Genes Involved In Mycobacterium Tuberculosis Persistence

Abstract

In 2017 about 10 million new tuberculosis (TB) cases and 1.6 million TB deaths were reported worldwide, making TB the leading cause of death by an infectious agent. Mycobacterium tuberculosis (Mtb), the causative agent of TB, has developed the ability to evade the host’s immune system and latently persist for years in the lungs of immunocompetent individuals. However, re-activation of the bacilli does occur, causing the development of active TB and the transmission of Mtb. Currently, the minimum duration of TB treatment is six months, which can result in patient non-compliance and selection for drug-resistant Mtb strains. The lengthy TB treatment is due in part to the formation of Mtb persisters, which are defined as phenotypic antibiotic-tolerant bacteria. Understanding how Mtb can evade the immune system and form persisters has the potential to provide information that is useful for the development of TB vaccines and therapies. Mtb primarily survives in the macrophages of a host’s lungs. Macrophages are activated by the cytokine interferon-gamma (IFN-), which stimulates antimicrobial functions, but Mtb can evade elimination. In Chapter 2, we describe the identification of additional Mtb counter-immune mechanisms. Using transposon-sequencing (Tn-seq) and a mouse infection model, we identify specific Mtb factors that counteract IFN-−dependent antimicrobial effects. We selected several transposon mutants of interest and confirmed their role in counteracting host immunity by performing individual infections. Furthermore, in Mtb the phosphate-specific transport (Pst) system controls expression of phosphate (Pi)-responsive genes by negatively regulating SenX3-RegX3, a two-component regulatory system, in Pi-rich conditions. In Escherichia coli the Pst system inhibits a homologous two-component system through the negative regulator, PhoU. The work done in Chapter 3 demonstrates that the two phoU homologs in Mtb, PhoY1 and PhoY2, function redundantly to mediate inhibition of SenX3-RegX3 by the Pst system during growth in abundant Pi conditions. We also showed that this regulatory function is essential for promoting persister formation. Our data suggest that disrupting Pi signal transduction mediated by the PhoY proteins can enhance the susceptibility of Mtb to antibiotics. Chapter 4 defines our attempts to determine what unique role in mycobacterial physiology each PhoY protein may have. We used RNA-seq to identify changes in the transcriptome in the phoY mutants and have initiated a mycobacterial two-hybrid assay to test whether the PhoY proteins directly interact with the Pst and SenX3-RegX3 systems. Quantitative reverse transcription PCR experiments examining the transcription trend of RegX3-regulated genes during the transition of Mtb from Pi-rich to Pi-limited conditions have indicated that PhoY2 may be more effective than PhoY1 in inhibiting RegX3 activation. In addition, an ethidium bromide uptake experiment showed that PhoY2 might be involved in maintaining Mtb cell envelope integrity. These data suggest that PhoY1 and PhoY2 have differing effectiveness in regulating RegX3 and different functions besides controlling SenX3- RegX3 activity in response to Pi availability. Supplementary materials included with this thesis are tables showing TnseqDiff data for mutants attenuated in immune-deficient mice in comparison to 24hr (Table S2.1) or in vitro input (Table S2.2). Additionally, differential gene expression in the ∆phoY1∆phoY2 mutant (Table S4.1) compared to the WT from RNA-seq.