Human Immunodeficiency Virus (HIV) infection is characterized by immune dysfunction that predisposes infected individuals to opportunistic infections such as Mycobacterium Tuberculosis (MTB). The result of this is an exacerbation of HIV-TB related deaths annually. Therefore there is an imperative need for HIV-TB focused research that aims to identify immunological factors that are involved in the control of MTB and HIV in both mono- and co-infected individuals. The CD161+ CD4+ T cell subset is linked to a distinct phenotypic and functional profile. Importantly, these CD161+ T cells may act as an important component of immunological defense and provide protection in infected tissues. CD161+ CD4+ T cells have also been identified as the precursor population of Th17 cells and it has been previously reported that reduction of CD161+ CD4+ T cells during HIV infection may limit Th17 reconstitution (Prendergast et al., 2010). This may ultimately contribute to impairment of mucosal immunity leading to the acquisition of opportunistic infections such as MTB and disease progression in HIV infected individuals. Our study aimed to comprehensively characterise the impact of HIV and MTB infection on the CD161+ CD4+ T cell subset and to assess the frequency, phenotype and function of these cells. The study also aimed to correlate the longitudinal variation in frequency, phenotype and function with markers of HIV disease progression. Methods The frequency, phenotype and function of the CD161+ CD4+ T cell subset was measured by flow cytometry. For the frequency and phenotypic assessment, whole blood was collected from HIV negative and HIV/MTB mono and co-infected subjects (n = 17 per patient group). Whole blood was surface stained with antibodies specific to CD3, CD4, CD8, CD161 and chemokine receptors CD103, CCR6, CXCR4, CCR5 and CXCR6. The percentage positive expression of CD161 on CD4+ T cells and chemokine receptor expression was measured. The functional assessment of CD161+ CD4+ T cells involved PBMC stimulation with antigenic stimulant, phorbol 12-myristate 13-acetate (PMA) and ionomycin or ESAT-6/CFP-10, GAG, TB10.4 and Ag85a followed by intracellular cytokine staining for IFN-γ, IL-17A, IL-22 and TNF-α. A subgroup of HIV negative (frequency and phenotype, n = 10, function n = 7) and HIV mono-infected subjects (frequency and phenotype, n = 10, function n = 7) were longitudinally followed to assess variations in the frequency, phenotype and function of CD161+ CD4+ T cells over time. Results The CD161+ CD4+ T cell subset demonstrated high-level expression of chemokine receptors CCR5, CCR6, CXCR4 and low-level expression of CD103 and CXCR6. The subset also demonstrated the ability to produce cytokines IFN-γ, IL17A, IL-22 and TNF-α in healthy subjects. Analysis of HIV infected samples revealed a significant reduction in the frequency of the CD161+ CD4+ subset (median = 06.86%, p < 0.0001) compared to that of healthy individuals (median = 14.75%). Correlation of the subset frequency to markers of disease progression revealed a positive trend to CD4 count (r = 0.2590, p = 0.0787) and a significant negative correlation to viral load (r = -0.3522, p = 0.0152). Unlike with HIV infection, no significant changes in CD161+ CD4+ T cell frequency was observed in individuals with LTBI (mono- or HIV co-infected) or active TB disease compared to that of the healthy patient group. However, the exception to this was HIV infected individuals with active TB disease (co-infected) (median = 03.80%, p < 0.0001). Decreased CCR6 expression on CD161+ CD4+ T cells was observed in HIV monoinfected (p = 0.0065) and HIV infected individuals with active TB disease (p = 0.007). No functional changes were observed in both the HIV and MTB mono- and co-infected cohorts following non-specific stimulation. An interesting positive trend in correlation between IFN-γ production and CD4 count (r = 0.2727, p = 0.0733) was demonstrated with a significant negative correlation between IFN-γ production and viral load observed following non-specific antigenic stimulation (r = -0.3705, p = 0.0133). CD161+ CD4+ T cells demonstrated antigen-specific T cell responses to peptides ESAT-6/CFP-10, TB10.4, Ag85a and GAG in a small proportion of 69 study participants with variable ranges in magnitude of the responses observed. The longitudinal assessment of CD161+ CD4+ T cell frequency and phenotype demonstrated low-level proportion of CD4+ T cells expressing CD161 and CCR6 expression longitudinally maintained in HIV mono-infected compared to that of healthy individuals. Conclusion The phenotypic and functional profile of the CD161+ CD4+ T cell population indicates that it may be an important component of immunological defense that may provide mucosal defense and protection at epithelial sites and tissues e.g. expression of tissue homing markers like CCR6 and the production of cytokines such as IL-17A and IL-22 (important in mucosal immunity). HIV infection is associated with a reduced frequency of CD161+ CD4+ T