published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The global emergence of drug resistance is posing increasing difficulties in the public health control and treatment of tuberculosis (TB). Fluoroquinolones (FQs) are regarded as having a pivotal role among the antimicrobial agents in multidrug regimens against multidrug-resistant tuberculosis (MDR-TB). Thus, early diagnosis of fluoroquinolone-resistant (FQr) MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) by molecular tests has predictive value for the guidance of TB therapy. The pharmacokinetic (PK) and pharmacodynamic (PD) indices are valuable parameters to evaluate the activity and efficacy of fluoroquinolones (FQs) based upon the bactericidal effect and prevention of the emergence of resistance. In the first part of this study, the potencies of ofloxacin (OFX) and moxifloxacin (MXF) against clinical isolates of MDR-TB in terms of their PK/PD indices (Cmax/MIC90, AUC/MIC90, Cmax/MPC90 and AUC/MPC90) were investigated and compared. The results revealed that MXF displays higher ratios of PK/PD in vitro and could serve as a promising agent for the treatment of MDR-TB. Molecular tests on resistance genes are reliable and rapid technology for diagnosis of drug-resistant TB which facilitates timely patient management and public health control of TB. In the second part of the study, the feasibility of a PCRsequencing assay for the examination of mutations in the quinolone-resistance-determining- region (QRDR) of the gyrase A (gyrA) gene in FQ-resistant (FQr) Mycobacterium tuberculosis in direct clinical specimens was evaluated. As determined by gyrA QRDR DNA sequencing analysis, complete concordance of phenotypic and genotypic outcomes was demonstrated. The results indicate that the molecular assay is an accurate and effective method for the diagnosis of FQr TB and allows identification of mixed resistant variants in the same patient. GyrA mutations that associated with FQr in clinical isolates of M. tuberculosis were clustered in hotspot codons 88, 90, 91 and 94, corroborating other reports. We also detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from the respiratory specimens by using the PCR-DNA sequencing assay. In the third part of this study, the functional effect of the Ala74Ser mutant was verified through study of the DNA supercoiling inhibitory activities of OFX and MXF against the recombinant DNA gyrase. Fifty percent inhibitory concentrations (IC50) of FQs against the DNA supercoiling activities of the recombinant DNA gyrase complex reconstituted with gyrA Ala74Ser were eight-fold and 14-fold greater than the wild-type H37Rv reference strain, and results correlated well with their phenotypic drug susceptibilities. Besides, a combination of gyrA mutations (Glu21Gln, Ser95Thr and Gly668Asp) was also characterized to be non-functional polymorphisms. The impact of the gyrA Ala74Ser mutation on drug binding affinity was elucidated through a crystal structure model of the gyrA-MXF-DNA cleavage complex. Alanine at position 74 of gyrA in M. tuberculosis, which corresponds to the alanine at position 67 of gyrA in Escherichia coli, is an amino acid lying in the α3 helix domain which forms a hydrophobic interface between the gyrA-gyrA dimer. Perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative drug binding pocket, and leads to the reduction of the binding affinity of FQ due to the distance effect. This is the first report verifying that gyrA Ala74Ser mutation alone is responsible for FQr in M. tuberculosis. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Tuberculosis is an enormous global health problem. Despite decades of research, the mechanism(s) by which Mycobacterium tuberculosis (Mtb) mediates virulence remains incompletely understood. The ESX-1 secretion system is critical for Mtb to survive and cause disease in vivo, but its primary function and mechanism of action are unclear. The many inherent challenges of working with this slow-growing pathogen often limit the experimental approaches that can be used to address these questions. Thus, we have developed a model system in the nonpathogenic bacterium Bacillus subtilis to study ESX-type secretion systems. Here, we demonstrate that the B. subtilis yuk operon encodes an ESX-type secretion system responsible for the secretion of YukE. Additionally, we demonstrate that the yuk system is active in B. subtilis during conditions of nutrient deprivation and is required for normal biofilm formation. Interestingly, this is similar to our findings that the Mtb ESX-1 system plays dual roles in protein secretion and modulating cell wall integrity. One defining feature of all ESX loci is the presence of an FtsK/SpoIIIE family ATPase. Interestingly, these ATPases have a domain structure unique to ESX-associated ATPases, where each protein contains multiple (2-3) enzymatic domains. We used our B. subtilis system to dissect the mechanism of action of this unique class of motor proteins. We find that the yuk-encoded ATPase YukBA dimerizes to form a hexamer of enzymatic subunits that are differentially required for secretion. Strikingly, we find a unique requirement for rotational symmetry in the nucleotide binding activity of the subunits. Finally, we compared the energy requirements of the Mtb ESX-1 system and the B. subtilis yuk system. We find that these systems have some overlapping ATPase requirements for protein secretion and cell wall integrity/biofilm formation, suggesting that there is a conservation of function among ESX-type systems. We also find that some ATPase domains are differentially required for function between these two systems, which we postulate is due to the split protein architecture of the ESX-1-encoded ATPases. Together, these findings highlight the power of using a B. subtilis model system to understand the function and mechanism of action of ESX-type secretion systems.