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thesis entitled
THE USE OF CHITIN SYNTHASE GENE SEQUENCES
TO STUDY THE PHYLOGENY OF THE OOMYCETE
PATHOG EN PYTHIUM INSIDIOSUM
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THE USE OF CHIT IN SYNTHASE GENE SEQUENCES TO STUDY THE
PHYLOGENY OF THE OOMYCETE PATHOGEN PY T HI UM INSIDIOSUM

By

Chia-Ju Lin

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Clinical Laboratory Science Program

2005

ABSTRACT

THE USE OF CHITIN SYNTHASE GENE SEQUENCES TO STUDY THE
PHYLOGENY OF THE OOMYCETE PATHOGEN PYT HI UM INSIDIOS UM

By

Chia-Ju Lin

Pythium insidiosum, an emerging pathogen, causes life-threatening infections in
humans and animals. The infections it causes are termed pythiosis. P. insidiosum has
been classified into three phylogenetic clusters by restriction fi'agment length
polymorphism (RFLP) and phylogenetic analysis using the ribosomal DNA (rDNA)
internal transcribed spacers (ITS). A forth cluster was found in P. insidiosum studied
from an African dog. Besides DNA analysis, protein data, such as actin, chitin synthase,
elongation factor, and tubulin, are often used to study and confirm the phylogeny of
microorganisms. In this study, the gene sequence encoding chitin synthase was used to
investigate the phylogeny of P. insidiosum in twenty-five isolates of this organism. A 500
bp DNA sequence of the gene encoding chitin synthase was amplified and then used in
phylogenetic analysis using maximum parsimony and neighbor-joining methods. The
investigated isolates were separated into four clades. Clade I was composed of isolates
fi'om America and Southeast Asia; Clade 11 displayed a similar pattern; clade III was an
isolate from America; and clade IV contained isolates from Africa, America, and
Oceania. This study showed some support with previous studies using ITS gene
sequences. The placement of the Asian strains of P. insidiosum with the American strains
might suggest that the 500 bp fragment of chitin synthase gene does not possess enough

sequence variation to geographically group the strains of P. insidiosum used in this study.

ACKNOWLEDGEMENTS

I would like to acknowledge all the people who supported me during my time
writing this thesis: My major professor, Leonel Mendoza, for his unwavering support of
this research project and kind patience to me especially when the experiments did not go
well; Kathleen Hoag and C. Adinarayana Reddy for serving on my guidance committee;
David Thorne for his genial personality; Richard Garcia and Debora Rodrigues for
helpfully providing advice and the Medical Technology Program faculty and staff for

their kindness and helpfulness.

iii

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................... vi
LIST OF FIGURES ........................................................................................ vii
1. INTRODUCTION ....................................................................................... 1
1.1 History .................................................................................................. l
1.2 Distribution ........................................................................................... 2
1.3 Life cycle ............................................................................................. 2
1.4 Pythiosis in humans and animals .................................................................. 4
39% ................................................................................................. 4
M ..................................... y ............................................................ 5
Dogs and Cats ......................................................................................... 5
C_att_1§ .................................................................................................... 6

1.5 Diagnosis .............................................................................................. 7
1.6 Treatment ............................................................................................ 10
1.7 Phylogeny of Pythium insidiosum ............................................................. 13
1.8 Chitin and Chitin Synthase ....................................................................... 14
2. OBJECTIVES ........................................................................................... 19
3. MATERIALS AND METHODS ................................................................... 20
3.1 Pythium insidiosum strains ....................................................................... 20
3.2 DNA extraction ..................................................................................... 20
3.3 RNase treatment .................................................................................... 21
3.4 PCR ................................................................................................... 22
3.5 Cloning ............................................................................................... 22

iv

3.6 Cracking gel ....................................................................................... 23

3.7 Plasmid DNA extraction ......................................................................... 24
3.8 Sequencing ....................................................................................... 25
3.9 Phylogenetic analysis .................................................................. . ......... 25
4. RESULTS ............................................................................................ 27
4.1 DNA extraction from twenty-five strains of Pythium insidiosum ......................... 27
4.2 Amplification of a 500 bp product of chitin synthase gene of P. insidiosum ........... 27
4.3 Cloning of the 500 bp DNA amplicon into E. coli cells ................................... 31
4.4 Purification of the vector from E. coli cells and sequencing .............................. 31

4.5 Phylogenetic analysis using neighbor-joining and maximum parsimony

methods ........................................................................................... 31
5. DISCUSSION ........................................................................................ 37
6. FUTURE DIRECTION ............................................................................ 42
REFERENCES ......................................................................................... 43
APPENDIX A Twenty-five strains of Pythium insidiosum’s gene sequences
encoding chitin synthase (498 bp) ................................................. 52
APPENDIX B The predicted amino acid sequences of chitin synthase based
upon DNA sequence in twenty-five strains of Pythium insidiosum ........... 58

LIST OF TABLES

Table 1: List of twenty-five P. insidiosum species used in this study for sequencing
and phylogenetic analysis ............................................................... 26

vi

Figure 1:

Figure 2:
Figure 3:

Figure 4:
Figure 5:
Figure 6:

Figure 7:

LIST OF FIGURES

DNA extracted from of Pythium insidiosum ......................................... 28
RNAse treatment ........................................................................ 29
PCR using primers CH8] and CHSZ to amplify the gene encoding chitin

synthase .................................................................................... 30
Cracking gel to determine the plasmid with DNA insertion ....................... 33
Plasmid purification ..................................................................... 34
Phylogenetic analysis using neighbor-joining method ............................. 35
Phylogenetic analysis using maximum parsimony method ........................ 36

vii

1 . INTRODUCTION

1.1 History

Pythium insidiosum is an emerging pathogen causing life-threatening infections in
humans and animals. The findings in the last ten years on its epidemiology, treatment,
and molecular aspects have shaped the way we approach this pathogen. The following are
some of the most important aspects of the infections caused by this pathogen.

P. insidiosum is classified in the Kingdom Stramenopila, Phylum Oomycota,
Class Oomycetes, Order Pythiales and Family Pythiaceae (18, personal communication).
Pythium sp. are common pathogens of plants. There are more than two hundred reported
species to date (23, 68). Among these, P. insidiosum is the only mammalian pathogen.
The infections caused by P. insidiosum are termed pythiosis (18), which is characterized
by the formation of cutaneous, subcutaneous, and arterial granulomas. The infections
caused by P. insidiosum have been reported in captive camels (95), jaguars (15), and
spectacled bears (Grooters and Lamberski; unpublished data; 27), and also in cats (5, 27),
cattle (63, 77), dogs (27, 60, 69, 75), horses (1, 17, 50, 74), sheep (84), and humans (7, 35,
89, 92, 94).

According to a recent review (49), the first case of the disease was reported in
India. This report described cutaneous granulomas in equines in the middle of the 19th
century. However, it was not until the end of the 19'“ century that the fungal-like nature of
the disease was established. In 1901, de Haan and Hoogkamer first successfully isolated
this organism from equines with cutaneous granulomas. In 1924, Witkampt published
two important papers regarding equine pythiosis. His work, however, was ignored

because the clinical aspects of the Habronema species mimicked those of pythiosis. In

1974, the organism was isolated from horses in Papua New Guinea by Austwick and
Copland. Because this isolate formed biflagellate zoospores, they classified it in the
Oomycete genus Pythium. In 1980, Ichitani and Amemiya concluded that the isolate
studied by them was Pythium gracile based on the smooth oogonia and aplerotic
oospores it produced. Later in 1987, de Cock et al., who worked with several isolates
from horses, suggested that those isolates should be classified in a new species, and the
term P. insidiosum was introduced (49). In 2003, Schurko et al., using molecular tools,

confirmed that P. insidiosum is the only mammalian pathogen (79).

1.2 Distribution

Pythiosis has been reported in Argentina, Australia, Brazil, Colombia, Costa Rica,
Guatemala, Haiti, India, Indonesia, Japan, New Zealand, Nicaragua, Panama, Papua New
Guinea, South Korea, Thailand, the United States, and Venezuela. In Europe, the only
case was published in France in 1896. The first case of pythiosis fi'om Afiica in an eight-
month-old dog has been recently diagnosed (75). In the Americas, the disease has been
reported in several countries. In the United States, infection is common in the states near
the Gulf of Mexico, including Alabama, Florida, Louisiana, Mississippi, and Texas. Also,
some cases have been reported in Georgia, Missouri, North Carolina, South Carolina,

Tennessee, and even in Illinois, Indiana, New Jersey, New York, and Wisconsin (49).

1.3 Life cycle
P. insidiosum is widely distributed in soil and aquatic tropical and subtropical

environments. However, cases reported in Japan and northern America indicate that this

organism could be found in cooler environment as well. In culture, P. insidiosum
develops aseptate fungal-like hyphae similar to those produced by the Zygomycetes.
Hence, P. insidiosum was once considered as a member of the Kingdom Fungi. However,
in recent years, P. insidiosum has been proven to be phylogenetically closely related to
Stramenopiles.