cells. The correlation between CD161+ CD4+ T cell frequency and markers of disease progression suggests that the observed low-level frequency in HIV infected individuals may in part be a result of non-specific HIV-mediated depletion of CD4+ T cells. However, lower levels of CD161+ CD4+ T cells in HIV infected individuals could also be a result of naturally lower levels being present in individuals prior to infection, thereby making these individuals more susceptible to HIV infection. The significantly reduced levels of CCR6 expression on CD161+ CD4+ T cells in HIV monoinfected individuals may also be an indication of cell subset migration to gut associated lymphoid tissue (GALT, target site of HIV replication) during HIV infection. Given their potential role in mediating signals that are essential for immune responses to microbes and microbial products, migration of CCR6+ CD161+ CD4+ T cells to target sites of HIV infection could serve as a protective measure in the fight against HIV infection. Although there were no observable changes in the functional capacity of the CD161+ CD4+ T subset in HIV infection, we believe that the reduction in frequency may contribute to HIV disease progression and susceptibility to opportunistic infections such as MTB or active TB disease. Unlike with HIV infection, infection with MTB appeared to have no significant impact on CD161+ CD4+ T cells as there were no observable differences in frequency or the functional capacity of the cell subset following PMA stimulation. However, MTB and HIV antigen-specific responses were observed in a small proportion of the total 69 subjects tested. This therefore indicates that a subset of CD161+ CD4+ T cells may act in an HIV and MTB-specific manner. Additional MTB and HIV-specific responses may be present in this CD161+ CD4+ population and may only be identified through stimulation with additional antigenic targets. Further investigation of CD161+ CD4+ T cells should be performed at the actual sites of infection to investigate if CD161+ CD4+ T cells are concentrated at sites of disease. Also it may be important to investigate the polyfunctionality of CD161+ CD4+ T cells to understand the multifunctional capacity of the cell subset in providing immunological defense to pathogens such as HIV and MTB. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

<p>Many studies have reported low cure rates for multidrug-resistant tuberculosis (MDRTB) patients and MDR-TB patients co-infected with human immunodeficiency virus (HIV). However, little is&nbsp / known about the effect of HIV infection and antiretroviral therapy on the treatment outcomes of MDR-TB in South Africa. Therefore, the objectives of the study are: to find out whether HIV infection&nbsp / and interactions between ARVs and second line anti-TB drugs have an impact on the following MDR-TB treatment outcomes: cure rate and treatment failure at Brewelskloof Hospital. MDR-TB&nbsp / patients were treated for 18-24 months. The study was designed as a case-control retrospective study comparing MDR-TB treatment outcomes between HIV positive (cases) and HIV negative&nbsp / patients (controls). Patients were included in the study only if they complied with the following criteria: sensitivity to second line anti-TB drugs, MDR-TB infection, co-infection with HIV (for some&nbsp / of them), male and female patients, completion of treatment between 1 January 2006 and 31 December 2008. Any patients that presented with extreme drug-resistant tuberculosis (XDR-TB)&nbsp / were excluded from the study. Data were retrospectively collected from each patient&rsquo / s medical records. There were a total of 336 patients of which 242 (72%) were MDR-TB patients and 94&nbsp / (27.9%) MDRTB co-infected with HIV patients. Out of the 242 MDR-TB patients, 167 (69.2%) were males and 75 (30.7%) were females. Of the 94 patients with MDR-TB co-infected with HIV, 51&nbsp / (54.2%) males and 43 (45.7%) females. Patients with multidrug-resistant tuberculosis co-infected with HIV who qualify for antiretroviral therapy were treated with stavudine, lamivudine and&nbsp / efavirenz while all MDR-TB patients were given kanamycin, ethionamide, ofloxacin, cycloserine and pyrazinamide. The cure rate of MDR-TB in HIV (+) patients and in HIV (-) patients is 34.5%&nbsp / and 30 % respectively. There is no significant difference between both artes (pvalue = 0.80). The MDR-TB cure rate in HIV (+) patients taking antiretroviral drugs and in HIV (+) patients without&nbsp / antiretroviral therapy is 35% and 33% respectively. The difference between both rates is not statistically significant. The study shows that 65 (28.0%) patients completed MDR-TB treatment but&nbsp / could not be classified as cured or failure, 29 (12.5%) patients failed, 76 (32.7%) defaulted, 18 (7.7%) were transferred out and 44 (18.9%) died. As far as treatment completed and defaulted is concerned,&nbsp / there is no significant statistical difference between HIV (+) and HIV (-) The number of patients who failed the MDR-TB treatment and who were transferred out is significantly higher in the HIV (-)&nbsp / group than in the HIV (+) group. Finally the number of MDR-TB patients who died is significantly higher in the HIV (+) group). The median (range) duration of antiretroviral therapy before starting&nbsp / anti-tuberculosis drugs is 10.5 (1-60) months. According to this study results, the MDR-TB treatment cure rate at Brewelkloof hospital is similar to the cure rate at the national level. The study also&nbsp / hows that HIV infection and antiretroviral drugs do not influence any influence on MDR-TB treatment outcomes.</p>

