instructions. Replicated plasmids were isolated using the Qiagen plasmid isolation kit (Qiagen Inc.) before submitting samples for DNA sequencing. Each mutant plasmid was used to transform E.coli BL21(DE3) for protein production and purification following the same method described above for YscFA4 (the wild type in this case). Analysis of protein expression and purification was done by denaturing gel electrophoresis (32) and standard protein assays (33) respectively. Analytical gel filtration chromatography. The hydrodynamic radius assay of the Y65AYscFA4 mutant was carried out by analytical gel filtration chromatography at pH 5.5 and pH 8.0 on a HiPrep 26/60 S-100 HR sephacryl column (Amersham Biosciences). This enabled us to determine the effective molecular weight of this mutant at both pH values. The column had been calibrated with bovine pancreatic ribonuclease A, chymotrypsinogen A, ovalbumin, and albumin by plotting the logarithm of molecular weight versus elution volume. The effective molecular weight of the protein was determined from the following equation: 45 Ve — Vo = -43.9 In (mol. wt.) + 208.25 where V., is the elution volume and V0 is the void volume. Circular dichroism (CD) spectroscopy. Circular dichroism experiments were done at protein concentrations ranging between 10 uM to 40 pM using a 1 cm path-length cell. The protein was prepared in 10 mM NaHgPO4, pH 7.5, 10 mM NaF. The acquisition of CD spectra was done at 24°C over the far-UV range of 190 nm - 250 nm on a Chirascan CD spectrophotometer (Applied Photophysics) in continuous scanning mode at 15-20 nm/min. Spectra were acquired in triplicate and averaged. The temperature was controlled by a circulating water bath and the data analyzed with DICHROWEB server (34). Needle formation in vitro and electron microscopy. For each protein, needle assembly was induced through concentration by ultrafiltration to 10 mg/mL in 50 mM NaHzPO4, pH 7.5, 300 mM NaF. In case of Y65AYscFA4, we went a step further to analyze the effect of pH on its needle assembly process. For this protein, we also used 50 mM NaHzPO4, pH 5.5, 300 mM NaF to compare needle assembly at pH 8.0 and 5.5. All samples were frozen at -20°C for a minimum of 24 hours and thawed at 4°C for 12 hours before transmission electron microscopy (TEM) studies. A 2% uranyl acetate solution in 50% ethanol was used for negative staining. The pictures were taken between 80000x and 140000x magnification on a JEOL (Japan Electron Optics Laboratories) 100CXII microscope. 46 RESULTS Multiple sequence alignment. The YscF conserved residues targeted for site-directed mutagenesis were chosen based an atomic structures of three YscF homologs, MxiH (Figure 1.2, chapter 1) (10), BsaL (Figure 1.3, chapter 1) (11), and Prgl (Figure 1.4, chapter 1) (18). These three present the only available T3SS needle protein structures and show high sequence identity to YscF. More information was also deduced from the structure of YscEFG complex (Figure 1.5, chapter 1) (9). The multiple-sequence alignment results show a high degree of conservation in the C-terminal region. YscF structure prediction. Results from all four secondary structure prediction programs show that YscF has high helical content with four o-helices (Figure 2.1) labeled (:1 (residue 13-24), 02 (residue 30-42), (13 (residue 48-57) and 04 (residue 69-85). These results are consistent with the four helices observed in the BsaL structure although they vary slightly from the MxiH structure, which had only 3 helices. A portion of the N-terminal region was not solved in the MxiH structure probably because the fourth helix was not stable enough to be solved by X-ray crystallography. As shown in the secondary- structure predictions, the YscF structure might also have the two helix bundle fold around the PDNP coil. This suggestion is further confirmed by our YscF homology model (Figure 2.2) that confirms atleast 3 of the predicted four a- helices. During our homology modeling of YscF, MxiH was picked by the server as the best parent structure due to the high sequence similarity between them. Since the MxiH structure shows only three a helices, it limits the YscF model to 47 three a helices (02, a3 and 04). We would have probably seen 4 helices in our YscF model if BsaL was used as the best parent structure. In this study, all the mutated residues except Y65 are located in the region corresponding to predicted helix 4 (C-terminal region). The conserved residue, Y65 is located in the hinge region between (13 and 04. Comparing the YscF homology model to both MxiH and BsaL structures show that all the seven conserved residues in this study (Y65, N66, K76, D77, I82, Q84 and F86) are exposed on the surface of the YscF monomer because they all point away from the two-helix bundle. Comparison of de novo protein structure predictions for YscF with experimental structures of its homologs and the YscEFG complex. The predicted tertiary structures fell into three main families, which were denoted as cluster 0, cluster 1 and cluster 2 (Figure 2.3). All three families have the 4 helices (01, 02, as, and (14) (Figure 2.4) found in the secondary-structure predictions (Figure 2.1). All three families of predicted structures also form a hairpin between 02 and 03. The N-terrninal region of a1 (roughly residues 1-15) appears to be unstructured in all three clusters. In clusters 0 and 2, a4forms a three-helix bundle with the 02-03 hairpin. Specifically, in cluster 2, the N-terminal 01 joins with (12 to form one long initial d-helix, whereas in cluster 0, a1 is roughly perpendicular to the bundle, running parallel to the loop connecting a3 and (14. By contrast, in cluster 1, a1 is positioned between 04 and the 02-03 hairpin; in this case, the a-helices are short and often bent. When the predicted structures are compared to the experimentally determined structures of three T3SS proteins BsaL (11), MxiH (10) and Prgl (18) (Figure 2.5), they show a common feature, the predicted 02-03 hairpin structure. 48 In all three experimental T3SS protein structures, (14 does not form a bundle with the 02-03 hairpin; rather, it projects away, but not in a straight line with (13. Both MxiH and Prgl structures (Figure 2.5) lack 01, whereas BsaL (Figure 2.5) has a partially unstructured (11 forming a bundle with the 02-03 hairpin. Finally, the YscEFG complex (Figure 2.5) shows YscF having an 03-04 hairpin, as predicted in clusters 0 and 2. The positions of the YscF helices oz and (13 agree across the T3SS protein structures (Table 2.2), suggesting that the o2-a3 hairpin forms the folding core of the T388 pilin. The 03-04 hairpin found in the YscEFG complex may also occur in YscF when it folds to form a pilus. Alternatively, given the minimal hydrophobic core of the predicted YscF structures (Figure 2.4), YscF could refold (including the possibility of domain swapping) when it assembles to form pill. 49 Helix PsiPRED _ SAM ~ PROFsec SABLE2‘,. STRAAI) PgPRED' * SAM PROFsec i SABLE2 aw E—F-rl-r- .H-i-fl 4m »~.-*‘ ~-.---. LOOP PSIPRED' 1‘ rt 31‘!