Characterization of map3773c, ferric uptake regulator protein, in iron metabolism of mycobacterium avium subsp. paratuberculosis
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a major hurdle to diagnosis (Bannantine et al., 2002). Currently, no current vaccine can protect animals against JD or prevent shedding of MAP (Garcia and Shalloo, 2015). These challenges establish a need for a better understanding of the MAP physiology during infection. Unlike other mycobacteria, MAP has a unique requirement for supplementation of an iron-binding siderophore (mycobactin J) for optimal growth in laboratory media. A whole genome sequence of MAP K10 revealed a truncation of the mbtA gene that was speculated to have led to its mycobactin dependency (Li et al., 2005). Zhu et al (2008) showed that MAP is able to transcribe all mycobactin synthesis genes in an intra-macrophage environment. Furthermore, several genes responsible for iron acquisition in infected tissues, including genes responsible for mycobactin biosynthesis have been shown to be upregulated in naturally infected tissues (Janagama et al., 2010). Its known that iron plays an important role in vital biological processes; however high intracellular concentration of free iron can lead to toxicity to the bacteria. As such, bacteria activate expression of specific group of genes that are controlled by a metal-sensing regulatory transcription factor. In 2009, Janagama and others identified and characterized the iron dependent regulator (IdeR) in MAP. In addition, in a MAP-specific genomic island, MAP carries three putative ferric uptake regulator (Fur) boxes, an iron regulated transcriptional control motif, (Stratmann et al., 2004). To date, nothing is known about the role of Fur in MAP. To elucidate the mechanisms of iron homeostasis in MAP, we investigated LSP15 using a transposon mutant MAP3776c. We demonstrated a phenotype for LSP15 genes in a culture model of cell entry and survival and suggest a function in epithelial cell pathogenesis, however further functional analysis with complementation by a MAP3776c::Tn strain would be necessary to confirm these findings. Additionally, full characterization of a Fur-like protein (MAP3773c) was performed. Using PRODORIC for computational analysis, 23 different pathways that were likely regulated by MAP3773c were identified. These findings were confirmed using a chromatin immunoprecipitation assay followed by high-throughput sequencing (ChIP-seq), that revealed 58 regions where Fur binds under iron replete and deplete conditions. From those, three were directed related to iron regulation MAP3638c (hemophore-like protein), MAP3736c (Fur box) and MAP3776c (ABC transporter). Using the Fur box consensus sequence, we confirmed binding specificity and Mn2+ availability by a chemiluminescent electrophoresis mobility shift assay (EMSA). A transcriptional profile of the parent MAP K10, deletion mutant of MAP3773c and the complemented strains was developed under iron replete and depleted conditions. However, under the current experimental conditions, we are unable to conclude if the lack of transcriptional responses in our study was indicative of a lack of FUR activity.
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Miyagaki Shoyama, Fernanda
- Thesis Advisors
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Sreevatsan, Srinand
- Committee Members
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Mansfield, Linda
Coussens, Paul
Abramovitch, Robert
- Date Published
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2020
- Program of Study
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Comparative Medicine and Integrative Biology - Doctor of Philosophy
- Degree Level
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Doctoral
- Language
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English
- Pages
- 126 pages
- ISBN
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9798698540670
- Permalink
- https://doi.org/doi:10.25335/q680-ve60