Tuberculosis continues to be a major threat in public health; 8.8 million incidence of TB has been reported and 2 million deaths every year. Diagnosis of TB is impeded by slow growth of an organism with a generation time of 21 days. The emergence of multidrug-resistant TB isolates which are resistant to rifampicin and isoniazid worsened the treatment programme. Furthermore, surfacing of extensively drug-resistant TB isolates especially in HIV positive patients has led to a treatment failure. Currently available diagnostic methods are time consuming and laborious. Polymerase chain reaction-based assay proved to have a better resolution for TB strain discrimination, nevertheless are costly and cannot be routinely employed in resource poor settings. As a result there is an urgent need of cheap, cost effective and rapid diagnostic methods that will reduce a turnaround time. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry potentially offers an alternative rapid and cheaper method for discrimination of TB isolates. Proper discrimination of TB isolates depends on the sample preparation method that is capable of yielding high protein content. Conventional formic/ethanol sample preparation was investigated for mycobacteria MALDI-TOF mass spectrometric analysis. Poor quality of spectra was obtained due to a complex cell wall structure of mycobacteria which released less amounts of proteins. Further attempts were made to optimize the sample preparation method by introducing glass beads for maximum cell wall disruption. Non-consistent spectra were obtained in some mycobacterial strain; therefore it was not used as a method of choice. Introduction of delipidation step using chloroform/methanol (1:1, v/v) before formic/ethanol sample preparation step, led to a generation of reproducible and consistent spectra. This newly developed method was selected to extract protein content from large number of clinical TB isolates. With MALDI-TOF MS and chloroform/methanol-based method, all mycobacterial isolates used in the proof-of-concept were correctly identified and clustered. Fifty six of sixty clinical TB isolates were correctly identified using Biotyper software. Four were incorrectly identified; it might be possible that they carry mutations in unknown regions in their genome which led to a translation of proteins that affected the overall spectra profile. MALDI-TOF MS showed the potential to be used in the clinical laboratories for discrimination of TB isolates at lower costs. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder characterized by proliferation of cells expressing BCR-ABL fusion protein. In the BCR-ABL+ leukemia murine model, 12B1, we explored the therapeutic applicability of chaperone-rich cell lysate (CRCL) in the context of syngeneic hematopoietic cell transplantation (HCT) to treat pre-existing leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL immunized donors compared to animals receiving HCT from non-immunized donors. CRCL immunization post-immune HCT further hindered tumor growth when compared to immune HCT without post-transplant vaccination. The magnitude of the immune response was consistent with the anti-tumor effects observed in vivo. We also demonstrated that cured mice had developed long-term tumor specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and NK cells contributed to the anti-tumor effect of CRCL vaccination as depletion of either subset hampered tumor growth delay. Thus, our results suggest that CRCL represents a promising vaccine capable of generating specific immune responses. This anti-tumor immunity can be effectively transferred to a host via HCT and further enhanced post-HCT with additional tumor CRCL immunizations.CD4+CD25+ regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4+ or CD8+ T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4+CD25+ FoxP3+ regulatory T cells may modulate immunity against human tuberculosis (TB). Ourresults indicate that the number of CD4+CD25+FoxP3+ Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4+CD25+FoxP3+ Treg in pleural fluid inversely correlates with local MTB-specific immunity(p<0.002). These CD4+CD25+FoxP3+ T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-γ and IL-10 production in TB patients. Therefore, CD4+CD25+FoxP3+ Treg expanded in TB patients suppress Mycobacterium tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.

