Browsing by Subject "Mycobacterium tuberculosis"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Design, Synthesis, and Evaluation of Pyrazinoic Acid-Derived Antituberculars for Drug-Resistant Mycobacterium tuberculosis
    (2022-01) Cole, Malcolm
    Tuberculosis (TB), an infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb), is a major cause of suffering worldwide. The impact of this disease has been exacerbated by the ongoing coronavirus pandemic, reversing much recent progress that had increased diagnosis and treatment rates in the preceding decade. In addition to ongoing issues related to public health shortcomings and lack of access to treatment, the emergence and spread of resistant strains is an increasing cause for concern. While the antitubercular pipeline has produced a few new antibiotics in the 21st century, more novel treatments are urgently needed to keep abreast of resistant strains. This dissertation describes efforts to create new therapeutic options for resistant TB, centered around pyrazinoic acid (POA), the active form of pyrazinamide (PZA), an important first-line TB drug.Recently, a growing number of reports have highlighted the promise of β-lactam conjugates in selectively targeting resistant organisms. β-lactams are a widely-employed class of antibiotics that target cell wall biosynthesis. Bacteria can evade this activity through expression of β-lactamases, powerful enzymes that destroy the electrophilic β-lactam warhead. However, researchers have learned to take advantage of this resistance mechanism, designing β-lactam conjugates that release a molecular payload following β-lactamase cleavage. This strategy, referred to here as β-lactamase-mediated fragmentation, is explored in great detail in Chapter 1, including descriptions of its discovery and applications in a variety of fields, including diagnostics, cellular imaging, and antibiotic design. Chapter 2 describes our own work in this space, designing β-lactam conjugates bearing POA as a Mtb-selective warhead. This strategy circumvents the most common resistance mechanism against PZA, imparting activity in an Mtb macrophage infection model (where conventional β-lactams are typically ineffective). We also provide preliminary mechanistic evidence that our conjugates act as codrugs, achieving antibacterial activity through action of the β-lactam scaffold as well as the POA warhead. In Chapter 3, we remove the β-lactam scaffold and focus on POA itself, reporting a series of new analogues featuring substitutions on the pyrazine ring. We identify several analogues with improved activity over POA, and use biochemical techniques to demonstrate they are inhibitors of PanD, a putative target of POA. We use the structure of our most active lead and recent structural insights into PanD to design additional inhibitors with comparable antimycobacterial activity, providing proof-of-concept for future structure-based design of new PanD inhibitors.
  • Thumbnail Image
    Item
    Genes Involved In Mycobacterium Tuberculosis Persistence
    (2019-09) Namugenyi, Sarah
    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.
  • Thumbnail Image
    Item
    The mechanistic basis of susceptibility and resistance to the antitubercular drug para-aminosalicylic acid
    (2019-05) Kordus, Shannon
    Tuberculosis (TB) is responsible for the deaths of 1.6 million people worldwide each year and is the leading cause of death by the pathogen Mycobacterium tuberculosis. The treatment regimen for M. tuberculosis involves lengthy, intensive drug therapy that causes severe side-effects. The antimicrobial para-aminosalicylic acid (PAS) is used to treat drug-resistant M. tuberculosis infections. Despite the use of PAS to treat M. tuberculosis for over 70 years, the biochemical mechanisms which govern PAS susceptibility and resistance in M. tuberculosis are incomplete. The focus of this dissertation is to determine these mechanisms and can be summarized into three interrelated studies: 1) The mechanism of action of PAS on the M. tuberculosis folate metabolic pathway; 2) Understanding the mechanisms of PAS resistance; and 3) Examining the drug-drug interactions between PAS and other anti-folate drugs that are used to treat opportunistic infections in patients with HIV-M. tuberculosis co-infections. Chapter 2 shows that PAS is a pro-drug and is converted, via the M. tuberculosis folate biosynthetic pathway, to hydroxy-dihydrofolate. The