Tuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide blocker by riboswitch. To accomplish this, promoter induction studies were used to find stronger promoters for the M-PFC, optimization of the riboswitch as a method for inducible protein expression within this system, and the addition of both elements to the existing version of the M-PFC. This M-PFC targets DosR homodimerization; this process is known to be essential for survival within the host. This study optimizes a system that may be used to screen for drugs that are capable of interrupting this interaction.

Mycobacterial species are diverse organisms, classified into tuberculous and nontuberculous mycobacteria (NTM). Mycobacterium tuberculosis has been thoroughly investigated, but pathogenic NTM have not. The identified technology gap for studying potential antibiotic targets across mycobacteria is that there is not a tool developed for efficiently creating bacterial clones containing these genes with a reporter system to evaluate CRISPR interference (CRi) knockdowns. CRi is a quick and simple way to silence genes. In this study, Golden Gate (GG) cloning compatible Lux reporter plasmids were engineered for the efficient cloning of target genes as transcriptional fusions with luxAB, a luminescent reporter, for use with CRi. Additionally, a CRi plasmid was designed with a Giles integration site so that it could be integrated into the mycobacterial genome with the reporter plasmid, but at a different location. Based on current research, it seems that mycobacterial L,D-transpeptidase enzymes (Ldts), involved in peptidoglycan synthesis, are potential targets for the drug class known as β-lactams and should be further explored. Ldt 2 is of particular interest as research indicates that it may be involved in pathogenicity; therefore, GG cloning of M. smegmatis (Msm) Ldt 2 was performed using the designed GG plasmid. Constructing the GG plasmid (pMV306hsp+luxG13, GG pMV) as well as the CRi + Giles integration plasmid (pLJR962 + pML1357, CRi + Giles) was successful; however, the evaluation of the luminescent reporter with CRi knockdown has yet to be performed.

No description available.

Mycobacteria are atypical bacteria possessing unusual cell envelopes comprised of an outer membrane, covalently linked to an arabinogalacatan-peptidoglycan structure via waxy mycolic acids, in addition to the conventional inner membrane. This thick and highly impermeable cell envelope is a major deterrent to antibiotic treatment of clinically relevant mycobacterial pathogens, including Mycobacterium tuberculosis (Mtb), which infects a third of the world’s population and kills millions each year. Thus, the regulation of mycobacterial cell envelope biosynthesis is of great interest for the development of more effective therapeutics for treating Mtb infections. Using the model organism Mycobacterium smegmatis (M. smegmatis), we identified a novel protein, Spe2, with an unknown role in the biosynthesis of the cell envelope glycolipids lipomannan (LM) and lipoarabinomannan (LAM). Based on the observation that Δspe2 mutants produce truncated LM/LAM, I speculated Spe2 might enhance the elongation of these products. Here, I use biochemical assays to show Spe2 is localized to the periplasm where it can directly interact with the LM/LAM biosynthetic pathway. I further utilize a genetic approach to demonstrate that Spe2 acts at the stage in which the mannosyltransferase MptA mediates periplasmic LM elongation. Moreover, native polyacrylamide gel electrophoresis (PAGE) and co-immunoprecipitation techniques failed to reveal Spe2 protein binding partners. Together, these data suggest Spe2 is a periplasmic protein involved in regulating LM/LAM biosynthesis, perhaps through direct interactions with LM intermediates.

Water and soil samples collected from the mouth to origin of the Savannah and Tombigbee Rivers were treated for enumeration of organisms of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) complex. Their numbers in soil and water were compared to physiochemical characteristics of these samples. Further, the number of MAIS organisms recovered from acidified and alkalinized soil samples, measurement of possible movement of soilborne MAIS organism into water and the numbers in soil and sediment at one location in the James River were investigated. Most MAIS organisms in soil (95%) could not be recovered because of irreversible binding. Appreciable numbers of MAIS organisms from soil were recovered along the entire length of both rivers, while high numbers in water were recovered toward the mouth and from turbid water samples. However the numbers in soil were much greater than those in water and numbers in sediment were greater than those in soil. The number recovered from soil was highest in acidic samples and in those with high organic matter content. High soil conductivity was associated with high recovery of MAIS organisms in the Tombigbee but not the Savannah River. The recovery of MAIS organisms from acidic soil samples could not be decreased by increasing soil pH, nor recovery from basic or neutral soils increased by acidification. Waterborne MAIS organisms were recovered from soil in a laboratory simulation of percolation of water through soil. / M.S.

