Immunological and ultrastructural changes in mononuclear cells exposed to Mycobacterium avium serovar-specific glycopeptidolipid (GPL) and the chemically derived R-lipid (lipopeptide fragment) were examined.

The aim of this study was to investigate the diversity and prevalence of non-tuberculous mycobacteria (NTM) in cattle, African buffaloes and their environments in South Africa and the potential of these NTM to elicit cross- reactive immune responses in these animal species which may in turn lead to false diagnosis of bovine tuberculosis. A total of 40 NTM species were identified during a countrywide survey. Mycobacterium terrae, Mycobacterium nonchromogenicum, Mycobacterium vaccae/ Mycobacterium vanbaalenii and a group of isolates closely related to Mycobacterium moriokaense (M. moriokaense-like isolates) were the four most frequently isolated species. Further characterization of M. moriokaense- like isolates revealed two novel NTM species which were named Mycobacterium malmesburii sp.nov. and Mycobacterium komanii sp.nov. respectively. Genomes of M. nonchromogenicum, M. malmesburii sp. nov., M. komanii sp. nov., and M. fortuitum ATCC 6841 were elucidated and investigated for genes encoding homologues of M. bovis predominant immunogenic proteins. These included genes encoding for the Esx family proteins (esx genes), mpb70, mpb63, mpb64, hspX, tpx, Rv1120c, canA and dnaK. The esx gene orthologs encoded in ESX-1 (esxA and esxB), ESX-3 (esxH and esxG), esxR, and ESX-4 (esxT and esxU) loci were identified in the NTM genomes while those encoded in ESX-2 locus were absent in all the four NTM genomes and only esxN (encoded in the ESX-5 locus) and its homologue, esxK were present in M. nonchromogenicum. Gene orthologs encoding for MPB70 (M. malmesburii sp.nov. and M. komanii sp.nov.), DnaK (all four NTM species), CanA (all four NTM species), MPB64 (all four NTM species), Rv1120c (in all four NTM species), TpX, MBP63 and HspX (all in M. nonchromogenicum and M. fortuitum), were found in the NTM genomes. In contrast orthologs of mpb83 and espC were not detected in any of the four NTM. We could not judge just based on the overall protein sequence homologies of the antigens whether the NTM homologues will give rise to cross-reactive immune responses. We consequently checked the existence in NTM of epitopes shown to be immunogenic in M. bovis and M. tuberculosis. Amino acid sequence alignment of the EsxA and EsxB of the NTM sequenced in this study as well as M. smegmatis, M. bovis and M. tuberculosis respectively was done to investigate their similarities at “immunogenic” epitope level. In this analysis, we found that the six bovine T-cell recognized epitopes of M. bovis ESAT-6 described by Vordermeier et al., 2003 and 2007 had similarities to those of M. fortuitum and M. nonchromogenicum (showing sequence similarity of as high as 81.28% and as low as 52.9% ). Likewise a certain degree of sequence similarity between the six M. bovis CFP 10 immunogenic epitopes and those of the NTM species (highest similarity of 75% observed between all NTM and M. bovis and lowest similarity of 50% between M. komanii sp.nov, M. malmesburii sp.nov and M. bovis.) was observed. Still, with sequence homologies of less than 100% between the M. bovis immunogenic epitopes and those of the NTM, it was difficult to unambiguously predict T-cell cross-recognition. Comparison of the EsxR and EsxH amino acid sequences at immunogenic epitope level, revealed higher sequence similarities in the epitopes of NTM and those of M. bovis than the predicted protein sequences of EsxA and EsxB. A sequence similarity of 100% was observed between two of the five M. bovis immunogenic epitopes of EsxR and those of M. fortuitum, M. malmesburii sp. nov. and M. komanii sp.nov. Full cross- recognition of these NTM EsxR epitopes is therefore highly likely, and may lead to misdiagnosis of bovine Tuberculosis (BTB). The other three EsxR/EsxH epitopes shown to be immunogenic in M. bovis also exist in the three NTM showing similarity of as low as 77.7%. / Thesis (PhD)--University of Pretoria, 2015. / WOTRO Science for Global Development / Genomics Research Institute (GRI) / Veterinary Tropical Diseases / PhD / Unrestricted