— _—r“--.:‘ “-31! " r-‘r—‘Itg SAM I I ‘ " ‘ ‘ g ‘ . If i; Iv ' ' ' . _ ". "I" ' I PROFsecI 0 I _ j . . Figure 2.1. Secondary structure predictions for YscF by four different methods, PsiPRED, SAM, PROFsec and SABLE2. Four helices are predicted (structured regions of the sequence are highlighted in red. 50 Figure 2.2. YscF homology model based on the MxiH crystal structure. Some of the selected conserved residues are shown on predicted oz and 03-04 helices. Blue represents N terminus while red represents C terminus. Some of the residues are involved in 02 — 03 hydrophobic interactions to keep the two core helices close to each other. Site-directed mutagenesis. To demonstrate the effect of some conserved residues on the YscFA4 secondary structure and in vitro needle assembly, the corresponding codons were each altered to alanine. The expression of all the mutant proteins was comparable to YscFA4 (Figure 2.6) except Y65AYscFA4 (Figure 2.7) and l82AYscFA4 (some of the data not shown here). Expression of Y65AYscFA4 was much more than YscFA4, while l82AYscFA4 was much less than YscFA4. There was a noticeable variation in solubility of the mutants compared to the wild type, with Y65AYscFA4 being the most soluble. Protein expression and purification. The 6 M GnHCl was added to the extraction buffer because YscFA4 and all its mutants (except Y65AYscFA4) did not bind onto the nickel column without the denaturant. This is probably due to 51 polymerization that occurs at high protein concentrations thereby shielding the Hiss-tag. However, the Y65A mutant binds to the nickel column in absence of the denaturant (Figure 2.8). Native gel analysis (Figure 2.9) shows polymerization of A19YscFA4 confirming that the N-terminal region does not affect YscF polymerization. The gel shows a higher molecular weight band (~1040 kDa) yet the corresponding MALDI-TOF-MS results show the molecular weight of A19YscFA4 (7.7 kDa). In their work, Matson et al. (35) also observed higher molecular weight bands representing multimers of YscF. Analytical gel filtration chromatography. Unlike purification of YscFA4 and other mutants that require a denaturant, Y65A binds onto the nickel column without 6 M GnHCl (Figure 2.8). These results prompted us to analyze Y65AYscFA4 further by analytical gel filtration chromatography. We monitored the protein at pH 5.5 and 8.0 to analyze the effect of pH on its polymerization. Each buffer resulted in a different elution volume for the same protein as depicted by the two peaks (Figure 2.10) representing a shift in elution volume. At pH 8.0 the effective molecular weight is ~ 70 kDa while the same protein has an effective molecular weight of 22 kDa at pH 5.5. The protein elutes as a dimer at the lower pH, but it polymerizes further (7-mer) at a higher pH. Circular dichroism spectroscopy. The CD spectra for YscFA4, A19YscFA4 and all the mutant proteins analyzed except Y65FYscFA4 show a high helical content with a double minimum (ellipticity) at around 208 nm and 222 nm corresponding to the o-helix (Figure 2.11) (some data not shown here). We used the Y65FYscFA4 mutant to further understand the characteristics of 52 Y65AYscFA4. It gave a spectrum with a single minimum at around 204 (Figure 2.12) corresponding to a random coil. The DICHROWEB server (34) was used for deconvolution and analysis of all the spectra and the results were tabulated (Table 2.3). The helical content is comparable in YscF and YscFA4 (61% and 60% respectively). All the mutants have a high helical content except Y65FYscFA4 that has 55% random coils. All proteins analyzed had low IS-strand content except Y65FYscFA4 that had 19%. Residue Y65 seems to be important for YscF folding. Needle formation in vitro. In vitro needle formation was induced in YscFA4, A19YscFA4 and in all the mutants except N66AYscFA4 and Y65FYscFA4 (Figure 2.13). The N66AYscFA4 mutant did not form needles probably due to the importance of the N66 residue in needle assembly. However, the inability of Y65FYscFA4 to form needles may indicate the importance of the hydroxyl group of Y65 in needle assembly. The A19YscFA4 protein formed needles (Figure 2.13) even though it lacks the first 19 residues. This supports earlier results in YscF/YscG system (9) or in the MxiH system (10), showing that the N-terrninal region does not affect polymerization (needle assembly). In all the cases where needles formed, the process took 24 hours or less. This trend was not observed in Y65AYscFA4 because the formation of detectable needles took almost 10 days. We analyzed Y65AYscFA4 needle formation at pH 5.5 and pH 8.0 and the two results were different. At pH 5.5, the protein did not form needles even after 14 days. At pH 8.0, the needle-formation process was much slower than in YscFA4, but needles were eventually formed (Figure 2.14). Lowering the 53 pH from 8.0 to 5.5 had an effect of completely hindering needle assembly in Y65AYscFA4. This result complements the analytical gel filtration results that showed Y65AYscFA4 as a dimer at pH 5.5, and a 7-mer at pH 8.0. 54 6_34 __._ _ _.. 12_25 55 8_30 l3_24 1 7 17_21 7 3 11_28 149 53 4_49 20_2o__, 1123.3 9_30 720 14_24 l9_20 18_20 7 57 5 37 10_30 101 Figure 2.3. YscF structural families from de novo predictions. (a) Each family has similar conformations resulting in three main families (cluster 0, cluster 1, and cluster 2). (b) Cluster 0, showing contacts between (12 and 03 and between 03 and 04. (c) Cluster 1, showing contact between 02 and 03. (d) Cluster 2, showing contact between 02 and as and between 03 and a4. 55 Figure 2.3 (cont’d) 57 Figure 2.4. De novo YscF structure predictions showing the side chains for Y65, N66, K76, D77, I82, Q84 and F86 in: (a) cluster 0, (b) cluster 1, and (c) cluster 2. The coloring of the main-chain ribbons is as follows: blue - (11, green - C12, yellow - (13 and red - (14. 58 Figure 2.4 (cont’d) 59 o q a 02 03 1 2 3 w °3 “4 a a4 4 . YscF (inactive form) Prgl and Mer BsaL (YscEFG complex) Figure 2.5. TBSS secondary structure models based on the atomic structures of Prgl (18) (PDB ID: 2JOW), MxiH (10) (PDB ID: 2CA5), BsaL (11) (PDB ID: 2GOU) and the inactive form of YscF in the YscEFG complex (9) (PDB ID: 2P58). 6O 24.9 kDa 18.3 kDa 13.9 kDa If I 5.7 kDa . « 5,, Figure 2.6. 12% SDS-PAGE gel of purified YscFA4. 24.9 kDa 18.3 kDa 13.9 kDa 5.7 kDa - Figure 2.7. (a) 12% SDS-PAGE gel of purified Y65AYscFA4 ~ 10 kDa. Note: small amounts of trimer visible ~ 30 kDa. 61 YscFA4 Y65AYscFA4 Figure 2.8. Analysis of the Nickel column binding properties of YscFA4 and Y65AYscFA4 in absence of GnHCl. (a-c) Soluble protein extracts following sonication and centrifugation. Both proteins are highly soluble in the extraction buffer. Note: The extraction buffer does not contain GnHCI. (d and e) Nickel column loading flow through. Note: YscFA4 flows through without binding, while Y65AYscFA4 binds. (f) Column wash. (g) Protein elution. Note: Y65AYscFA4 binds onto the column even in absence of the denaturant. 1040 kD 242 kD 146 kD 66 kD 20 kD Figure 2.9. Polymerization of A19YscFA4. Native gel electrophoresis results showing polymerization in absence of residues 1-19 of YscF. 62 150 100 0 pH 8.0 IJgH 5.5 mL Ve — Vo = - 43.9 In (mol. wt.) + 208.25 Figure 2.10. Analytical gel filtration of Y65AYscFA4 under two different pH conditions. The protein elutes as a dimer at pH 5.5 while it elutes as a multimer at pH 8.0. 63 7 208 218 228 238 I O.) ._L (D m Ellipticity &> -13 - -18 Wavelength (nm) O r r r I ‘ . |\ z””:;;; -5 198 208 218 228 238 -10 _ -15 _ -20 - -25 _ -30 _ Ellipticity Wavelength (nm) b Figure 2.11. CD spectra showing d-helical structures: (a) YscF, (b) YscFA4, and (c) N66AYscFA4. 64 -1O 1 98 208 218 228 233 248 Ellipticity Wavelength (nm) Figure 2.11 (cont’d) 65 Ellipticity Wavelength (nm) -10 199 209 219 229 239 249 Ellipticity Wavelength (nm) b Figure 2.12. CD spectra comparing Y65AYscFA4 and Y65FYscFA4: (a) Y65AYscFA4 showing o-helical structure, and (b) Y65FYscFA4 showing a random coil structure. 66 b Figure 2.13. In vitro needle formation: (a) YscFA4 formed needles, (b) A19YscFA4 formed needles even with the N-terminal truncation, (c) N66AYscFA4, and (d) Y65FYscFA4 did not form needles. 67 Figure 2.13 (cont’d) 68 b Figure 2.14. In vitro needle formation for Y65AYscFA4 in different buffers: (a) At pH 8.0 needles formed, and (b) at pH 5.5, Y65AYscFA4 does not form needles. 