Human clinical (n=131) and environmental (n=226) isolates of the Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) complex were screened for plasmids in an effort to increase knowledge about the genetics and epidemiology of these pathogenic bacteria. Approximately 50% of the clinical MAIS isolates from New York, Maryland, Virginia, South Carolina, and Georgia contained one or more plasmids. On the basis of plasmid content, aerosol MAIS isolates more closely resembled human MAIS isolates than did MAIS isolates from the other environmental sources examined (dust, soil, sediment, and water). Plasmid profiles were remarkably heterogenous, and isolates with identical profiles were rarely encountered. However, a 115 megadalton (Md) plasmid was detected in 15 mercury resistant human and environmental isolates. In one of these isolates (M. scrofulaceum W262) the presence of the 115 Md plasmid was shown to correlate with the presence of an NAD(P)H dependent mercuric reductase. Plasmids with molecular weights of 8.8, 11.2, 14.2, 16.9, 17.9, and 18.3 Md were also common among both human and environmental isolates. On the basis of molecular weight, 36 distinct plasmids were detected; their sizes ranged from 7 to 230 Md. It was concluded that human and environmental MAIS isolates share a number of plasmids with identical molecular weights and that plasmids can serve as useful entities in genetic and epidemiologic studies of this group of extremely slow-growing, poorly understood human and animal pathogens. / Ph. D.

