Emergence of drug resistance is a major problem in the treatment of many diseases including tuberculosis. To tackle the problem, it is essential to obtain a global perspective of the molecular mechanisms by which bacteria acquire drug resistance. Systems biology approaches therefore become necessary. This work aims to understand pathways to drug resistance and strategies for inhibition of the resistant strains by using a combination of experimental genomics and computational molecular systems approaches. Laboratory evolution of Mycobacterium smegmatis MC2 155 by treatment with isoniazid (INH), a front-line anti-tubercular drug, resulted in a drug-resistant strain (4XR), capable of growth even at about 10-times the minimum inhibitory concentration of the drug. Whole genome sequence of the 4XR was determined, which indicated only 31 variations in the whole genome, including 3 point mutations, 17 indels and 11 frame-shifts. Two mutations were in proteins required for the pharmacological action of the drug, albeit in regions distant from the drug binding site. The variations however were insufficient to explain the observed resistance to isoniazid. For a better understanding of the global changes associated with drug resistance, whole genome-wide gene expression data was obtained for the resistant strain and compared with that of the WT strain. 716 genes were found to be differentially regulated in 4XR, spanning different biochemical, signaling and regulatory pathways. From this, some explanations for the emergence of drug resistance were obtained, such as the up-regulation of the enzymes in the mycolic acid biosynthesis pathway and also of the drug efflux pumps. In addition, enrichment analysis indicated that up-regulated genes belong to functional categories of response to stress, carbohydrate metabolism, oxidation-reduction process, ion transport, signaling as well as lipid metabolism. The differential gene regulations seemed to be partially responsible for conferring the phenotype to the organism. Alterations in the metabolic pathways in 4XR were characterized using the phenotypic microarray technology, which experimentally scanned the respiratory ability of the resistant bacteria under 280 different nutrient conditions and 96 different inhibitors. Phenotypic gain, where the resistant strain grows significantly better than the wild type and phenotypic loss, where the growth of the resistant strain is compromised as compared to the sensitive strains were derived from the comparison of the phenotypic responses. Differences in survival ability and growth rates in different nutrient sources in the resistant phenotype as compared to the wild type were observed, suggesting rewiring in the metabolic network of the drug-resistant strain. In particular, the pathways of central carbon metabolism and amino acid biosynthesis exhibit significant differences. The strain-specific metabolic pathway differences may guide in devising strategies to tackle the drug-resistant strains selectively and in a rational manner. Scanning electron microscopy indicated the morphology of the drug-resistant strains to be significantly altered, as compared to the control drug-sensitive strain. It is well-known that isoniazid acts by inhibiting mycolic acid biosynthesis. The pathway turns out to be a target for many other anti-tubercular drugs also, since mycolic acids are major components of the cell wall. It is therefore important to understand what changes occur in the mycolic acid and the associated pathways in the drug-resistant variety so that strategies to tackle the latter can be chosen more judiciously. The lipidome of the cell wall was therefore quantitatively characterized by mass spectrometric analyses, which indeed confirmed that the 4XR strain has a significantly different composition profile. Among the six categories of lipids, the members of the glycerophospolipids category were abundant while the fatty acyls, polyketides and saccharolipids were lower in the 4XR strain as compared to the WT. The lipidomic data derived from the cell wall of INH-resistant strain shows that it results in the mycolic acid pathway function restoration, which would otherwise be lost upon drug exposure in the sensitive strain. Understanding the precise changes that occur in the lipidome in the drug-resistant strains is expected to be useful in developing new ways to tackle resistance. Next, to understand the implications of altered gene expression profiles, protein-protein interaction networks are constructed at a genome-scale that captures various structural and functional associations mediated by proteins in the mycobacterial cell. Using transcriptome data of 4XR, a response network is computed. Using an algorithm previously developed in the laboratory, the networks have been mined to identify highest differential activity paths and possible mechanisms that are deployed by the cells leading to drug resistance. Known resistance mechanisms such as efflux, cytochromes, SOS, are all seen to constitute the highest activities for achieving drug resistance in 4XR. Interestingly, such paths are seen to form a well-connected subnet, indicating such differential activities to be orchestrated. This clearly shows that multiple mechanisms are simultaneously active in the 4XR and may together generate drug resistance. Mechanisms of detoxification and antioxidant responses are seen to predominate in the 4XR subnet. Overall the analysis provides a shortlist of