Conventional drug susceptibility testing techniques, the ‘gold standard’ for M. tuberculosis are slow, requiring about 3-6 weeks from a positive culture. This diagnostic delay, before initiation of appropriate treatment, contributes to increased transmission rates. Molecular techniques provide rapid results and therefore present an alternative to conventional tests. The aim of this project was to develop an inhouse reverse line blot hybridization assay (RIFO assay) that could detect mutations associated with Rifampicin resistance directly in clinical specimens of patients in KwaZulu Natal. A 437 bp region of the rpoB gene was sequenced to ascertain the most frequently occurring mutations conferring resistance to rifampicin in isolates in KwaZulu-Natal. Wildtype and mutant probes designed to target these mutations, were immobilized on a Biodyne C membrane. Hybridization conditions were optimized using biotin labeled PCR products from culture. Detection was performed with peroxidase labeled streptavidin using enhanced chemiluminescence. Four DNA extraction methods were evaluated on sputum specimens to determine the one with the least inhibitory effect on amplification. A total of 11 mutations were found in 236 clinical isolates: 531TTG (109, 58.3%), 516GTC (26, 13%), 533CCG/516GGC (20, 10%), 533CCG (18, 9.6%), other mutations < 5% each. The chelex extraction method was found to be optimal for removing inhibitors in sputum specimens. Sputum specimens of 404 patients hospitalized at King George V Hospital between 2005 and 2006 were rifoligotyped. The RIFO assay was optimised on clinical isolates and then applied to sputum specimens. The RIFO assay on culture and sputum correlated well with the DST (sensitivity 92% and 94% respectively). However, the specificity was very low in both culture and sputum specimens compared to DST (38% and 35% respectively). This could be attributed to the presence of silent mutations, mixed infections, mixed populations of bacteria or the small number of susceptible strains used in this study. The in-house RIFO assay can be used directly on sputum specimens to predict Rifampicin resistance and therefore MDR-TB in less than a week compared to the gold standards. A total of 43 samples can be tested simultaneously at low cost and the membrane is reusable compared to commercial kits such as the Hains test that is expensive and strips are not reusable. A similar assay can be designed to target mutations for the detection of XDR-TB. Future studies should be conducted in a clinical setting on patients with sensitive strains to increase the specificity. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

Mycobacterium tuberculosis (MTB) is an airborne pathogen that is easily transmitted from person to person. An intact immune system prevents the organism from causing disease in most individuals. In South Africa, the prevalence of human immunodeficiency virus (HIV) has reached astronomical levels and is now fuelling the tuberculosis (TB) epidemic. Drug resistant MTB strains combined with a weakened host immune system is a lethal combination. Multi-drug resistant (MDR) including extensively drug resistant (XDR) tuberculosis is on the increase, with Tugela Ferry in KwaZulu-Natal South Africa, reporting the largest cluster of XDR cases in the world. It is unknown whether a single clone of the drug resistant strain is circulating in this area or whether there are multiple strains at play. Using 2 complementary genotyping methods, we showed that the MDR strains present are the result of clonal spread associated with the F28 family, as well as de novo resistance which manifests as unique patterns. The XDR epidemic in Tugela Ferry is the result of clonal spread of a strain belonging to the F15/LAM4/KZN family. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

Le séquençage intégral du génome de Mycobacterium tuberculosis a permis de mettre en évidence l'existence de onze Sérine/Thréonine Protéine-Kinases (STPKs) chez cette bactérie. Bien que la quasi-totalité des STPKs aient été biochimiquement caractérisées, très peu de substrats endogènes ont pu être identifiés. Par conséquent, le rôle physiologique de ces couples kinase/substrat reste à élucider. Tout d'abord, les études réalisées au cours de ce travail ont concerné la caractérisation biochimique de la protéine-kinase PknL, ainsi que l'identification de ses substrats potentiels, et notamment la protéine Rv2175c. En effet, l'analyse de l'environnement génétique du gène pknL de la kinase a révélé la présence du gène adjacent rv2175c, pouvant ainsi représenter un substrat éventuel de PknL. Les différentes approches mises en oeuvre ont permis d'identifier cinq sites de phosphorylation sur PknL, et de mettre en évidence le caractère essentiel des résidus K48, T173 et T175 dans les mécanismes d'autophosphorylation de PknL et de phosphorylation de Rv2175c, confirmant ainsi Rv2175c comme substrat spécifique de PknL. Par ailleurs, la caractérisation par RMN de la structure de Rv2175c a permis de déterminer la fonction de cette protéine. Rv2175c possède toutes les