Le microbiote désigne l'ensemble des microorganismes (bactéries, virus, champignons) vivant dans un environnement spécifique, en particulier chez un hôte (humain, animal ou végétal). La relation symbiotique existant entre le microbiote et son hôte a été mise en évidence dans de nombreux contextes. Le rôle protecteur du microbiote a été démontré chez l'homme, dans diverses pathologies, dont des infections bactériennes. Le microbiote colonise l'ensemble des muqueuses, dont l'intestin, où il est le plus abondant. Bien que le rôle du microbiote intestinal ait été beaucoup décrit, l'existence de bactéries commensales dans les poumons a été mise en évidence plus récemment. D'abord sujette à controverse, l'existence d'un microbiote pulmonaire, dont la composition est distincte de celle de l'intestin, et qui peut être altérée en conditions pathologiques, est maintenant bien établie. Il est également établi que le microbiote d'un organe peut agir sur la physiologie d'autres organes ; ainsi on parle par exemple d'un axe " intestin-poumons " pour désigner l'action de composés solubles produits par le microbiote intestinal, ainsi que de cellules immunitaires ou cytokines de l'intestin, véhiculés par le sang ou la lymphe, sur la physiologie du poumon. Les poumons sont une cible majeure pour la colonisation par des pathogènes. La tuberculose (TB), une inflammation chronique pulmonaire causée par la bactérie Mycobacterium tuberculosis, est encore aujourd'hui la pathologie respiratoire due à un agent étiologique unique la plus meurtrière. A ce jour la complexité des mécanismes mis en jeu pour expliquer la différence de susceptibilité à la TB entre les individus n'est pas encore complètement comprise. Il est proposé que la balance entre virulence de la souche de M. tuberculosis, et statut immunitaire de l'hôte pourrait expliquer l'inégalité entre les individus face au développement de la maladie. Ici nous avons émis l'hypothèse que le microbiote de l'hôte serait un facteur influençant l'interaction hôte-pathogène dans la TB via i) la modulation de l'immunité antituberculeuse et/ou ii) la physiologie (métabolisme, virulence) du pathogène. Mon travail de thèse a permis de montrer que l'élimination du microbiote par un traitement antibiotique à large spectre conduit à une colonisation plus importante des poumons par M.[...] / The microbiota refers to all microorganisms (bacteria, viruses, fungi) living in a specific environment, especially in a host (human, animal or plant). The symbiotic relationship existing between the microbiota and its host has been demonstrated in many contexts. In particular, it is now well-established that the microbiota plays a protective role during different human pathologies, including bacterial infections. The microbiota colonizes all the mucosal membranes of the body; particularly the intestine where it is more abundant. While role of the gut microbiota has already been widely studied, the existence of bacteria in the lungs has been described more recently. Even if at first controversial, the existence of a pulmonary microbiota, whose composition is distinct from that of the intestine, and which can be altered in pathological conditions, is now well established. It is also well-established that the microbiota of an organ can act on the physiology of other organs; for instance, a "gut-lungs" axis is used to designate the action of soluble compounds produced by the intestinal microbiota, as well as immunes cells or cytokines from the gut, carried by the blood or the lymph, on the physiology of the lung. The lungs are one of the major colonization site for different pathogens. Tuberculosis (TB), a chronic pulmonary inflammation caused by the bacteria Mycobacterium tuberculosis, is still today the most lethal respiratory disease due to a single etiological agent. To date, the complexity of the mechanisms explaining the difference in susceptibility to TB between individuals has not been fully understood yet. It has been suggested that the balance between virulence of the strain of M. [...]

