Thesis (M.S.)--University of North Texas, Aug., 2007. / Title from title page display. Includes bibliographical references.

No description available.

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Thesis (M. Sc. (Chemistry) -- University of Pretoria. 2006. / Includes bibliographical references (leaves 111-117).

This study was focused on the promoter and non-coding regions of the ribosomal RNA (rrn) operon(s) of mycobacteria; namely, the leader and the intergenic spacer regions. Two clones containing the promoter sequences of M .leprae and M. tuberculosis rrn operon were sequenced, their promoter elements were identified by primer extension experiments and by comparison with E.coli consensus promoter sequences. Their function was tested in E.coli and M. smegma tis . The sequences of the leader and intergenic spacer regions from eight and six species respectively were established after amplification by means of peR. Both leader and spacer regions contain antitermination elements and RNaseIII processing sites. The sequences established for these two regions also showed greater variability than the 168 rRNA gene and are suitable for phylogenetic studies. The sequences of the two rrn operons of M.smegmatis upstream from the 168 rRNA gene were cloned and sequenced. Their sequences showed that rrnI has a Box B element which is typical of slow-growers and that rrnII does not. Primer extension studies revealed that the rrn operon of slow-growers has a single promoter. In contrast multiple promoters were identified in the faster-growing M.smegmatis. Distinctive features, which are absent from slow-growers, were identified in the intergenic spacer regions of M.smegmatis.

No description available.

Prosthetic joint infection with Mycobacterium tuberculosis usually involves the hips or knees and can result from either local reactivation, or less often from hematogenous spread. Predisposing conditions include rheumatoid arthritis, chronic steroid use and pulmonary diseases. The most common symptom at presentation is pain, and the most common physical finding is joint swelling and/or a draining sinus tract. The sedimentation rate is helpful when elevated but is nonspecific, and initial skin testing is only helpful when positive. The diagnosis depends on culture and histologic examination of tissue. Removal of the joint combined with oral antituberculous treatment is necessary when the infection is discovered greater than six weeks post joint replacement. Early diagnosis leads to decreased morbidity. Tuberculous infection of prosthetic joints is a rare disease and its diagnosis depends on a high degree of clinical suspicion.

No description available.

Mycobacterium tuberculosis (MTB) encodes three enzymes that are dependent on vitamin B12–derived cofactors for activity, including a B12-dependent methionine synthase (MetH). Previously, work in the Molecular Mycobacteriology Research Unit (MMRU) demonstrated vitamin B12 auxotrophy in a mutant strain disrupted in the alternative, B12-independent methionine synthase, MetE. This observation established the ability of MTB to transport corrinoids despite the absence of an identifiable B12-specific transporter. In addition, it suggested that MTB does not synthesize vitamin B12 in vitro. Notably, bioinformatic analyses identified PPE2 as the only B12-related transport candidate in MTB, though as a putative B12-regulated cobalt transporter. PPE2 is unusual in possessing directly upstream of its predicted start codon one of only two B12-dependent riboswitches in the MTB genome, and it lies in a putative operon with B12 biosynthetic genes, cobU and cobQ1. In this study, the possibility that PPE2 functions in the transport of vitamin B12 or cobalt was investigated. Transcriptional and phenotypic data suggested that PPE2 was not involved in B12 transport. Instead, it was shown that cobalt can supplement the growth of an MTB metE mutant in liquid medium, strongly supporting the ability of MTB to synthesize B12 de novo. Moreover, the ability to utilise exogenous cobalt was dependent on functional PPE2, thereby establishing a role for a PPE-family member in cobalt assimilation in MTB. Vitamin B12 comprises a central corrin ring co-ordinated to 5,6-dimethylbenzimidazole (DMB) as α-axial ligand. Substituting DMB with adenine yields the alternate form, pseudo-B12. The ability of mycobacteria to utilize pseudo-B12 precursors (cobinamide and adenine) to support full function of B12-dependent metabolic pathways was evaluated. Although the pseudo-B12 precursors appeared to complement chemically the mycobacterial B12 auxotrophs, growth of the mutants on cobinamide alone complicated this interpretation. To address this limitation, DMB synthesis was targeted by disrupting the MTB bluB homologue, Rv0306. Neither site-directed mutagenesis of key Rv0306 residues, nor full-gene deletion was sufficient to eliminate growth on cobinamide. Instead, this observation highlights the need to establish biochemically the nature of the active B12 form synthesized and utilized by MTB under different conditions. In combination, the results presented here support the inferred flexibility of vitamin B12 biosynthesis in MTB, and reinforce the potential role of B12-dependent metabolism in mycobacterial pathogenesis.

Lipoarabinomannan (LAM) and the mycolyl-arabinogalactan (mAG) complex are two major entities found in the cell wall of Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. Given their important roles in the viability and virulence of the pathogen, enzymes involved in these pathways represent a rich source of potential therapeutic drug targets. As fundamental understanding of substrateenzyme interactions is often essential in the drug discovery process, the purpose of this study was to investigate the substrate specificities of an -(16)-mannosyltransferase (ManT) and a -(15,6)-galactofuranosyltransferase (GlfT2), two key enzymes involved in the biosynthesis of LAM and mAG, respectively. Although the ManT activity had been detected using an established radioactive assay, its substrate specificity remained poorly defined. The current study focused on the design, synthesis and evaluation of acceptor substrate analogs of ManT. Among those analogs prepared were those containing methoxy-, hydrogen-, and amino-substituted carbohydrate residues as well as epimeric derivatives. A homologous series of oxygen- and sulfur-linked mannosides were also prepared. Evaluation of these analogs revealed the steric requirements and hydrogen bonding interactions of the enzyme, and the effect of acceptor length on mannosyltransferase activity. Also, these results provided additional insight into the role of ManTs and allowed the current proposed pathway of LAM to be further revised. Another objective of the current study was to understand how GlfT2 catalyzes the alternating -(15) and -(16)-galactofuranosyl transfers in a single active site. A panel of mono- and dideoxy trisaccharide derivatives was synthesized, in which hydroxyl groups at either or both C-5 and C-6 positions on the sugar residues at the reducing ends were selectively removed. Biological evaluation of these analogs using a spectrophotometric assay, and structural analysis of some of the enzymatic products, showed that the removal of the hydroxyl group(s) in the acceptors appeared to have no dramatic effect on either GlfT2 activity or the regioselectivity of its galactosylation. These results suggest that groups other than the C-5 and C-6 hydroxyl groups of the acceptors are more critical for the enzyme catalysis. The identification of these key elements would be the further objective of this project. The results from these fundamental studies provide important information about how these enzymes interact with their substrates at the molecular level. More importantly, this work will serve as the basis for the further design of potential inhibitors, which are potential lead compounds for novel therapeutic agents that are active against tuberculosis.