Understanding and limiting public health threats resulting from exposure to opportunistic pathogens (OPs) in domestic water (i.e., hot/cold water for human use) will be one of the grand challenges for water safety in the 21st century. This dissertation anticipates some of the complexities in balancing stakeholder goals and developing building standards to limit OP growth, and advances scientific understanding of OP survival and proliferation in domestic plumbing systems. In a cross-sectional survey of water- and energy-efficient buildings, domestic water age ranged from 8 days to 6 months and resulted in pH and temperature fluctuations, rapid disinfectant residual decay up to 144 times faster than municipal water delivered to the buildings, and elevated levels of OP gene markers. This motivates future work to determine how to maintain high quality and safe water while preserving the sustainability goals of these cutting-edge buildings. Head-to-head pilot-scale experiments examining OP growth in recirculating hot water systems revealed that elevated temperature had an overarching inhibitory effect on L. pneumophila growth where temperatures were maintained. However, control was undermined in distal branches, especially when density-driven convective mixing gradients maintained ideal growth temperatures and delivered nutrients to the otherwise stagnant branches. These results resolve discrepancies reported in the literature regarding the effects of flow, and identify important system design and operational conditions that facilitate OP growth. Advancements were also made in understanding how corrosion can trigger OP growth. In Flint, MI, corrosive Flint River water damaged iron pipes, releasing iron nutrients, consuming chlorine residual, and supporting high levels of L. pneumophila in large building systems. This likely triggered two unprecedented clusters of Legionnaire's disease. In pilot-scale systems, copper released from copper pipes, but not dosed as soluble cupric, triggered release of >1,100 times more H2 into the water due to deposition corrosion. The organic carbon fixed by autotrophic hydrogen oxidation has the potential to facilitate OP growth, but more work is needed to understand the limits of this mechanism. Finally, well-controlled laboratory experiments confirmed past reports from field surveys that the use of chloramines trigger a trade-off between controlling Legionella and allowing non-tuberculous Mycobacteria to persist. / Ph. D. / Understanding and limiting public health threats resulting from exposure to opportunistic pathogens (OPs) in domestic water (i.e., hot and cold water intended for human use) will be one of the grand challenges for water safety in the 21st century. Unlike traditional fecal-based waterborne pathogens that have all but been eliminated through advanced treatment applied at water treatment facilities, OPs are native microbial members of drinking water and tend to proliferate in domestic plumbing. In addition to the complexity and technical nature of engineering controls applied in buildings to limit OP growth, there are many stakeholder groups with varied responsibilities and expertise in preventing, diagnosing, and/or remediating problems. Stakeholders sometimes present additional challenges when their goals have direct or indirect trade-offs with limiting OP growth in buildings. This dissertation anticipates some of the challenges to come, and advances scientific understanding of how OPs survive and proliferate in domestic plumbing systems. Water- and energy-efficient buildings, while nobly seeking to preserve precious natural resources, potentially create unintended consequences with respect to water quality. In a cross-sectional survey of green building designs, water remained within domestic plumbing for over a week to months before being used by consumers, and resulted in water quality changes that facilitated the growth of OPs. While short-term solutions exist, such as flushing water to decrease water stagnation and introduce “fresh” water into the system, this work motivates future research for how to maintain high quality and safe water while preserving the sustainability goals of these cutting-edge buildings. Systematic experiments were conducted on water heaters with a recirculating pump, which are marketed as a “green” technology for water and energy savings, to determine the effect of system design and operation on the growth of OPs. Elevated temperature was found to have an overarching inhibitory effect on growth of <i>L. pneumophila</i>, the most commonly reported OP. However, when the water heater temperature was not sufficient to completely eliminate <i>L. pneumophila</i> (51 °C), higher water temperatures actually supported high levels of <i>L. pneumophila</i> growth in infrequently used pipes by periodically disinfecting other microorganisms that are more susceptible to thermal disinfection and decreasing competition for nutrients. System design also impacted <i>Legionella</i> growth. In pipes that slowly mixed with the recirculating line (simulating a pipe running upward to a shower head from a recirculating line in the floor, for instance), <i>L. pneumophila</i> were consistently elevated relative to pipes that did not convectively mix (simulating a pipe running downward to a kitchen tap from a recirculating line in the ceiling, for instance). The slow mixing maintained ideal <i>Legionella</i> growth temperature in the pipes with mixing, even when water heaters were maintained well above thermal disinfection levels for <i>Legionella</i> (i.e., at 60 °C). This is due to continuous delivery of nutrients to the upward pipes with mixing, but not the downward pipes without it. This result is significant because it outlines scenarios encountered in real buildings where even the most effective thermal disinfection strategy can be undermined in distal branches within the building. This work also outlines the importance of corrosion in potentially triggering OP proliferation. In Flint, MI, when the water utility began distributing very corrosive Flint River water, the new water source damaged iron water pipes, releasing iron (which is a nutrient for <i>Legionella</i>) and eliminating disinfectant residual in the distribution system (which is needed to prevent <i>Legionella</i> regrowth). As a result, the corrosion supported increased <i>Legionella</i> levels and likely triggered two unprecedented clusters of Legionnaires’ disease. In the pilot-scale systems, corrosion of the water heater anode rod caused trace nutrients to evolve into the water. However, this only occurred when low levels of copper released to the water from the natural corrosion of copper pipes were present. Ionic copper, which is sometimes used to disinfect <i>Legionella</i>, did not have the same effect when it was dosed to the experiment at similar concentrations. These trace nutrients generated from copper-enhanced corrosion of the water heater anode rod are a potential source of carbon for OP growth, and may help explain the variable effects of copper that have been reported in the literature. More work is needed to fully understand this potential growth mechanism. Finally, this work confirmed past field observations that there is a trade-off between controlling <i>Legionella</i> and allowing <i>Mycobacteria</i>, another OP, to persist when using chloramines disinfectant residual. Reproducing this phenomenon in controlled laboratory settings is an important step in understanding, and ultimately preventing it.