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Tuberculosis and malaria are among the most deadly infectious diseases in the world. The prevalence in regions without well-established public health causes economical and financial burdens for both society and patients. There is an urgent need to find effective treatments due to the emergence of drug-resistant strains. The aim of the studies reported here was to gain knowledge from the protein structures that can lead to the elimination of these pathogens. In these studies, protein crystallography is the main method used to solve protein structure. Based on the protein structure, we used different methods to characterize the protein function of three lipid-binding proteins (LprG, LprA, and gp232), and to identify potent inhibitors against Plasmodium falciparum enoyl-ACP reductase (PfENR), a drug target protein involved in central lipid metabolism. To characterize the function of two lipid-binding proteins (LprG and LprA), liquid chromatography-mass spectrometry (LC-MS) was used to analyze the ligand extract. In the study of tail fiber protein from mycobacteriophage, we used protein sequence alignment to identify gp232 as a major tail fiber protein, which potentially binds to lipids on the cellular surface of mycobacteria. A pull-down assay and imaging methods (fluorescence microscopy and electron microscopy) were conducted to confirm the function of gp232. In the structural study of PfENR, the structure-activity relationships method was used to find potent inhibitors against PfENR, which would show stronger inhibition than the known inhibitor triclosan. The triclosan-like analogs with modification at the 5-position revealed a new binding site in PfENR that has great potential for improving the potency of inhibition. We found that two inhibitors containing the core structure of piperidine and tetrahydroquinoline reached this new binding site and were 10-fold more potent than triclosan. The structural study of PfENR provides structural insights into the inhibitor-binding site that can lead to the discovery of new drugs. The comprehensive knowledge that we gained from the structural studies of these lipid-binding proteins provide new information that could lead to a greater understanding of pathogen physiology or guide the discovery of effective treatments to eliminate the pathogens.

Background Mycobacterium tuberculosis isolates such as the Beijing and F15/LAM4/KZN families dominate in patients. The emergence of extensively drug resistant (XDR) M. tuberculosis isolates raises concern. The need to better understand the pathogenesis of M. tuberculosis isolates resulted in this work. Methods M. tuberculosis clinical isolates that belonged to the Beijing and F15/LAM4/KZN families, isolates with unique DNA fingerprints and laboratory strains were used. Isolates were grown in the presence of oxygen and then exposed to A549 alveolar and BBM bronchial epithelial cells. The number of bacilli that adhered to the epithelial cells were viewed and counted using light microscopy. Isolates grown in the presence of oxygen and under oxygen deprivation were used for subsequent assays. Invasion of A549 and BBM cells by isolates grown under these different circumstances was investigated. Based on the results, the remaining assays were performed with A549 cells only. Cytotoxicity was quantified using the Cyto Tox96 Non-Radioactive Cytotoxicity Assay kit. Morphological changes in A549 cells after exposure to the isolates were observed using the scanning electron microscopy (SEM). Real-time quantitative PCR was performed to assess the relative expression levels of four genes potentially associated with virulence (hbhA; mdp1; fdxA; hspX). Results were normalized against 16S rRNA and ftsZ gene transcription and reported as fold difference as compared to H37Rv. Results All isolates adhered to and invaded A549 cells in significantly higher numbers than BBM cells (P<0.0029). Isolates grown under oxygen deprivation displayed higher levels of virulence than their aerobic phenotype. Grouped together, the isolates belonging to the Beijing and F15/LAM4/KZN families of strains showed greater adhesion capacity (28%) than isolates with unique DNA fingerprints (5%) (P<0.05%). Three F15/LAM4/KZN isolates (two XDR-variants), were at least twice as invasive (>33%) as the most invasive Beijing isolate (15%) (P<0.05). The highest cytotoxicity level (35.7%) was produced by an XDR-F15/LAM4/KZN strain. SEM revealed bleb-like structures on bacterial cells grown under oxygen deprivation. Beijing and XDR-F15/LAM4/KZN isolates had the highest number of projections (16+5 per bacillus. The expression levels of all four genes were highest in Beijing and F15/LAM4/KZN isolates grown under oxygen deprivation and exposed to A549 cells. Conclusions Beijing and F15/LAM4/KZN strains are more virulent and their successful spread might be related to their interaction with alveolar epithelium. M. tuberculosis pathogenesis studies should include isolates grown under oxygen deprivation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

