Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Please see fulltext for abstract. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming.

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2006. / The focus of this study was the pathways for β-alanine production in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The major pathway for β-alanine production is the decarboxylation of L-aspartate by L-aspartate-α-decarboxylase (PanD). This enzyme is not essential for the survival for Mtb which implies that an alternative pathway for β-alanine production must exist. We postulated that such a secondary pathway may be based on the oxidation of various polyamines by a polyamine oxidase to give the β-alanine precursor 3-aminopropanal, and therefore set out to find data in support of this hypothesis. Based on sequence homology to the FAD-dependent Saccharomyces cerevisiae polyamine oxidase Fms1, Mtb AofH was identified as a likely candidate. The soluble expression and purification of AofH proved troublesome and lead to the investigation of various techniques to increase protein yield. These methods include fusion to various tags, coexpression with the protein chaperones, addition of scarce codon tRNA’s to the translation mixture and protein refolding. AofH was eventually purified as fusions to the Nus and MBP proteins and its activity determined by analysis of the enzymatic reactions by TLC, reverse phase HPLC, ESI-MS and LC-MS. TLC analysis indicated that 3- aminopropanol formed as a product during polyamine oxidation, but this could not be confirmed by any of the more sensitive analytical techniques. We set out to confirm the presence of the FAD cofactor in the enzyme by various methods and concluded that the AofH fusions did not contain FAD. Efforts to refold the protein in the presence of FAD also failed. From this study it is clear that the biochemical confirmation of the presumed activity of AofH will remain elusive until the enzyme can be purified in its active form, i.e. with FAD bound. A genetic test for activity based on functional complementation studies of Escherichia coli ΔpanD strains proved inconclusive since no difference in growth rate was found between cell transformed with the aofH gene and the negative control. We continued our studies of β-alanine biosynthesis by attempting the design of mechanism-based inhibitors for the PanD enzyme. Various structural analogues were identified and tested by qualitative and quantitative methods. Our results show that β- substituted aspartate analogues may be good potential inhibitors of Mtb’s PanD protein and can thus be used in rational drug design.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / Dissertation presented for the degree of Doctor of Philosophy at Stellenbosch University. / ENGLISH ABSTRACT: This study describes the molecular epidemiology of Mycobacterium tuberculosis strains with the Beijing genotype. This genotype has received clinical prominence due to its global distribution and the hypothesis that these strains have acquired the ability to evade the protective effect of BCG vaccination, spread more readily, acquire drug resistance and cause severe forms of disease. Molecular biological techniques were used in a series of studies to elucidate the genetic evolutionary mechanisms underlying the success of this genotype in Cape Town, South Africa. Using a collection of 40 different markers it was possible for the first time to construct a phylogenetic history of Beijing genotype strains. This phylogeny was characterized by the consecutive evolution of 7 sublineages. Analysis of epidemiological data in relation to these sublineages showed an association between more recently evolved Beijing strains and an increased ability to transmit and cause disease. From these findings it was hypothesized that the pathogenic characteristics of the Beijing genotype were not conserved but rather that strains representative of the different sublineages had evolved unique properties. In order to determine whether these socalled unique properties were associated with either the host population or the genetic background of strains from sublineage 7, a meta-analysis of published Mycobacterial Interspersed Repetitive-Unit (MIRU) typing data (East Asia) was compared with MIRU typing data from the South African strains in the context of their phylogenetic histories. This study showed that Beijing genotype strains in South Africa originated in East Asia following their introduction during the early 18th century. A significant association was observed between the frequency of occurrence of strains from defined Beijing sublineages and the human population from whom they were cultured (p <0.0001). Based on these findings it was