Streptococcus pneumoniae (the pneumococcus) is the most common cause of childhood pneumonia. Nasopharyngeal (NP) colonization by the pneumococcus is a necessary first step in the pathogenesis of pneumonia and yet the dynamic nature of pneumococcal colonization remains incompletely understood. In children, asymptomatic colonization of the nasopharynx by the pneumococcus is common and also serves as a reservoir for person-to-person transmission. We aimed to investigate in detail, the dynamics of pneumococcal nasopharyngeal carriage over the first year of life, in a cohort of South African children, particularly after implementation of the 13-valent pneumococcal conjugate vaccine (PCV-13). The study will further elucidate the interaction of S. pneumoniae with other respiratory pathogens and how such interactions may contribute development of severe disease.

Human breast milk is a complex species-specific biological fluid universally known as the optimal postnatal source of nutrition for infants and therefore, recommended by the World Health Organization as the exclusive food for infants in the first six months of life. Despite the importance of human breast milk in infant health, study of its composition, especially the bacteriome (bacterial communities) and metabolome (complete set of metabolites), and their relationship to infant health and growth have not yet been comprehensively characterized. This is particularly true in low- and middle-income countries. We therefore conducted a cross-sectional study, nested within an existing birth cohort, the Drakenstein Child Health Study, to describe the bacteriome and metabolome of human breast milk samples collected between 6-10 weeks postpartum from lactating women living in South Africa. The determinants of these components of human breast milk and their role in infant growth were also investigated. Four commercial DNA extraction kits were compared for DNA extraction from human breast milk samples. The kit showing the best results, including quality and quantity of DNA, as well as best reproducibility, was chosen for further extractions. Using 16S rRNA gene amplicon next generation sequencing, a reproducible bioinformatics sequencing pipeline, and robust multivariate statistical analysis, we confirmed the presence of a diverse bacterial community in human breast milk and identified a core bacteriome, present in 80% of the samples. The bacteriome was shown to cluster into three different profile groups (biotypes) according to the predominant bacterial genus present. Bacterial interactions were suggested by the finding of positive correlations between the relative abundances of bacteria usually found in the oral or skin microbiota. Apart from study site (a proxy for ethnicity in this study), infant birth length and maternal age, no other associations were found between potential sociodemographic and psychosocial determinants and the composition of the human breast milk bacteriome. Using Nuclear Magnetic Resonance spectroscopy, we quantified forty-nine metabolites in all human breast milk samples. A subset of women with low levels of lactose concentrations were identified. Low lactose was associated with an increase in metabolites associated with mixed acid fermentation and microbial dysbiosis (staphylococcal-predominant biotype). Low-lactose (vs normal lactose) human breast milk correlated with a reduced median duration of exclusive breastfeeding and reduced infant growth (reduced weight and length z-scores) during the period of exclusive breastfeeding. These results suggest that bacterial fermentation of lactose results in low-lactose breast milk, which in turn impacts on breastfeeding outcome. Taken together, the results presented in this thesis provide a better understanding of human breast milk composition among lactating mothers living in South Africa, their potential determinants and their role in infant growth. Knowledge about the composition of human breast milk may provide opportunity for diagnostic and therapeutic interventions and help promote (exclusive) breastfeeding for the recommended period to improve infant health.

Includes abstract. / Includes bibliographical references. / The genome structure of Mycobacterium tuberculosis is strongly clonal, in the absence of horizontal gene transfer. Thus it is feasible that clonal lineages may exhibit particular phenotypic characteristics, which may, in turn, result in differences in virulence or influence their association with particular host populations. Indeed, the global distribution of M. tuberculosis strains is not uniform and certain strain lineages predominate in particular geographical areas. Further, there is evidence that some strain lineages are emerging, suggesting differences in virulence. Firstly, we investigated the association between strain genotype of M. tuberculosis and in vitro correlates of virulence such as growth phenotype and cytokine induction in the monocyte-derived macrophage (MDM) model.

Studies on Mycobacterium tuberculosis (MTB) antigens are important to improve immunodiagnostics and vaccine efficacy. A novel genome based strategy for antigen discovery is to relate what is highly expressed by bacilli in vivo or in vitro, to what is recognized by human T cells as antigens. As hypoxia is a relevant stimulus that MTB encounters in vivo, whole genome based transcriptional profiles of M. tuberculosis subject to prolonged hypoxia (described as the Enduring hypoxic response (EHR) were analyzed, to guide the discovery of novel potential anitgens, by a combined bioinformatic and empirical approach and to determine evidence of infection stage specific recognition.

