published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Persistence in human macrophages is central to the virulence of Mycobacterium tuberculosis, which is the causative agent of tuberculosis. Although the intracellular parasitism is apparent, molecular determinants of mycobacterial virulence are not well understood. The current investigation identified virulent genes of M. tuberculosis by measuring survivability of Mycobacterium smegmatis recombinants inside a human monocytic cell line THP-1 after acquiring various virulent gene candidates of M. tuberculosis. These gene candidates included nine virulent gene candidates suggested by other studies, five genomic polymorphisms identified in hypervirulent strains of M. tuberculosis using microarray-based comparative genomic hybridization, and ten single nucleotide polymorphisms identified in the hypervirulent strains using full genome sequencing. Interestingly, only recombinants harboring a truncated Rv2820c and a known virulent gene mce1A survived significantly better than vector control after six hours of ex vivo infection. As nucleotide sequencing indicated that the truncated Rv2820c loses around 60% of gene at 3’ end, ex vivo survivability of M. smegmatis recombinants harboring the last 60% of Rv2820c as well as the intact Rv2820c was measured, but was similar to that of vector control. The 3’ truncated portion itself did not alter mycobacterial survivability ex vivo, but its presence did compromise the survival advantage gained due to the truncated Rv2820c. To determine whether the truncated and the intact Rv2820c could enhance mycobacterial virulence in vivo, these two alleles were transformed into Mycobacterium marinum and their recombinants were used to infect zebrafish. In vivo infection showed that zebrafish infected with the recombinant harboring truncated Rv2820c died significantly faster than vector control, whereas the recombinant harboring intact Rv2820c behaved similarly to vector control. Results indicated that the truncated Rv2820c, but not the intact Rv2820c, could enhance mycobacterial virulence both ex vivo and in vivo. Additional nucleotide sequencing revealed that the 3’ truncation in Rv2820c is caused by a Beijing/W-defining deletion RD207 and is commonly found in Beijing/W strains of M. tuberculosis. Non-Beijing/W strains possess the intact Rv2820c conversely. Since Beijing/W strains have proven to be more virulent than non-Beijing/W strains both ex vivo and in vivo, the truncated Rv2820c may be one of the Beijing/W-specific virulence determinants. To confirm that Rv2820c of Beijing/W strains really enhances M. tuberculosis survival in human macrophages, the truncated Rv2820c was transformed into non-Beijing/W M. tuberculosis strains and their recombinants were used to infect THP-1 cells. Ex vivo infection confirmed that the truncated Rv2820c could enhance M. tuberculosis survival inside human macrophages, but is unlikely to induce a different profile of cytokine secretion from infected macrophages. In conclusion, the current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence both ex vivo and in vivo. Enhanced phenotypic virulence, however, was not observed for the intact Rv2820c of non-Beijing/W strains. The truncated Rv2820c may be one of the Beijing/W-specific virulence determinants and collaboratively contribute to the high phenotypic virulence of this family. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Tuberculosis (TB) is a highly infectious disease that causes the second highest mortality rate in human worldwide. The emergence of multi-drug resistance tuberculosis (MDR-TB) leads to a major public health problem in controlling TB-caused mortality. Pyrazinamide (PZA) is an important first-line drug in the treatment of MDR-TB. However, since the challenge in performing susceptibility test on PZA, World Health Organization has not published any data on the prevalence of PZA resistance in Mycobacterium tuberculosis (M. tuberculosis). Since the occurrence of PZA resistance makes MDR-TB more difficult to treat with poor prognosis, rapid detection method in PZA resistance is urgently needed. Since pncA mutation is highly associated with up to 98% PZA resistant M. tuberculosis strains, it is worthwhile to develop rapid molecular method for detecting PZA resistance. This study aims to identify the mutations in PZA resistant M. tuberculosis strains. The first part of this study aims to characterize the pattern of pncA mutation among PZA-resistant and PZA-susceptible M. tuberculosis using Sanger sequencing method. Among all clinical isolates, 12 out of 29 cases of M. tuberculosis were resistant to PZA. All PZA-resistant M. tuberculosis strains harbored pncA mutation, whereas no known mutations were found among those PZA-susceptible strains, giving the positive predictive value to be 100%. Eight mutation patterns were found among 12 resistant isolates. Four of these pncA mutations have not been described previously by other studies. Study also characterizes the pattern of pncA mutation in 19 sputum specimens, with 2 mutation patterns found. Overall 10 mutation patterns were found in this study. Results show that the mutation of pncA gene is highly associated with PZA-resistant M. tuberculosis. Results also suggest the scattered and more extensive mutations in pncA gene that confer PZA resistance to M. tuberculosis. The second and the last part of this study aims to evaluate the possibility of using molecular method to detect PZA resistance in routine clinical laboratory. Results show that using molecular sequencing to detect PZA resistance can shorten the turnaround time to about 3-4 working days. Since mutation of pncA was scattered along the entire pncA gene, using DNA sequencing approach may be the best strategy for the rapid detection of PZA resistance in M. tuberculosis. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

No description available.

The eis gene of M. tuberculosis is believed to play a role in the intracellular survival of this pathogen. Significantly higher levels of antibodies to Eis were detected by ELISA in the sera of patients with tuberculosis as compared to healthy controls. PBMCs from recovered TB donors were also found to demonstrate significantly higher levels of proliferation in response to stimulation with the Eis protein than PBMCs from either active TB cases or healthy controls. Neither the active TB population nor the healthy controls showed significant levels of IFN- or IL-4 secretion in response to stimulation of PBMC with Eis or ESAT-6. Far Western analysis determined that Eis interacts with a ~65 kDa protein that localizes to the cytoplasmic fraction of M. tuberculosis lysate. Real-time PCR analysis of M. tuberculosis infected U-937 macrophages showed that the eis gene is constitutively expressed during infection. Using immunofluorescence microscopy (IF), the Eis protein was detected within the cytoplasm of M. tuberculosis infected macrophages indicating that the protein was being released/secreted from the mycobacterium containing phagosomes. Western blot analysis of the cytoplasm of macrophages infected with M. tuberculosis expressing green fluorescent protein confirmed these results. Western blot analysis also detected the presence of native Eis both in the culture supernatant of infected macrophages and vesicles released from the macrophages. IF also detected the presence of Eis in uninfected macrophages. The Eis protein in the cytoplasm of M. tuberculosis infected macrophages was also found to colocalize with EEA1, an endosomal marker, indicating a possible association of the protein with early endosomes. Eis was also shown to elicit higher levels of IL-10 secretion than PPD in human monocytes. Infection of monocytes from healthy tuberculin reactors with M. tuberculosis wild type and eis mutant demonstrated that eis plays a role in modulation of IL-10/TNF- secretion in response to infection. Bioinformatic analysis of the amino acid sequence of Eis indicates that Eis is an acetyltransferase of the GCN5 related family of N-acetyltransferases. Further work is required to determine the role Eis plays in the survival of M. tuberculosis within the macrophage.

Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (MScMed)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.