Browsing by Subject "ESX-5"

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    Investigating the RegX3-Dependent Regulation of Protein Release from Mycobacterium tuberculosis
    (2020-07) White, Dylan
    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. As a facultative intracellular pathogen, Mtb is well-adapted to survive within the host and establishes a persistent infection in humans despite a strong immune response. As part of its survival strategy, Mtb releases a wide array of proteins that aid in nutrient acquisition and immune evasion. Broadly, the work presented in this thesis focuses on how an Mtb response regulator, RegX3, regulates protein export by the bacteria, and how export of these proteins impacts Mtb viability. In Chapter 2 we describe that a ∆pstA1 mutant, in which RegX3 is constitutively activated, hyper-secretes a variety of proteins. In previous work, we found that RegX3 induces secretion through the specialized ESX-5 secretion system. We also found hyper-secretion of LpqH, a lipoprotein associated with membrane vesicles (MV) produced by Mtb. Further investigation into this phenotype revealed that ∆pstA1 bacteria release significantly more MV than wild-type (WT) bacteria. We showed that this is a RegX3-dependent phenotype, suggesting a gene(s) within the RegX3 regulon controls MV production. Furthermore, MV release does not depend on ESX-5 activity, so RegX3 induces protein export by at least two different mechanisms. Work in Chapter 3 builds upon the observation that ∆pstA1 bacteria produce more MV. Because this strain constitutively activates RegX3, we leverage it as a tool to identify factors that drive MV release. We hypothesized that the deletion of a specific RegX3-induced gene in the ∆pstA1 background would restore MV production to WT levels, and that the product of this gene would provide information about the physical process of MV release through the Mtb cell wall. We demonstrate that only the deletion of lppF or whiB3 reduce MV production. While WhiB3 was previously reported to transcriptionally regulate the production of cell wall lipids, we found no evidence to support this regulatory mechanism in our ∆whiB3 strains. We conclude that no single gene drives the increased MV production by the ∆pstA1 strain; activation of WhiB3 is responsible for a portion of the phenotype and additional RegX3-dependent factors also contribute. Chapter 4 focuses on the biological functions of ESX-5 activity, specifically to determine how ESX-5 affects Mtb growth and what proteins are secreted by the system. Creating strains harboring tetracycline-repressible copies of ESX-5 components allowed us to circumvent ESX-5 essentiality while still controlling its activity. Using these strains, we found that Mtb requires ESX-5 activity for growth when either glycerol or glucose was the sole carbon source. Additionally, ESX-5 activity was required for optimal growth and survival in both resting and interferon-gamma activated murine macrophages in vitro. It remains to be investigated whether this intracellular survival is connected to ESX-5-dependent nutrient acquisition. These strains also allowed us to perform the first full proteomic analysis to identify protein substrates secreted via ESX-5. Our results uncovered that ESX-5 may play a role in the release of a wider range of proteins than was previously appreciated. We report that release of proteins associated with the cell membrane/cell wall and also proteins secreted via other systems rely on ESX-5 activity to reach the culture filtrate. Taken together, the results presented in Chapter 4 uncover new roles of the ESX-5 system and highlight the importance of this system to Mtb viability.