caractéristiques structurales d'une protéine capable de fixer l'ADN. Des études fonctionnelles ont permis de confirmer la capacité de Rv2175c de fixer l'ADN et ont mis en évidence le mécanisme de régulation via phosphorylation régissant son activité de fixation. Ensuite, nous avons mis en évidence la phosphorylation des protéines chaperonnes mycobactériennes et, plus particulièrement, caractérisé GroEL1. Nous avons démontré que GroEL1 était phosphorylée par PknF, et identifié les résidus T25 et T54 comme étant les sites de phosphorylation de GroEL1. L'ensemble de cette étude nous a donc permis de caractériser de nouveaux substrats de phosphorylation chez M. tuberculosis, de mieux appréhender les interactions kinase/substrat et d'impliquer la phosphorylation dans la régulation de l'activité de ces substrats

Tuberculosis (TB) is the fifth largest cause of death in South Africa, with one in ten cases being resistant to treatment due to the development of multidrug-resistance and extensively drug-resistance in the agent responsible for this disease, Mycobacterium tuberculosis. This pathogen has developed mechanisms to evade killing by immune cells such as macrophages. Mycobacterium smegmatis, a non-pathogen, that does not evade killing by the macrophage, is often used to gain a better insight into the bacteriocidal pathways used to kill mycobacteria, and those potentially blocked by M.tuberculosis. In such studies nitric oxide and “lysosomal” proteases have emerged as major bacteriocidal pathways. Studies on the role of aspartic protease, cathepsin D, in killing green fluorescent protein- (GFP-) tagged-M.smegmatis in J774 macrophages required antibodies that would not cross-react with mycobacterial antigens. These were raised in chickens, using alum and saponin as adjuvants, and porcine and human cathepsin D. Using such antibodies, quantitative colocalization analysis using ImageJ and the JACoP colocalization plugins showed a greater degree of colocalization between cathepsin D and LysoTracker Red DND-99 in M.smegmatis-infected J774 macrophages than in uninfected cells. This indicates the possible presence of active, bacteriocidal cathepsin D in acidic, and hence matured phagosomes. A higher colocalization between cathepsin D and LAMP-1 and cathepsin D and LAMP-2 in uninfected cells possibly indicates the recycling of these two markers from vesicles not containing killed bacteria. Propidium iodide (PI) labelling and loss of GFP fluorescence appeared reliable indicators of M.smegmatis death or viability, respectively, as myobacteria that took up PI also lost green fluorescence, while M.smegmatis that exhibited green fluorescence (viable) were not observed to take up propidium iodide (dead). Faint colocalization between cathepsin D, LAMP-1 and -2 with dead, and to a lesser extent with live M.smegmatis occurred. Besides intensity correlation values other colocalization programs indicate the absence of colocalization between these markers and dead M.smegmatis, but, together with in vitro killing experiments (cathepsin D, 0.0098 units/ml resulting in 59% killing in 4 h) these appear to indicate a possible role of cathepsin D in killing of M.smegmatis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria. The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosynthetic pathways in M. tuberculosis for drug development. The methylerythritol phosphate (MEP) pathway synthesizes a group of compounds called isoprenoids. These compounds have essential roles in all living organisms. The fact that humans utilize a different pathway for isoprenoid synthesis makes the MEP pathway enzymes attractive targets for drug development. We have determined the structures of two essential enzymes from this pathway by X-ray crystallography: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). These are the first structures of these enzymes from M. tuberculosis. Additionally, structures of the IspD enzyme from the related bacteria Mycobacterium smegmatis were determined. We have characterized these enzymes and evaluated the efficiency of a number of inhibitors of the DXR enzyme by biochemical methods. Crystal structures of DXR in complex with some of these inhibitors were also determined. The second pathway of interest for drug development is the universal pathway for Coenzyme A biosynthesis. Enzymes in this pathway have essential roles in all living organisms. However, the bacterial enzymes have little similarity to the human homologues. We have determined a number of structures of the M. tuberculosis pantothenate kinase (PanK), the regulatory enzyme of this pathway, in complex with two new classes of inhibitory compounds, and evaluated these by biochemical methods. The structures and biochemical characterization of these enzymes provide us with detailed information about their functions and broadens our knowledge of these bacteria. Biochemical and structural information about new inhibitors of these enzymes serve as a starting point for future development of antibiotics against tuberculosis.

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship