Abstract not available

Mycobacterial cell wall components have been shown to elicit a range of immunological responses in mammalian hosts. A family of cell wall antigens, the phosphatidylinositol mannans (PIMs), have been shown to reduce allergic response in a murine model of allergic airway disease and have been suggested as potential therapeutic agents. Isolation and characterisation of these compounds is not facile. To confirm the structure of PIMs a number of phosphatidylinositols (PIs), 1a-c, PIM1s 2a, 2d and 2e, and AcPIM1s, 2g and 2f, were prepared to allow assignment of the acylation pattern of natural products and for evaluation in immunological assays. As the natural products include 19:0 acylation in the form of (R)-tuberculostearoyl residues, a source of (R)-tuberculostearic acid was needed. To this end, an efficient synthesis of (R)-tuberculostearic acid from (S)-citronellol, utilising a copper-catalysed cross-coupling reaction and a modified Julia olefination, was developed. This material was incorporated into diacylglycerols prepared from (R)-benzyl glycidol. A protected myo-inositol derivative, 188, and two protected pseudo-disaccharides, 10 and 241, were prepared from myo-inositol via desymmetrisation utilising a camphylidene acetal. These were coupled with diacylglycerols via a phosphate ester and deprotected to give PIs, PIM1s and AcPIM1s. Mass spectrometry studies were undertaken on the PIs, 1a-c, PIM1s 2a, 2d and 2e, and AcPIM1s, 2g and 2f which structures that have been established by chemical synthesis. Comparison of these data with those reported for natural PIs and PIMs containing 19:0 ((R)-tuberculostearoyl) and 16:0 (palmitoyl) acyl groups unequivocally established that the 19:0 residue was located at the sn-1 and the 16:0 at the sn-2 position of the glycerol moiety in nature.

Mobile inteins and introns are genetic elements capable of self-propagation by “homing” into host genes and occur in entire taxonomy: eubacteria, eukarya, archaea and viruses. The process of “homing” is promoted by an endonuclease encoded by the open reading frame (ORF) embedded within the genetic element. Homing endonucleases are encoded by group I and group II introns, archaeal introns, inteins, and free standing ORFs. They are believed to play a central role in rearrangement of organelle as well as nuclear genomes. Inteins are genetic elements present within protein-coding genes with dual function: protein-splicing and homing endonuclease activities. One hallmark of homing endonucleases is their ability to recognize and cleave extended degenerate asymmetric sequences (14 - 40 bp) in intein- or intron-less alleles. Homing endonucleases are classified into four families based on the presence of LAGLIDADG, GIY-YIG, His-Cys box, or H-N-H conserved motifs. Among these, LAGLIDADG family is the largest, widespread and well-studied class. Structural and biochemical studies have demonstrated that homing endonucleases with one LAGLIDADG motif act as homodimers, whereas enzymes with two such motifs function as monomers during catalysis. In vitro, these enzymes are extremely specific for their recognition sites and they prefer Mg2+ as the metal-ion cofactor. Unlike Escherichia coli, recA of Mycobacterium tuberculosis and Mycobacterium leprae contain in-frame insertion of an intein-coding sequence. In addition to recA,scrutiny of M. tuberculosis genome revealed that intein-coding sequences are present in the ORFs of dnaA and Rv1461 (pps1). M. tuberculosis recA encodes a 85 kDa precursor protein. Amino acid sequence comparison between M. tuberculosis RecA precursor and the prototype E. coli RecA displayed high degree of homology at the amino-terminal (1 -254 amino acid residues) and carboxyl-terminal (694 - 790 amino acid residues)domains of the RecA precursor. The central domain comprising 440 amino acid residues showed significant homology to the members of the LAGLIDADG super-family of intein homing endonucleases. Following the synthesis of precursor protein, RecA intein and active RecA are generated by protein splicing reaction. The protein splicing reaction of RecA intein has been studied extensively; however, its endonuclease activity remained obscure. To identify the biochemical function of M. tuberculosis RecA intein (PI-MtuI), the intervening sequence from recA was cloned, overexpressed in E. coli and purified to homogeneity. The identity of PI-MtuI was ascertained by sequencing 10 amino acid residues at the amino-terminal end and by Western blot analysis using polyclonal antibodies raised against precursor RecA.