Tuberculosis remains one of the oldest diseases known to mankind but still persists as a very major risk. Discovery of several antimycobacterials was marked by a steady decline in the active cases of pulmonary tuberculosis. However, in the recent past there has been a surge in its clinical incidence. The reasons for this failure are the emergence of multi drug resistance and the ability of the organism to survive under extreme condition or for a very long period of time known as ‘persistence’. The latter one established itself as a major hindrance in the effective control of tuberculosis. The latent bacilli are confined for a very long period after the infection in caseous cavities, called granulomma, inside the host and gets reactivated any time when the host becomes immuno-compromised. Latency has been successfully simulated in vitro by various models which mimic the in vivo condition by depleting O2 (Wayne, 1977), nutrients (Nyka, 1974) or the carbon source (Ojha et al., 2000). Stationary phase is exemplary of a stage in bacterial growth where the organism is exposed to various stresses like nutrient starvation, accumulation of toxic metabolites, low temperature, high osmolarity and acidity to name a few. Some evidences suggest that cells survive in nutrient deprived stationary phase. The present investigation was pursued with an objective to further our understanding on the mechanism of adaptation that the persistent mycobacterium may undertake to survive during the stationary phase of growth. The fast growing M.smegmatis, a nonpathogenic member in the non-tuberculous genera, however, with a genetic and metabolic similarity to M.tuberculosis has been used as a model for this study. Chapter 1 introduces the challenges in tuberculosis therapy and discusses the reason for drug tolerance and persistence of M.tuberculosis and M.avium complexes that were uncovered recently. It focuses on the intricate lipid rich cell wall which forms the first barrier for drug delivery with an emphasis on the cell surface antigenic glycolipids, the glycopeptidolipids. A detail account of their structure, biosynthetic pathway, intracellular function and their implications on biofilm formation has been provided. The evolution of the genetic approaches currently used in mycobacterial research is highlighted. The transcription apparatus and the regulation of gene expression in mycobacteria at different environmental condition and stages of growth are also discussed. The need for a detail investigation of the stationary phase RNAP in mycobacteria is stressed. Chapter 2 observes the changes in the cell surface of M.smegmatis at different ambience of growth. It focuses on the composition of glycopeptidolipid, one of the non-covalently attached free lipids and the mycolic-acids which are covalently attached to the inner layer of the cell wall. Composition of the mycobacterial cell wall with respect to the glycopeptidolipids and mycolic acids during biofilm mode of growth is also addressed. This chapter examines the conditional synthesis of a novel class of polar glycopeptidolipid in carbon starved cultures of M.smegmatis and determines their molecular structure. Chapter 3 revisits the biosynthetic pathway of the glycopeptidolipids and justifies a need for a fresh perspective. It identifies a glycosyltransferase responsible for the transfer of an extra rhamnose to the existing rhamnose linked to the terminal alaninol in the new polar glycopeptidolipid. This chapter also identifies a conserved Polyketide synthase in the glycopeptidolipid biosynthetic cluster. Characterization of the domains present in its two distinct modules with a correlation to the structure of the fatty acylchain together with the formation of a hydroxy fatty acyl chain is delineated. Chapter 4 deals with the construction of a suicide vector which when recombines to the mc2155 genome, incorporates a hexa-histidine tag at the C’ of the β΄ subunit of the RNAP. It details the strategy for the construction of the strain and established the genetic exchange event both genotypically and phenotypically. A single step procedure for purification of the holo-RNAP is also described in this chapter. In chapter 5 the role of the mycobacterial principal likes sigma factor, SigB, at the stationary phase of growth is highlighted. An approach of expression proteomics involving differential display of the total protein was undertaken to investigate the genes that are under the SigB regular during the stationary phase of growth. This chapter also examines the stationary phase induced changes in the RNAP. Various proteins that interact with the assembly are identified and their role in conferring rifampicin resistance to the RNAP is described. Appendix 1 details the preparation of the partially methylated deoxy monosaccharide using the esoteric reactions of organic synthesis. They were used extensively for glycosyl linkage analysis of the glycopeptidolipids by mass spectrometry, where they acted as standards.

