Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. The high incidence is still found in Taiwan. There is strong evidence that host genes influence individual susceptibility to tuberculosis. Young children, like immunocompromised patients, once infected are at increased risk for TB disease and progression to extrapulmonary disease. Thus far, to identify the genes responsible for the variation in the human susceptibility/resistance to TB has remained elusive. Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays an important role in innate immunity in the regulation of inflammatory cytokine release by monocytes. It is one of the molecules that have been suggested to have a link to human susceptibility or protection against infection. According to some studies (mostly conducted in adult populations) , low levels of MBL associated with variant alleles at the promoter and exon 1 regions of MBL protect against tuberculosis. Other investigators instead claim that protection against the disease is associated with high levels of MBL. In this study we aimed to investigate the relationships between the susceptibility to TB and MBL gene polymorphisms in children with cervical mycobacterial lymphadenitis infected by M. tuberculosis.139 case patients with cervical mycobacterial lymphadenitis and 102 unrelated healthy control subjects were tested by real-time PCR for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of mycobacterial lymphadenitis infected by M. tuberculosis, based on findings of pathological examination of the lymph nodes, was confirmed by acid-fast stain and TB PCR.The frequency of A allele was significantly higher in TB+ patients compared with TB- controls (82.7% vs 72.6%; odds ratio 1.813; p=0.007). The frequency of high-producer MBL2 genotypes (A/A) was higher in TB+ patients than in TB- subjects (70.5% vs 45.1%, odds ratio 2.91, p<0.001), while patients carried the B alleles (A/B and B/B) that have decreased levels of MBL was inversely associated with mycobacterial infectivity (29.5% vs 54.9%; odds ratio 2.910; p<0.001). The frequencies of MBL promoter -550 genotypes also revealed a significant difference between TB+ and TB- groups (p = 0.046), but in contrast, with significantly higher frequency of L/L genotype (of low MBL level) in TB+ patients (34.5% vs 21.6%; odds ratio 1.918; p=0.029). The frequencies of MBL promoter -221 genotypes (X and Y) was similar in TB+ and TB- groups.This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the host¡¦s haplotype pair.

Thesis (Ph. D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Nov. 25, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry, University of Alberta." Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 41-49).

Zugl.: Giessen, Univ., Diss., 2009

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.