69 Table 2.1. Oligonucleotides used to generate YscF mutants. Mutated codons are underlined. Y65A 5'- AATAAATGGTCGGTAATTQLEAATATAAACTCAACCATA -3' Y65F 5'- AATAAATGGTCGGTAA'ITflAATATAAACTCAACCATA -3' N66A 5'- AAATGGTCGGTAAT'ITACQQIATAAACTCAACCATAGTT -3' K76A 5'- ACCATAGTTCGTAGCATGQQAGACTTAATGCAAGGCATC-3' D77A 5'- ATAGTTCGTAGCATGAAAflTTAATGCAAGGCATCCTA -3' |82A 5'- AAAGACTTAATGCAAGGCGCCCTACAGAAGTTCCCATAA -3' Q84A 5'- TTAATGCAAGGCATCCTAGCGAAGTTCCCATAATATGAA -3' F86A 5'- CAAGGCATCCTACAGAAGGCCCCATAATATGAAATATAA -3' Table 2.2. Comparing positions of the predicted YscF helices 01, oz, 03 and (14 in relation to the atomic structures of Prgl (18) (PDB ID: 2JOW), MxiH (10) (PDB ID: ZCA5), BsaL (11) (PDB ID: 2GOU) and the inactive form of YscF in the YscEFG complex (9) (PDB ID: 2P58). Note: All the residues are numbered according to the YscF sequence in the multiple sequence alignment (Figure 1.6, chapter 1). Helix (11 02 (13 a4 YscF (proposed) I13 - L24 A30 — K44 P48 — I64 T70 — K85 YscF _ _ L50 — I64 N68 — P87 (YscEFG complex) . _ K24 — K44 A49 — W61 $62 — Q84 MXIH prgl — A27 — K42 P48 - $69 077 - Q84 BsaL K9 — P26 D28 — K44 A49 — N66 869 — |82 7O Table 2.3. Summary of results from the deconvoluted far-UV CD spectra into various components of secondary structure in YscF, YscFA4 and its mutants. Note: All experiments were done at 24°C. Reported values are percentages of the total structure. Protein Helix Strand Random YscF 61 8 3O YscFA4 6O 7 32 Y65A YscFA4 73 2 25 N66A YscFA4 65 5 29 Y65F YscFA4 26 19 55 K76A YscFA4 62 6 32 D77A YscFA4 68 4 27 l82A YscFA4 59 7 34 71 DISCUSSION Our work has used circular dichroism to show that YscF secondary structure has a high q-helical content similar to MxiH and BsaL. We confirmed that the N-terminal region of YscF does not affect needle assembly, just as previously hypothesized in YscF/YscG complex (9) and MxiH (10). Protein bands corresponding to oligomers are evident through native gel electrophoresis even after cleaving off the first 19 residues. This was further confirmed by observation of needle formation in solution. Despite the high sequence identity among YscF and its two homologs, MxiH and BsaL, there is a significant difference. The two atomic structures were solved after deletion of five C-terminal residues and introduction of a Hise-tag (16) because this modification totally prevented polymerization in BsaL and MxiH, thereby improving the solubility of the protein. In our work, the same modification (YscFA4) significantly reduced polymerization in YscF and improved its solubility. However, we noticed polymerization in YscFA4 especially at higher protein concentrations. Generally, we were able to extract and purify YscFA4, A19YscFA4 and all the mutant proteins mainly because of this modification. It was also noticeable that polymerization rate was much slower in Y65A. Results from this work confirm a previous hypothesis (18) that differences exist in the electrostatic surfaces of the type III secretion needle proteins Prgl, BsaL, MxiH and YscF. Indeed YscF has a much higher theoretical pl (~7.77) compared to Prgl (4.76), BsaL (4.76) and MxiH (4.47). These three homologs are acidic and their electrostatic maps show large areas of negatively charged surfaces (18). The locations of negatively charged surfaces are reported 72 to be radically different among Prgl, BsaL, and MxiH (18). All these differences indicate that YscF needles might have a unique pattern of assembly. Despite the 30% sequence identity between YscF and both MxiH and BsaL, YscF might have some different polymerization characteristics. This opinion is also supported by Sun et al. (9) based on their reported YscEFG structure. The CD spectrum shows that Y65FYscFA4 is a random coil and therefore not expected to form needles because it does not form a—helices that are essential for needle assembly. Most of the proteins we looked at show a high helical content. This is in line with the findings of Kenjale et al. (16) that show the importance of q-helix in T3SS needle formation. The high helical content is a common characteristic among the T388 needle proteins like MxiH (50%) and Prgl (59%) (17). These values represent a significant loss in helical content because the studies were done at 25°C as opposed to 10°C that gave 53% (MxiH) and 69% (Prgl) (17). Such results show that lower temperatures promote/stabilize formation of d-helical structures. It may also mean that these proteins are relatively unstable especially at higher temperatures (17). We apply this principle (low temperatures) to induce in vitro needle formation because proper needle assembly requires d-helical structures. Although the CD spectrum for N66AYscFA4 shows d-helical structures, the protein does not form needles in vitro like YscFA4 and other mutants. This mutant might be a monomer since it prevents needle assembly completely. Our YscF model shows these conserved residues (Y65, N66, K76, D77, I82, 084 and F86) pointed away from the two helix bundle. We propose that these residues 73 are not needed in stabilizing the hydrophobic core of the two helix bundle because mutating them to alanine does not disrupt the a-helical structures. Needle assembly results also show that mutating these residues (K76A, D77A, l82A, Q84A and F86A) does not interfere with needle assembly. We propose that these conserved residues don’t participate in needle monomer-monomer contact, but rather play a role in the interaction at the needle-base interface. Although Y65AYscFA4 forms needles, it slows down the process significantly. One of our mutants, Y65AYscFA4 has tremendously slowed down the rate of polymerization. A combination of this mutation and lowering the pH to 5.5 has abolished oligomerization and prevented needle assembly by maintaining it as a dimer. The hydroxyl group of Y65 might be involved in hydrogen bonding that is destroyed in the mutant, thereby interfering with needle monomer-monomer contact. This is supported by evidence from another mutant, Y65FYscFA4 that we analyzed for more evidence. The resulting protein has much lower solubility and gives a CD spectrum corresponding to a random coil. The CD spectrum for this mutant supports the corresponding results from electron microscopy because a random coil structure cannot support needle formation. This highlights the importance of the hydroxyl group as opposed to just hydrophobic interaction. 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BMC Microbiology 5, 38. 78 CHAPTER 3 In vivo Analysis of the Functions of YscF Variant Proteins 79 ABSTRACT The effect of YscF protein variants on YopQ secretion in Yersinia pseudotuberculosis was analyzed using GFP-YopQ18 and GFP-YopQS3 hybrid proteins. We transformed wild-type Y. pseudotuberculosis cells with a parent plasmid stcF (YscF in pET22b) and each of 8 stcF mutants to determine their effect on wild-type YopQ secretion. We used A red recombinase, a helper plasmid to knock out the yscF gene from Y. pseudotuberculosis virulence plasmid pW. The yscF ' model resulting from complete deletion of the gene was used to analyze secretion. We used the same plasmids and mutants as described above to transform the YscF knockout model and analyze their effect on GFP-Yop053 secretion. We detected and analyzed the secreted GFP- YopQ53 by Western blot using anti-GFP antibodies. Through this method, we have been able to identify anomalous phenotypes in secretion. We have combined results from these assays with those from chapter 2 of this thesis to identify two YscF mutants, Y65A and Y65F that display a dominant negative phenotype in wild-type Y. pseudotuberculosis. Based on our findings, we also propose that the conserved residue l82 of YscF may be involved in the interaction at the needle-base interface. 80 INTRODUCTION Yersinia pathogens invade the eukaryotic system by binding to host cells (1) using different adhesion factors like invasin (2) and YadA (3). The adhesion factors used may vary depending on environmental conditions such as temperature. Efficient injection of Yersinia outer proteins (Yops) such as YopE and YopH into host cells requires the pathogens to properly stick to the target cells (1, 5). These pathogens use toxins to evade oxidative burst, phagocytosis and killing by polymorphonuclear leukocytes (6). In Yersinia, these virulence factors are encoded by a 70-kb plasmid called pYV (7, 8). Yops were historically called Yersinia outer membrane proteins at the time when they were believed to be strictly membrane proteins, but over time they have been identified as secreted proteins (9). Their secretion involves the use of Ysc (Yersinia secretion) structure that is encoded by the same virulence plasmid (9, 10). The Ysc system comprises many proteins including YscF (described in chapter 1 and 2 of this thesis), the needle protein that is essential for Yop secretion. Due to their environmental sensitivity, Yersinia can secrete toxins in vitro (culture medium) when Ca2+ is depleted from the medium at 37°C. In vivo (eukaryotic