proposed that either the host population (South African) had selected for a particular Beijing sublineage (i.e. sublineage 7) or that strains from that sublineage had adapted to be more successful in the South African population. In a subsequent study, using the methodology developed in the above studies, it was shown that strains from the ancestrally positioned lineage (termed “atypical” Beijing genotype) were over-represented in drug resistant isolates in the Eastern Cape region. This contradicts current dogma which suggests that “atypical” Beijing genotype strains are attenuated in their ability to transmit. However, this phenomenon may be ameliorated in immune-compromised patients as review of the clinical records showed that transmission was associated with HIV co-infection. These findings highlight the need to improve tuberculosis control in vulnerable populations as strains which would normally not contribute significantly to the epidemic now become a cause for concern especially if they are associated with drug resistance. To improve our understanding of the evolution of the Beijing genotype, the genomic stability of an additional 27 polymorphic markers were analysed. These markers have recently been proposed as the new standard in molecular epidemiological studies and were based on MIRU-Variable Number Tandem-Repeats (VNTR) sequences. Superimposition of the MIRU-VNTR data onto the phylogenetic tree showed excellent concordance thereby demonstrating that these alleles were largely stable over time. It is currently not known how the alleles that do change could influence pathogenicity. The results of this study also demonstrated discordance between strains defined by IS6110 DNA fingerprinting and those defined by MIRU-VNTR typing thereby demonstrating that these markers evolve independently and at different rates. Furthermore, the MIRU-VNTR typing method was unable to predict transmission of drug resistant strains which contradict previous reports from low incidence settings. This has significant implications for the use of this typing method in high incidence settings. Using an improved PCR-based method it was possible for the first time, to identify the 5 most prominent phylogenetic lineages in primary cultures of adult tuberculosis patients resident in a high HIV/TB co-infection setting. The results of this study showed that 15% of the study population was infected with two or more strains and Beijing genotype strains were over-represented in these mixed infections. Furthermore, drug susceptibility tests showed that one patient was co-infected with both a drug sensitive and a drug resistant strain. Since mixed infections have been implicated in treatment failure, these findings demonstrate the epidemiological importance of detecting mixed infections in vulnerable populations. This PCR-based method was further applied to cultures of paediatric tuberculosis patients to classify strains which spoligotyping was unable to define. The result of this study showed three mixed infections which otherwise would have been missed. In order to determine whether clinical disease presentation of patients infected with strains of the Beijing genotype were different from that of patients infected with non-Beijing genotype strains, clinical and demographic data of these two groups were analysed. This study showed that patients infected with strains of the Beijing genotype were highly infectious as defined by the increased bacterial load in sputum specimens. However, this finding could not be validated by lung pathology according to chest radiographs of infected patients. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf die molekulêre epidemiologie van Mycobacterium tuberculosis rasse met die Beijing genotipe. Hierdie genotipe is van groot kliniese belang weens hul globale verspreiding en die hipotese dat hierdie rasse die vermoë ontwikkel het om die beskermende effek van BCG vaksinasie te vermy, om meer geredelik te versprei, middelweerstandigheid te ontwikkel en erger vorms van siekte te veroorsaak. Molekulêre biologiese tegnieke is gebruik in ‘n reeks studies om die genetiese evolusionêre meganismes onderliggend tot die sukses van hierdie genotipe in Kaapstad, Suid-Afrika te verklaar. Deur ‘n versameling van 40 verskillende merkers te gebruik, was dit moontlik om vir die eerste keer ‘n filogenetiese stamboom van die Beijing ras genotipe te skep. Hierdie filogenie word gekenmerk deur die opeenvolgende evolusie van 7 ras sublyne. Met die analise van epidemiologiese data in verhouding tot hierdie ras sublyne, is ‘n assosiasie tussen die mees onlangs ontwikkelde Beijing rasse en die verhoogde vermoë om te versprei en siekte te veroorsaak, getoon. Vanweë hierdie bevindinge, is ‘n hipotese