Mycobacterium are important human pathogens and their strength lies in establishing acute infections, latent infections as well as co-existing with other dreadful infectious agents like HIV. The success of mycobacterium infection often relies in its ability to evade immune-surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immune regulatory molecules. In this scenario, the role of pattern recognition receptors (PRRs) in orchestrating host immune responses assumes central importance. Of the PRRs, the Toll-like receptors (TLRs) or intracellular surveillance receptors such as retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) govern key immune-surveillance mechanisms in recognition as well as control of mycobacterial or viral infections. The first part of this study illustrates the role of SHH signaling in macrophage induced neutrophil recruitment during mycobacterial infections. The present investigation demonstrates that, in response to mycobacterium infection, macrophages displayed robust activation of TLR2 dependent SHH signaling. By utilizing the well-documented experimental air pouch model, we show that the ability of pathogenic mycobacterium infected macrophages to recruit polymorph nuclear leukocytes (PMNs) like neutrophils to the infected site was dependent on SHH signaling. The activated SHH signaling differentially regulated the expression of proteolytic enzymes, MMP-9 and MMP-12 that would contribute to PMN migration. Interestingly, SHH-responsive krüppel-like family (KLF) of transcription factors, KLF4 and KLF5 were found to modulate these chemokine effectors to regulate neutrophil recruitment. Subsequent chapters describe novel functions of SHH signaling during RIG-I mediated anti-viral immunity and RIG-I mediated modulation of TLR2 anti-inflammatory signature in mycobacteria infected macrophages. In this perspective, we demonstrate that RIG-I ligand robustly induces the activation of SHH signaling via the phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. Furthermore, we show that the sustained inhibition of PKA-GSK-3β-SUFU negative regulatory axis upon RIG-I engagement with 5'3pRNA is critical for the activation of SHH signaling. Gain or loss of function studies implicate the necessity of RIG-I triggered MAVS-TBK1 canonical axis in the inhibition of PKA-GSK-3β-SUFU negative regulatory axis that contributes to SHH signaling activation. The RIG-I activated SHH signaling drives the production of anti-viral type 1 interferons leading to the inhibition Japanese encephalitis virus (JEV) replication. Further, RIG-I-mediated anti-viral type 1 interferon production and subsequent control of viral replication suggested the involvement of two transcriptional factors, IRF3 and YY1 in the response along a SHH axis. Further, mounting evidence clearly depicts a significant cross talk among the molecular events initiated by given TLRs and RLRs like RIG-I. Clearly, these studies present an interesting challenge in delineating the events during polymicrobial infection of host immune cells like macrophages or DCs. Altogether, our results improve our understanding of mycobacteria associated confections’ and may add significantly to the current knowledge of the delicate balance that determines a successful mycobacterial infection.

Molecular typing is increasingly essential to tuberculosis (TB) control programmes, providing public health practitioners with a tool to characterize transmission patterns, track the emergence and spread of strains of M. tuberculosis complex (MTC) in populations. While molecular typing is already used extensively as a tool for TB control in many developed settings across the globe, its use in resource-poor settings is still limited. Moreover, information on the role, contribution and burden of nontuberculous mycobacteria (NTM) and other pathogens in aetiology of TB-like syndromes is also lacking in such settings. The broad objective of this dissertation was to determine the genetic diversity of MTC and their drug resistance profiles as well as the prevalence of NTM and other potentially pathogenic bacteria among TB suspects in Northeastern, Tanzania in order to generate insights that may inform the design of a rational TB control programmes. A total of 18 distinct spoligotypes were identified in this study area, with CAS1-KILI and EAI8 being the most predominant families. Major lineages prediction by conformal Bayesian network (CBN) revealed that 70% of TB infections in this area is due to modern lineages, whereas 30% of TB infections is due to the ancestral lineages mainly of Indo-oceanic lineage. The study also revealed that the overall proportions of any drug resistance and MDR-TB were 12.7% and 6.3% respectively. With the prevalence of any drug resistance and MDR-TB among new cases being 11.4% and 4.3% respectively, among previously, treated cases were 22.2%. The prevalence of NTM was found to be 9.7 %, with HIV being a significant predictor of NTM detection (P < 0.001). Four out of 30 patients with NTM diagnosed by culture received 1st line anti-TB treatment suggesting that a proportion of patients diagnosed by smear microscopy (4/65, 6.2%) were mistreated as TB patients. Our findings further showed that 17 (4.6%) out of 372 TB suspects were due to pulmonary nocardiosis. Overall this dissertation has revealed that TB is still a major problem in Tanga and is characterized by a diverse array of MTB strains. Additionally, modern MTB strains contribute significantly to TB infections in this area. High proportions of anti-TB drug resistance among new treated cases observed suggest that more efforts need to be done to identify individual cases at facility level for improved TB control programmes. Inefficient screening of TB patients and a prevalent increase of NTM may contribute to both unrealistic and mismanagement of TB cases. A diverse array of pathogenic Nocardia species among TB suspects further indicates that they are likely cause of human disease in this population. Therefore, need to integrate NTM and pathogens causing TB-like syndromes in diagnosis and management of TB is urgent. Results of these investigations contribute to the understanding of the dynamics of TB transmission in resource poor settings of Tanzania and highlight key factors that should be considered in the development of rational approaches to design effective TB prevention and control programmes in the country.