strategies for targeting the drug resistant strain. Next, the phenotypic microarray platform was used for screening for growth in Msm in the presence of various drugs. Data analysis and clustering resulted in identification of conditions that lead to phenotypic gain or loss in the 4XR as well as those that lead to differential susceptibility to various drugs. Drugs such as cephalosporins, tobramycin, aminotriazole, phenylarsine oxide, vancomycin and oxycarboxin were also found to inhibit growth in the resistant strain selectively. In other words, the 4XR is found to be collaterally sensitive to these drugs. The top-net formed by the highest differential activity paths, identified from the network described earlier has already indicated the involvement of proteins that generate antioxidant responses. Insights from the two methods, first from the targeted approach and second, from the phenotypic discovery approach were combined together to select only those compounds to which the 4XR strain was collaterally sensitive and targeted proteins responsible for antioxidant responses. These compounds are vancomycin, phenylarsine oxide, ebselen and clofazimine. These were further tested against the virulent M. tuberculosis H37Rv strain in a collaborator‘s laboratory. 3 of these compounds such as vancomycin, ebselen and phenylarsine oxide were found to be highly active in combinations with isoniazid against all tested Mtb strains, showed high levels of inhibition against H37Rv and 3 different single drug resistant, MDR and XDR strains. Moreover, they were observed to be highly potent when given in combinations. Clofazimine on the other hand, in combination with isoniazid showed activity but no significant synergy in the virulent drug-resistant strains of M. tuberculosis though synergistic to the sensitive strain. Thus, experiments with M. tuberculosis provide empirical proof that four different compounds, all capable of blocking antioxidant responses, are capable of inhibiting growth of single-, multiple- and extremely-drug-resistant clinical isolates of M. tuberculosis. Using transcriptome data from literature for M. tuberculosis exposed to six different drugs, similar drug specific response networks were constructed. These networks indicate differences in the cellular response to different drugs. Interestingly, the analysis suggests that different drug targets and hence different drugs could trigger drug resistance to various extents, leading to the possibility of prioritizing drug targets based on their resistance evolvability. An earlier study from the laboratory suggested the concept of target-co-target pairs, where-in the co-target could be a key protein in mediating drug resistance for that particular drug and hence for its target protein. Top ranked hubs in multiple drug specific networks such as PolA, FadD1, CydA, a monoxygenase and GltS, can possibly serve as co-targets. Simultaneous inhibition of the co-target along with the primary target could lower the chances of emergence of drug resistance. Such analyses of drug specific networks provide insights about possible routes of communication in the cell leading to drug resistance and strategies to inhibit such communication to retard emergence of drug resistance. Since mutations in the target proteins are known to form an important mechanism by which resistant strains emerge, an understanding of the nature of mutations in different drug targets and how they achieve resistance is crucial. Sequence as well as structural bases for the resistance from known drug-resistant mutants in different drug targets is deciphered and then positions amenable to such mutations are predicted in each target. Mutational indices of individual residues in each target structure are computed based on sequence conservation. Saturated mutagenesis is performed in silico and structural stability analysis of the target proteins has been carried out. Critical insights were obtained in terms of which amino acid positions are prone to acquiring mutations. This in turn suggests interactions that are not desirable, thus can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. Finally, the work presented here describes application of the systems biology approaches to understand the underlying mechanisms of drug resistance, which has provided insights for drug discovery on multiple fronts though target identification, target prioritization and identification of co-targets. In particular, the work has led to a rational exploration of collateral drug sensitivity and cross-resistance of the drug-resistant strain to other compounds. Combinations of such compounds with isoniazid were first identified in the M. smegmatis model system and later tested to hold good for the virulent M. tuberculosis strain, in a collaborative study. The combinations were found to be active against three different clinical drug-resistant isolates of M. tuberculosis. Therefore, this study not only reveals the global view of resistance mechanisms but also identifies synergistic combinations of promising drug candidates based on the learnt mechanisms, demonstrating a possible route to exploring drug repurposing. The combinations are seen to work at a much reduced dosage as compared to the conventional tuberculosis drug regimens, indicating that the toxicity and any associated adverse effects may be greatly reduced, suggesting that the combinations may have a high chance to succeed in the next steps of the drug discovery pipeline.

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No. of bitstreams: 2 Dissertação - Fábio Muniz de Oliveira - 2014.pdf: 12287155 bytes, checksum: 58eb4d1e227a17283f27cf610577402a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-28 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / For hundreds years tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (Mtb), has been a global public health problem. Even after the development of the vaccine BCG, in 1921, tuberculosis control continues on slow pace. This comes to be as a result of the variable efficacy (from 0 to 80%) presented by the vaccine in the protection against TB in adults. Therefore, the development of a new vaccine against TB is necessary. In this study, it was evaluated a recombinant vaccine composed of Mycobacterium smegmatis expressing the CMX fusion protein (mc2- CMX), formed from three antigen epitopes of Mtb: Ag85C, MPT51 and HspX. M. smegmatis mc2 155 was transformed with pLA71-CMX by electroporation, and the presence of the CMX protein was confirmed by imuno blotting. BALB/c mice were distributed in four groups: saline, infection, BCG and mc2-CMX. The groups were immunized with their respective vaccines in two moments with an interval of fifteen days and the animal blood was collected fifteen days after the last immunization. Thirty days after the last immunization, the animals were challenged with Mtb H37Rv (intravenously) and thirty days after the challenge, the blood was collected to perform ELISA test. Seventy days after the challenge, the lungs from all mice were collected to obtain cells for flow-cytometry, histological analysis and also to determine the bacillary burden. The immunization with mc2-CMX induced higher levels of antibodies of IgG1 (1,910±0,70) and IgG2a (0,139±0,020) class anti-CMX when compared with BCG group (0,646±0,19 and 0,413±0,24; respectively, p<0,05). These results demonstrated the relevance of CMX antigen in the immunogenicity of the recombinant vaccine. Seventy days after the challenge, the amount of T CD4 cells in the lung producing Th1- type cytokines was assessed. It was observed a significant increase in the percentage of T CD4 cells positive for IFN-γ and TNF-α in the immunized mice with mc2-CMX vaccine, when compared with the group immunized with BCG. Mice challenged with Mtb presented significant higher percentage of IL-2 producer cells when compared with the non-immunized group. However, only the mice immunized with the vaccine mc2- CMX presented significant higher percentage when compared with the infection group. The immune response induced by the vaccine was effective in the control of Mtb infection, confirmed by the histological analysis and the bacillar burden determined. The groups vaccinated with mc2-CMX and BCG presented a significant reduction of the lung lesion induced by the Mtb infection, and also lung bacterial load, when compared with the infection group. Thus, the recombinant vaccine mc2-CMX presents potential characteristics to be used in the prevention of TB. / Há séculos a tuberculose (TB), doença infectocontagiosa causada por Mycobacterium tuberculosis (Mtb), vem sendo um problema de saúde pública mundial. Mesmo após o surgimento da vacina BCG em 1921, o controle da tuberculose continua a passos lentos. Isso se deve à eficácia variável de 0 a 80% apresentada pela vacina na proteção contra TB em indivíduos adultos. Deste modo, o desenvolvimento de uma nova vacina contra a TB é necessário. Neste estudo, avaliou-se uma vacina recombinante composta por Mycobacterium smegmatis expressando a proteína de fusão CMX (mc2-CMX), formada por três antígenos do Mtb: Ag85C, MPT51 e HspX. M. smegmatis mc2 155 foi transformado com pLA71-CMX por eletroporação, sendo a expressão da proteína CMX confirmada por imunoblot. Camundongos BALB/c foram distribuídos em quatro grupos: salina, infecção, BCG e mc2-CMX. Os grupos foram imunizados com suas respectivas vacinas em dois momentos com intervalos de 15 dias, e o sangue de todos os animais coletado quinze dias após a última imunização. Trinta dias após a imunização, os animais foram desafiados com Mtb H37Rv (via endovenosa) e trinta dias após o desafio, o sangue foi coletado para realização de ELISA. Setenta dias após desafio, o pulmão e o baço de todos os camundongos foi coletado para obtenção de células para realização de citometria, histopatológico e determinação da carga bacilar. A imunização com o mc2-CMX induziu níveis maiores de anticorpos da classe IgG1 (1,910±0,70) e IgG2a (0,139±0,020) anti-CMX quando comparado com o grupo BCG (0,646±0,19 e 0,413±0,24, respectivamente, p<0,05). Estes resultados demonstram a relevância do antígeno CMX na imunogenicidade da vacina recombinante. Após setenta dias do desafio, a quantidade de células T CD4 produtoras de citocinas do tipo Th1 foi analisada no pulmão. Foi observado um aumento significativo na porcentagem de células T CD4 positivas para IFN-γ e TNF-α nos camundongos imunizados com vacina mc2-CMX, quando comparado com o grupo BCG. Camundongos desafiados com Mtb apresentaram porcentagens maiores de células produtoras de IL-2, quando comparado com o grupo não desafiado. Todavia, somente os camundongos imunizados com a vacina mc2-CMX apresentaram porcentagens significativamente maiores em comparação ao grupo infecção. A resposta imune observada foi efetiva no controle da infecção por Mtb, sendo isto confirmado quando os pulmões dos camundongos foram analisados histologicamente e a carga bacilar determinada. Os grupos vacinados com as vacinas mc2-CMX e BCG apresentaram uma redução significativa da lesão pulmonar induzida pela infecção por Mtb, e também da carga bacilar no pulmão, quando comparados com o grupo infecção. Conclui-se que mc2-CMX tem um bom potencial para ser explorado como vacina contra a TB.