cell culture or mouse model) secretion is also turned on during contact with a eukaryotic cell. Regulation of these secretion genes is controlled mainly by the VirF factor (11). Yersinia relies on YopE and YopH for evasion of phagocytosis (12) and Rosqvist et al. (13) have shown that lack of YopH leads to phagocytosis of pathogens by macrophages. Some secreted toxins have been shown to cause apoptosis in a cell-specific manner in vitro (14) whereas others are known to play 81 a critical role in preventing the oxidative burst of immune cells (15). Some Yops like Lch, YopM, YopQ/YopK, and YopR are readily soluble in culture medium while others like YopH, YopE, YopO/kaA, YopB, YopD, YopP/YopJ, and YopN/LcrE are insoluble (9). Under conducive environmental conditions, pathogens can secrete large amounts of toxins which vary in stability; the unstable ones can be broken down by proteases over a short period of time (16, 17). Most Yops including YopQ (9) that we use in this study have a minimum region necessary for secretion and translocation, e.g., the first 15 codons for YopN (18) and the first 17 codons for YopH (19). For this reason, secretion of most of the Yops is controlled by their N-terminal sequences. Another secretion signal based on chaperones has been described (20) in some Yops, although the short N-terminal sequence secretion signal is more important. Translocation of Yops into target cells is unidirectional (polarized) in such a way that toxins are channeled into the host-cell cytosol (21). YopB and YopD bind host-cell membranes (22) to make the pore at the translocon and are believed to interact with Lch (23), another regulatory protein involved in calcium response (24, 25). Due to regulation by Lch, in vitro Yop secretion will only occur at 37°C in the absence of Ca2+ (Ca2+- dependency) (26). It is believed that secretion and assembly of YopB and YopD at the translocon pore is regulated by Lch; together, these three proteins play an important role in translocation of toxins to the target cell cytosol (22, 23). In Yersinia, YopQ/YopK (Y. enterocolitica/ Y. pseudotuberculosis) control translocation (27) by regulating the pore size at the translocon (28). 82 Jacobi et al. (29) constructed truncated yopE (23 kDa cytotoxin) genes fused to gfp (encoding the green fluorescent protein) (30, 31) to study yopE gene expression and GFP-YopE hybrid protein translocation of Y. enterocolitica in cell culture and mouse infection models. Since full-length GFP is required for fluorescence, it was necessary to use only the minimum YopE sequence necessary for secretion and translocation. This enabled them to use a much smaller protein because the secretion and translocation signals of YopE are found within the first 53 residues. They used the low-copy-number plasmid pACYC184 for their GFP-YopE construct. Their method was successful in applying gfp as a reporter gene for the study of protein translocation by protein T3SS and differential virulence gene expression in vivo. Other methods have used different reporter-gene technologies for the analysis of virulence gene expression of microorganisms (29). Such methods include the YopK—IacZ fusions (32) and quAB operon fusions (33). However, these methods assay enzyme activity of the reporter-gene product, thereby limiting their use because they cannot be applied in: (i) live microorganisms or target cells and (ii) single bacterial cells (29). The use of GFP is more advantageous because it is more stable against shifts of pH, ionic strength or temperature (29). They used a conventional fluorescence microscope to detect GFP production. GFP fluorescence detection (unlike other reporter genes, for instance CAT, b-galactosidase or luciferase) does not require additional gene products or secondary substrates and occurs, with a few known exceptions, in a species-independent fashion (29). It has been shown that both 83 N- and C-terrninal protein fusions remain fluorescent just like the native GFP (34). Induction of yopE transcription and translocation was achieved by shifting the bacterial culture from 27°C to 37°C in a Ca2+-depleted medium (29). However, their secretion assays showed that the amount of secreted GFP-YopE hybrid protein is less than that of YopE probably because GFP folds into its mature barrel structure of 24A° by 42A° in the cytosol (35). The formed barrel structure may be too large to pass the secretion pores hence accumulates in the cytoplasm whereas unfolded GFP-YopE may be predominantly secreted (29). They offered another possible explanation that binding of Sch (YopE chaperone) to GFP-YopE may be impaired. This would favor rapid maturation of GFP-YopE followed by degradation of the N-terminal secretion domain of YopE resulting in a secretion-deficient hybrid protein. Additional explanations pointed to the fact that a portion of GFP-YopE, which is not co-translationally secreted (36), will accumulate in the cytosol as protease-resistant GFP (29). Despite the difference in amounts of YopE and GFP-YopE secreted, this method remains the best for in vivo assays because the hybrid proteins are secreted in quantities large enough for complete assays. Trcek et al. (37) has studied YopQ (18 kDa) expression and secretion in different Yersinia strains using a similar method with a GFP-YopQ construct. While some toxins like YopE and YopN have a second chaperone-dependent secretion and translocation signal (38), YopQ secretion and translocation depends on one signal located within the first 53 N-terrninal residues. Lack of a 84 chaperone-dependent signal in YopQ solves one of the problems experienced in the GFP-YopE secretion assays (29). While this construct showed a remarkable improvement, it should also be noted that Trcek et al. (37) could not demonstrate translocation of GFP-YopQ fusion protein into eukaryotic cells. This restricts the use of GFP-YopQ fusion protein to secretion assays alone. Successful in vivo secretion assays require gene knockout models and Datsenko and Wanner (39) have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s) (Figure 3.1). Unlike yeast and a few naturally competent bacteria, most bacteria are not readily transformable with linear DNA (39) because linear DNA is prone to degradation by intracellular exonucleases (40). In their method (Figure 3.1), a chromosomal sequence is replaced with a selectable antibiotic resistance gene that is generated by PCR using primers with 36— to 50-nt homology extensions (H1 and H2). This process (39) is accomplished by Red-mediated recombination in these flanking homologies. Following the antibiotic-mediated selection, the resistance gene is also eliminated by using a helper plasmid expressing the FLP (flippase) recombinase, which acts on the directly repeated FRT (FLP recognition target) sites flanking the resistance gene. The Red and FLP helper plasmids are cured by growth at 37°C because they are temperature-sensitive replicons (39). The Red system is used in this process because it has three genes: y, l3, and exo, whose products are called Gam, Bet, and Exo, respectively (41). Gam inhibits 85 the host RecBCD exonuclease V so that Bet and Exo can gain access to DNA ends to promote recombination. Here, we use GFP-YopQ secretion assays (37) to identify YscF mutants that affect Yop secretion in Y. pseudotuberculosis. We examine the effect of Y65A, Y65F, N66A, K76A, D77A, I82A, 084A and F86A on GFP-YopQ secretion. We use a truncated yopQ (Yersinia outer protein Q) gene fused to gfp (encoding the green fluorescent protein) to study GFP-YopQ protein secretion in Y. pseudotuberculosis. Through homologous recombination, we used A red recombinase to knock out the yscF gene from Y. pseudotuberculosis virulence plasmid pYV in a method (39) described elsewhere in this work. We used the yscF model resulting from complete deletion of the gene to analyze GFP-YopQ protein secretion. The parent plasmid stcF (wild type) and each of the stcF mutants mentioned above were individually transformed into the yscF model to reconstitute Yop secretion. We used this method to look for anomalous phenotypes in secretion, such as non-secreting by detecting GFP-YopQ secretion using Western blot analysis (42). In this work, we show that introduction of some stcF (YscF in pET22b) mutants to wild type Y. pseudotuberculosis leads to reduced GFP-Yop053 secretion. We also report that GFP-YopQ53 secretion is not totally eliminated by knocking out the yscF gene from Y. pseudotuberculosis, but the yscF strain shows decreased secretion instead. Our work shows that some YscF mutants restore normal GFP-Yop053 secretion in the YscF knockout while other mutants don’t show similar restoration. 