daargestel dat die patogeniese kenmerke van die Beijing genotipe nie in alle raslyne voorkom nie, maar eerder dat verteenwoordigende rasse van die verskillende sublyne unieke eienskappe deur evolusie ontwikkel het. ‘n Metaanalise van gepubliseerde MIRU tipering data van Oos-Asië is vergelyk met MIRU tipering data van Suid-Afrikaanse rasse in die konteks van hul filogenetiese geskiedenis om te bepaal watter van hierdie sogenoemde unieke eienskappe geassosieer is met die gasheerpopulasie en watter eienskappe geassosieer is met die genetiese agtergrond van die sublyn 7 rasse. Hierdie studie het getoon dat die Beijing ras genotipe van Suid-Afrika hul oorsprong gekry het van Oos-Asië en vir die eerste keer waargeneem is in die vroeë 18de eeu. ‘n Betekenisvolle assosiasie is waargeneem tussen die frekwensie waarteen die rasse van ‘n bepaalde Beijing sublyn voorkom en die menslike populasie van wie hulle geïsoleer is (p < 0.0001). Gebaseer op hierdie bevindinge is dit voorgestel dat die menslike populasie (Suid-Afrikaners) vir ‘n spesifieke Beijing sublyn geselekteer het (bv. Sublyn 7) of dat rasse van hierdie sublyn aangepas het om meer suksesvol te wees in die Suid-Afrikaanse populasie In ‘n daaropvolgende studie is, deur gebruik te maak van die metodiek wat ontwikkel is vir die bogenoemde studies, getoon dat die voorouerlike sublyn (bekend as die“atipiese” Beijing genotipe) die mees verteenwoordigende sublyn was onder middelweerstandige isolate van die Oos-Kaap gebied. Dit is teenstrydig met die bestaande dogma wat bepaal dat die “atipiese” Beijing genotipe rasse hulle vermoë om te versprei verloor het. Hierdie verskynsel kan egter versterk word in immuun inkompetente pasiënte aangesien hersiening van die kliniese rekords aangedui het dat verspreiding geassosieer was met HIV ko-infeksie. Hierdie bevindinge bring die behoefte om TB beheer in vatbare populasies te verbeter, na vore, omrede rasse wat gewoonlik `n onbetekenisvolle bydrae tot die epidemie lewer, nou ‘n rede vir kommer is veral as hulle met middelweerstandigheid geassosieer is. Om ons insig rakende die evolusie van die Beijing genotipe te verbeter, is die genomiese stabiliteit van ‘n addisionele 27 polimorfiese merkers geanaliseer. Daar is onlangs voorgestel dat hierdie merkers, wat gebaseer is op MIRU-VNTR volgordes,die nuwe standaard vir molekulêre studies is. Die MIRU-VNTR data is op die filogenetiese boom geplaas en het uitstekende ooreenstemming getoon wat die allele se stabiliteit oor tyd gedemonstreer het. Dit is tans nie duidelik hoe van die allele wat wel verander, die patogenisiteit beïnvloed nie. Die resultate van die studie wys ook onenigheid tussen rasse wat deur IS6110 DNA tipering gedefinieer is en dié wat deur MIRU-VNTR tipering gedefinieer is. Dit impliseer dus dat die evolusie van merkers onafhanklik van mekaar plaasvind en teen verskillende tempos. Verder was die MIRU-VNTR tipering metode nie in staat om verspreiding van middelweerstandige rasse te voorspel nie, wat teenstrydig is met vorige verslae waar lae insidensie omgewings bestudeer is. Dit het noemenswaardige implikasies vir die gebruik van hierdie tipering metode in hoë insidensie omgewings. ‘n Verbeterde PKR-gebaseerde metode is vir die eerste keer gebruik om die 5 mees prominente filogenetiese sublyne in primêre kulture van volwasse tuberkulose pasiënte van ‘n hoë MIV/TB ko-infeksie omgewing, te identifiseer. Die resultate van hierdie studie het gewys dat 15% van die studiepopulasie geïnfekteer is met twee of meer rasse en dat die Beijing genotipe ras die meeste voorgekom het in gemengde infeksies. Verder het middelweerstandige toetse gewys dat een pasiënt geïnfekteer was met beide ‘n middelsensitiewe en ‘n middelweerstandige ras. Gemengde infeksies is al vantevore gekoppel aan onsuksesvolle behandeling en dus demonstreer hierdie bevindinge die epidemiologiese belang van die opsporing van gemengde infeksies in vatbare populasies. Hierdie PKR-gebaseerde metode is verder gebruik om rasse wat voorkom in kulture van pediatriese pasiënte, wat spoligotipering nie kon klassifiseer nie, te klassifiseer. Die resultate het drie gemengde infeksies gewys wat sonder die PKR-gebaseerde metode, nie geïdentifiseer sou gewees het. Om te bepaal of die kliniese beeld van pasiënte wat geïnfekteer is met rasse van die Beijing genotipe verskil van dié van pasiënte wat geïnfekteer is met rasse van die nie-Beijing genotipe, is die kliniese en demografiese data van die twee groepe pasiënte geanaliseer. Hierdie studie wys dat pasiënte wat geïnfekteer is met rasse van die Beijing genotipe hoogs aansteeklik is (gedefinieer op grond van hoë bakteriële lading in sputum monsters). Hierdie bevindinge kon egter nie met behulp van long patologie op borskas X-strale bevestig word nie.