Includes bibliographical references (leaves 120-133). / Ergothioneine and mycothiol are the two major low molecular weight thiols present in mycobacteria. The generation of mycothiol-deficient mutants has demonstrated its role in protecting M tuberculosis against oxidative and nitrosative stress. To date, no ergothioneine-deficient mutants have been identified and the role of ergothioneine in mycobacteria remains unknown. The work in this thesis was performed with the aim of better understanding the function of ergothioneine in mycobacteria, by studying its biosynthesis and the conditions affecting its production.

The intrinsic resistance of Mycobacterium tuberculosis ( Mtb ) to antibiotics is generally attributed to multiple factors, most significantly the low permeability of the mycobacterial cell wall, the operation of various drug inactivating systems, and the activity of efflux pumps. This study aimed to investigate the role of various components of the "intrinsic resistome" that limit the efficacy of antitubercular agents. The DNA damage response: the SOS response was hypothesized to play a role in antibiotic- mediated cellular death, and that disabling the mycobacterial SOS response, by generating non-cleavable LexA mutants (lexA Ind-), could be used as a tool to validate antibiotic-mediated cell death. To this end, the M. smegmatis (Msm) cleavable LexA was shown to be essential for induced mutagenesis and damage tolerance and that an intact DNA damage repair system is required to respond to antibiotic-mediated DNA damage. In contrast, Mtb cleavable LexA was required for induced mutagenesis but not necessarily damage sensitivity. In addition, the Mtb SOS response does not contribute significantly to remediation of antibiotic-mediated DNA damage. Collectively, these data suggest that DNA repair mechanisms differ between the mycobacterial species and despite effectively inactivating the LexA-dependent DNA repair mechanism(s) in Msm and Mtb, these organisms are able to circumvent this pathway and successfully remediate damaged DNA sustained under various conditions. Furthermore, Mtb auto-bioluminescent reporter strains were generated by introducing the lux operon downstream of the recA or radA promoters. Analysis of a panel of antimicrobials against these strains allowed for the identification of true DNA-damaging agents and the evaluation of the kinetics of the DNA-damage response, in a concentration- and time-dependent manner. Efflux-mediated drug resistance: This study aimed to evaluate the interactions between pairwise combinations of selected antimicrobials and efflux pump inhibitors (EPIs), in vitro and ex vivo, and to identify a novel verapamil (VER)-analogue with improved efficacy against Mtb. Subsequently, a candidate EPI was identified, with equivalent in vitro synergistic effects to VER when used in combination with various antibiotics but with reduced cytotoxic effects, ex vivo, when compared to VER. Mycothiol-mediated protection : It was hypothesized that undetectable levels of mycothiol ( MSH ) in Mtb would potentiate the use of current antibiotics. To investigate the contribution of the cellular antioxidant, MSH, to the mitigation of antimicrobial efficacy, this study aimed to disrupt MSH production by conditionally knocking-down expression of the essential gene, mshC. The mshC knock-down mutants (in all configurations) were not anhydrotetracycline (ATC)-regulatable in liquid or on solid medium, which was subsequently validate d with quantitative gene expression analysis. These data suggest that a tetracycline (Tet)-based conditional expression system may not be applicable to mshC. In conclusion, Mtb has a multitude of inherent mechanisms to subvert the effects of antimicrobial treatment. This study has contributed to the understanding of certain aspects of the intrinsic resistome and in doing so, established tools that can be used in future drug discovery programmes.