86 EXPERIMENTAL PROCEDURES Site-directed mutagenesis. We used site-directed mutagenesis [QuikChange® XL Site-Directed Mutagenesis Kit (Stratagene)] to introduce the following mutations in the parent plasmid stcF (yscF in pET22b): Y65A, Y65F, N66A, K76A, D77A, l82A, 084A and F86A. Each of the listed forward primers (Table 3.1) was used in a PCR reaction (18 cycles of 50 s at 95 °C, 50 s at 50 °C and 5 min 30 s at 72 °C) with its complement that is not listed (mutant codons are underlined). The rest of the mutagenesis, DNA replication and plasmid isolation processes were done following the same procedure described in chapter 2 of this thesis. We used E. coli DH-50 (43) (Hanahan, 1983) as the primary host organism in mutagenesis. The correct mutants were verified by DNA sequencing (MSU genetics core facility). Y. pseudotuberculosis Competent Cells and Electroporation. We obtained wild type Y. pseudotuberculosis cells (CB2587) from Dr. Michael Bagdasarian (MSU microbiology). The culture was incubated at 37°C overnight with shaking, then 2 mL of this starter culture was used to inoculate 100 mL LB medium. The culture was grown at 37°C to an 00600 of ~ 0.6; then it was chilled on ice for 30 minutes. Cells were pelleted at 4000 g for 15 minutes at 4°C and the supernatant discarded. The cells were resuspended in 40 mL sterile ice-cold ddiH20, then pelleted as described above. The supernatant was discarded and pellet resuspended in 40 mL of GYT medium (10% glycerol, 0.125% yeast extract and 0.25% tryptone). The cells were centrifuged again as described above, the 87 supernatant was discarded. The pellet was resuspended in 200 uL of GYT medium and immediately stored at -80°C. We isolated the helper plasmid pKD119 (containing 3 helper genes, I3, y and exo) from E. coli cells (032646 obtained from Dr. Bagdasarian) using the Qiagen (Hilden, Germany) plasmid-isolation kit. Electroporation process was done as follows: A 60 pL aliquot of the competent cells prepared as described above were added to an ice-chilled electroporation cuvette. Approximately 100 ng (1 pL) of the isolated pKD119 plasmid was added and thoroughly mixed with the cells. Electroporation was done on a Gene PulserTM (BIO-RAD) at a voltage of 2.5 kV, resistance of 200 OHMS and capacitance of 25 uF. Shocked cells were added to 1 mL of LB medium containing 1 mM arabinose, then incubated at 30°C (pKD119 is temperature-sensitive) for 1 h before plating 100 uL onto LB agar containing 10 pg/mL tetracycline and 1 mM arabinose. The plate was incubated at 30°C for 30 h before selecting tetracycline-resistant transforrnants for preparation of new competent cells (named C82588). yscF knockout. The PfuTurbo® master mix (Stratagene) was used in the PCR reaction (30 cycles of 15 s at 94 °C, 45 s at 48 °C and 1 min 30 s at 68 °C) to amplify the chloramphenicol-resistance gene from a pKD3 plasmid template. The following primers with 36-nt homology extensions (underlined) (H1 and H2) (Figure 3.1) were used in the amplification process: 5'- _C_CATTA'I'TQAT|'AT§TAGCAGGAGACCTAAAATAAGTGTAGGCTGGAG CTGCTTC - 3' (forward) 88 5'- CTCTGCTAACAGTACG'I'I'GAGTTTATATI'I'CATTI'ATGGGAA'ITAGCCAT GGTCC - 3' (reverse) The PCR-amplified DNA was purified from the gel using the Qiagen (Hilden, Germany) gel-extraction kit, but DNA was eluted with sterile diH20 instead of the elution buffer in the kit. Following DNA purification, Dpnl was added, then the sample was incubated at 37 °C for 1 h to digest any existing template plasmid that could contaminate the amplified DNA. The DNA was concentrated by ethanol precipitation as follows: Two microliters of pellet paint/co-precipitant was added, then 3 M sodium acetate was added (1/10 - v/v). The sample was mixed before adding cold 100% ethanol (twice the volume) followed by 20 minute centrifugation at 4°C (maximum speed on a microfuge). After the supernatant was discarded, the pellet was resuspended in cold 70% ethanol. After being centrifuged for 10 minutes at room temperature, the supernatant was discarded and the pellet air-dried for 30 minutes. The precipitated DNA was resolved in 10 uL of sterile diHZO ready for electroporation. The Y. pseudotuberculosis strain containing the helper plasmid pKD119 (CBZ588) was transformed with the amplified chloramphenicol-resistance gene (described above) following the same electroporation process as described above. The chloramphenicol-resistant transforrnants (named CB2754) were selected using 25 ug/mL chloramphenicol and made electrocompetent using the method described above. At this point, 082754 is a Y. pseudotuberculosis strain that has lost the yscF gene (yscF), but still contains the helper plasmid (pKD119), meaning that this strain is both chloramphenicol- and tetracycline-resistant. 89 Eliminating the helper plasmid pKD119. After knocking out the yscF gene, the helper plasmid was eliminated. The yscF ' Y. pseudotuberculosis strain (082754) containing the helper plasmid was grown in 10 mL LB medium containing 25 ug/mL chloramphenicol at 40°C overnight. Based on the ODsoo, the cells were diluted by 106 before plating to get a good colony spread. The cells were plated on LB agar containing 25 ug/mL chloramphenicol, then incubated at 37°C for 30 h. Approximately 50 colonies were picked from the above plate and each used to patch onto two different plates, one tetracycline- and the other chloramphenicol-resistant. The two plates were labeled to enable matching for identification of colonies that had lost the helper plasmid. We picked strains that grew on the chloramphenicol plate, but did not survive tetracycline because they had lost the helper plasmid. The yscF knockout strain was confirmed by PCR in which the new yscE-chloramphenicoI-yscG gene was amplified. For more confirmation, we did a PCR reaction using primers flanking the yscF gene to ensure that the yscF gene did not exist in the knockout. To ensure that the virulence plasmid pW was not lost, another gene, yopE on the plasmid was also amplified. In each PCR reaction, a bacterial colony was picked and resuspended in 6 pL of H20 followed by a 10 min heating at 94°C to provide the virulence plasmid template. Glycerol stock solution of the yscF knockout strain was prepared and stored at -80°C. The cells were also made electrocompetent ready for GFP-YopQ secretion studies. GFP-YopQ secretion studies. The GFP-yopQ gene construct was designed in a low-copy-number plasmid pACYC184 that is resistant 90 chloramphenicol (42). We obtained two GFP-YopQ constructs from Dr. Jiirgen Heesemann (Max von Pettenkofer-lnstitut - Munich, Germany) through Dr. Bagdasarian. The two constructs contain GFPmut2 (45, 46) that was generated by exchanging three amino acids (Ser—65 to Ala; Val-68 to Leu; Ser-72 to Ala) in its chromophore. The first construct, GFP-YopQ18 contains only the first 18 residues of YopQ while the second construct, GFP-YopQ53 has the first 53 residues of YopQ. The former can only be secreted, while the latter can be secreted and translocated into eukaryotic cells by Y. enterocolitica (37). Based on our preliminary assays in Y. pseudotuberculosis, GFP-YopQ18 secretion is low compared to GFP-YopQS3; hence, we use the former as a control in this study. We carried out the first assay in wild-type Y. pseudotuberculosis strains to compare the secretion of both GFP-YopQ18 and GFP-YopQ53. The two constructs were used to transform wild-type Y. pseudotuberculosis cells (CB2587) in separate experiments screened by 25 pg/mL chloramphenicol. In each case, a single colony of the transfonnants was used to inoculate 20 mL of brain-heart infusion (BHI, Difco) medium with the following additives: 4.0 mM CaCIz, 1.0 mM cysteine, 6.25 pg/mL Biotin (vitamin H), 0.5 mM histidine, 0.2 mM tryptophan and 16.0 mM MgCl2. The cultures were grown at 28°C overnight after which the .ODeoo was determined for each culture. Approximately 500 uL (volumes were adjusted based on the recorded ODsoo to ensure uniformity) of each sample was pelleted on a microfuge; after discarding the supernatant, the cells were resuspended in 500 uL of PBS buffer (pH 7.2). Each sample was 91 again pelleted and resuspended in fresh PBS buffer. Cells were plated on cellophane overlaid on BHI agar medium. The BHI medium used in making the plates had a different composition of additives compared to the previous BHI medium described above. The new medium had the following additives: 1.0 mM cysteine, 6.25 ug/mL Biotin (vitamin H), 0.5 mM histidine, 0.2 mM tryptophan and 16.0 mM MgCl2. It was also supplemented with 0.2% glucose and 5 mM EGTA (Ca2+ depletion) to induce Yop secretion. The plates were incubated at 37°C for 24 hours. PBS buffer (2 mL) was poured onto each plate to wet the cells for easy scraping using a razor blade. Cells from each plate were resuspended in a total of 5 mL PBS buffer", the ODsoo was determined for each sample before centrifugation. Each culture (before centrifugation — refered to as pellet) and supernatant was analyzed by SDS-PAGE and Western-blot using anti-GFP antibodies. During SDS-PAGE analysis, the amount of bacterial supernatant or pellet loaded was standardized/normalized based on the recorded ODsoo. All samples were dissolved in a modified SDS sample buffer (37) consisting of 0.1 M MgCIz, 4% SDS, 10% glycerol, 5% B-mercaptoethanol, 0.001% bromophenol blue and 100 mM Tris/HCI, pH 9. Addition of MgClz was necessary to precipitate DNA of whole-cell lysates (47). It was necessary to change the pH from 6.8 to 9 because this process eliminates hydrolysis of aspartyl-prolyl peptide bonds (48). We analyzed the effect of each one of the 8 stcF mutants on GFP- YopQ53 secretion in Y. pseudotuberculosis by transforming the cells (CB2587 - wild type) with each of the mutants separately. Cells were also transformed with the parent plasmid, stcF to form a control for comparison with the mutants. In 92 each case, cells were grown and GFP-Yop053 secretion induced as described in the method above. In each case, the pellet and supernatant was analyzed by SDS-PAGE and Western blot using anti-GFP antibodies. The yscF knockout strain (CBZ754) was transformed with each of the two constructs (GFP-YopQ18 and GFP-YopQS3) separately, but selection of transfonnants was more detailed because the knockout and GFP-YopQ constructs are resistant to chloramphenicol. We used SDS-PAGE and Western blot (anti-GFP antibodies) to identify the correct transformants. The two strains were made electrocompetent using the method described earlier in this work. By this point, we have two yscF knockout strains, one containing GFP-YopQ18 and the other containing GFP-Yop053. yscF reconstitution and the effect of 8 YscF mutants. The two yscF knockout strains described above were each transformed with the parent plasmid stcF (yscF in pET22b plasmid) to reconstitute YscF. The GFP-Yop053 containing strain was also transformed with each of the 8 stcF mutants (described in site-directed mutagenesis above) separately. The right transforrnants were selected by 100 ug/mL ampicillin because pET22b is ampicillin-resistant. The aim is to compare each one of the mutant-reconstituted strains to the stcF-reconstituted one. Cultures were grown for each strain and Yop secretion induced using the same method described earlier in this work. All samples were analyzed by SDS-PAGE and Western blot using anti-GFP antibodies. 93 Table 3.1. Oligonucleotides used to generate YscF mutants. Mutated codons are underlined. Y65A 5'- AATAAATGGTCGGTAATTflAATATAAACTCAACCATA -3' Y65F 5'- AATAAATGGTCGGTAATTMAATATAAACTCAACCATA -3' N66A 5'- AAATGGTCGGTAATTTACQCJATAAACTCAACCATAGTT -3' K76A 5'- ACCATAGTTCGTAGCATGflGACT-I'AATGCAAGGCATCQ' D77A 5- ATAGTTCGTAGCATGAAAG_CC'|TAATGCAAGGCATCCTA -3' l82A 5'- AAAGACTTAATGCAAGGOG—CCCTACAGAAGTTCCCATAA -3' Q84A 5'- TTAATGCAAGGCATCCTAG_CGAAGTTCCCATAATATGAA -3 F 86A 5- CAAGGCATCCTACAGAAGGCCCCATAATATGAAATATAA -3' 94 RESULTS Site-directed mutagenesis. Seven YscF conserved residues were each replaced with alanine in a similar method described in chapter 2. One of the residues, Y65, was also replaced with phenylalanine. However, there is a difference between the two cases because in chapter two the yscF gene was modified to yscFA4 to enable successful purification of the protein. In this chapter, we carry out site-directed mutagenesis on an untruncated yscF gene. We also have 084A and F86A in this chapter, but in chapter 2, 084 and F86 are cleaved off during the construction of yscFA4. Each mutant in this chapter has the full length yscF gene so that any observable changes in the analysis is due to the respective point mutation. All mutants were verified by DNA sequencing. Y. pseudotuberculosis competent cells and electroporation. Previous Yop0 secretion and translocation studies (29, 37) were performed in Y. enterocolitica. Here we present GFP-YopQ secretion study results from Y. pseudotuberculosis. We use Y. pseudotuberculosis because it shares greater than 90% DNA homology with Y. pestis and yet they differ in pathogenicity. They cause different diseases and are transmitted differently. Y. pestis is very dangerous and difficult to handle because it causes bubonic plague as mentioned in chapter 1, whereas Y. pseudotuberculosis is relatively safer to handle (see chapter 1). Y. enterocolitica is slightly more distant from the two species as mentioned in chapter 1. We therefore use Y. pseudotuberculosis instead of Y. enterocolitica as a good model to investigate secretion in Y. pestis. 95 Using the helper plasmid pKD119 required addition of 1 mM L-arabinose to the growth medium and temperatures had to be maintained at 30°C at all times. This helper plasmid was modified from pKDZO and pKD46 (39) that contain the A, IS, and exo genes from phage A. Therefore, all three plasmids have optimized ribosome-binding site for efficient translation of A and they all express A, I3, and exo from the arabinose-inducible ParaB promoter (39). The three plasmids are also temperature-sensitive thus, they are eliminated (cured) by growing cells at 40°C. yscF knockout. We amplified the chloramphenicol-resistance gene by PCR and introduced 36-nt homology extensions (H1 and H2) (Figure 3.1) on both ends of the gene. The PCR product is 1103 bp long including the 72 bp extension. We opted not to eliminate the chloramphenicol-resistance gene after knocking out yscF because we use it for selection. It can easily be removed because it is flanked by the two FLP recognition targets (FRT) that are recognized by flippase recombinase (FLP). In such a process, the two target sites are joined together hence eliminating the chloramphenicol-resistance gene. During the knockout process, chances of contamination by the template plasmid were minimized by extracting the PCR product right from the gel followed by a 1 h 37 °C Dpnl treatment. This eliminated possible contaminants and was verified by using E. coli strains as control because they lack pYV. The PCR product was concentrated through ethanol precipitation to enhance chances of recombination. Successful yscF knockout strains were verified by PCR using primers complementary to genes flanking the knockout region (YscE and YscG) 96 (Figure 3.2). These PCR analyses were performed alongside the wild type (CBZ587) for comparison. Results show that the knockout process was successful because PCR results for the knockouts show the size of DNA corresponding to the new YscE-chloramphenicol-YscG (1570 bp) (Figure 3.2). In contrast, PCR results for the wild type cells show DNA corresponding to YscE- YscF-YscG (810 bp) (Figure 3.3). We tried knocking out yscF using both Kanamycin- and chloramphenicol-resistance genes, but the latter worked first, so we ended up with a new problem: The GFP-YopQ constructs were provided to us in a