Memoria para optar al Título Profesional de Médico Veterinario / Con el fin de pesquisar y describir las lesiones pulmonares en Lobos marinos (Otaria flavescens), se realizó un estudio radiográfico simple de tórax a 20 ejemplares cachorros y juveniles. También se realizaron lavados traqueales para la obtención de muestras que fueron analizadas para la búsqueda de bacterias del Complejo Mycobacterium tuberculosis, mediante la prueba de Reacción de Polimerasa en Cadena (PCR), con el objetivo de investigar la presencia de tuberculosis pulmonar. Para ambos estudios los animales se encontraban bajo anestesia general mediante la utilización de Isofluorano, con buena respuesta a la inducción y recuperación. Se logró estandarizar la técnica radiográfica, kVp y mAs, y se extrapoló técnica de lavados traqueales de caninos a lobos marinos. Los resultaron mostraron que 11 animales (55%) presentaban algún tipo de lesión pulmonar, en 10 individuos (50%) se observó un infiltrado del parénquima pulmonar, en 5 de ellos este infiltrado es de tipo difuso, mientras que en los 5 restantes es de tipo celular. En cuanto a la presencia de broncogramas aéreos positivos (BAP), éste estuvo presente en 9 de los individuos en estudio (45%). En lo referente la prueba de PCR, ésta arrojó resultados negativos en la totalidad de los animales, probablemente, debido a que los animales no se hayan encontrado infectados con alguna bacteria del complejo M. tuberculosis

Submitted in fulfillment for the Degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2016. / Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has infected approximately one-third of the world population, with 9.6 million TB cases in 2014. The emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of MTB has further complicated the problem of TB control. It is now imperative that novel antimycobacterial compounds are discovered in order to treat infections and reduce the duration of current TB therapy courses. For centuries, medicinal plants have been used globally worldwide for the treatment and prevention of various ailments. This occurs particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years, the use and search for plant drug derivatives have been fast-tracked. Ethnopharmacologists, botanists, microbiologists, and natural product chemists are trying to discover phytochemicals which could be developed for the treatment of infectious diseases, especially TB. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimycobacterial activity. In the search for new lead compounds, nine medicinal plant species, Buddleja saligna, Capparis tomentosa, Carpobrotus dimidiatus, Dichrostachys cinerea, Ekerbergia capensis, Ficus Sur, Gunnera perpensa, Leonotis leonurus and Tetradenia riparia were collected in Kwa-Zulu Natal (KZN) following report of their therapeutic use in traditional medicine to treat symptoms and infections related to TB. They were tested in vitro for their activity against Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv (ATCC 25177) and three well-characterized clinical isolates of MDR-TB and XDR-TB using the agar incorporation method. The minimum inhibitory concentration of the active plant extracts was determined using the broth microdilution method. Our findings show that five of the nine plants screened have antimycobacterial activity with concentrations ranging from 125 µg/ml to 1000 µg/ml. The aqueous extracts of G. perpensa and T. riparia; and the methanolic extracts of B. saligna, C. tomentosa, and C. dimidiatus possessed significant activity against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177) and the three well-characterized clinical isolates of MDR-TB and XDR-TB. The cytotoxic effect of the active plant extracts was evaluated against the mouse BALB/C monocyte-macrophage (J774.2) and peripheral blood mononuclear cells (PBMCs). The toxic effects of the active plant extracts were evaluated using the brine shrimp lethality assay. Except for a high concentration of G. perpensa none of the other plants which possessed antimycobacterial activity showed any toxic or cytotoxic activity. The active plant extracts were thereafter assessed to determine if they had any effect on the survival or death of mycobacterial species, M. smegmatis, bound within the macrophage (J774.2) cell line at a concentration of 100 µg/ml. B. saligna had inactivated most of the phagocytosed bacilli after 24 hours of treatment therefore, it has a bactericidal effect on the mycobacteria located within the mouse macrophage. A phytochemical investigation of the leaves of B. saligna led to the isolation of two isomeric pentacyclic triterpene compounds namely Oleanolic Acid (OA) and Ursolic Acid (UA) using thin layer chromatography followed by silica gel column chromatography. The structures of these compounds were fully characterized by detailed NMR investigations, which included 1H and 13C NMR. Ursolic acid was isolated from this plant for the first time. Two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationship (QSAR) studies were carried out to provide insight on the interaction of the compounds with the enzyme. Molecular docking studies predicted the free binding energy of the triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of Mycobacterium tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177), clinical isolates of MDR-TB and XDR-TB using the Microplate Alamar Blue Plate (MABA) assay. The present study has scientifically validated the traditional use of medicinal plant B. saligna. / D