The colonies of P insidiosum are colorless to white with short aerial filaments on

the cornmeal agar, and when it is incubated in water culture at 28~37°C, it produces
zoosporangia. At maturity, biflagellate zoospores are produced from zoosporangia, which
play an important role in the life cycle of this pathogen. TWO flagella arise from a groove
on the zoospore. Using electron microscopy, it has been found that after being released,
the zoospores are attracted by the host tissues, and then they bind to the tissues by
secreting a sticky material on the surface of the encysted zoospores (55). The encysted
zoospores penetrate into the plant or animal tissues by developing germ tubes. This sticky
amorphous material helps P. insidiosum to remain attached to the plant and animal cells;
thus, this material could be considered a potential virulence factor in P. insidiosum
infections. Besides P. insidiosum, Mendoza et al. also found that the other Oomycetes
possess the same special chemotactic factors and they also express this sticky material
when the encysted zoospores attach to the hosts (55).

In addition to asexual zoospores, a few strains of P. insidiosum develop oogonia
to proceed sexual cycle. However, the mechanism of oogonia formation is still under

investigation (49).

1.4 Pythiosis in humans and animals
Borges

The first case of pythiosis in horses was reported in the 19'“ century and has been
well described by several researchers. Although it was described more than 100 years ago,
cases of pythiosis in horses are still being published (49). The disease is characterized by
the formation of fistulating and ulcerating granulomas, predominantly on the lower limbs,
but could display on any part of the infected host anatomy. In horses, the granuloma
contains firm, yellowish coralloid masses of necrotic tissues, which are known as
“kunkers”. The metastasis to lymph nodes, lungs, and bones has been reported. Similar
kunkers are also seen in habronemiasis, which displays identical appearances. Therefore,
in horses, pythiosis is often misdiagnosed as cutaneous habronemiasis. However, the
kunkers from cases of habronemiasis are smaller and usually the disease regresses with
cold weather (14). Two similar fungal pathogens, named Basidiobolus haptosporus and
Conidiobolus comnatus, which cause subcutaneous zygomycosis and
rhinoentomophthoromycosis, have both been reported to infect horses and with the
identical clinical manifestations as in pythiosis. These three infections could be
distinguished by the anatomical locations where they occur (62). However, it is difficult
to differentiate them by histological methods; hence, some investigators have developed
serological methods for the accurate diagnosis of this disease. For instance, complement
fixation test was introduced by Miller and Campbell in 1982 and immunodiffusion test
was established by Mendoza, Kaufman, and Stanford in 1986 (52). Later in 1992,
Mendoza et a1. published a paper regarding a new immunoblot analysis (54). These

investigators found that the serum from infected horses or those cured by immunotherapy

showed three immunodominant bands (32 kd, 30 kd, and 28 kd), while the serum from
healthy horses or those with other infections did not. This method is proved to be useful
for the diagnosis of P. insidiosum infections (54).

Hm

The first case of human pythiosis was reported by Thianprasit et al. in Thailand in
1985. Since then, more than one hundred cases have been diagnosed and ninety cases
were from Thailand. The other cases were from Australia, Haiti, Malaysia, New Zealand,
and the United States. Within this group, most patients were farmers. Thalassemia was
also a common associated finding. Because of the small number of cases, scientists
concluded that pythiosis in humans is rare. Since the main occupation of Thai people is
farming and 40% of Thai populations haVe an abnormal hemoglobin gene, which makes
thalassemia a common disease in Thailand, it is not possible to conclude that farmers and
people with thalassemias have higher infection rates of pythiosis (73).

The clinical features of P insidiosum infection in humans differ from those in
animals and can be mainly classified into three forms: ophthalmic, cutaneous or
subcutaneous, and systemic or vascular (86). Cutaneous or subcutaneous pythiosis in
humans has some similar symptoms to animal pythiosis. However, the major form of
human pythiosis is the systemic or vascular form, which has not yet been reported in
animals. In addition, the finding of misdiagnosed cases of orbital pythiosis in five young
U.S.A. boys were discussed by Mendoza et a1. (57).

Dogs and Cats
Compared to horses, pythiosis in dogs and cats is relatively rare. In dogs, this

disease often infects young, male, large-breed animals, and especially outdoor working

breeds. It is a reasonable expectation that those dogs are at higher risk because they are
more likely to contact swampy water contaminated with P. insidiosum (88). TWO clinical
signs of canine pythiosis are cutaneous pythiosis (75) and gastrointestinal pythiosis (69).
Cutaneous or subcutaneous lesions are commonly found in extremities, tail, head,
abdomen, and perineum. Gastrointestinal pythiosis usually comes with chronic weight
loss, vomiting, diarrhea, and hematochezia. Lesions often occur within the stomach and
small intestine. The two forms of the infection rarely take place in the same host (88).
Early in 2005, an eleven-month-old male dog was diagnosed using serological and
sequencing methods. It is the first case of dog pythiosis found in Venezuela (60).

The first report of cat pythiosis was published in 1991 (5). Bissonnette and
coworkers diagnosed a three-year-old, male domestic cat in North Carolina, USA. as
infected by P. insidiosum. The cat presented with a protrusion of the right eye, and a soft-
tissue mass within the nasal chambers and nasopharynx extending into both orbital
cavities. They confirmed this case by immunofluorescent antibody staining,
immunodiffusion test, and culture. Treatment with oral ketoconazole for six weeks
resulted in clinical improvement. The cat had no signs of active infection eighteen
months after treatment (5). Two cats were diagnosed with subcutaneous masses on the
distal limbs, which was a different presentation than that seen in other species (88).

m

In 1985, cutaneous pythiosis in beef calves was reported (63). Miller et al.
conclude that the disease mainly occurs in the late summer and fall because of the
ecology of this aquatic organism and the requirement of high temperatures for rapid

growth and asexual reproduction. Several years later, two more cases of pythiosis in beef

calves were confirmed based on the morphological aspects, immunohistochemical
findings and culture of P. insidiosum (77). More recently, several cases of cattle pythiosis
were described in Venezuela (personal communication; unpublished data, 49). Overall,

pythiosis is not a common disease in cattle.

1.5 Diagnosis

A rapid diagnosis of pythiosis is necessary for its successful therapy. However, P.
insidiosum is difiicult to diagnose by its clinical features alone. Scientists have developed
several different methods to identify this pathogen, including culture, wet mount
preparations, histopathological methods, serological methods, and molecular tools (49).

P. insidiosum is sensitive to low temperatures; therefore, the tissue sample should
be transported in distilled water with antibiotics at room temperature. The use of ice to
transport the specimen decreases the chance to isolate the pathogen. Kunker is the most
suitable tissue from horses for diagnosing pythiosis. The tissue should be placed on the
Sabouraud dextrose agar plate and incubated at 37°C for several days to grow. However,
in recently years, it is found that kunkers could be stored at either room temperature or
4°C up to 5 days without affecting further culture procedure (29).

In wet mount preparations, the mass is cut into small pieces and placed in 10%
KOH for direct light microscopy examination (49). If sparsely coenocytic, septated
hyphae 4.0 ~ 9.0 pm in diameter with approximately 900 angle branches are observed, it
indicates that P. insidiosum or other fungal pathogenic agents may be present. After being
cultured over boiled grass leaves for 24 hours at 37°C, zoosporangia containing zoospores

could be observed at the edge of the leaves under microscopic examination (51). This is

not an accurate way to identify this pathogen, but it could provide keys for the diagnosis.

Histopathology examinations of tissue biopsy from infected cases stained with
hematoxylin and eosin would show eosinophilic inflammatory reactions. Silver stain and
Periodic Acid-Schiff stain are also adequate for the observation and identification of the
hyphae of P. insidiosum in tissue. However, the hyphae of P. insidiosum are easily
confused with those found in zygomycosis caused by fungal species of the orders
Mucorales and Entomophthorales. Thus, some other more accurate methods have been
developed to diagnose pythiosis.

Serology is often used to diagnose pythiosis in the infected hosts. There are
several serological methods to diagnose P. insidiosum infection in humans and animals,
including immunohistochemical methOds, immunodiffusion tests, enzyme-linked
immunosorbent assay and western blot (49).

The immunohistochemical methods include immunofluorescenoe test and
immunoperoxidase test. The immunofluorescence test was first introduced in 1987 and
has been confirmed to be a specific means for diagnosing P. insidiosum in infected dogs,
horses, and humans (53). In 1988, the immunoperoxidase test was developed to diagnose
pythiosis when culturing was not practical or as an alternative way to diagnose this
infection (10). These researchers applied indirect immunoperoxidase technique to
forrnalin-fixed tissue embedded in paraffin. This methodology successfully distinguished
P. insidiosum in tissue sections from infected cases of Conidiobolus sp, Basidiobolus sp,
and a case of mucormycosis. This assay was also performed on the tissue of human with
pythiosis and had been proven to be specific (89).

The immunodiffusion test was performed using culture filtrate antigen (CFA)

from P. insidiosum and antiserum from rabbits. This method was proven to be specific,
but with low sensitivity, not only in diagnosing the disease in cats, cattle, dogs, horses,
and humans, but also in monitoring the efficiency of treatment (36, 52, 72).

