Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 73-91).

Thesis--Ph. D., University of Hong Kong, 1984.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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.