The mycobacterial DNA damage and repair pathways involved in the emergence of drug-resistance during host infection remain poorly understood, yet are critical to any efforts to develop novel "anti-evolution" drugs aimed at reducing the capacity of Mycobacterium tuberculosis to adapt genetically during tuberculosis (TB) treatment. The thesis presented here aimed to investigate the contribution of the DNA damage (SOS) response in adaptive mutagenesis, and focused on two specific components: the role of the specialist translesion synthesis DNA polymerase, DnaE2, in mutagenesis under stress and, secondly, the function of the mycobacterial homologue of a putative SOS response associated peptidase (SRAP) protein which has been identified in comparative genomics analyses of organisms possessing a DnaE2-type C family DNA polymerase. This work focused on the putative SRAP protein which was predicted to form part of the mycobacterial DNA damage response as a functional switch by binding DNA in an autoproteolytic dependent manner. To this end, SRAP deletion mutants were generated for both M. smegmatis (MSMEG_1891) and M. tuberculosis (Rv3226c). Despite the fact that SRAP was upregulated in both M. smegmatis and M. tuberculosis following genotoxic stress, no DNA damage phenotype was detected in any SRAP deletion mutant using a variety of DNA damaging agents. In parallel, an eGFP-tagged M. smegmatis SRAP allele was constructed to enable visualisation of SRAP upregulation and sub-cellular recruitment using fluorescent microscopy; however no eGFP expression could be visualised after MMC treatment. It was not clear whether this was due to faulty eGFP expression in the fusion protein, or to low-level induction of SRAP. In a biochemical approach to elucidate SRAP function, soluble M. smegmatis SRAP protein was expressed and purified using a N-terminal hexa-histidine tag. No proteolytic activity was detected in gelatine or casein zymography, perhaps indicating that SRAP has a very specific substrate. Moreover, while it was predicted that autocatalytic cleavage of the C-terminus was required for activation of SRAP, no such cleavage was detected using hexa-histidine tag staining, possibly pointing to a set of very specific conditions for activation. In combination, therefore, neither microbiological nor biochemical assays could elucidate a definitive role for SRAP in the mycobacterial DNA damage response. DnaE2 has been directly implicated in induced mutagenesis to rifampicin (Rif) resistance in Mycobacterium tuberculosis following exposure of bacilli to genotoxic stress. In previous work in our group, a vitamin B₁₂-sensitive ΔmetH strain was found to form "B₁₂-resistant" suppressor mutants at a frequency higher than could be explained by spontaneous mutagenesis alone. The first part of this thesis investigated the potential role of DnaE2 in the high-frequency emergence of B₁₂-resistance by mutating DnaE2 in the ΔmetH background. Whereas elimination of polymerase function in a DnaE2ᴬᴵᴬ mutant abrogated DNA damage-induced mutagenesis to Rif resistance, no change in B₁₂ sensitivity was detected in a ΔmetH dnaE2ᴬᴵᴬ double mutant. PCR sequencing of spontaneous B₁₂-resistant mutants revealed mutations in genes previously associated with the suppressor phenotype; moreover, there was no apparent difference in the nature of mutations observed in both parental and dnaE2ᴬᴵᴬ mutant strains. Instead, these results suggest that an alternative mechanism must exist to enable adaptive mutagenesis in methionine-starved mycobacteria.

The emergence of antibiotic resistant strains of Mycobacterium tuberculosis, coupled with the time it takes to perform phenotypic drug susceptibility testing of this organism, makes the treatment of tuberculosis increasingly difficult. Several genotypic assays for the rapid detection of drug resistance in M. tuberculosis have been developed, but the sensitivity with which these assays identify resistance differs geographically. Additionally, the identification of phenotypically resistant isolates with no identifiable genotypic marker suggests that other factors, such as differential gene expression, may play a role in the development of drug resistance in M. tuberculosis. This investigation aims to both develop and evaluate rapid genotypic assays for the detection of resistance to both first- and second-line drugs in M. tuberculosis, and to investigate the role of alternative sigma factors in the progression to multidrug resistant M. tuberculosis.

Includes bibliographical references / Of the 35 million people living with HIV-1 globally, approximately 71.4% are in the resource-limited sub-Saharan Africa. The immense sequence diversity of HIV-1, even within subtypes, makes it challenging to develop effective vaccines that target a wide range of HIV subtypes. Mosaic immunogens have been computationally designed to specifically overcome this hurdle by maximizing the inclusion of common T cell epitopes. When compared to consensus immunogens, polyvalent mosaic immunogens of HIV-1 group M have shown increased breadth and depth of antigen-specific T-cell responses. More than 90% of HIV positive individuals in sub-Saharan Africa are infected with HIV-1 subtype C (HIV-1C). We therefore designed, constructed, and evaluated candidate vaccines expressing HIV-1C mosaic Gag (GagM) in a proof of concept study. Gag was chosen as the most appropriate target for a T cell-based vaccine as there are many studies correlating control of HIV viral load with T cell responses to Gag. The immunogen was designed by Fischer et al., 2007 (1). Three different vaccine platforms were chosen based on their different strengths to be used in prime-boost regimens to determine the immunogenicity of HIV-1C GagM in mice. The first was a pantothenic auxotroph of the tuberculosis vaccine Mycobacterium bovis Bacille Calmette Guérin (BCG). The second was a DNA vaccine vector with enhanced expression of transgenes due to a novel enhancer element from porcine circovirus type 1, which has been demonstrated to increase gene expression. The third vaccine vector selected was the well characterised poxvirus modified vaccinia Ankara (MVA).

Bibliography: leaves 103-118.