folate biosynthesis pathway is an essential metabolic pathway used maintain the production of DNA, RNA, and proteins. Results showed that hydroxy-dihydrofolate acted as a potent inhibitor for dihydrofolate reductase and confirm the biochemical mode of action of PAS. Although PAS was originally found to be effective at inhibiting M. tuberculosis and showed no activity against other bacterial species. PAS activity was tested against other bacterial pathogens. While M. tuberculosis was extraordinarily sensitive to PAS, other bacteria resisted PAS-mediated killing. Chapter 2 found these bacterial species could utilize PAS as a fully functional folate analog into one-carbon metabolism. The folate biosynthesis precursor, para-aminobenzoic acid (PABA), is a known antagonist of antifolates, namely sulfonamides. Since PAS was shown to act similarly to PABA in folate biosynthesis, PAS-sulfonamide interactions were tested against a panel of bacterial pathogens. PAS could antagonize sulfonamides in all of these organisms. HIV-infected individuals are given numerous drugs to both treat the HIV infection and prophylactically treat and prevent opportunistic infections. The most commonly prescribed prophylactic drugs are the sulfonamides. These findings strongly support that PAS and sulfonamides should cease to be used in combination in individuals with HIV-M. tuberculosis co-infections. Many patients discontinue treatment of PAS because of the side effects, namely, gastrointestinal distress. Bacteria must make their own folates and many bacteria in the human colon excrete folate to supply human enterocytes with folates. All rapidly dividing human cells require folates for the synthesis of new DNA, RNA, and proteins. All human cells contain folate receptors which recognize the metabolites dihydrofolate or folate. Purified human dihydrofolate reductase enzyme and could use hydroxy-dihydrofolate but much less effectively than the native substrate. Since enterocytes require a large amount of folates for rapid cell growth, enterocytes using hydroxy-dihydrofolate as a source of folates may not be able to grow as fast leading to severe gastrointestinal distress. Indeed, hydroxy-dihydrofolate, not PAS, was cytotoxic to enterocytes and hepatocytes. These findings will allow us to ultimately to design better ways to administer PAS to prevent patients from discontinuing PAS treatment. In Chapter 3, we hypothesized mice could be co-treated with sulfonamides to prevent PAS bioactivation and resulting PAS toxicity. Surprisingly, sulfonamides antagonized the anti-mycobacterial action of PAS in mice, resulting in the unrestricted growth of M. tuberculosis in the lungs and dissemination of M. tuberculosis into the liver and spleen. Taken together, these data indicate that combining PAS with sulfonamide in the clinic would not be useful, could be detrimental to patient outcome, and further highlights the need for mechanistic studies of drug-drug interactions. . Chapter 4 established that PAS resistance in M. tuberculosis primarily mapped to the folate biosynthetic pathway. The most prevalent mutations mapped to thyA and folC, a thymidylate synthase and dihydrofolate synthase, respectively. We hypothesized that folC mediated resistance occurred through an increase in PABA biosynthesis. Indeed PABA biosynthesis genes were upregulated in folC resistant mutants but not in thyA resistant mutants. The folC mutants had restored susceptibility to PAS when PABA biosynthesis was disrupted. Furthermore, it was found that disruptions in PABA biosynthesis were bactericidal in M. tuberculosis. These data represent a novel intrinsic mechanism of resistance to PAS and highlights a novel drug target in M. tuberculosis. This dissertation is the first to determine that PAS selectively inhibits M. tuberculosis dihydrofolate reductase enzyme and subsequently, the folate biosynthetic pathway. The work presented in this dissertation found that using PAS and sulfonamides together prevented both drugs from working correctly in M. tuberculosis and in other bacterial pathogens. The results generated from this dissertation will be used to inform the current clinical practices in combination therapy and foster a paradigm shift in the treatment regimen administered to HIV-M. tuberculosis co-infected patients, leading to decreased mortality rates among this population.
  • Thumbnail Image
    Item
    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.