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.