The DNA glycosylases function in the first step of the base excision repair (BER) process, that is responsible for removing base lesions resulting from oxidation, alkylation or deamination. The DNA glycosylases that recognize oxidative base damage fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis. While Fpg proteins are widely distributed among the bacteria and plants, Nei homologs are sparsely distributed across phyla, and are only found in γ-proteobacteria, actinobacteria and metazoans. Interestingly, M. tuberculosis H37Rv harbors two proteins (Rv2464c and Rv3297) from the Nei clade and two (Rv2924c and Rv0944) from the Fpg clade. All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 (Rv2924c) has a substrate specificity similar to that of EcoFpg and recognizes oxidized purines. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpg1 has a substantially increased opposite base discrimination compared to EcoFpg. The Rv0944 gene encodes MtuFpg2, which contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5- OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (5S) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Escherichia coli, the protein decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M. tuberculosis against oxidative stress, as has been observed with other pathogens .

P450s are heme containing enzymes which affect oxidation of substrates via catalytic intermediates having transient lifetimes. These oxidative catalytic intermediates are formed by a sequential interplay of electrons and protons at the active site of the enzyme bearing molecular dioxygen. The proton transfer to the active site from bulk solvent is coordinated by an “acid-alcohol” pair of active site residues which are conserved in all P450s. Sterol 14α-demethylases (CYP51) are P450 enzymes which catalyze oxidative deformylation of lanosterol in the cholesterol/ergosterol biosynthetic pathway. Both cholesterol and ergosterol are important regulators of membrane fluidity. CYP51 differs from other P450s in that the acid in the acid-alcohol pair in the active site is replaced by a His residue. This enzyme is present in tuberculosis (TB) causing pathogen Mycobacterium tuberculosis (Mtb). This finding was significant for primarily two reasons. The first one being the baffling presence of CYP51 in Mtb, as Mtb is not known to have any endogenous sterol biosynthetic pathways. The second being that CYP51 is a validated drug target in treating fungal infections. Thus given the global resurgence of multidrug resistant strains of Mtb and the deadly coexsistence of Mtb in immunocompromised HIV patients, CYP51 may be an ideal drug target for new generation of antimycobacterial drugs. The Mtb CYP51 enzyme was chosen to study the proton transfer pathways in the active site based on the outcome of explicit solvent molecular dynamics and hybrid quantum mechanics/molecular mechanics calculations performed in our laboratory. Based on these calculations of CYP51 catalysis, Glu173 was implicated to be the proton source. Proton transfer to the active site occurred by a coordinated shuttling via four water molecules, His259 and Thr260. To experimentally verify the roles of Glu173, His259 and Thr260 they were mutated to alanine and biophysically characterized. Ferredoxin, an accessory protein required to shuttle electrons from NADPH to the CYP51 active site for catalysis, was also cloned using ligation independent cloning. We were successfully able to reconstitute the electron transport chain for CYP51. The mutants were found to differentially bind type I and type II enzymes. Based on biophysical characterization, Thr260 can be implicated to have a role in modulating the spin state of the enzyme. The Mtb CYP51 enzyme was chosen to study the proton transfer pathways in the active site based on the outcome of explicit solvent molecular dynamics and hybrid quantum mechanics/molecular mechanics calculations performed in our laboratory. Based on these calculations of CYP51 catalysis, Glu173 was implicated to be the proton source. Proton transfer to the active site occurred by a coordinated shuttling via four water molecules, His259 and Thr260. To experimentally verify the roles of Glu173, His259 and Thr260 they were mutated to alanine and biophysically characterized. Ferredoxin, an accessory protein required to shuttle electrons from NADPH to the CYP51 active site for catalysis, was also cloned using ligation independent cloning. We were successfully able to reconstitute the electron transport chain for CYP51. The mutants were found to differentially bind type I and type II enzymes. Based on biophysical characterization, Thr260 can be implicated to have a role in modulating the spin state of the enzyme.

