Acquired Immunodeficiency Syndrome (AIDS) and tuberculosis (TB) have posed diagnostic and therapeutic challenges globally. Nowadays, it is estimated that 34 millions people are living with Human Immunodeficiency Virus (HIV). About 2 millions of people die from AIDS-related causes currently in each year. Tuberculosis is the most common presenting illness and leading cause of death among AIDS patients. Emerging studies suggest that HIV and Mycobacterium tuberculosis (Mtb), the causative pathogen of TB, act synergistically to accelerate decline of immune functions and cause the death. Mtb infection usually remains latent. Only small portion of infected individuals develops active TB. However HIV infection boosts the risk of reactivation of TB and susceptibility to new Mtb infection. In contrast, Mtb infection dysregulates cytokines production and induces HIV viral replication. Although it is well-known that HIV and Mtb potentiate each other in disease development, mechanisms of interaction of the two pathogens remain not well-elucidated. The aim of this study is to investigate the interaction of HIV viral protein Tat with mycobacteria infection, which may provide insights in the interplay between HIV and Mtb infections. HIV viral transactivator protein, Tat, plays a critical role in HIV replication; and its induction of apoptosis in CD4+ T cells contributes to immune defects. In this study, Tat was demonstrated to dysregulate immune responses against mycobacteria such as autophagy, a tightly regulated bacterial clearance mechanism. With pretreatment of the primary human blood monocyte-derived macrophages with Tat, the interferon-γ (IFN-γ)-induced Signal Transducer and Activator of Transcription-1 (STAT-1) phosphorylation was suppressed. Inhibition of STAT-1 phosphorylation ultimately led to downregulation of autophagy-associated gene, microtubule-associated protein light chain 3 (LC3) expressions. Of note, Tat was demonstrated to inhibit the colocalization of Bacillus Calmette Guerin (BCG) and IFN-γ-induced autophagosomes under fluorescent microscopy examination. In addition to the inhibition of bactericidal autophagy, Tat was found to manipulate cytokines production. Tat was demonstrated to enhance mycobacteria-induced tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production. TNF-α and IL-1β have been well-demonstrated in literatures that can limit bacterial growth. They, however, have been also shown as important contributors to the increase of HIV viral replication in HIV and mycobacteria coinfection. Mtb-induced TNF-α production can induce transcriptional activation of the HIV long terminal repeat (LTR) promoter while blocking of IL-1β production decreases HIV replication. Tat enhancement of these cytokines production may therefore contribute to the knowledge of the increased viral replication in HIV and mycobacteria coinfection. Furthermore, new microRNAs, up-and-coming fine-tuners of innate immunity, were discovered. MicroRNAs, a family of non-coding RNAs, can regulate gene expressions post-transcriptionally and control various developmental and cellular processes. They can target mRNAs of cellular signaling molecules, transcription factors or cytokines as to regulate the immunity. Herein, microRNA-1303, originally with unknown function, was shown to regulate mycobacteria-induced TNF-α production and affect the Tat enhancement of TNF-α production. Taken together, the results of this study demonstrated that HIV viral protein, Tat could dysregulate immune responses to mycobacteria. The study of the dysregulation may further elucidate the interplay between HIV and mycobacteria infections. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

No description available.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "August 1996" Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Please read the abstract on page 4 of this document / Dissertation (MSc (Chemistry))--University of Pretoria, 2007. / Chemistry / MSc / unrestricted

Glycopeptidolipid (GPL) antigens isolated from Mycobacterium intracellulare were used to sensitize BALB/c mice. Primary footpad challenge of mice sensitized with GPL antigens suspended in phosphate buffered saline demonstrated an increased footpad swelling reaction.