No description available.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Background: Elevated levels of iron impair immune defence mechanisms specifically cell mediated immunity and macrophage function, favors infection with Mycobacterium tuberculosis (M.tb), its replication, progression to clinical disease and death form tuberculosis (TB). Chelation of iron in individuals with an excessive iron burden may restore host defence mechanisms, decrease M.tb viability and replication and could find application in the prevention and treatment strategies in a setting where both iron overload and TB are prevalent. Aim: The aim of this study was to investigate the effect of iron chelation on mycobacterial replication, host viability and defence mechanisms in iron-loaded monocyte-macrophages during M.tb infection. Materials and Methods: Mycobacterial replication was monitored using the microplate AlamarBlueTM assay (MABA) for M.tb strains H37Ra and H37Rv, or detection of green fluorescent protein (GFP) expression by BCG (GFP-BCG). Mitochondrial membrane potential (MMP), phosphatidylserine (PS) exposure and plasma membrane integrity of premonocytic U937 cells differentiated by vitamin D3 served as indicators of host cell viability after treatment with 500M FeSO4.7H2O alone or in combination with 500 M desferrioxamine (DFO) or silybin, and infection with M.tb at 1:1 infection ratio. Superoxide anion radical (O2-•) and total nitrate/nitrite generation was monitored as host defence mechanisms by absorption spectroscopy and fluorimetry respectively. Results: Iron supplementation enhanced intra- and extracellular growth of M.tb and BCG. Chelation of iron with DFO prevented the enhanced replication of mycobacteria promoted by iron. Iron overload increased host cell death of H37Ra-infected monocyte-macrophages through increased PS exposure and decreased MMP and plasma membrane integrity, while increasing O2-• production and decreasing NO production. DFO restored the iron-related increase in PS exposure, plasma membrane integrity, O2-• and NO production to levels similar in H37Ra-infected, iron-normal cells, while MMP remained suppressed. In contrast, infection of iron-loaded cells with H37Rv reduced the already suppressed monocyte-macrophage MMP while not affecting cell death or NO production. DFO restored the iron-related suppression of O2-• in H37Rv-infected cells, and induced the production of NO while not affecting host cell death. Conclusion: These results confirm the replication enhancing effect of iron-loading on M.tb. Its suppressive influence on macrophage viability and defence mechanisms by increasing cell death and decreasing NO production during infection, is also highlighted. The beneficial effect of iron chelation by DFO through the inhibition of mycobacterial replication and restoration of host viability and defence mechanisms are suggested. / Prof. Liza Bornman

The global re-emergence of TB and other mycobacterial infections have underscored the need for a thorough investigation of the biology of the causative agent, Mycobacterium tuberculosis, at the molecular level. The peculiar features of the bacterium such as slow growth rate, dormancy, unique cell wall composition and resistance towards phagocytosis by macrophages demands a detailed understanding of different essential molecular processes including transcription in this genus. Sequencing of several mycobacterial genomes provided an impetus for understanding the gene function and regulation of this formidable pathogen. Transcriptional regulation is one of the major mechanisms controlling gene expression. While a number of transcription units, promoters, sigma factors, and gene functions were identified and characterized, key features of transcription process are yet to be understood. The current study aims to understand some of the facets of transcription initiation and elongation in mycobacteria. The thesis is divided into five chapters. Chapter 1 introduces the bacterial transcription process. It starts with the description of the central molecule in transcription -the RNA polymerase (RNAP) and its catalytic mechanism. In the next section, each step of the transcription initiation, elongation and termination has been discussed. The mechanistic details as well as the different cellular factors involved in the regulation of the transcription have been discussed. The final part gives an overview of the transcription machinery of the mycobacteria, describing the promoter specificity and regulation of different sigma factors and other transcription factors known till date in mycobacteria. The scope and the objectives of the thesis are presented at the end of this chapter. In Chapter 2, a method of purification of RNAP from mycobacteria for optimized promoter -polymerase interactions is described. In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNAP with high specific activity is necessary to carry out a variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low σA-promoter specificity in promoter -polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of σA required for house - keeping transcription. In vivo reconstitution of RNAP holoenzyme with σA and its purification procedure which resulted in a holoenzyme with stoichiometric σA content is described in this chapter. The reconstituted holoenzyme showed enhanced promoter -specific binding and transcription activity compared to the enzyme isolated using standard procedure. Chapter 3 is aimed at the comparison of promoter - specific events during transcription initiation in mycobacteria. DNA -protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNAP binds to promoters in a sequence -specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. The first detailed kinetic analysis of promoter - RNAP interactions during transcription initiation in the σA-dependent promoters PrrnAPCL1, PrrnB and Pgyr of M. smegmatis are presented in this chapter. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to the distinct kinetic patterns, each one of the house -keeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation. Moving the focus of the thesis from transcription initiation to elongation, a transcript cleavage factor of M. tuberculosis has been characterized in Chapter 4. After initiation of transcription, a number of proteins participate during elongation and termination by modifying the properties of the RNAP. Gre proteins are one such class of transcription elongation factors which are conserved across bacteria. They regulate transcription by binding near the secondary channel of RNAP, projecting their N-terminal coiled-coil domain into the active center and stimulating hydrolysis of the newly synthesized RNA by RNAP in the backtracked elongation complexes. Rv1080c is a putative gre factor homolog (MtbGre) present in M. tuberculosis.The protein enhanced the efficiency of promoter clearance by lowering the abortive transcription and also rescued the arrested and paused elongation complexes efficiently in the GC rich mycobacterial template. The Gre factor of M. smegmatis encoded by the gene MSMEG_5263 also showed biochemical properties similar to the M. tuberculosis protein. Although the mycobacterial Gre is similar in domain organization and shared the key residues for catalysis and RNAP interaction with Escherichia coli Gre proteins, it could not complement the E. coli strain deficient in Gre factors. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein - protein interactions for transcript cleavage. Decrease in the level of MtbGre also reduced the bacterial survival by several fold indicating its essential role in mycobacteria and suggesting that a single Gre copes up with the burden of transcription fidelity of the genome. Chapter 5 describes the studies carried out to identify Gre factor homologs in mycobacteria and deciphering their function during transcription. Gre factors are members of a growing family of proteins which regulate RNAP through secondary channel. Apart from the Gre factor, putative members of this class of proteins are identified in both M. smegmatis and M. tuberculosis.The closest homologue of the canonical Gre factor of M. tuberculosis in its genome is Rv3788. The protein has Gre factor like domain organization and possess the key acidic residues required for transcript cleavage activity and the putative hydrophobic RNAP interacting residues in the C-terminus similar to MtbGre. Despite having these common features, Rv3788 did not stimulate transcript cleavage. In contrast, it turns out to be a transcription inhibitor by preventing the binding of NTPs to the enzyme. The transcription inhibition is not promoter specific, and is mediated by its binding to RNAP through the secondary channel with its N-terminus coiled coil domain. Like M. tuberculosis, the fast growing non-pathogenic mycobacteria M. smegmatis also has an ORF (MSMEG_6292) which is homologous to its canonical Gre factor and it interacts with RNAP in a similar manner. However, this protein did not exert any transcript cleavage or inhibitory activities but could compete with the Gre factor for binding to RNAP. The Gre factor homologs in mycobacteria may be involved in regulation by inhibiting transcription or by blocking the RNAP secondary channel from other RNAP active site modulators.

The increasing prevalence of bacterial infections has made it necessary to find novel methods of combatting the resistance of bacteria to conventional antibiotics. Mycobacterium abscessus is an increasingly prevalent pathogen that is intrinsically drug resistant, therefore difficult to treat. The use of phytochemicals as a source of alternate antibiotics has been explored, however, the poor solubility of phytochemicals in water makes it difficult to effectively deliver them to bacterial biofilms. In this study, I investigated the efficacy of nanosponge-emulsified phytochemicals in killing M. abscessus biofilms. The nanosponge technology was used to improve the solubility and stability of the phytochemicals, allowing for improved bioavailability. Results showed that the nanosponge-emulsified phytochemicals effectively reduced the viability of M. abscessus biofilms, compared to non-emulsified phytochemicals. The findings of this study contribute to a development of new strategies for the treatment of bacterial infections and demonstrate the potential of nanosponge-emulsified phytochemicals as a promising alternative to conventional antibiotics.

No description available.

Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are opportunistic pathogens that can be transmitted via drinking water, when tiny droplets containing the bacteria are aerosolized and inhaled during activities such as showering. The resulting respiratory illnesses, Legionnaires' Disease and NTM lung disease, are among the leading sources of drinking water associated disease in the United States and other parts of the world. Lp and NTM are both difficult to control, because they establish as part of natural biofilms that form within the interiors of pipes and fixtures that deliver drinking water to the point of use. These pathogens are especially problematic within premise (i.e., building) plumbing, where intermittent use throughout the day leads to long periods of stagnation, increased water age, warmer temperatures, and depleted disinfectant residuals that exacerbate bacterial growth. The recent advent of high throughput DNA sequencing has led to the discovery that drinking water microbiomes are diverse, complex, and largely comprised of non-pathogenic microbes. This has further led researchers to hypothesize that the microbial ecology of this diverse microbiome could be harnessed as a natural means to control Lp and NTM, i.e., a "probiotic" approach, but such an approach has not yet been demonstrated. The objective of this study was to assess this hypothesis by utilizing biologically active granular activated carbon (GAC) filters, which are already a widely used drinking water treatment both at the municipal and household scale, as a means to naturally shape the microbial ecology of downstream premise plumbing and inhibit Lp and NTM proliferation. GAC has an extremely high surface area that aids removal of organic carbon via adsorption but also provides an ideal habitat for establishment of biofilms, which removes organic carbon from the water via biodegradation. Convectively-mixed pipe reactors (CMPRs) were used for replicable simulation of premise plumbing distal taps. The CMPRs consisted of four-foot-long closed polyvinyl chloride (PVC) pipe segments with the sealed bottom portion resting in a ~48 °C water bath and with the top portion plugged and exposed to the cooler, ambient atmosphere (25 °C in this study), inducing convective mixing and resulting in an internal water temperature of 37 °C. PVC was chosen because it is common in premise plumbing and generally leaches the least organic carbon among the different types of plastic pipe. Four different influent water conditions were implemented in the experimental design: 1) Untreated, dechlorinated municipal tap water with high organic carbon and low biomass; 2) GAC-treated tap water with low organic carbon and elevated, viable biomass; 3) GAC-treated + 0.22-m pore size membrane-filtered tap water to remove both nutrients and biomass; 4) GAC-treated tap water pasteurized at 70 °C with low nutrients and elevated, killed biomass. The 0.22-m pore size membrane filter simulated the use of a building scale particle filter, while pasteurization simulated water passing through a hot water heater at an elevated temperature recommended for pathogen thermal disinfection. To understand the influence of these experimental conditions on older pipes containing mature biofilms versus new pipes that leach more organics and are being freshly colonized, a set of older pipes colonized with mature ~4-year-old biofilms were compared to newly purchased pipes. Each set of pipes was tested in triplicate for the four different experimental conditions with the full volume replaced three times a week for eight months, simulating infrequently used taps containing warm, continuously mixing water thought to create conditions at a very high risk for opportunistic pathogen growth. In the aged CMPR bulk water effluents, droplet-digital-polymerase-chain-reaction measurements showed a one-log reduction of Lp and NTM when receiving GAC-treated or GAC-treated + particle-filtered influent water versus receiving dechlorinated municipal tap water or GAC-treated + pasteurized water. These findings suggest that decreased biodegradable dissolved organic carbon achieved by GAC filtration acted to suppress Lp and NTM growth, while the additional step of biomass removal by particle filtration provided a more modest benefit. In the CMPRs consisting of new pipes, concentrations of Lp and NTMs in the effluent bulk water were similar among the experimental conditions, except that the CMPRs receiving the GAC-treated + particle-filtered influent water experienced a two-log reduction in NTMs. These results demonstrate that the colonization and proliferation of NTM within premise plumbing can be significantly controlled by limiting nutrients and biomass in the influent water. This work demonstrates the potential of harnessing GAC-treatment as a means to Control Lp and NTM in premise plumbing via nutrient removal. In scenarios where chemical disinfectants have been depleted, off-the-shelf GAC-treatment used as point-of-entry treatment to large buildings with recirculating plumbing could provide benefits that have previously been unrecognized. Alternatively, pasteurization in very hot water heaters could provide a short-term disinfection benefit, but eventually the nutrients embodied in the dead biomass undermine the positive influence of the nutrient removal provided by the GAC-treatment. Improved