The enzyme-linked immunosorbent assay (ELISA) was also used to diagnose
pythiosis in humans, cats, dogs, and horses (30, 42, 59). Soluble antigen from the broken
hyphae of P. insidiosum was used to perform this test. It is as specific as immunodiffusion
test and much more sensitive because ELISA could detect some serum samples which
were shown to be negative in immunodiffusion test (59). With a suitable cutoff value, the
sensitivity and specificity could reach 100%. ELISA combined with clinical observations
could be used as a powerful tool for monitoring pythiosis (42).

In addition, in recent years, some molecular based methods were developed to
diagnose pythiosis (2, 28, 92, 97). Badenoch et a1. (2) were the first group to use a DNA
fragment to diagnose pythiosis from a human case with keratitis. Nested polymerase
chain reaction (PCR) using primers specific for P. insidiosum could amplify a 105 bp
amplicon in P. insidiosum, but not in other species like Lagenidz‘um sp, Lagenidium
giganteum, Basidiobolus ranarum, Conidiobolus coronatus, and Aspergillus terreus (28).
PCR was also used to detect P. insidiosum DNA in frozen and ethanol-fixed animal
tissues (97). A PCR method with amplicon of 188 rRNA was proven to be useful for this
purpose as well (92). These studies indicate that molecular based methods have become a
trend for identifying P. insidiosum infection, and are convenient for the rapid
identification of pythiosis. In 2005, an intestinal canine pythiosis in Venezuela was

successfully confirmed through serological testing and sequencing analysis. It was the

first confirmed dog pythiosis in Venezuela (60). The combination of serological and

molecular methods could increase the reliability of the diagnostic results.

1.6 Treatment

Three methodologies have been used to treat P. insidiosum infections: surgery,
chemotherapy, and immunotherapy (49). Surgery has been used to remove the lesions
caused by P. insidiosum in horses and humans. This method is very popular within
veterinary practitioners, although it is not always practical. The response to the surgery is
limited. The lesions on the limbs of horses have an intricate anatomic structure for
eradicating by surgery. Moreover, recurrence is very common in equine cases whose
lesions containing the pathogen are not rMoved completely through surgery (1, 49). For
those reasons, surgical removal followed by other treatments has been investigated.
Amphotericin B has been used systemically or locally, or both, in the treatment of
pythiosis in ten equines after the infected tissues had been removed. It was concluded that
early surgical removal of the lesion followed by the use of amphotericin B would be an
ideal treatment for equine pythiosis (46, 73).

Surgery in human cases with arteritis has been used as well. However, the
mortality of patients because of disseminated abdominal arteritis showed that this method
was only partially successful (49). In the treatment of human pythiosis, surgery is the
most widely used method. In the cutaneous form, which is limited, surgery may be
sufficient. Super-saturated potassium iodide (SSKI) and the combination of amphotericin
B and S-fluorocytosine, or terbinafine and itraconazole after surgical removal have been

proven to be partially effective (35, 73). Therefore, removal of the infected tissues and

10

arteries by surgery is still used for the treatment of human pythiosis (35, 73).

Chemotherapy is another choice that has been used to treat pythiosis. Two drugs
are usually used: amphotericin B and iodine (49). Amphotericin B is usually used to treat
most mycotic infections. It mainly targets ergosterol, a compound present in the
cytoplasmic membranes of fungi (46). Interestingly, ergosterol is not present in the
cytoplasmic membrane of P. insidiosum (46). In humans and animals, amphotericin B
showed inconsistent results. For instances, some cases responded well to amphotericin B
while some reports indicated that amphotericin B was not an efficient therapy for
pythiosis. Two young immunocompetent males were reported with subcutaneous
pythiosis and responded well after treatment with amphotericin B (89). Nevertheless,
there are more cases showing poor success with amphotericin B. It was inefficient in
curing six thalassemic patients with systemic pythiosis in Thailand (94). Imwidthaya’s
review article pointed out that there is no drug of choice for pythiosis because P.
insidiosum does not need sterols for cell wall synthesis (35). Thianprasit et al. (86) also
indicated that amphotericin B was not practical in the treatment of pythiosis. Additionally,
the highly toxic side effects to the host and the cost of the therapy make amphotericin B
the last choice for chemotherapy (49, 73).

Iodine has also been used to treat pythiosis with contradictory results in equines
and humans. Some equine cases with intravenous injection of potassium iodide or sodium
iodide have been cured while others did not response (49). In humans, saturated solution
of potassium iodide has been used to cure two cases of subcutaneous human pythiosis
without recurrence after one year of therapy. However, the therapy failed to cure cases of

vascular human pythiosis. In ophthalmic pythiosis, it was ineffective as well (86).

In addition to the two drugs, other drugs have also been used and acquire
successful results. For example, a child with deeply invasive infection caused by P.
insidiosum has been treated by the combination of terbinafine and itraconazole. After
several months, the infection was solved and the patient remained in healthy condition
after 1.5 years (81).

Immunotherapy has been investigated for the treatment of pythiosis as well. Two
kinds of immunogens have been used: the Australian and Costa Rican vaccines. The
Australian vaccine by Miller (61) was prepared from sonicated hyphal antigens, whereas
the Costa Rican vaccine was made of precipitated proteins from culture filtrate antigens
(CFA) by Mendoza and Alfaro (50). Miller vaccinated thirty horses with clinical features
and ten horses with pythiosis following unsuccessful surgery and he got 53% cure rate in
animals using vaccination only and a further 33% cure rate in those using vaccination
after surgery (61). Later, Mendoza et a1. utilized CFA to treat infected horses and reported
a similar result (50). They also noted that this vaccine cures the early infections better,
but not the chronic stages (50).

Both vaccines cure horses with pythiosis. However, the age of the lesions is
important for the efficiency of immunotherapy. Horses with lesions two or more months
old do not respond to the vaccines. On the contrary, the vaccines cure horses with lesions
less than two months old. The Costa Rican vaccine significantly reduces the
inflammatory reaction at the site of injection, which is the main side effect of the

Australian vaccine. In addition, the Costa Rican vaccine is effective even 18 months after

its preparation, whereas the Australian vaccine loses its effectiveness after storage at 4°C

for two to three weeks (58).

12

Besides horses, immunotherapy has been successfully used in humans and other
animals. A fourteen-year—old boy fi'om Thailand with arthritic infection caused by P
insidiosum was treated by immunotherapeutic vaccine. He received one hundred
microliters of the vaccine through subcutaneous injection twice. After four weeks, his
symptoms vanished. One year after the vaccination, the boy was still in good condition
without recurrence (87). A dog with cutaneous pythiosis was also cured by
immunotherapy (33).

In 2003, Mendoza et a1. introduced a new P. insidiosum vaccine formulation with
enhanced immunotherapeutic properties (5 6). This new vaccine contained exoproteins
and endoproteins extracted from cultures of P. insidiosum and cured 72% of eighteen
equines and 33% of six canines with pythiosis. Most of the injected equines with strong
or mild inflammatory reactions at the site of injection were cured while dogs with acute
diseases (less than two months) were cured. After being injected into animals with
pythiosis, this vaccine triggers the production of lymphocytes and macrophages (Th1
immune response) instead of eosinophils, mast cells, IgE and precipitin IgG (Th2
immune response) activated by P. insidiosum infection. It is believed that the
transformation from Th2 immune response to Th1 immune response is crucial to this

vaccine’s curative property (56).

1.7 Phylogeny of Pythium insidiosum
To understand more about P. insidiosum, its phylogeny using rDNA sequences has
been investigated. The strains of P. insidiosum have been classified into three

phylogenetic groups using restriction fragment length polymorphism (RFLP) analysis of

13

the ribosomal DNA (rDNA) intergenic spacer (IGS) between the large subunit and small
subunit (78). In this study, thirty-one P. insidiosum isolates were examined. PCR was
performed with primers Q and P2 to amplify a DNA product with 4.8 to 5.2 kb for all
isolates. The amplified DNA was digested by restriction endonucleases including AluI,
HaeIII, HincII, Hinfl, Mbol, R501 and Tan and then analyzed by RFLP. A phenograrn
was created by using these restriction fragment patterns (78). This study found P.
insidiosum distributed into three groups while two of them corresponded to their
geographic origins. The RFLP assay provides a method to study the geographic origins of
P. insidiosum isolates.

Sequence analysis of the rDNA internal transcribed spacers (ITS) was later used
to generate phylogenetic relationships between twenty-one strains of P. insidiosum (79).
Neighbor-joining method and parsimony analysis were used to analyze those P.
insidiosum isolates, and the phylogenetic trees they revealed were almost identical (79).
Based on those studies, P. insidiosum has been divided into at least three groups.
Interestingly, these three genetic groups are related to their geographical areas.

In 2005, an African dog was diagnosed with pythiosis (75). The DNA sequence
from this infected dog was used to perform the phylogenetic analysis and it was found
that this dog belongs to a forth group which is far away from the other species in the

evolutionary relationships (75).
1.8 Chitin and Chitin Synthase

In addition to DNA sequence data, protein sequences have been used for

analyzing phylogenetic relations. Examples of these are actin, tubulin, elongation factor,

14

chitin synthase and other gene sequences. In this study, 500 bp of the gene encoding
chitin synthase from 25 isolates of P. insidiosum was used to challenge the finding of
Schurko et a1. (78, 79) using ribosomal DNA sequences.

Chitin is a polymer of N-acetylglucosamine residues (3) widely distributed in the
Kingdom Fungi, and present in the exoskeleton of insects (20, 93). It has been also
successfully isolated from shells of crustaceans using ethylenediaminetetra—acetic acid
(26). Chitin polymer is attached to the nonreducing end of the glucan chain. This unique
connection has been suggested as a target to develop anti-fimgal drugs against fungal
pathogens (41, 85). The length of a single strand of chitin varies from 5,000 to 8,000
residues in crabs to only about 100 residues in yeast cells (83). Based on the hydrogen
bond interactions between strands, chitin molecules are classified as alpha, beta, and
gamma forms. The alpha form is the only one found in true fungi (70).

The synthesis of chitin is catalyzed by the enzyme chitin synthase (CHS), a
membrane bound protein that has been suggested as another target for the development of
new anti-fungal drugs. In Saccharomyces cerevisiae, more than three chitin synthase
isoenzymes have been found, each with different functions. Chitin synthase 1 and chitin
synthase 11 together make 10% of the cellular chitin whereas chitin synthase 111 makes the
remaining 90% (11). In S. cerevisiae, chitin synthase II was originally reported as an
essential gene for septum formation and cell division (82). However, subsequent work
has found that chitin synthase 1 and chitin synthase II are nonessential for cell division
(12). Interestingly, the interaction of chitin synthase II and chitin synthase III is essential
for the viability of S. cerevisiae (80). Drugs such as nikkomycin, polyoxin-D, and

diflubenzuron are inhibitors of chitin synthase in fungi and insects (20, 24); thus, they

15

have been used to study the in vitm synthesis of CHS.

The majority of fungi have three to six CH8 and the genes encoding these
isoenzymes are important for their developments (6, 47, 48, 65). For instance, in
Aspergillus nidulans, five genes have been reported (chsA, chsB, chsC, chsD, csmA) (65,
96). chsA is involved in conidia formation. chsB is required in hyphal growth and
development (6). chsC is a nonessential gene. chsD together with chsA is important in
conidia formation (65). csmA is essential for the maintenance of cell wall integrity as
shown by the chsA chsC double mutant (96).

By aligning the S. cerevisiae 011s] and chs2 gene and the Candida albicans chsl
gene, two short, completely conserved regions were found by Bowen et a1. (8). They used
the conserved sequences to design PCR primers and successfully amplified chitin
synthase fragments (~600 bp) from the genomic DNA of different fungal species. Those
sequences were used for performing phylogenetic analysis (8). The ch31 gene sequences
of T richophyton mentagrophytons complex and T rubrum were analyzed to investigate
their phylogenetic relationship (40). Later, this gene was used to study the phylogenetic
relation of Epidermophyton floccosum to eight other derrnatophyte species belonging to
the genera Microsporum and Trichophyton. The result showed that these three genera
were genetically different from each other, which confirmed the previous analysis using
mitochondrial and ribosomal sequences (39). It was also used to genetically distinguish
Microsporum from Chrysosporium and T richophyton, verifying the same classification
reached by morphological characteristics in the past (34). These examples showed the
successful application of chs in the phylogeny of firngi.

Oomycetes was known to have cellulose and glucan in their cell walls, but not

16

chitin. However, in recent years, it was reported that the Oomycetes contain chitin as well.
It was found that chitin is present in the Oomycete Apadachlya sp. by using X-ray
diffraction (44). Chitin was found to occur in the cell walls of the Oomycete Pythium
uotimum by using wheat germ agglutitin (WGA) (19). Also, Bulone et al. demonstrated
that chitin exists in the cell wall of hyphae and of regenerating protoplasts of Sapralegnia
monoica using several assays (13). They obtained a residue that is hydrolysable by
chitinase using the chemical extraction procedure used to isolate chitin from hyphal cell
walls of S. monoica. They also found chitin in chemically extracted residues from hyphal
cell walls and from KOH-insoluble products of regenerating protoplasts using X-ray
diffraction, electron diffraction, and infrared spectra methods. In addition, chitin synthase
activity was identified from hyphal cell walls and regenerated protoplasts. The activity of
this enzyme was stimulated by trypsin, activated by magnesium chloride and N-
acetylglucosamine, and inhibited by polyoxin D (13). Later, the gene encoding chitin
synthase II was characterized from S. monoica by PCR using the degenerate primers
designed by Bowen et al. in 1992. This was the first time chitin synthase gene was ever
cloned and recognized from the Oomycetes (64). Those investigators amplified a 600 bp
fragment from the conserved regions of S. monoica. Cth gene from several different
fungi, including Rhizopus oligosporus, Candida albicans, Aspergillus nidulans,
Saccharomyces cerevisiae and Neurospora crassa were amplified, and a phylogenetic
tree was built based on those sequences. These researchers suggested that chitin is present
in Oomycetes and that Oomycetes and chitinous fungi have conserved chitin synthase
systems despite their divergent evolution (64).

In 1999, Perpich et al., using the same set of degenerate primers designed by

17

Bowen et al., successfully amplified a 600 bp amplicon of the ch32 gene from the
Oomycete Pythium insidiosum and the fungi Achlya Ambisexualis, Phytaphthara capsici,
and Saprolegnia monoica. They constructed a phylogenetic tree showing that the
Oomycete grouped together away from the other true fungi (71). They also designed a
pair of primers that was specific for P. insidiosum chs gene based on the sequences they
acquired in this research.

These studies showed that the use of chs to study the phylogenetic relations of P.
insidiosum should be useful to understand the evolution and the background of this

Oomycete.

l8

2. OBJECTIVES

(1) To amplify and sequence 500 bp of the chitin synthase gene in twenty-five isolates
of Pythium insidiosum by PCR using the in-house designed primers CHSI and
CHSZ.

(2) To use the chitin synthase gene sequences of P insidiosum for phylogenetic analysis.

(3) To compare the P. insidiosum phylogeny analyzed by chitin synthase sequences and

those published before using RNA sequences.

19

3. MATERIALS AND METHODS

3.1 Pythium insidiosum strains
Twenty-five strains of P. insidiosum were used to study phylogeny (Table 1).
Among them, twenty-four samples from Dr. Mendoza’s P. insidiosum collection were

transferred and cultured in the Sabouraud’s dextrose broth (2% glucose [Mallinckrodt

Baker, Inc., USA] and 1% tryptone [Difco Laboratories, USA] in distilled water) at 37°C
under shaking condition for 5-7 days. The last sample was DNA extracted directly from

the infected tissues in an Afi'ican dog with P. insidiosum infection.

3.2 DNA extraction

After incubation, the DNA was extracted as follows: 200 ml of P. insidiasum
culture was killed by the addition of merthiolate with 0.05% final concentration. The
hyphae were harvested by filtration, and then transferred to an autoclaved mortar. The
hyphae were ground by hand until homogeneous. The mixture was transferred into 1.5 ml
eppendorf tube and additional 400 pl of lysis buffer (3% SDS [Roche, USA], 50 mM
EDTA [Sigman, USA], 50 mM TrisHCl [Gibco BRL, USA], 1% 2-mercaptoethanol
[Sigma, USA] in distilled water) and 250ug/ul of proteinase K (Roche, USA) were added.
The tube was vortexed until the mixture became homogeneous, and then incubated in a
65°C water bath for 1 hour. 400 pl of phenol: chloroform: isopropanol (25: 24: 1) (Sigma,
USA) was added and vortexed briefly. Then the tube was centrifuged at 14,000 rpm for
15 minutes at room temperature to separate organic and aqueous phases. Following
centrifugation, the aqueous layer was removed to a new eppendorf tube. Two volumes of

100% ice cold ethanol (Pharmcom, USA) and 10 ul of 3 M ammonium acetate

20

(Mallinckrodt Baker, Inc., USA) were added into the tube. The tube was inverted gently
several times and incubated at —20°C overnight. The next day, the sample was centrifuged
at 14,000 rpm for 15 minutes at room temperature to pellet the DNA. The supernatant
was poured off and the pellet was rinsed with 1 ml of 70% cold ethanol. The pellet was

air dried for 30 minutes and resuspended with 200 ul of sterile water. The DNA was

stored at —80°C. A 0.8% agarose gel (FMC®, USA) stained with ethidium bromide was

run at 150 V for 30 minutes to evaluate the genomic DNA quality.

3.3 RNase treatment

A final concentation of 4pg/u1 RNase (Promega, USA) was added to the DNA
sample and the tube was incubated at 37°C for 20 minutes. To remove the enzyme, an
equal volume of phenol: chloroform: isopropanol (25: 24: 1) solution was added and the
tube was shaken. The sample was centrifuged at 14,000 rpm for 10 minutes at room
temperature and the upper aqueous layer was collected. Additional 0.5 volume of 1.5 M
ammonium acetate and 2 volumes of 100% ice-cold ethanol were added. Then the DNA
was incubated at —20°C overnight. The next day, the sample was centrifuged at 14,000
rpm for 15 minutes at room temperature to pellet the DNA. The upper layer was
discarded and the pellet was rinsed with 70% cold ethanol. The pellet was air dried for 30

minutes and resuspended with 100 pl of sterile water. The DNA was verified on a 0.8%

agarose gel at 150 V for 30 minutes and then stored at —80°C.

21

3.4 PCR

In an eppendorf tube, 2 mM MgC12 (Roche, USA), 1X buffer (Roche, USA),
1.25U Taq Gold Polymerase (Roche, USA), 20 pmole chitin synthase 1 primer, 20 pmole
chitin synthase 2 primer (CHSI, CHSZ) (Integrated DNA Technologies, Inc., USA), 0.3
mM dNTP (Roche, USA), 100 ng DNA template, and distilled water to a total volume of
25 pl were mixed. The set of primers was designed by Perpich et al. (71) to amplify a 500
bp of the gene encoding CHS of P. insidiosum. A negative control reaction containing all
the reagents except the DNA template, and a positive control using MT PI-27 DNA
sample were included in all PCR experiments. The thermal cycler (GeneAmp® PCR
System 9700, Perkin Elmer. USA) protocol was as follows: 95°C for 10 minutes, then 40
cycles of 94°C for 1 minute, 63°C for 2 minutes, 72°C for 3 minutes, and the final
extension reaction at 72°C for 7 minutes. The PCR products were used to run a 0.8%
agarose gel to visualize the amplicons. The remainder of the PCR products were kept

at —20°C.

3.5 Cloning
The PCR amplicons were cloned into pGEM®-T Easy Vector (Invitrogen, USA). 2

pl of PCR product, 1 pl of pGEM®-T Easy vector, 1 pl of T4 DNA Ligase, 5 pl of 2X

Rapid Ligation Buffer and 1 pl of sterile water were mixed and incubated at 4°C
overnight. The next day, the mixture was centrifuged briefly. 2 pl of the ligation reaction
was added to a sterile 1.5 ml tube on ice. JM109 High Efficiency Competent Cells were
placed on ice until just thawed. The cells were mixed by gently flicking the tube. 50 pl of

the cells were transferred to the ligation reaction tube. The tube was flicked gently and

22

incubated on ice for 20 minutes. Then the tube was heat-shocked at exactly 42°C for 50
seconds and immediately returned to ice for 2 minutes. 950 pl of room temperature SOC
medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, and 20 mM glucose) was added
and the tube was incubated at 37°C for 1.5 hours with shaking (~200 rpm). After
incubation, 50 pl and 100 pl of the culture were plated onto Luria and Bertani (LB) agar
(1% tryptone, 0.5% yeast extract, 2% agar and 1% NaCl) with ampicillin (400pg/ml),
isopropyl-beta-D-thiogalactopyranoside (IPTG), and 5-Bromo-4-Chloro-3 Indolyl-Beta-
D-thiogalactopyranoside (X-gal) plates. The plates were incubated at 37°C overnight. The

next day, 5 white colonies were selected to subculture onto a new plate. The new plate

was incubated at 37°C overnight.

3.6 Cracking gel

A small amount of the bacterial colony from the above LB/ampicillin/IPTG/X-gal
plate was transferred to a sterile eppendorf tube containing 50 pl of sterile 10 mM EDTA
(pH=8) with a sterile toothpick. 50 pl of 0.2 N NaOH, 0.5% SDS and 20% sucrose

freshly made solution was added. The mixture was vortexed for 30 seconds and then

incubated in a 70°C water bath for 15 minutes. Additional 1.5 pl of 4 M KC] and 0.5 p] of
0.4% bromophenol blue were added. The mixture was incubated on ice for 5 minutes and
centrifuged at 14,000 rpm for 3 minutes. 50 pl of the supernatant was loaded to run a

0.8% agarose gel at 140 V for 1 hour. Colonies with inserted PCR products were selected

and subcultured onto a new plate. The plate was incubated at 37°C overnight. The next

day, the colonies were transferred to a 6 m1 LB broth (1% tryptone, 0.5% yeast extract,

and 1% NaCl). The broth was incubated at 37°C under shaking condition (~200 rpm)

23

overnight.

3.7 Plasmid DNA extraction

Plasmid DNA was extracted with S.N.A.P. MiniPrep Kit (Invitrogen, USA)
following manufacture protocol as follows: 1.5 ml of the culture was transferred to a 1.5
m1 eppendorf and centrifuged at 14,000 rpm for 5 minutes to collect the cells. The broth
was poured off and the cells were resuspended with 150 p1 of Resuspension Buffer. 150
pl of Lysis Buffer was added and the tube was gently mixed. The tube was then incubated
for 3 minutes at room temperature. Additional 150 pl of ice-cold Precipitation Salt was
added and the tube was mixed. The tube was centrifuged at 14,000 rpm for 5 minutes at
room temperature. The supernatant was collected into a new eppendorf tube and 600 pl of
Binding Buffer was added to the new tube. The tube was mixed. The entire solution was
pipetted to the S.N.A.P. Miniprep Column/Collection tube and passed through the column
by gravity to increase the DNA binding rate. 500 pl of Wash Buffer was added and the
tube was centrifuged at 3,000 rpm for 30 seconds and the flow-through was discarded.
900 pl of IX Final Wash Buffer was added and the tube was centrifuged at 3,000 rpmhfor
30 seconds. The tube was centrifuged at 14,000 rpm for 1 minute at room temperature to
dry the resin. The S.N.A.P. MiniPrep Column was transferred to a sterile 1.5 ml
eppendorf tube. The plasmid DNA was eluted by adding 60 pl of sterile water and the

column was incubated for 3 minutes at room temperature. The tube was centrifuged at

14,000 rpm for 30 seconds to elute the plasmid DNA and the product was stored at -20°C.
A 0.8% agarose gel was run at 140 V for 30 minutes to evaluate the quality of the plasmid

DNA.

24

3.8 Sequencing
In a PCR tube, 4 pl of Template Ready Reaction Mix (TRRM) (Applied
Biosystems, USA), 1 pl of M13 forward or reverse primer (Invitrogen, USA), 2 pl of

distilled water, and 3 pl of the above plasmid DNA preparation were mixed. A PCR of 25

cycles at 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes was run. After
PCR, a clean-up step was performed to eliminate unincorporated Dye Terminator from
the reaction mixture. 6 pl of sterile water, 32 pl of 95% ethanol and 10 pl of PCR product
were mixed and incubated for 15 minutes at room temperature. The tube was centrifuged
at 14,000 rpm for 20 minutes at room temperature. The supernatant was poured off and
the pellet was centrifuged in a vacuum centrifuge for 10 minutes at room temperature to
dry the residues of ethanol. After that, 20 pl of Template Supression Reaction buffer

(Applied Biosystems, USA) was added. The tube was then mixed by vortex, briefly

centrifuged at 14,000 rpm for 5 seconds, heated at 95°C for 2 minutes, immediately
transferred to ice, and incubated for 5 minutes. The tube was mixed by vortex and then
briefly centrifuged at maximtun speed. A cap was added and sequence was performed in
an ABI Prism 310 genetic analyzer apparatus (Perkin Elmer, USA) using the software

DNA Sequence Analysis version 2.0 (Applied Biosystems, USA).

3.9 Phylogenetic analysis
The phylogeny was analyzed by Molecular Evolutionary Genetic Analysis
Software (MEGA3) (Written by Sudhir Kumar, Koichiro Tamura, and Masatoshi Nei,

2004)

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Species Country of Origin Host GenBank Accession No.
MTPI-O 1 USA Equine DQl 16403
MTPI-02 USA Canine Not submitted
MTPI-03 USA Equine DQl 16404
MTPI-04 USA Equine DQl 16405
MTPI-05 USA Equine DQl 16406
MTPI-06 USA Canine DQl 16407
MTP 1-07 USA Canine DQl 16408
MT PI-08 USA Canine DQl 16409
MTPI-09 USA Feline DQl 16410
MTPI- 1 0 USA Human DQl 16411
MTPI-ll USA Human DQl 16412
MTPI-12 Thailand Human Not submitted
MTPI-13 Thailand Human Not submitted
MTPI-14 Thailand Human Not submitted
MTPI-lS Thailand Human Not submitted
MTPI- l 6 Thailand Human Not submitted
MTPI-l 7 Costa Rica Equine DQl 16413
MTPI-18 Costa Rica Equine DQ116414
MTPI-l 9 Costa Rica Equine DQl 16415
MTPI-20 Costa Rica Equine DQl 16416
MTPI-21 Papua New Guinea Equine DQl 16417
MTPI-22 Haiti Human DQl 16418
MTPI-23 Australia Equine DQl 16419
MTPI-27 Brazil Human DQl 16420

Africa Dog Mali Canine DQ116421
Table 1

List of twenty-five P. insidiosum species used in this study for sequencing and
phylogenetic analysis (Some sequences were not submitted. They need further

verification).

26

4. RESULTS

4.1 DNA extraction from twenty-five strains of Pythium insidiosum

The hyphae of P. insidiosum were collected, usually after 5-7 days of incubation.
After the hyphae were ground, the DNA was extracted and then electrophoresed in a
0.8% agarose gel stained with ethidium bromide to evaluate its purity. A strong single
band at approximately 23 kb was observed in all samples (Figure l). The smeared lower
section contained ribosomal RNA, transfer RNA, mitochondrial RNA, and small nuclear

RNA, which were later removed by RNAse treatment (Figure 2).

4.2 Amplification of a 500 bp product of chitin synthase gene of P. insidiasum

Primers designed by Perpich et a1. (71) based on the chitin synthase gene from P.
insidiosum amplified by using primers designed by Bowen et a1. (8) amplified a 500 bp of
the gene encoding chitin synthase in all twenty-five P. insidiosum isolates. No amplicon
was observed in the negative control containing no DNA templates. A PCR reaction,
using MTPI-27 DNA sample as template, was performed as a positive control in all PCR

reactions (Figure 3).

27

10000 bp , , <— 23 kb

mRNA

rRNA
500 bp tRNA
250 bp snRNA

 

 

Figure 1

DNA extracted from Pythium insidiosum. The figure shows the typical extracted DNA
from six different P. insidiosum strains. The size of the genomic DNA is 23 kb. The lower
section is a mixture of RNA. Lane M — DNA ladder (Promega, USA), and lanes 1 to 6 —

genomic DNA extracted fi'om six P. insidiosum samples before RNAse treatment.

28

23 kb

23 kb

 

Figure 2
RNAse treatment. This figure shows the genomic DNA sample (1 to 17) after RNAse
treatment. A 23 kb DNA band with little or without RNA is observed in all samples. Lane

M — DNA ladder and lanes 1 to 17 — seventeen different isolates of P. insidiosum.

29

500 bp
250 bp

 

Figure 3

PCR using primers CHSI and CHS2 to amplify the gene encoding chitin synthase.
The genomic DNA from twenty-five P insidiosum isolates was used as DNA template for
PCR. In this figure, a single band of approximately 500 bp was amplified in all five DNA
samples including the positive control. No products were observed in the negative control
which had no DNA template. Lane M — DNA ladder, Lane 1 to 4 — four P. insidiosum

isolates, lane 5- positive control, and lane 6 — negative control.

4.3 Cloning of the 500 bp DNA amplicon into E. coli cells

The 500 bp DNA amplicons were cloned into plasmid vectors (pGEM®-T Easy
Vector) and then transfected into E. coli cells. The positive clones were confirmed by
cracking gel. The cells were broken to release their cytoplasmic contents, including the
genomic DNA and the plasmids. A gel was run to separate the plasmids by size. The
plasmid with insertion had bigger size than the plasmid without insertion. A negative
control (plasmid without insertion) was used as a marker to distinguish the plasmids with

and without insertions (Figure 4).

4.4 Purification of the vector from E. coli cells and sequencing

After confirming by cracking gel,‘plasmids with insertion were purified from the
E. coli cells using a commercial kit (S.N.A.P. MiniPrep Kit, Invitrogen, USA). A gel was
run to evaluate the quality and concentration of the plasmid DNA (Figure 5). The purified
plasmids were then used for sequencing. Each sample was analyzed twice by adding M13
forward and M13 reverse primers to get a complete nucleotide sequence. After all the
samples were sequenced, they were aligned with the sequence navigator program and a
DNA fragment with 498 bp was defined in all isolates (Appendix A). Those sequences
matched the chitin synthase gene sequence obtained by Perpich et a1. (71). After
alignment, all sequences were deposited to NCBI GeneBank (accession number

DQ116403 ~ DQ116421) (Table 1).

4.5 Phylogenetic analysis using neighbor-joining and maximum parsimony methods

These sequences were then used for the phylogenetic analysis. Phylogenetic trees

31

were constructed with the Molecular Evolutionary Genetic Analysis Software (MEGA3)
(Appendix A and B) using maximum parsimony and neighbor-joining methods with 1000
bootstrap, respectively, to confirm the accuracy between the different methods. Figure 6
and Figure 7 show the phylogenetic trees built by these two methods. In both methods,
the twenty-five isolates were mainly separated into four distinct genetic clades. Those
isolates from America and Thailand grouped together away from those from Africa,
middle Asia, and Oceania. However, the isolates from Thailand did not show any
differentiation from those from America, which was controversial with the previous result
(75, 78). The average genetic distance within these species was very low (3.3%), which
confirmed the previous conclusion that chitin synthase gene was very (conserved in the

Oomycetes (64).

32

 

15 161718. 9.26 N

 
 
 

 

Genomic
DNA
——->

Figure 4

Cracking gel to determine the plasmid with DNA insertion. All the tested plasmids
were higher in the gel than the negative control (N), indicating that all colonies have
plasmids with DNA insertions. Lane N — negative control (plasmid without DNA
insertion) and lanes 1 to 20 — plasmids from white colonies selected from the

LB/ampicillin/lPTG/X-gal plates.

33

Coiled p1asmids—+

Plasmids—v

 

Figure 5

Plasmid purification. This figure shows purified plasmids fi'om five different strains
containing 500 bp insertions from P. insidiosum. A strong band is observed in each
sample and a weaker band is shown above the plasmid band. Lanes 1 to 5 — plasmids

from strains MTPI-05 ~ MTPI-09.

34

MTP|13
MTPIZO

 
   
  
  
   
  
 

44

MTPI15

MTPI 03
83 MTPIO7
MTPI19

MTPI17

MTPI22
51_[_—MTPIOQ
96 50 [MTPIOB

52 MTPI18

—MTP|05

68

 

 

 

 

 

MTP127

 

 

9° MTPI01

 

56 MTP116

 

MTPI12

 

67 MTPI14
MTP106

 

MTPI 04
MTP|10

 

MTPI 02

 

MTP123

 

 

— MTPI 21

 

 

72 MTPI11

 

 

 

5‘ DOG

 

fl.
db

Figure 6
Phylogenetic analysis using neighbor-joining method. Twenty-five genes sequences
encoding chitin synthase from P. insidiosum were analyzed using neighbor-joining

method.

35

MTPI 13

  
  
   
     
 

MTP120

MTPI 15

MTP103
MTPI 07
MTPI 19

53 MTPI17

 

MTPI 22

 

 

MTPI O9

 

45
91 34 {MT-Pica
6° MTPIIB

MTPI 05

 

 

 

 

MTP127

 

 

MTPI16
84

MTPI 01

 

 

— MTPI 12
MTPI 14
MTPI 10

 

MTPI 04
MTPI O6

 

MTPI 02

 

DOG

65
MTPI11

 

 

 

 

MTP121

 

99

 

MTP123

 

-I-
‘-

Figure 7
Phylogenetic analysis using maximum parsimony method. Twenty-five gene
sequences encoding chitin synthase sequences from P. insidiosum were analyzed using

maximum parsinomy method to get the phylogenetic tree.

36

5. DISCUSSION

Phylogenetics—the reconstruction and analysis of evolutionary relationships
between different organisms—-is crucial, not only in evolutionary biology, but also for
related areas such as conservation genetics and epidemiology. Morphological or
physiological characteristics have been used as the basis to classify organisms. However,
just by using morphological data, it is difficult to distinguish very closely related
organisms or those with similar characteristics. In recent years, molecular analysis has
been used as a new tool combined with morphological observations to get more accurate
classifications. Small subunit ribosomal RNA (SSU rRNA) and internal transcribed
spacer (ITS) are the most common sequences used in molecular studies (22, 31, 43, 45,
66-68, 90, 91). In addition, protein sequences have been used. For instance, the gene
sequence encoding actin protein has been used in the analysis of the molecular
evolutionary and taxonomic relationships in a number of different eukaryotes, such as
amoebae (25), protozoa (9), fungi (21, 32), plants (4), and mollusks (l6). Chitin synthase
gene sequence has also been used to evaluate the evolutionary and phylogenetic
relationships in several organisms (76).

In eukaryotes, the animals, fungi, plants, most algae, and many protistan lineages,
such as ciliates, oomycetes, and hyphochytriomycetes are known as a “crown”. The SSU
rRNA sequences from organisms belonging to the crown were used by Van de Peer et al.
to construct an evolutionary tree (90). The results from other research using SSU rRNA
as well, along with Van de Peer et al.’s research, suggested that SSU rRNA is a practical
approach for the construction of phylogenetic trees between organisms within the

Kingdom Stramenopila (31, 45, 91). In recent years, the evolutionary history of the

37

Oomycetes gained increased awareness because of their unique characteristics; thus, the
phylogeny of the Oomycetes has been well studied (22, 37, 43). They were once
classified as fungi because of their filamentous growth, and because they feed on rotting
materials as fungi do. However, later molecular sequence data indicate that those
organisms belonging to the Stramenopila are far away from the fungi.

DNA sequence has been used for the identification and classification of newfound
species. Some Pythium species, such as P. segm'tium, P. carbonicum, and P. terrestris,
were isolated and then categorized in the genus Pythium. DNA data have suggested that
they are different species even though they possess similar morphological characteristics
(66-68). Besides distinguishing different organisms, phylogenetic analysis using DNA
sequences have also been used to construct phylogenetic trees of different clusters with
one organism. A good example is Arthroderma benhamiae, a member of dermatophytes,
in which the chitin synthase gene of eight isolates from different origins was investigated.
This study showed that A. benhamiae could be separated into two clusters with different
origins. One was the American-European cluster and the other the Africa cluster (38).
Schurko et al. also utilized the ITS gene sequences to explore the phylogenetic
relationship of P. insidiosum isolates fiom different origins (78, 79). According to their
studies, P insidiosum could be classified into four clusters highly related to their
geographic origins.

To confirm this original P. insidiosum phylogeny using the ITS sequences, a
phylogenetic study, using the gene encoding chitin synthase, was performed. PCR using
the primers designed by Perpich (71) amplified a single band at approximately 500 bp in

twenty-five P. insidiosum isolates, including twenty-four samples from Dr. Mendoza’s P.

38

insidiosum collection and a DNA sample extracted from a biopsy specimen collected
from the infected tissue of a dog diagnosed with pythiosis in Afiica (75). The DNA
segments of twenty-five samples were cloned and sequenced. The nucleotide sequences
were aligned and then used to perform phylogenetic analysis by maximum parsimony and
neighbor-joining methods.

Overall, the difference within the studied strains was slight, supported by a low
average genetic distance 3.3%. After translating to amino acid sequences, these studied
strains displayed almost identical sequences, confirming that chitin synthase is a highly
conserved protein in P. insidosum (Appendix B). The maximum parsimony and neighbor-
joining methods generated phylogenetic trees with nearly identical patterns. Those P.
insidiosum isolates were mainly separated into four clades. Clade I contained eleven
isolates from regions of the Americas and Southeast Asia. Clade II had nine strains with a
similar distribution. Clade III contained one isolate from the USA and clade IV was four
isolates from Australia, Afiica, and the USA.

Clade I grouped strains from Costa Rica, Haiti, Thailand, and the USA from
mammalian hosts including equine, feline, and human, respectively. All four Costa Rican
strains are included in this clade. Clade 11 contained strains from Brazil, Thailand and the
USA. Thai strains, which were clustered as a group and separated from the Asia strains in
the previous studies using ITS sequences, were divided into two clades along with other
strains from the Americas. These two clades were more closely related to each other than
the others. However, they were indeed two different groups, which was supported by a
high bootstrap value at 96%.

The isolate MTPI-02, from a dog in the USA, was the only member of clade III. It

39

was separated from the other isolates supported by nearly a 100% high bootstrap value.
The Afiican strain, which was separated as a 4th clade by Ricierre et al. (75), was grouped
with MTPI-11, MTPI-21, and MTPI-23 as clade IV. A special event previously reported
by Schurko et al. (78, 79) was a strain from a patient with keratitis in Pennsylvania, USA.
In this study, this strain (MTPI-11) showed genetic similarity with those from Afiica and
Oceania. A reasonable explanation for this situation proposed by those researchers was
that this man once lived in Afghanistan and he had previously received food products
from this country. Therefore the origin of his infection might be from Asia instead of
America (78, 79).

The result obtained in this study displayed partial consistent with the previous
studies (78, 79). Schurko et al. found that those isolates were classified into different
clades highly related to their geographic origins. However, in this study using chitin
synthase gene, this trend was not obviously observed. Basically, all the Thai strains were
grouped with the American strains into two groups with one exception, the MTPI-02. The
other strains from Afiica, Asia, and Oceania grouped together. It is possible that the CH8
gene sequences are so conserved that they could not perfectly discriminate strains of P.
insidiosum isolated from different geographic locations.

Besides this possibility, other reasons are considered. In this study, twenty-four
strains were selected from Dr. Mendoza’s P. insidiosum collection. Those collected
strains have been subcultured several times in the past years. It is possible that some
strains were mislabeled and do not represent the strains in the label. Thus those strains
marked from Thailand were actually from the USA. Another possibility is that those

strains might be contaminated with American strains. Also, contaminations might occur

40

during the experimental procedures, such as DNA extraction, PCR, or cloning. Another
hypothesis previously discussed is the possibility that chitin synthase gene is not
powerful enough to discriminate those samples from Thailand from those found in
America in P. insidiosum. Although it has been reported as a useful tool to do the
phylogenetic analysis in other strains, it might not be that usefiIl in P. insidiasum (8, 34,

39,40,64,71)

41

6. FUTURE DIRECTION

So far this result showed that the gene encoding chitin synthase might not have
enough sequence variation to perfectly discriminate the strains of P. insidiosum. However,
more samples and more sequences should be analyzed before we reach the conclusion
that chs is not suitable for P. insidiosum phylogenetic analysis. In addition, genes
encoding other proteins, such as actin, could also be investigated and compared with the
result obtained using chitin synthase gene sequences to reevaluate its usefulness for P.

insidiosum phylogeny.

42

10.

ll.

12.

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51

APPENDIX A

Twenty-five strains of Pythium insidiosum’s gene sequences encoding chitin synthase
(498 bp)

52

#MEGA

lTitle MTPI:

tFormat

DataType=Nucleotide CodeTable=Standard
NSeqs=25 NSites=498

Identical-.

1Domain-Data

#MTPI_01
#MTPI_02
tMTPI_03
#MTPI_04
#MTPI_05
tMTPI_06
#MTPI_07
#MTPI_08
tMTPI_09
tMTPI_1o
#MTPI_11
tMTPI_12
tMTPI_13
tMTPI_14
tMTPI_15
tMTPI_16
IMTPI_17
lMTPI_18
#MTPI_19
tMTPI_2o
tMTPI_21
#MTPI_22
9MTPI_23
0MTPI_27
#ooc

tMTPI_01
#MTPI_02
#MTPI_03
IMTPI_04
OMTPI_05
tMTPI_06
#MTPI_07
#MTPI_08
#MTPI_09
{MTPI_10
OMTPI_11
{MTPI_12
{MTPI_13
#MTPI_14
#MTPI_15
#MTPI_16
tMrPI_17
#MTPI_18
QMTPI_19
tMTPI_2o
#MTPI_21
OMTPI_22
#MTPI_23
#MTPI_27

GGA

property-Coding
ACA CTC ACA

0. 0.. 000 00.
0. 00 0..
0. 0 .0
0 0 0 0
0 0 0 000
0 0 0
0 00
.
0..
0 0 000
0 .0 0 0 .
0.0 .0 0 0 000
0 0 .0. 0..
.0 .I. . 0 0
000 000 000 0 0
.0 . . 0 0
0 0 .0. 0 0
O I . 0 O 0 . O
O 0 0 O . .00
. 0 000 000
0 0 O O 0 . 0.0
. 0 .0. 0 O . 0
. I 0.. 0 0 0 .
0.. 00. 000 0 0

.OT .0. 00. 0.0
0. 0 00C 0..
OT 0 .0. C.
O O I 00C 0..
0T . 0. 0..

Missing=? Indel=-;

CodonStart-l;

GGC ATC GCA

AGA

53

AAC

EDOG ...

{MTPI_01
snrpr_o2
OMTPI_03 ...
#MTPI_04 ...
tMTPI_05 ...
ourPI_06 ...
IMTPI_07 ...
#MTPI_08 ...
tnrpr_09 ...
#MTPI_10 ...
#MTPI_11
#HTPI_12 ...
#MTPI_13 ...
{MTPI_14 ...
tnrpr_15 ...
tMTPI_16 ...
tMTPI_17 ...
tMTPI_18 ...
tMTPI_19 ...
tMrPI_20 ...
OMTPI_21 ...
{MTPI_22
turpr_23
tMTpI_27
tooc

tMTPI_01
tnrpr_02 ...
turprgoa ...
tnrpr_04 ...
turpr_os ...
OMTPI_06 ...
tnrpr_o7 ...
tnrpr_oe ...
turer_o9 ...
tMTPI_10 ...
tMTPI_11 ...
turpr_12 ...
#MTPI_13 ...
tnrpr_14 ...
{MTPI_15 ...
tMTPI_16 ...
tMTPI_17 ...
tnrpr_18 ...
IMTPI_19 ...
tnrpr_20 ...
tnrpr_21 ...
tnrpr_22 ...
tMTPI_23 ...
OMTPI_27 ...
sooc ...

sMTPI_01
ourpr_o2 ...
tMTPI_o3 ...
turpr_04 ...
turpr_05 ...

..C

GGA

CGC ACC AAA GCG AGT GCT TCC TGC CTC

..T ..G ..G

. .. . ... .T .
T .G . G . . . . . ..
.0 ..G ..G 0 I. O 0.. O .0.
OT ..G ..G 0.. ..C O 0.. .0. OT
..T ..G ..G ... 0.. 00. 0.. ... .0.

GCC TTT GAC

..T

III IIE
" IE: II
III '2: 'IE
.3 IE:

. IE: 'I
III IE: ..
.I'IE ..
.. ..C ..
. .. .T
I'. .II Iii:

O. O 0.. 000
.00 .0

.00 .0. 0 O
0.0 00. 0.

0.. .00 ..C .0. 000 00.
0 .0 0C 0.. .0. .0

0 .0. O C ... 0.. .00
000 000 ..C 0.. 0 00
00. ... ..C .0. .0. 0..

CTT TTC GAA TCC ACG GT6

.0. .00 .0 0. 0.. .00
0.. 0. 00. 0 0.

.00 0 00 00. O.

0 .00 0. 0 .0

..T
..T
..T

..T

..T ..C

TAT CTT ACC AGT CTT

one ..C on. no. ..C
no. 000 000 .00 ..C
0.. 0.0 no. .00 ..C
o o ..C ..T on. ..C
o o oo- oo 00. ..C
no. one no. .00 ..C
on an. 0.. .00 ..C
on. oo- ooo on. ..C
no. co. no. 00. ..C
coo ..C no. .0. ..C
no 00. 000 .00 ..C
000 ..C 0'0. .0. ..C

AGC GCG GGT GTC GAC

..T ..C 00. 0.. .0.