chloramphenicol-resistant plasmid, so we had to distinguish between the two strains during selection. We solved this problem by running an SDS-PAGE of the respective lysate followed by Western blot using anti-GF P antibodies. Eliminating the helper plasmid pKD119. The helper plasmid was eliminated by growing the cells at 40°C. It is important to ensure that the virulence plasmid, pW is not lost during the study. To ensure that pW was not lost, another gene, yopE (657 bp) on the virulence plasmid was amplified by PCR (Figure 3.4). GFP-YopQ secretion studies. We used two different constructs (GFP- Yop018 and GFP-Yop053) (Figure 3.5) to study Yop0 secretion in Y. pseudotuberculosis. We started our preliminary work by comparing the secretion levels of the two constructs and results show that GFP-Yop018 is secreted in very low (almost negligible) quantities compared to GFP-Yop053 (Figure 3.6). In subsequent studies, we used GFP-Yop018 as a control to show that all GFP- Yop053 released in the medium is a result of secretion and not lysed cells. Both 97 proteins are well-expressed, as can be seen from the pellet samples analyzed by SDS-PAGE and Western blot (Figure 3.6). Both proteins are present in the pellet, but only Yop053 is visible in the supernatant. Secretion of GFP-Yop053 was successfully induced by addition of EGTA (Ca2+ depletion) because it is a better chelator of Ca2+ than EDTA. The amount of protein loaded during SDS-PAGE was normalized based on the ODeoo therefore the difference in secretion can be clearly seen. To achieve consistency during SDS-PAGE analysis, we modified the sample buffer by changing the pH (37). In their study, Jacobi et al. (42), monitored Yop0 secretion in solution using anti-GFP antibodies. Their method involves concentrating of protein from large amounts of solution and TCA precipitation because the protein is secreted in very small quantities. We have designed an easier way of monitoring secretion in which secretion is enhanced tremendously. Instead of using solution BHI medium, we plate the cells on a cellophane covered agar plate. This approach minimizes the background noise caused by many small peptides found in the BHI medium. Secondly, since secretion is many orders of magnitude higher, concentration by TCA precipitation is unnecessary. The SDS-PAGE results are also clear like a Western blot, meaning that we can analyze with SDS-PAGE alone. Both GFP-YopQ constructs appear at approximately the same mass (Figure 3.6) because they are 28 kDa (GFP-Yop018) and 32 kDa (GFP-Yop053) in mass. Secretion assay results show that introduction of two stcF mutants, Y65A and Y65F to Y. pseudotuberculosis (wild type) leads to reduced GFP- 98 Yop053 secretion (Figure 3.7). Each one of these two mutants has a negative impact on wild-type secretion. Other mutants don’t show an effect on wild-type secretion. Based on our GFP-Yop0 secretion assay results, it is evident that GFP-Yop053 secretion is not totally eliminated after knocking out the yscF gene from Y. pseudotuberculosis, the knockout strain shows decreased secretion instead (Figure 3.8). Results also show that normal GFP-Yop053 secretion is restored in YscF knockout following reconstitution by the stcF plasmid. Normal secretion was also restored in the knockout by the following stcF mutants: N66A, K76A, D77A, 084A, and F86A. However, three stcF mutants, Y65A, Y65F, and I82A did not restore normal secretion because their level of secretion is similar to the yscF ' strain. 99 H1 FRT FRT L 7 Chloramphenicol 7‘ L Q “2 Homologous recombination by A red recombinase H 1 f H2 YscE I YscF I YscG Selection of chloramphenicol- resistant transformants FRT ,, FRT Chloramphenicol " Figure 3.1. A simple gene disruption strategy. H1 and H2 refer to the homology extensions or regions. 100 1.5kb «--—H‘--~ 1.010).. 0.5 kb .0 Figure 3.2. Verification of yscF knockout by PCR using primers complementary to genes flanking the knockout region (YscE and YscG). Results show the YscE- ChloramphenicoI-YscG gene (1570 bp) from the knockout strain. Figure 3.3. Verification of yscF knockout by PCR using primers complementary to genes flanking the knockout region (YscE and YscG). Results show the YscEFG gene (810 bp) from wild-type Y. pseudotuberculosis. 101 700 bp 600 bp 500 bp 400 bp ’ Figure 3.4. PCR amplification of the yopE gene (657 bp) from the yscF knockout strain to verify the presence of the virulence plasmid pYV. YopQ GFP3 a 1-18 YopQ GFP3 aa 1-53 | m Figure 3.5. GFP-Yop018 and GFP-Yop053 constructs for in vivo secretion assays. 102 Yop0 Yop0 Yop0 Yop0 wt 18 53 wt 18 53 pellet supernatant a Yop0 YopQ Yop0 YopQ wt . 18 53 ......WI 1?. _-.§§._.- supernatant Figure 3.6. YopQ-GFP secretion assay results. (a) Westem-blot using anti-GFP antibodies. (b) The same secretion assay analyzed by SDS-PAGE. Yop018 is expressed, but not secreted. 103 '- J Pellet .- _... ..- n- - _.. “...-um- ' ...-.. f .9.-... Supernatant WT YsCF Y65A Y65F N66A K76A D77A I82A Q84A F86A Figure 3.7. Western blot analysis of differential secretion. Secretion assays in the wild-type Y. pseudotuberculosis. Results show that introduction of two stcF mutants, Y65A and Y65F to Y. pseudotuberculosis (wild type) leads to markedly reduced GFP-Yop053 secretion. I Pellet , Supernatant WI"I}]':S‘1-I'i 1""!!!1. :I T"I M ‘< ko YscF Y65A Y65F N66A k76A D77AI82A 664A F86A Figure 3.8. Complementation of the yscF knockout strain using YscF and different mutants. The knockout strain (lane 1) shows decreased secretion. 104 DISCUSSION We have determined that modification of the SDS sample buffer was necessary based on previous studies (37, 42) because both YopQ and YopK have aspartyl-prolyl peptide bonds which are prone to hydrolyse during heating in Laemmli sample buffer due to low pH (48). Other Yop secretion studies (49) have used TCA precipitation to concentrate the secreted toxins because of the minute amounts secreted. In the original design, Jacobi et al. (29) monitored secretion in solution using anti-GF P antibodies. Their method involves concentrating large amounts of solution and TCA precipitation because the protein is secreted in very small quantities. This process involves many steps and may compromise consistency. We have designed an easier way of monitoring secretion in which secretion is enhanced tremendously. Instead of using soluble BHI medium, we plate the cells on a cellophane-covered agar plate. Thus, we prevent background noise caused by many small peptides found in the BHI medium. Secondly, concentration of secreted Yop0 derivatives is higher because the protein remains on the cellophane membrane. It is possible to analyze just by simple SDS-PAGE or native gel electrophoresis. In this modified method, the process of concentrating protein by TCA precipitation is eliminated. By using our method, the protein can be recovered by cutting from the gel for further analysis by (for example) N-terminal sequencing and mass spectrometry. Our method provides a better way of concentrating cells and the secreted proteins. However, 105 it should be noted that some secretion assays can only be carried out in soluble medium. Our study shows that GFP-Yop053 is well secreted by Y. pseudotuberculosis in Ca2+-depleted medium, but secretion of GFP-Yop018 is negligible. This characteristic makes GFP-Yop018 a good control because it is well-expressed, but not well secreted. Although we used Yop0 constructs of Y. enterocolitica to assay secretion in Y. pseudotuberculosis, it is worth noting that YopK is the corresponding homolog in Y. pseudotuberculosis. It would be interesting to compare secretion of both Yop0 and YopK in Y. pseudotuberculosis. We have used the yscF model resulting from complete deletion of yscF to analyze GFP-YopQ secretion. The parent strain stcF (wild type) and each of the stcF mutants were transformed into the yscF ' model to determine their effect on GFP-YopQ secretion. This method enabled us to identify anomalous phenotypes in secretion (reduced secretion) caused by three mutants, Y65A, Y65F, and l82A. While previous studies have focused mainly on secretion analysis, our study goes a step further by looking at both the in vitro and in vivo needle formation by YscF variant proteins. If a given mutant is able to form needles in vitro but prevents Yop secretion, then we propose that such a residue might participate in linking the needle to the base. Since YscF I82A assembles into needle-like structures in vitro but does not support secretion in vivo, it must assemble nonfunctional needles that are possibly unable to interact with other 106 components of injectisome such as the base. Two other mutants, Y65A and Y65F seem to interfere with secretion in the wild type and don’t restore normal secretion in the knockout strains following reconstitution. 