Thesis (Ph.D.(Microbiology)) -- University of Limpopo, 2022 / Respiratory tract infections (RTIs) are frequent ailments among humans and are a high burden to public health. One strategy for the development of new therapies against pathogenic bacteria such as Mycobacterium tuberculosis is to target essential biosynthetic pathways of its metabolism. The aim of this study was to evaluate and target the biosynthesis of aromatic amino acids (shikimate pathway) of Mycobacterial spp using medicinal plant extracts. The selection of the plants in this study was based on their ethnopharmacological use for the treatment of tuberculosis infections and related symptoms. The leaves were dried at ambient temperatures and ground to fine powder. The powdered material was extracted with hexane, dichloromethane, acetone, methanol and water. Phytochemical screening was done using standard protocols that tested for tannins, saponins, terpenoids, alkaloids, flavonoids, steroids, anthraquinones, phlobatannins, quinones, and betacynins. Phytochemical fingerprints were established using thin layer chromatography (TLC) where three mobile phases varying in polarity were used to develop the chromatograms. Total Phenolics, flavonoids, flavonols, tannins, alkaloids and proanthocyanidin contents were quantified using UV/Vis spectrometry. Spectrometric quantification of the free radical (DPPH) scavenging activity and ferric (potassium ferricyanide) reducing power were performed. The heat-dependent bovine serum albumin and egg albumin denaturation assays were used to evaluate anti-inflammatory activity. Antimycobacterial activity was screened using bioautography assay in qualitative analysis. Quantitatively, broth microdilution assay was used to determine the minimal inhibitory concentrations. The Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 interference genetic editing technique was used to evaluate and validate the essentiality of the aromatic amino acids in Mycobacteria to further determine the vulnerability and draggability of the transketolase (tkt) and DAHPs (aroG) genes. Plasmid, PLJR962, was used for the CRISPRi/dCas9 gene knockdown experiments. The integrating CRISPRi plasmid expressed both sgRNA with the targeting region (for tkt or aroG) and the dCas9 handle which is under control of the anhydrotetracycline (ATC) inducible promoters. The spot assay and growth curves were used to for phenotypic characterisation and gene knockdown experiments. RNA microarray (qPCR) was used to evaluate the level of expression inhibition of tkt gene . Mechanism of action of plants extracts bioactive components were predicted based on synergy between gene knockdown, shikimate inhibitors and the plant extracts. To evaluate whether the shikimate intermediates may rescue gene depleted M. smegmatis hypomorphs, the cultures were grown in L-tryptophan, L-phenylalanine, L-tyrosine and shikimic acid and growth curves constructed. Cytotoxicity of the extracts was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero cell lines and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages. Phytochemical analysis showed that the various extracts had various polar and non-polar compounds which belonged to phenolics, saponins, steroids, terpenoids, alkaloids, cardiac glycosides and resins. Numerous non-polar compounds from Gardernia volkensii, Senna petersiana, Ficus sur had antimycobacterial activity against M. smegmatis in bioautography. Remarkably, acetone extracts from S. petersiana, Acacia senegal, Carissa bispinosa, P. africanum and C. gratissimus that had moderate to low antimycobacterial activity against wild type M. smegmatis (mc2 155) demonstrated improved inhibitory activity against the tkt PAM1 M. smegmatis CRISPRi mutant. Only the acetone Clerodendrum glabrum, Croton gratissimus, Peltophorum africanum and Gardenia volkensii demonstrated activity against M. tuberculosis H37Rv. These results suggest that the employment of CRISPRi in M. tuberculosis to develop screening models may increase changes of obtaining bioactive chemical species because the tkt gene knockdown was showed to possess the ability to potentiate the antimycobacterial activity of the plant extracts. An added advantage of the plant extracts is their antioxidant and anti-inflammatory activities which may benefit the host immune system during treatment of infection by reducing free radicals and pro-inflammatory agents that perpetuate the infection. Non polar compounds were found to generally have higher anti-inflammatory activity than the polar counterpart for all the plant extracts. These results suggest that the non-polar compounds from the tested extracts may not only confer antimycobacterial effects, but also anti-inflammatory activities. A. senegal, G. volkensii, F. sur, S. petersiana and C. glabrum were found to be toxic to the Vero cell line. However, purification techniques may circumvent their toxic effects. This study demonstrated that the amino acid biosynthesis is a potential antimycobacterial drug target because it was found to be essential, vulnerable and druggable by medicinal plant extracts / University of Limpopo and National Research Foundation (NRF-DAAD In-Country Doctoral Scholarship Programme)

Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.

Clinical genomics/metagenomics is a rapidly developing field and it makes genomic, transcriptomic, and epigenomic evaluations of clinically relevant samples possible due to decreasing sequencing costs and large volumes of sequence datasets. Applications of comprehensive protocols for clinical metagenomic analysis is rapidly moving from the research laboratories to the clinical laboratories in healthcare settings. It has not only improved medical interventions but also continue to contribute to precision treatments. In this dissertation, I am going to discuss (i) the applications of clinical genomics/metagenomics protocols in several clinical cases of infections and (ii) the application of comparative genomics in Mycobacterium riyadhense clinical isolates which provide insights into ancestry and adaptive evolution in MTBC (Mycobacterium tuberculosis complex); The research questions are systematically addressed in four chapters. Briefly, Chapter 1 provides a brief introduction of conventional clinical microbiology and clinical metagenomics and a research summary of the thesis; Chapter 2 presents an analysis of Mycobacterium riyadhense from an evolutionary genomics point of view, followed by functional genomics experiments to look for clues obtained from the comparative genome analysis of M. riyadhense and other mycobacteria including members of the MTBC. The third chapter describes an imported case of Mycobacterium leprae found in Riyadh, Saudi Arabia, revealed by metagenomic sequencing and bioinformatic analysis, which was challenging for the clinicians to treat due to lack of timely diagnosis and occurrence of drug resistance during the course of treatment. The fourth chapter describes how applications of NGS facilitate rapid pathogen discovery in an imported case of Naegleria fowleri from the Cerebrospinal fluid (CSF) of a resident in KSA with recent travel history to Pakistan. The fifth chapter presents how we have monitored an ongoing outbreak by drug-resistant strains of the human pathogenic yeast Candida auris in Saudi Arabia from King Faisal Medical City, Riyadh, Saudi Arabia. In this thesis, I have shown several applications of NGS and bioinformatic analysis protocols in the clinical genomics/metagenomics fields. I believe some of the main clinical applications of NGS will lead to the adoption of these methodologies in clinical settings in Saudi Arabia in the forthcoming future.

No / With the general increase in the worldwide incidence of tuberculosis there is increasing interest in the use of upper room ultraviolet germicidal irradiation (UVGI) systems to disinfect air. A number of researchers have demonstrated experimentally the ability of such systems to inactivate airborne microorganisms. However, relatively little theoretical work has been done to explain the results observed and few models exist to describe the performance of upper room UVGI systems. This paper presents a new model, which can be used both to design such systems and to evaluate their germicidal effectiveness. A theoretical study is undertaken, which indicates that although upper room UVGI systems work well at lower ventilation rates, they are of limited benefit in highly ventilated applications. The paper also demonstrates and quantifies the relationship between inter-zonal air velocity and room ventilation rate. In particular, the paper shows that under steady-state conditions the number of passes made by bioaerosol particles through an upper room UV field is independent of the ventilation rate.

Bacteria of the <i>Mycobacterium avium, M. intracellulare, and M. scrofulaceum</i> group (MAIS) are opportunistic human pathogens and widespread in the environment. The first objective of this study was to demonstrate that plasmids from clinical isolates are closely related to plasmids from environmental MAIS isolates. A 12.9 kb plasmid, pVT2, from a clinical <i>M. avium isolate</i>, MDl, was cloned and used a a DNA probe to examine the relationship of MAIS plasmids. The pVT2 probe hybridized with plasmids isolated from MAIS strains from the environment, from patients without AIDS with pulmonary infections, and from AIDS patients with disseminated MAIS infections. Similar results were seen with a second probe derived from pLR 7, a 15.3 kb plasmid from clinical <i>M. intracellulare</i> strain LR 113. The similarity of plasmids from environmental and clinical isolates supports the hypothesis that the environment is a source of MAIS infection in humans. / Ph. D.

Mycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and detoxification includes various promising drug targets. Several metalloenzymes are involved in this pathway, such as a deacetylase (MshB) and mycothiol S-conjugate amidase (MCA). MshB catalyzes the deacetylation of GlcNAc-Ins to form GlcN-Ins and acetate. Mycothiol S-conjugate amidase (MCA) cleaves the amide bond of mycothiol S-conjugates of various drugs and toxins. The identification of the native co-factor is critical for the design of potent and effective inhibitors. Therefore, in this study, we identified the possible native co-factors of MshB and MCA from M. smegmatis and M. tuberculosis. To reach our aim, we used a pull-down method to rapidly purify halo-MshB and halo-MCA under anaerobic conditions. Our data indicates that the metal bound to MshB and MCA anaerobically purified from E. coli grown in minimal medium is mainly Fe(II), while proteins purified under aerobic conditions contain bound Zn (II) and Fe(II) that varies with the metal content of the medium. For a further clarification of the metal ion preferences of MshB and MCA, we determined the MshB and MCA affinity for Zn(II) to be in the picomolar range and Ms MshB affinity for Fe(II) in nanomolar range. These results indicate that MshB and MCA can be found bound with either iron or zinc and this is independent to their affinities for these metal ions. / Master of Science

Yes / The geographically restricted Mycobacterium africanum lineages (MAF) are primarily found in West Africa, where they account for a significant proportion of tuberculosis. Despite this phenomenon, little is known about the co-evolution of these ancient lineages with West Africans. MAF and M. tuberculosis sensu stricto lineages (MTB) differ in their clinical, in vitro and in vivo characteristics for reasons not fully understood. Therefore, we compared genomes of 289 MAF and 205 MTB clinical isolates from the 6 main human-adapted M. tuberculosis complex lineages, for mutations in their Electron Transport Chain and Central Carbon Metabolic pathway in order to explain these metabolic differences. Furthermore, we determined, in silico, whether each mutation could affect the function of genes encoding enzymes in these pathways. We found more mutations with the potential to affect enzymes in these pathways in MAF lineages compared to MTB lineages. We also found that similar mutations occurred in these pathways between MAF and some MTB lineages. Generally, our findings show further differences between MAF and MTB lineages that may have contributed to the MAF clinical and growth phenotype and indicate potential adaptation of MAF lineages to a distinct ecological niche, which we suggest includes areas characterized by low oxygen tension. / European Research CouncilINTERRUPTB starting grant nr. 311725 (to BdJ, FG, CM, LR, BO, MA) and The UK Medical Research Council and the European & Developing Countries Clinical Trials Partnership (EDCTP) Grant No. CB. 2007. 41700.007. / Research Development Fund Publication Prize Award winner, January 2020.