mechanistic understanding of probiotic strategies to opportunistic pathogen control would be needed to overcome inherent limitations to the approaches examined herein, if more effective control is desired in the absence of thermal or chemical disinfection. / Master of Science / Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are bacterial pathogens that are the leading source of drinking water-associated disease in the US. Unfortunately, they are not effectively controlled by protections put in place by the US Safe Drinking Water Act (SDWA). Firstly, they cause respiratory infections, which are spread when tiny droplets of water are inhaled during activities such as showering, whereas the SDWA is specifically designed to protect against ingested pathogens. Secondly, unlike fecal-derived organisms (e.g. E. coli) that are the focus of the SDWA, Lp and NTM grow naturally in drinking water distribution systems, especially in premise (i.e., building) plumbing, where water is warmer and more stagnant. Therefore, even if water leaving the treatment plant is devoid of Lp or NTM, this does not guarantee that the consumer's tap water will be Lp- or NTM-free. Also, even though chlorine or other chemical disinfectant is required by the SDWA to be added to the water leaving the treatment plant to control downstream microbial growth, the disinfectant can be depleted or absent within the premise plumbing itself. Additionally, both Lp and NTM tend to more naturally resist chemical disinfectants than fecal-derived organisms. This research is aimed at overcoming these challenges, opening the door to new approaches to controlling Lp and NTM in premise plumbing. Historically, any microbial growth occurring in drinking water has been viewed as problematic, as it usually indicates the chemical disinfectant is inadequately protecting consumers. However, this work explores whether having an abundant community of beneficial bacteria could improve microbial water quality by competing against pathogens for limited space for attachment and nutrients. Such an approach would be analogous to the use of probiotics in humans, to establish a beneficial gut flora that is less susceptible to pathogen invasion. Granular activated carbon (GAC) filters are often used at drinking water treatment plants and by consumers as a point-of-use (e.g., installed on the kitchen tap or in a refrigerator) or whole-house treatment to remove any contaminants of concern and improve the taste and odor of tap water. The granules within GAC filters have a high surface area that helps remove contaminants, but also provides an environment where microbes can live and thrive. As water enters the filter, beneficial microbes can break down any remaining nutrients in the water (e.g., organic carbon and nitrogen). Additionally, the water leaving the filter carries high levels of microbes that grow on the GAC filter that are shed as water passes through. The resulting water with reduced nutrients and higher concentrations of potentially beneficial microbes could create a competitive environment that alters growth of harmful bacteria, like Lp and NTM, in downstream portions of plumbing. The incoming cold water is also warmed by the building envelope, which increases bacterial growth rates. Thus, the underlying hypothesis of this research is that GAC treatment could provide a combination of reduced nutrients and competitive microbes as water enters downstream premise plumbing and reduce the growth of Lp and NTM. However, GAC-treated water within a building can be further altered by other treatments, like a very hot water heater, which would heat and kill the microbes flowing through it, or a particle filter, which could remove the microbes in the water. This work also seeks to understand how these additional treatments might improve or interfere the nutrient reduction and addition of competitive microbes provided by GAC treatment. This research explores how all these different scenarios affect the growth of Lp and NTM using a lab-scale simulated premise plumbing system constructed out of polyvinyl chloride (PVC) pipe that is a common plumbing material used in homes. Water that was added to the pipes was prepared in four different ways to test the probiotic control hypothesis across distinct experimental conditions that replicate the different influent water scenarios. The four conditions were implemented over the course of eight months with regular chemical and biological analyses conducted to understand the effects of the different influent waters on Lp and NTM. It was discovered that premise plumbing with mature biofilms receiving GAC-treated water or GAC-treated + particle-filtered water contained ~90% less Lp and NTM than premise plumbing receiving non-filtered municipal tap water. However, if the GAC-treated water passes through a water heater, the capacity to limit Lp or NTM growth was lost. While GAC filters are currently thought of as an instantaneous treatment that removes contaminants from water, this work demonstrates how GAC treatment might provide prolonged benefits to water, after it has passed through the filter on its journey to a shower head or faucet. Increased understanding of the exact mechanisms of limited pathogen growth gained by this research can lead to new and effective approaches to protect people from contracting diseases caused by Lp and NTM.