<p>Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), is among the leading causes of death from infectious disease world-wide. An intracellular pathogen, M. tuberculosis infects phagocytic cells, and subverts the host immune response, preventing eradication once infection has been established. Even after successful chemotherapy, exogenous re-infection occurs, indicating that sterilizing immune responses are not generated during natural infection. While a TB vaccine exists, it does not alter M. tuberculosis infection rate, rather it prevents the progression from latent TB infection to active TB disease. Vaccines against Haemophilus influenzae and Streptococcus pneumonia protect from bacterial colonization and infection through the induction of antibodies to capsular surface components. This dissertation explores if antibodies to the surface of M. tuberculosis can alter the initial interaction between a bacterium and host cell, leading to a reduction in infection rate. </p><p> When pre-mixed with M. tuberculosis prior to in vitro infection of macrophages, or retropharyngeal instillation of mice, monoclonal surface-binding, but not non-surface-binding antibodies, decrease bacterial burden and the number of infected cells within the first twenty-four hour of infection. If administered retropharyngeally prior to aerosol exposure, surface-binding antibodies decreased pulmonary bacterial burden at twenty-four hours post infection in an Fc&#947;R independent manner. Despite decreasing early bacterial burden, pre-administration of surface-binding antibodies prior to ultra-low dose aerosol infection did not alter infection rate compared to mice instilled with PBS (Chapters 4 and 5). </p><p> Infected humans do not produce high-titer, high-avidity surface-binding antibodies. Plasma from uninfected controls, individuals with latent TB infection, and active TB disease was assayed by ELISA to determine the titer, avidity and IgG/IgM ratio for antibodies to the surface and additional bacterial fraction. In contrast to antibodies to bacterial fractions, individuals with active TB disease had decreased avidity, and no augmentation of the IgG/IgM ratio for antibodies to the live M. tuberculosis surface, as compared to uninfected controls (Chapter 3). </p><p> Overall these findings demonstrate that surface-binding monoclonal antibodies alter early infection events, both in vivo and in vitro, though the magnitude of protection was not sufficient to decrease M. tuberculosis infection rate. Additionally, the failure of humans to generate high-titer, high-avidity surface-binding antibodies after infection indicates and that induction of surface-binding antibodies may be an appropriate target for future vaccines.</p> / Dissertation

Mycobacterium tuberculosis est la cause d’une des maladies infectieuses les plus fréquentes dans le monde causant la mort de plus de 1,2 millions de personnes chaque année selon l’organisation mondiale de la santé (OMS). Actuellement, la tuberculose à M. tuberculosis émerge chez d’autres espèces comme l’a montré notre revue bibliographique des cas de tuberculose à M. tuberculosis chez les primates non-humains, les éléphants d’Asie, les animaux de ferme, les animaux de compagnie et certains animaux sauvages. Nous avons montré que ces trois espèces survivent dans le sol pendant au moins 12 mois et restent pathogènes dans un modèle souris. Egalement nous avons montré que le sol infecté par M. tuberculosis est une source potentielle de contamination pour les animaux. Nous avons ensuite développé un protocole de culture rapide de M. tuberculosis, incluant un nouveau milieu de culture solide, des conditions optimisées d’incubation, et la détection des microcolonies par autofluorescence. Notre travail de thèse a permis de mettre en place des techniques et protocoles qui révolutionnent la culture et l’isolement de M. tuberculosis en réduisant les délais de culture et des antibiogrammes, un point déterminant pour la lutte contre la tuberculose notamment dans les pays à ressources limitées et les pays à forte émergence de souches de M. tuberculosis de plus en plus résistantes. Ces protocoles sont en cours de transfert pour la routine de laboratoire. / Mycobacterium tuberculosis is the cause of one of the most common infectious diseases in the world killing more than 1.2 million people each year according to the World Health Organization (WHO). Currently, M. tuberculosis tuberculosis emerges in other species like non-human primates, Asian elephants, farm animals and some wild animals. We have shown that three species of M. tuberculosis complex survive in the soil for at least 12 months and are pathogenic in a mouse model and M. tuberculosis-infected soil is a potential source of infection for animals. We then developed a protocol for rapid culture of M. tuberculosis, including a new solid culture medium, optimized conditions of incubation, and detection of microcolonies by autofluorescence. Our thesis has helped develop techniques and protocols that are revolutionizing the culture and isolation of M. tuberculosis by reducing delays culture and susceptibility testing, a crucial point for the fight against tuberculosis, especially in countries limited resources and countries with strong emergence of M. tuberculosis strains more resistant. These protocols are being transferred to the routine laboratory.