In this study we investigated the impact of exogenous reinfection on genetically resistant and genetically sensitive sub populations. We qualitatively analysed the dynamics of TB by assuming that TB is transmitted in two ways namely homogeneous and heterogeneous modes of transmission. Analytically, we computed the fundamental thresholds used to measure disease persistence; the basic reproduction number R₀; and found that the exogenous reinfection parameters do not appear in the basic reproduction number. Hence, basic reproduction number derived in presence of exogenous reinfection does not adequately predict the course of a TB epidemic. We obtained the exogenous reinfection threshold which indicated that exogenous reinfection complicates TB dynamics. Both analytical and simulation results disclosed that when exogenous reinfection is above exogenous reinfection threshold TB dynamics were governed by a backward bifurcation implying TB may continue to invade the population despite basic reproduction number being less than one. We computed critical value of basic reproduction numbers Rᴄ and found that TB can only be eradicated if basic reproduction number is reduced below critical value Rc. Furthermore, we incorporated TB therapy in heterogeneous model among individuals with clinically active TB and performed sensitivity and uncertainty analysis using Latin Hypercube Sampling. The sensitivity and uncertainty results showed that transmission rates, reactivation rates and proportion that is genetically resistant greatly infuenced outcome variables of our TB model. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Molecular modelling has become widely used in chemistry and biology. The aim of this project is to use a range of molecular modelling techniques to study enzymic actions. This thesis consists of two parts. Part A of this thesis describes computational studies conducted for the calpain-calpastatin system. Calpain is a cysteine protease. Over-expression of calpain is associated with many diseases. Calpastatin is the naturally occurring specific regulator of calpain activity. In this part of the thesis, the dynamic conformational preferences of region B of the inhibitory domain in calpastatin were examined in detail by using molecular dynamics simulations and stochastic dynamic simulations with Monte Carlo sampling. Part B of the thesis explores the structure and function of the enzyme 3-dexoy-D-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis (MtuDAH7PS). MtuDAH7PS catalyses the first reaction of the shikimate pathway and is a target for the development of anti-tuberculosis drugs. MtuDAH7PS is found to be synergistically inhibited by combinations of aromatic amino acids (Trp+Phe or Trp+Tyr), but not by any single aromatic amino acids. In this part of the thesis, this unique mechanism of allosteric regulation in MtuDAH7PS was investigated by using a range molecular modelling techniques. Firstly protein crystal structure refinements were conducted and those crystal structures of MtuDAH7PS in complex with various ligand molecules are described in Chapter 4. Secondly, the reaction mechanism and roles of active site residues were investigated in Chapter 5, through docking calculations (both rigid docking and induced fit docking) of a series of designed active site inhibitors. Finally, Chapter 6 discusses the molecular basis of the communication mechanism of allosteric regulation in MtuDAH7PS.

The ESX family of genes (esxA-W) in Mycobacterium tuberculosis (Mtb) encodes 23 effector molecules influencing immunogenicity and pathogenicity. This study was aimed at identifying and evaluating variations in ESX sequence and protein expression profiles in clinical isolates and examining how diversity might influence immune responses. 23 ESX genes from 55 clinical isolates (20 Beijing, 25 KZN and 10 Other) and 3 Laboratory strains (H37Rv, H37Ra and BCG) were sequenced. 482 single nucleotide polymorphisms (SNPs) were identified in 12 ESX genes relative to H37Rv. Majority of the identified 363 nsSNPs occured in Beijing isolates. No mutations were observed in esxA, B, C, E, G, H, J, R, S and T. Six unique nsSNPs were identified in the Beijing isolates: esxI (Q20L), esxO (E52G), 2 in esxP (T3S; N83D), esxU (P63S) and esxW (T2A). Three unique nsSNPs were identified in the KZN isolates: esxK (A58T), esxL (R33S). The esxL polymorphism resulted from a dinucleotide change. ESX gene transcription levels were evaluated using RT-qPCR. Varying expression levels were observed for esxA, B, C, F, M and Q across all clinical isolates with lowest levels seen amongst the Beijing isolates. This correlated with immunoblots with confirmed decreased esxAB protein expression relative to the other strains. The Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) spectral protein profiles were quantitatively compared within and between Mtb clinical and laboratory isolates. Protein spectral profiles within the mass range of the CFP-10 protein with variations in peak intensities were observed across all isolates. QILSS and Mtb9.9 peptides were tested individually for immune responses in TB infected patients. Healthy patients displayed no responses to QILSS and Mtb9.9, strong but variable immune responses were detected for specific regions of QILSS and Mtb9.9 in TB infected patients. These findings demonstrate that differences in sequence, transcriptional profiles and protein expression patterns in ESX secreted proteins exist between clinical isolates, and may translate into differences in human immune responses. Further research is needed to correlate human host immune responses to the phenotype and genotype of the infecting strain of Mtb to determine the consequences of specific variations of the other ESX members. These studies are important for the development of improved immune diagnostics and vaccines. / Thesis (Ph.D.)-University of University of Natal, Durban, 2013.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.