No description available.

Mycobacterial infection is a major health concern. High drug resistivity of the mycobacterium is due to its multi-layered, thick hydrophobic waxy cell wall components, consisting of cross-linked peptidoglycan (PG), mycolyl arabinogalactan (mAG) and lipoarabinomannan (LAM) polysaccharides. These polysaccharides are composed of arabinose and galactose in the furanose form and mannose in the pyranose form. The high waxy hydrophobic components of the mycobacterial cell wall acts as a barrier for most hydrophilic antibacterial agents. Enzymes responsible for the biosynthesis of polysaccharides of mAG and LAM are arabinosyl transferase (AraT), galactosyl transferase (GlfT) and mannosyl transferase (ManT). In the absence of furanoside derivatives of D-arabinose and D-galactose in mammalian systems, inhibitors based on these sugars arise an interest. Upon realizing structural characteristics of cell wall polysaccharides, the chemical syntheses of such polysaccharides were reported. Biological studies of synthetic arabinomannan and arabinogalactan oligosaccharides were performed, in order to identify their effects in enzymatic, as well as, mycobacterial growth assays. Chapter 1 of the thesis describes the structural features of mAG and LAM polysaccharides. Chemical synthesis of oligosaccharides related to mycobacterial cell wall components and their effects of mycobacterial growth and enzymatic assays are discussed. In my research program, synthesis and studies of oligosaccharides pertaining to mycobacterial cell wall components were undertaken. Monovalent and bivalent glycolipids 1 and 2 (Figure 1), containing arabinofuranoside trisaccharide as the sugar head group, were synthesized and their effects on the growth of M. smegmatis strain were studied. In the presence of arabinan glycolipids, retardation of the growth of M. smegmatis was observed and the inhibitory activity was found to be specific with glycolipids containing arabinofuranoside head groups. Glycolipid with maltosyl sugar and arabinofuranoside trisaccharide without lipid chains, did not affect the mycobacterial growth. Continuing the effort, tri- and tetrasaccharide of arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that 3 was inhibiting the growth of the mycobacterium, whereas in the case of 4, inhibition was found to be less when compared to 3. Relative inhibitions of mycobacterial growth by synthetic glycolipids 1-4, at a concentration 200 µg/mL, were found to be in a varying degrees, ranging from 16 % in the case of 4 and 65 % in the case of 3. Figure 1. Molecular structures of arabinan and arabinomannan oligosaccharides 1-7. Following mycobacterial growth inhibition studies, surface plasmon resonance studies of synthetic oligosaccharides were performed, in order to identify their interactions with mycobacterial cell lysates. Amine tethered glycosides 5-7 (Figure 1) were synthesized and immobilized onto SPR sensor chip through amine coupling methodology. From SPR studies, it was found that the binding affinity was higher with cell lysates from motile strains than non-motile strain. Among various arabinomannans, glycoside 5, presenting two mannose units showed higher affinity than 6 and 7, having no or one mannose unit, respectively. Chapter 2 of the thesis provides details of synthesis, biological and biophysical studies of arabinan and arabinomannan glycolipids. Continuing the synthesis and studies with arabinose oligosaccharides, a linear tetra-, hexa and octasaccharide glycolipids, containing α-(1→5) linkages (10-12), as well as, a branched heptasaccharide containing α-(1→2) and α-(1→5) linkages (14) between the arabinofuranoside units (Figure 2) were synthesized. In addition to glycolipids, oligosaccharides without alkyl chains (8, 9 and 13) were also prepared. Synthesis was performed using trichloroacetimidate and Figure 2. Molecular structures of linear and branched arabinan derivatives 8-14. thioglycosides as glycosyl donors. Synthesis of linear oligosaccharide derivatives 8-12 was achieved by iterative glycosylation and deprotection strategies. Branched heptasaccharide derivatives 13 and 14 were synthesized by using block glycosylation method, wherein two fold excess of arabinose disaccharide was reacted with a suitably protected arabinose trisaccharide. Upon synthesis, molecular modeling studies were performed to identify the conformational behavior of arabinan glycolipids. Conformational studies were performed in three steps, namely, (i) dihedral scan (ii) conformational search and (iii) molecular dynamics. Dihedral scan was performed to assess favorable torsion angles at each glycosidic linkage with respect to overall conformation of the molecule. Monte-Carlo conformational search was performed to obtain the lowest energy structure of arabinan glycolipids. Relative orientations of lipidic portions and sugar portions were identified for linear and branched arabinan glycolipids. The least energy conformations of 10, 11, 12 and 14 are shown in Figure 3. In the case of linear molecules 10, 11 and 12, alkyl chains and arabinofuranoside portion did not phase segregate, whereas in the case of branched glycolipid 14, the alkyl chains were observed to move away from the sugar moieties. Molecular dynamic calculations were performed for the lowest energy structure, in order to evaluate the torsion angles in the trajectory. Following the synthesis and conformational analysis of the arabinan glycolipids, surface plasmon resonance studies were performed to assess their interactions with a host protein, namely, pulmonary surfactant protein-A (SP-A). For the interaction studies, SP-A was immobilized on to the CM-5 sensor chip using amine coupling method. Varying concentrations of arabinan glycolipids 10, 11, 12 and 14 and oligosaccharides 8, 9 and 13 were used as analytes. Responses from the surface of SP-A were subtracted from that of ethanolamine to eliminate the non-specific interactions. Primary sensorgrams were fitted using 1:1 Langmuir model to obtain the kinetic parameters of the interactions. Specificities and relative binding affinities of arabinan oligosaccharides interacting with SP-A are presented in Table 1. The affinities between Figure 3. Lowest energy structures of glycolipids 10, 11, 12 and 14 derived from molecular modeling studies. arabinan oligosaccharides and SP-A were found in the range of 4.9-47x103 M-1. Among the series, branched arabinan oligosaccharides 13 and 14 showed higher Ka values than the linear arabinan glycolipids. The association rate constants (kon) were generally higher for the oligosaccharides without lipidic chain, whereas, the dissociation rate constants (koff) were slower with oligosaccharides having lipidic chains. Faster kon was also associated with a faster koff for oligosaccharides without the lipidic chains. For the glycolipids, a relatively slower koff was found to be the trend. In the case of branched heptasaccharide derivatives, glycolipid 14 showed higher binding constant than heptasaccharide with a thiocresyl group at the reducing end 13. Chapter 3 of the thesis presents the synthesis, conformational analysis and SPR studies of linear and branched arabinan glycolipids. Table 1. Kinetic parameters of the interactions between arabinose derivatives 8-14 and SP-A. Compound kon (M-1s-1) kd (s-1) (104) Ka (M-1) (10-3) χ2 12 3.9 7.91 4.9 8.3 11 1.5 3.98 3.77 2.9 10 0.384 0.22 17.5 6.7 14 27.3 5.79 47.2 4.5 8 11.3 6.14 18.4 2.3 9 23.3 11.6 20.1 2.4 13 53.6 17.9 29.9 5.4 Upon assessing the biophysical studies of the α-arabinofuranoside glycolipids, an effort was undertaken to prepare glycolipids containing β-arabinofuranoside linkages and to study their conformational and biophysical properties. Arabinan glycolipids 15 and 16 (Figure 4), containing β-(1→2), β-(1→3) and β-(1→5) linkages between furanoside units were synthesized to compare the properties with the corresponding synthetic α-arabinan glycolipids. Incorporation of β-arabinofuranoside linkages in 15 and 16 was achieved using low temperature activation of silyl substituted glycosyl donor 17 (Figure 4), with NIS and AgOTf. The configurations in 15 and 16 were confirmed through 1H-1H COSY, 1H-13C HMQC NMR techniques. During the synthesis of 15 and 16, stereoselective incorporation of two β-Araf linkages on a single furanoside unit was achieved for the first time. Conformational studies of 15 and 16 were conducted similar to α-arabinan glycolipids, as above, to identify most favorable conformations of inter-ring, as well as, overall conformation of the molecule. The interactions between the SP-A and β-arabinofuranoside glycolipids 15 and 16 were also assessed with the aid of SPR technique. The analysis showed that the affinities of glycolipids 15 and 16 to SP-A were found to be relatively lower when compared to α-arabinofuranoside glycolipids. Synthesis and studies of β-arabinofuranoside glycolipids are described