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    Regulation of the ESX-5 Secretion System in Mycobacterium tuberculosis
    (2017-05) Elliott, Sarah
    Mycobacterium tuberculosis (Mtb) is one of the most prolific bacterial pathogens in the history of human disease. Robert Koch discovered that Mtb was the causative agent of the disease tuberculosis in 1882, and despite intensive research and major advances, Mtb represents a major global health threat today. Worldwide in 2015, there were 10.4 million newly diagnosed active cases and 1.8 million deaths attributed to this infection. Bacterial pathogens often secrete factors to promote survival during infection, and Mtb is no exception. Mtb has evolved a unique, diderm cell membrane, which contributes to the ability of the bacterium to resist host immune responses. However, this hydrophobic barrier also presents an obstacle for the export of factors critical to success of the organism. Mycobacteria, including Mtb, have evolved the Type VII ESX secretion systems to facilitate protein export across the complex membrane. Three ESX systems have been implicated in Mtb pathogenesis, ESX-1, -3 and -5. While the regulatory mechanisms and biological functions for both ESX-1 and ESX-3 are well-defined, little was known about ESX-5 aside from a general role in Mtb virulence. The work described in chapter 2 reveals that ESX-5 secretion is directly regulated at the transcriptional level by the Pst/SenX3-RegX3 system in response to inorganic phosphate (Pi) limitation. RegX3, the response regulator, is normally activated when Pi is scarce. Disruption of a transmembrane component of the Pst Pi uptake system, through deletion of pstA1, causes constitutive activation of RegX3. We observed overexpression of esx-5 transcripts and hyper-secretion of the ESX-5 substrates EsxN and PPE41 in the Mtb ΔpstA1 mutant, and this response requires RegX3. In wild-type Erdman (WT) Mtb, transcription of esx-5 genes and secretion of ESX-5 proteins was induced by Pi limitation in a RegX3-dependent manner. Using electrophoretic mobility shift assays (EMSA), we found that RegX3 directly binds to a segment of DNA within the esx-5 locus, demonstrating that regulation of ESX-5 mediated by the Pst/SenX3-RegX3 system occurs directly. Experiments outlined in chapter 3 expand on the work reported in chapter 2. Using in vitro EMSAs, we defined the RegX3 binding sequence located within the intergenic region between ppe27 and pe19 in the esx-5 locus. RegX3 is a global response regulator, and targeted mutation of the esx-5 binding site sequence uncouples the secretion system from the myriad effects mediated by RegX3 throughout the cell. We found that mutating the esx-5 RegX3 binding site sequence reversed expression of esx-5 transcripts and secretion of EsxN and PPE41 in WT Mtb during Pi limitation. Similarly, esx-5 overexpression and ESX-5 hyper-secretion was suppressed in the ΔpstA1 mutant when the RegX3 binding site sequence was mutated or deleted. We then tested the importance of RegX3-mediated regulation of ESX-5 for Mtb virulence by infecting C57BL/6 and IrgM1-/- mice with a binding site mutant. Notably, deletion of the esx-5 RegX3 binding site partially restored virulence to the attenuated ΔpstA1 mutant. Our findings demonstrate that precise regulation of ESX-5 is critical for full Mtb virulence. Further, hyper-secretion of antigenic ESX-5 substrates sensitizes ΔpstA1 bacteria to host responses, suggesting that one or more of these aberrantly secreted proteins is responsible for attenuation. We next sought to determine whether aberrant hyper-secretion of one ESX-5 secreted factor, EsxN, sensitizes ΔpstA1 bacteria to host immune responses. Previous work has shown that the ΔpstA1 mutant is attenuated in immune competent C57BL/6 mice and immune compromised IrgM1-/- and Nos2-/- mice, while experiments described in chapter 3 demonstrated that attenuation of the ΔpstA1 mutant in C57BL/6 and IrgM1-/- mice was specifically due to constitutive activation of esx-5. Experiments in Chapter 4 evaluate the contribution of EsxN to Mtb virulence. Deletion of esxN did not reverse ΔpstA1 mutant sensitivity to reactive oxygen species, acidic pH or cell wall stress in vitro. However, WT bacteria were more sensitive to reactive nitrogen stress when esxN was deleted, suggesting a role for EsxN in resistance to reactive nitrogen species (RNS). We found that esxN does not suppress the ΔpstA1 mutant virulence defect in C57BL/6 or IrgM1-/- mice. However, deletion of esxN in the ΔpstA1 mutant did partially reverse the replication and virulence defect in Nos2-/- mice, indicating hyper-secretion of EsxN sensitizes ΔpstA1 bacteria to immune responses other than RNS production in these mice. Aberrant hyper-secretion of EsxN may influence sensitivity to other host responses in the ΔpstA1 mutant. EsxN seems to be required for survival during RNS stress in vitro in WT Mtb. Further work will be required to tease apart the potential role EsxN plays in RNS resistance. The work described in this thesis expands the knowledge of ESX-5 secretion system biology, and Mtb protein secretion in general. We have uncovered the mechanism of regulation, and also revealed a relevant environmental signal, Pi limitation, that activates this system. We have demonstrated that regulation of ESX-5 by the Pst/SenX3-RegX3 system occurs directly by identifying the RegX3 binding site sequence within the esx-5 locus. Using targeted mutation of the RegX3 binding sequence, we have shown that dysregulation of ESX-5 activity has a detrimental impact on Mtb virulence. These findings highlight the importance of proper regulation of the ESX-5 system to Mtb pathogenesis. An understanding of the regulatory mechanism and environmental signals that activate ESX-5 during infection provides an important frame-work for future studies to elucidate the functional role of this system.