..C
..C
..C

..T
..T
..T

DOA 0.0 .0.
00A .0. 0..

..T ..C .0. 0.. 0.0
..T ..C 0.. 0.. .0.
..T ..C .0. ... ..0
..T ..C 00A .0. 0..
..T ..C .0. 0.. .0.
..T ..C .00 0.0 0..
..T ..C 00A 000 .0.
000 00. 00C 0.. ...
... ..T 0'0. .0. .0.

{MTPI_06
§MTPI_07
tMrPI_oe
tnrpr_09
turp1_1o
#MTPI_11
#MTPI_12
tMTPI_13
#MTPI_14
turpr_15
#MTPI_16
#MTPI_17
#MTPI_18
surpr_19
tMTPI_20
#MTPI_21
#MTPI_22
tMTPI_23
{MTPI_27
VDOG

{MTPI_01
rMTPI_02
tnrpr_03
lMTPI_04
tMrPI_05
#MTPI_06
tMTPI_07
rarpr_oa
QMTPI_09
#MTPI_10
tMTPI_11
#MTPI_12
{MTPI_13
tMrPI_14
#MTPI_15
tMTPI_16
sMrPI_17
surpr_1a
tnrer_19
turpr_2o
iMTPI*21
tMTpI_22
tMTPI_23
#MTPI_27
#poc

QMTPI_01
tnrer_02
tMTPI_o3
IMTPI_04
tMTPI_05
tMrPI_06
tMTPI_07
tMTPI_oa
turpr_09
OMTPI_10
tMTPI_11
{MTPI_12

.
...
0.0
00
...
000
.0
.
0
I.
0
0
.

0
.0
..
. .
00.

0.0
.
.0
...
..
.0
0
.
.
0
0.
.
0
0
.
00.

000
..0
.0.
.
...
. .
O
.
0 00
0..
0

.0 . .0 ..G
. ... .G
000 000 0.0 ..G

. . .. ..G
. ... . ..G
0. 0. 0 .G

O O O O I O O O. O. O
O O O O O I O . o.
O . O . O O O O
o c o o o o o
O O O O I . .
o o o O o o o
o o o o .0 00 o
0 C O O O O
O. O I O O O O
O. O O O o O O O
0 o O O O O. O

..
0
.
. 0
0 .
0
.0
0
0.
.0.
.
000
.
.
...
..
.00
0.0

..C

C00
CO.
C0.

..G
..G

..G
..G
..G

tMTPI_13
{MTPI_14
iMTPI_15
tMrPI_16
tMTPI_17
tMTPI_18
tMTPI_19
rMTPI_20
tMTPI_21
tMTPI_22
tMTPI_23
#MTPI_27
{DOG

tMTPI_01
rMTPI_02
tnrpr_03
tMTPI_04
QMTPI_05
9MTPI_06
tnrpr_07
#MTPI_08
tnrpr_09
tMTPI_1o
tMTPI_11
#MTPI_12
OMTPI_13
#MTPI_14
tMTPI_15
§MTPI_16
onrpr_17
turPI_1a
IMTPI_19
#MTPI_20
tMTPI_21
tMTPI_22
iMTpr_23
sMTPI_27
{DOG

tMTPI_01
tMTPI_o2
turpr_03
tMTPI-O4
tMTPr_os
IMTPI_06
tMTPI_o7
turpr_08
§MTPI_09
tMTPI_1o
tMTpI_11
¢MTPI_12
turpr_13
tnrpr_14
#MTPI_15
{MTPI_16
turer_17
tMTPI_18
#MTPI_19

..G

56

tMTPI_2o
IMTPI_21
OMTPI_22
IMTPI_23
OMTPI_27
tooc

OMTPI_01
OMTPI_02
turpr_o3
OMTPI_O4
OMTPI_05
tMTPI_06
IMTPI_07
#MTPI_08
OMTPI_09
IMTPI_10
IMTPI_11
IMTPI_12
turpr_13
lMTPI_14
OMTPI_15
OMTPI_16
tMTPI_17
IMTPI_18
tnrpr_19
IMTPI_20
tMTPI_21
tMTPI_22
rMTPr_23
lMTPI_27
tooc

{MTPI_01
OMTPI_02
surpr_o3
§MTPI_04
#MTPI_05
tMTPI_06
IMTPI_07
tMTPI_08
tMTPI_09
tMTPI_10
turpr_11
IMTPI_12
turpr_13
{MTPI_14
onrpr_1s
IMTPI_16
tnrpr_17
tnrpr_1e
lMTPI_19
IMTPI_20
lMTPI_21
turpr_22
tMTPI_23
IMTPI_27
0006

00A

000
.0.
0.
0..
0.
00

.. .G .C
000 0G 00C
000 ..G ..C

GGA GGC
..T ...

0.0 ..T .00
00. ..T .0
0.0 ..T 0 0
.. ..T . .
0. .0. 0C
.. ..T ...
0 0 ..T O 0
0.0 ..T 000
.. ..T . .
00 ..T 0.0
00. ..T .00
00. 00. 00C

.0. ..G 0.0
000 .00 00C

57

00.
00
00

00.

000

000

0..

000

0..

...

000

00.

000

0.0
.0

00.

.00

...

.00

.00

0.0

0.0

.00

.0.

0 0 00A
00. 00A

.00 00A
. 0 00A

. 0 . 0A
. 0 0 0 0A
0 0 0 . 0A
0 . . 0A
0 0T 0 0 0

..T ..T 0.0
..T ..T 000

..T ..C .00

ATT GCA GTT
..G

000 0.. ..G
... ... ..G
.00 000 00G
.00 0.. ..G
0.. 0.. ..G
... ... ..G
.00 0.. ..G
.0. 0.0 ..G
000 0.0 00G
.00 000 ..G
0.. 0.0 00G
.00 .0 ..G
... ... ..G

..G 0.0
..G 0.0

APPENDIX B

The predicted amino acid sequence of chitin synthase based upon DNA sequence in
twenty-five strains of Pythium insidiosum

58

EMEGA

lTitle MTPI;

lFormat

DataType-Protein

NSeqs-ZS NSites=166

Identica1-.

lDomain-Data;
#MTPI_01 GTLTGIARNL AYMAEVWGER AWENVAVAIV SDGRTKASAS

#MTPI_02
#MTPI_03
#MTPI_04
tMTPI_05
#MTPI_06
#MTPI_07
#MTPI_08
#MTPI_09
£MTPI_10
tMTPI_11
OMTPI_12
#MTPI_13
#MTPI_14
#MTPI_15
#MTPI_16
§MTPI_17
sMTPI_18
sMTPI_19
#MTPI_20
{MTPI_21
#MTPI_22
#MTPI_23
#MTPI_27
soos

#MTPI_01
#MTPI_02
#MTPI_03
#MTPI_04
#MTPI_05
iMTPI_06
#MTPI_07
#MTPI_08
#MTPI_O9
#MTPI_10
#MTPI_11
#MTPI_12
*MTPI_13
iMTPI_14
#MTPI_15
IMTPI_16
iMTPI_17
IMTPI_18
#MTPI_19
lMTPI_20
#MTPI_21
#MTPI_22
#MTPI_23
sMTPI_27
ODOG

Missing-? Indel=-;

ESTVQFMRDD

59

CLDYLTSLGA

#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
fMTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
#MTPI_
tMTPI_
{MTPIP
#MTPI_
#MTPI_
tMTPI_
#MTPI_
IMTPI_

EDOG

tMTPI_
tMTPI_
#MTPI_

01 GKLNSHLWFF NAFSEQLLPT YTVLVDVGTI PGPDSIFRLV RSMDRNPQIG
02 ............................................. .....
O3 ..................................................
04 ........................................ ..........
05 ........................................ .... ......
06 ........................................ . ....... ..
07 ...... .... . ......... C .............................
08 ........ .. ........................................
09 .......... .. ......................................
10 ..................................................
11 . .................................................
12 .............................. . ........ . ..... .....
13 ................................................ ..
14 ..................................................
15 ..................................................
16 ........................................ ..... .....
17 ................................................. .
18 ............................ .. .......... . .........
19 ................................................ ..
20 ............................ .. ...... .... ..... .....
21 ..................................................
22 ........................... ... .. ..................
23 .. ...................................... ..... .....
27 .................... ...P ..........................

01 GVAGEIAVEQ PNYFNP
02 ..... ...........
03 .......... ......

.sMrPI_04 ................

EMTPI‘

05 ................

#MTpI_06 ................
tMTPI_07 ................
#MTPI_08 ................

#MTPI_
tMTPI_
#MTPI_
tMTPI_
{MTPI_
{MTPI_
#MTPI_
#Mrpr_
#MTPI_
#MTPI_
{MTPI_
#MTPI_
#MTPI_
#MTPI_

09 ................
10 .......... ......
11 ................
12 ................
13 ................
14 ................
15 ................
16 ................
17 ................
18 ................
19 ................
20 ................
21 ................
22 ................

OMTPI_23 . ...............

tMTPI_

#006

27 ................

60

11111111111111111111111 111111711

79666