L’agent pathogène responsable de la tuberculose, Mycobacterium tuberculosis, utilise principalement les lipides comme source d’énergie lors du processus infectieux. Cela suggère que les enzymes lipolytiques (ELs) sont des facteurs d’une importance cruciale, qui jouent un rôle essentiel pour la survie et la persistance du bacille au sein des granulomes tuberculeux. Dans ce contexte, nous nous sommes intéressés au rôle physiologique de la protéine LipY (Rv3097c), une TAG-hydrolase apparentée à la lipase Hormono-sensible humaine. En utilisant un modèle expérimental de « foamy » macrophages, nous avons démontré que cette protéine sécrétée par le système de sécrétion ESX-5 était responsable de l’hydrolyse des triacylglycerols (TAG) de l’hôte dans la lumière phagosomale. Dans une seconde étude, nous avons développé un modèle expérimental robuste et réversible basé sur la biodisponibilité en azote et en carbone, deux facteurs essentiels qui gouvernent la formation et l’hydrolyse de TAG sous la forme d’inclusions lipidiques intracytoplasmiques (ILI) chez les mycobactéries. La simplicité d’utilisation de ce modèle s’avère être un excellent système biologique permettant d’étudier les propriétés phénotypiques des bacilles riches en ILI. L’ensemble des résultats obtenus au cours de ces travaux de thèse confirme que certaines ELs sont directement impliquées dans la virulence et/ou la survie des mycobactéries pathogènes. De plus, une très grande partie de ce travail ouvre de nouvelles perspectives sur la caractérisation et l’inhibition d’acteurs protéiques essentiels au métabolisme des lipides in vivo, offrant ainsi de nouvelles opportunités pour lutter contre M. tuberculosis. / Mycobacterium tuberculosis, the pathogenic agent responsible for tuberculosis (TB), mainly uses lipids as a source of energy during the infectious process. This suggests that lipolytic enzymes (LEs) are crucial metabolic agents that play a critical role in the survival and persistence of the bacillus within TB granulomas. In this context, we investigated the physiological role of the LipY protein (Rv3097c), a TAG-hydrolase related to the human Hormono-sensitive lipase. Using an experimental model of "foamy" macrophages, we demonstrated that this protein which is secreted by the ESX-5 secretion system, was responsible for the hydrolysis of host-derived triacylglycerols (TAGs) within the phagosomal compartment. In a second study, we developed a robust and reversible experimental model based on the bioavailability of nitrogen and carbon, two essential factors that govern the formation and hydrolysis of TAGs in the form of intracytoplasmic lipid inclusions (ILI) in mycobacteria. The simplicity of this model proves to be an excellent biological system for studying the phenotypic properties of ILI-rich bacilli. All the results obtained during this thesis confirm that some LEs are directly involved in the virulence and/or survival processes of pathogenic mycobacteria. In addition, we truly believe that this work opens up new perspectives on the characterization and inhibition of protein, that are essential actors for lipid metabolism in vivo, thus offering new opportunities to better control M. tuberculosis.