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Immunochemical analysis of plasmid-encoded proteins released by enteropathogenic Yersinia sp. grown in calcium defficient media. Infect Immun 54, 561-567. Cornelis, G. R., Boland, A., Boyd, A. P., Geuijen, C., lriarte, M., Neyt, C., Sory, M., and Stainier, I. (1998) The Virulence Plasmid of Yersinia, an antihost genome. Microbiology and Molecular Biology Reviews 62, 1315- 1352. 112 Chapter 4 Conclusions and Remaining Questions 113 CONCLUSIONS AND REMAINING QUESTIONS The studies presented in this thesis aimed to identify YscF point mutants that affect GFP-YopQ secretion in Yersinia pseudotuberculosis without affecting in vitro YscF needle assembly. These results were used to identify YscF residues that are likely to be involved in the interactions at the needle-base interface in Yersinia type III secretion system. Secretion and translocation of Yops into target cells by the Yersinia T3SS requires proper assembly of the needle and the base. The interaction between these two structures of the T388 depends on residues at the interface. Previous studies have focused mainly on secretion analysis, but our work went a step further by looking at both the in vitro needle assembly and Yop secretion. If a particular YscF mutant is able to form needles in vitro but prevents Yop secretion, then we propose that the mutated residue might be involved in the interactions at the needle-base interface. We performed a detailed analysis of YscF sequence conservation, predicted secondary structure and homology modeling based on the published structures of YscEFG complex (1), MxiH (2), BsaL (3), and Prgl (4). From that preliminary analysis, our study focused on seven YscF residues: Y65, N66, K76, D77, l82, 084 and F86. In chapter 2, we performed a structural analysis of YscF (YscFA4) and its in vitro needle assembly studies by a combination of secondary-structure prediction, homology modeling, electrophoresis, mutagenesis, circular dichroism spectroscopy, and transmission electron microscopy. We performed a similar study on 8 YscF point mutants: Y65A, Y65F, N66A, K76A, D77A, I82A, 084A 114 and F86A. Results show that all mutants have a-helical secondary structures except Y65F, which appears to be unstructured. Six mutants were able to assemble into needles in vitro, but Y65F and N66A did not. The needle-assembly process was much slower for Y65A compared to the rest of the mutants. Lowering the pH from 8.0 to 5.5 prevented needle formation in Y65A. We have also shown that the N-terminal region of YscF does not participate in needle formation. A similar finding had been reported earlier for MxiH and Prgl (2, 4). Chapter 3 uses GFP-Yop053 to study secretion in Y. pseudotuberculosis. A yscF‘ knockout model was created through homologous recombination using A red recombinase to be used for this study. The parent plasmid stcF (wild type) and each of the stcF mutants were separately transformed into the yscF ‘ strain to reconstitute secretion and monitor their effect on GFP-YopQ secretion. A similar study was carried out to monitor the effect of stcF and its mutants on secretion in the wild-type Y. pseudotuberculosis. This method enabled us to identify anomalous phenotypes in secretion (reduced secretion) caused by three mutants, Y65A, Y65F, and l82A. These mutants failed to restore normal secretion in the yscF ' strain following reconstitution. Results also show that secretion was not totally abolished in the yscF‘ strain as some GFP-Yop053 was detected by Western blot. Two mutants, Y65A and Y65F, showed a semi-dominant negative phenotype because they lead to a significant reduction in secretion when transformed into wild-type Y. pseudotuberculosis. Results also show that N66A abolished needle polymerization, but allowed secretion. 115 By comparing results from chapter 2 and chapter 3, we propose that the YscF residue I82 might be involved in the interaction at the needle-base interface. These results contribute to a better understanding of the structure and assembly of the Yersinia T3SS. Such insight is useful because in the T383, needles are a good target for vaccine development. Some questions are still unanswered about YscF, the needle-forrnation process and Yersinia T3SS in general. Here, I propose some experiments that could help in elucidating the YscF structure and understanding TBSS further. a. Self-polymerization remains the biggest hinderance to solving the YscF atomic structure. Unlike MxiH and Prgl structures that were solved following C-terminal modification, YscF still shows some polymerization after this modification. However, my work has shown that replacing residue Y65 with alanine can stop polymerization of YscFA4 at pH 5.5. The lower pH maintains this protein as a dimer based on my results from analytical gel filtration. Since the CD results show that the protein is folded, it might be possible to solve the atomic structure through crystallography or by solution NMR. The homology model shows residue Y65 pointing away from the two-helix bundle, therefore it might not be involved in folding of the monomer, but participates in YscF self-polymerization. This mutant could be useful in solving the YscF structure. 116 b. Results of this work show that mutant N66A prevented in vitro needle formation. This mutant is o-helical based on the CD results. Its quaternary structure should be analyzed by analytical gel filtration with the possibility of using it for structural determination. c. Secretion studies have shown that both Y65A and Y65F display semi- dominant negative phenotypes. An in vitro needle assembly study should be performed by mixing Y65F with the wild type YscF in equal proportions. The in vitro needle assembly process can then be induced to assess the effect it has on YscF needle assembly. This study can confirm the dominant negative characteristics of this mutant if it prevents wild-type YscF needle assembly. We choose not to use Y65A in this study because it formed needles in vitro albeit at a very slow rate. An in viva needle assembly assay should also be carried out by separately introducing each mutant plasmid into the wild-type Y. pseudotuberculosis as described in chapter 3. However, needle formation in this case should be analyzed by transmission electron microscope instead of assaying for secretion. Thus, formed needles can be visualized. d. Secretion study results show that knocking out YscF did not completely abolish secretion, as GFP-Yop053 was detected by Western blot in the yscF ‘ knockout model. The detected secretion might be caused by an alternative secretion system or it could be due to leaking of the GFP-Yop053 protein as it accumulates in the bacterial cytosol following induction (Ca2*-depletion). An in viva needle formation assay should be carried out by inducing secretion in the yscF ‘ model. The growth solution should be analyzed for presence of 117 needles using transmission electron microscopy. Alternatively, presence of YscF can be analyzed by Western blot using anti-YscF antibodies. If needles form and/or YscF is detected, this will mean that an alternative YscF gene exists in addition to the one on the virulence plasmid. 118 REFERENCES Sun, P., Tropea, J. E., Austin, B. P., Cherry, 8., and Waugh, D. S. (2008) Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG. J Mol Biol 377, 819-830. Deane, J. E., Cordes, F. S., Roversi, P., Johnson, 8., Kenjale, R., Picking, W. 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