Thesis (MScMedSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The plasmid pAL5000 is a mycobacterial plasmid isolated from Mycobacterium fortuitum. It is a low copy number plasmid, which replicates in both fast growing (e.g. M. smegmatis) and slow growing (e.g. M. bovis BCG) mycobacteria. Most mycobacterial-E. coli shuttle vectors utilise the pAL5000 origin of replication. The minimum replicon consists of ORF1 (RepA), ORF2 (RepB) and the origin of replication. Dr W.R. Bourn created an E. coli-mycobacterial vector based on the pAL5000 origin of replication (pORI) and then subjected it to semi-random mutagenesis. A high copy number mutant was identified (pHIGH) and the causative mutation was tentatively identified as a 3bp deletion situated just upstream of repB. This work describes the further characterisation of the mutant plasmid. Firstly, it was shown by retransforming M. smegmatis with both the original and mutant plasmids (pORI and pHIGH), that the mutation causing the increased copy number was plasmid-encoded and not on the chromosome. Following this, it was demonstrated by simple subcloning of the region that carries the 3 bp deletion, that other pAL5000-based vectors could be converted to high copy number. In addition to this, the subcloned region was sequenced and the nature of the mutations was confirmed. The subcloning experiment confirmed that the 3bp deletion caused the high copy number phenotype. Following this, the exact copy number of pHIGH and the relative increase in copy number was determined. From this, the copy number of pORI could also be determined. The plasmid pHIGH has a copy number of approximately 54, compared to the 8 of pORI (a relative increase by a factor of 7). Because it is important for researchers to know the characteristics of the vectors that they use, especially the influence it will have on its host, stability tests and growth curves were also performed. It was seen that the higher copy number did not markedly increase the stability, however, this is because pORI is already extremely, and unexpectedly, stable in the host M. smegmatis. According to the growth curves, the increased copy number has little effect on the growth of the host M. smegmatis. Possible mechanisms for the increased copy number were then investigated. By using a promoter probe vector, the possible existence of a promoter situated between the two open reading frames of pAL5000 (repA and repB) was investigated. It was thought that the mutation might have created, or changed an existing promoter, situated between repA and repB. The results showed, however, that in both pORI and pHIGH there might be a very weak promoter upstream of repB, but the mutation did not cause any change that was measurable by the method that was used. A further possibility was that the mutation caused a change in the RNA secondary structure, which might then have an effect on the translational efficiency of RepB. It was found that the 3bp deletion in pHIGH causes a change in the local RNA secondary structure around the ribosomal binding site and the start codon, when compared to pORI (wild type). This change may cause the translation initiation rate of RepB to be different between pHIGH and pORI. Ultimately it would lead to a different ratio of RepA and RepB in the cell. / AFRIKAANSE OPSOMMING: Die plasmied pAL5000 is ‘n mikobakteriele plasmied wat vanuit Mycobacterium fortuitum gei'soleer is. Dit is ‘n lae kopie-getal plasmied wat in beide vinnig groeiende (bv. M. smegmatis) en stadig groeiende (bv. M. bovis BCG) mikobakteriee kan repliseer. Die meeste mikobakteriele-E. coli shuttle vektore gebruik die pAL5000 oorsprong van replisering. Die minimum replikon bestaan uit ORF1 (RepA), ORF2 (RepB) en die oorsprong van replisering. Dr. W.R. Bourn het ‘n E. coli-mikobakteriele vektor gemaak wat gebaseer is op die pAL5000 oorsprong van replisering (pORI), en dit onderwerp aan semi-random mutagenese. ‘n Hoë kopie-getal mutant is gei'dentifiseer (pHIGH) en die mutasie hiervoor verantwoordelik was tentatief gei'dentifiseer as ‘n 3bp delesie, net stroomop van repB. Die projek beskryf die verdere karakterisering van die mutante plasmied. Eerstens, deur M. smegmatis te hertransformeer met die plasmied DNA (pORI en pHIGH), is dit bewys dat dit mutasie wat die toename in kopie-getal veroorsaak, deur die plasmied gekodeer word, en dat dit nie ‘n mutasie op die chromosoom is nie. Hierna is dit deur eenvoudige subklonering bewys dat die gedeelte wat die 3bp delesie dra, ander pAL5000-gebaseerde vektore ook kan verander in ‘n hoër kopie-getal. Die sub-klonerings eksperiment het ook bewys dat die 3 bp delesie die oorsaak is vir die hoë kopie-getal fenotipe. Volgende is die presiese kopie-getal van pHIGH en die relatiewe toename in kopiegetal bepaal. Die kopie-getal van pORI kon vanaf hierdie data bepaal word. Die plasmied pHIGH het ‘n kopie-getal van ongeveer 54 in M. smegmatis, in vergelyking met die 8 van pORI (‘n relatiewe toename met ‘n faktor van 7). Aangesien dit vir navorsers belangrik is om die eienskappe van die vektore wat hulle gebruik, te ken, en veral die invloed wat dit op die gasheer sal hê, is stabiliteits toetse, en groeikurwes gedoen. Die hoër kopie-getal het nie die stabiliteit werklik verbeter nie, maar dit is omdat pORI alreeds uiters stabiel is in die gasheer M. smegmatis. Volgens die groeikurwes het die toename in kopie-getal ‘n minimale effek op die groei van die gasheer M. smegmatis. Moontlike meganismes vir die hoër kopie-getal is ook ondersoek. Die moontlike bestaan van ‘n promoter tussen die twee oop-leesrame van pAL5000 (repA en repB) is ondersoek deur gebruik te maak van ‘n “promoter probe” vektor. Die mutasie kon moontlik ‘n promoter geskep het, of ‘n bestaande een tussen repA en repB verander het. Die resultate het gewys dat daar in beide pORI en pHIGH moontlik ‘n baie swak promoter stroomop van repB is, maar die mutasie het nie enige veranderinge veroorsaak wat meetbaar was met die metode wat gebruik is nie. ‘n Verdere moontlikheid was dat die mutasie ‘n verandering in die RNA sekondere struktuur kon veroorsaak het, en dit mag ‘n effek hê op die translasie effektiwiteit van RepB. Daar is gevind dat, in vergelyking met pORI, het die 3bp delesie in pHIGH ‘n verandering in die lokale RNA sekondere struktuur rondom die ribosomale bindings posisie en die begin-kodon veroorsaak. Die verandering mag veroorsaak dat die translasie inisiasie tempo van RepB verskillend is vir pORI en pHIGH. Uiteindelik sal dit lei tot ‘n heeltemal ander verhouding van RepA en RepB in die sel.

Mycobacterial populations are known for the heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions. Such population heterogeneity had been observed in TB patients, in animal models, and in in vitro cultures. Also, the physiological relevance of population heterogeneity under nutrient starvation has been studied. However, the physiological significance of population heterogeneity in oxidative and nitrite stress has not been addressed yet. Our laboratory had earlier shown that a subpopulation of mycobacterial mid-log phase cultures divide by highly deviated asymmetric division, generating short cells and normal-sized/long cells. This proportion has been found to be consistent and reproducible, and has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum, which is known to have high levels of oxidative stress. The highly deviated asymmetric cell division has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, the physiological significance of the population heterogeneity generated by the highly deviated asymmetric division remained to be addressed. Therefore, in the present study, we addressed the physiological significance of the generation of population heterogeneity in terms of cell size in Mycobacterium smegmatis and Mycobacterium tuberculosis. In this regard, we explored whether the minor subpopulation of short cells generated in the population has any relevance in the response of mycobacteria to oxidative and nitrite stress for survival. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on the phenotypic heterogeneity in diverse bacterial systems and the physiological significance of such heterogeneity. Subsequently, an account of the phenotypic heterogeneity reported in mycobacteria is given, with examples of its significance implicated for survival under nutrient stress. Then an account of various studies on the oxidative and nitrite stress response of mycobacteria and on the genes involved in those processes are given. Further, the present study is justified by stating that so far there has not been any study to find out the physiological relevance of phenotypic heterogeneity on oxidative and nitrite stress response in mycobacteria. Finally, the Introduction is concluded by stating that the present study investigates and reports for the first time the physiological significance of the minor subpopulation of short cells for survival under oxidative and nitrite stress conditions. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the separation of the short cells, their characterisation, stress response, and so on are given in great detail. The Chapter 3 forms the first data chapter that presents results on the nature of response of Mycobacterium smegmatis and Mycobacterium tuberculosis against oxidative and nitrite stress. Here the cell size natural distribution, in terms of short cells and normal-sized/long cells in the mid-log phase population, the fractionation and enrichment of these subpopulations, differential susceptibility of the cells in the fractions to the stress conditions, the enhanced survival of the population upon mixing of these cell populations at the natural proportion, and the decreased survival upon mixing them at unnatural proportion are presented. The differential survival of the short cells and normal-sized/long cells was studied at a variety of stress concentrations for oxidative (H2O2) and nitrite (acidified sodium nitrite, pH 5), cell densities and exposure time to show the robustness of the phenomenon. Enhanced survival upon extended exposure to stress also has been documented. Essentially the data in this chapter shows that although the different sized populations show differential stress susceptibility to the stress conditions, their combined presence at the proportion that naturally exists in the mid-log phase population enhances the survival of the population, at the cost of the highly susceptible short cells for the enhanced survival of the less susceptible normal-sized/long cells, kin selection and altruism. The Chapter concludes with a discussion on the results. The Chapter 4 delineates the mechanism of the altruistic phenomenon that results in the enhanced survival of the population at the sacrifice of the minor subpopulation of short cells. Here we present evidence that hydroxyl radical generated through Fenton reaction is responsible for the enhanced survival through the induction of the synthesis of catalase-peroxidase (KatG) for the degradation of H2O2. The free iron deficient short cells acquire more iron, which in turn becomes stoichiometrically detrimental to them due to the high levels of hydroxyl generation in the presence of H2O2. On the contrary, the free iron containing normal-sized/long cells do not acquire iron and hence the hydroxyl radical produced in the population becomes stoichiometrically beneficial to them. Thus, the deficiency of free iron which consequentially necessitates the short cells to acquire more iron becomes a maladaptive trait in the presence of H2O2 but gets co-opted in kin selection, for the survival of the normal-sized/long cells that form major proportion of the population – a phenomenon reminiscent of altruism. The Chapter concludes with a model depicting the entire phenomenon and a discussion on the results and their implications.