in chapter 4 of the thesis. Figure 4. Molecular structures of β-arabinofuranoside glycolipids 15 and 16. In summary, the present thesis describes synthesis, conformational and biophysical studies of synthetic arabinan and arabinomannan glycolipids. Monovalent and bivalent arabinan, tri- and tetrasaccharide arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that arabinan and arabinomannan glycolipids inhibited the growth of the mycobacterium. The inhibitory activity is specific with the arabinan and arabinomannan glycolipids and the glycolipids with higher arabinose composition were found to be better inhibitors for mycobacterial growth. The interactions of mycobacterial cell lysates with arabinomannan compounds were evaluated through SPR technique. Linear tetra-, hexa-, octa- and branched heptasaccahride arabinan glycolipids containing α-Araf linkages between furanoside units were synthesized. Molecular modeling studies of arabinan glycolipids were performed, in order to identify their lowest energy conformations. Biophysical studies of linear and branched arabinan glycolipids were conducted to assess their interactions with pulmonary surfactant protein-A (SP-A) through surface plasmon resonance technique. Syntheses, conformational and biophysical studies were extended further to β-arabinofuranoside glycolipids. Overall, the thesis provides synthesis, conformational, biological and biophysical studies of a series of lipoarabinomannan oligosaccharides. The results provide a possibility to evolve newer types of glycolipids that can act as inhibitors of mycobacterial growth. (For structural formula pl see the hard copy)

Mycobacterium tuberculosis (Mtb) is one of the most virulent and prevalent bacterial pathogens across the world. As Mtb infects millions of people a year, it remains essential to study its physiology with the goal of developing new therapeutic interventions. A critical part of the bacteria’s ability to propagate is through successful cell division. Although the process of bacterial cell division and the key proteins therein are well understood in Escherichia coli, much remains to be understood about division in mycobacteria. Genetic and cell biological approaches have recently begun to identify key divisome components in Mycobacterium smegmatis. However, questions remain regarding the role and function of one divisome protein in particular, the DNA translocase FtsK. In this dissertation, I investigated the necessity of FtsK for the growth of mycobacteria. Using an inducible knockdown of FtsK, I present evidence that complete loss of FtsK is required to inhibit growth in both Mtb and M. smegmatis, and that these orthologs share a homologous function. Additional work suggests extended loss of FtsK may be lethal to bacteria. These observations support that FtsK is an essential member of the divisome in mycobacteria, facilitating the processes of growth and division.

<p>Ever since the invention of antibiotics nearly a century ago,the threat of antibiotic resistance has been gradually increasing. As antibiotics are continually prescribed, the rate at which bacteria are becoming resistant to antibiotics is increasing as well. It is projected that antibiotic resistance is one of the largest threats to overall world health, and bacteriophage therapy is one of the leading strategies to combat it. Bacteriophages are viruses that infect and kill specific host bacteria andcan potentially be utilized to kill desired bacteria causing infections that are resistant to antibiotics.</p> <p><br></p> <p>The purpose of this research project is to learn more about the bacteriophage-host interaction through mass spectrometry and bioinformatic tools. This is done through the analysis of proteins and lipids that are produced when the bacteriophage infects the host bacteria. The growth curve of a Passage One From Frozen (P1FF) and a Passage Two From Frozen (P2FF) sample of Mycobacterium smegmatiswas calculated to determine to optimum time for bacteriophage infection. Twobacteriophages were chosen, PotatoSplit and Zalkecks, the Mycobacterium smegmatis samples were infected, samples collected, and mass spectrometry performed. A large portion of this research project is based on the analysis of the proteins and lipids that are produced during each bacteriophage’s infection. Proteomic and lipidomic strategies can be implemented to understand more about the bacteriophage-host interaction and discover any proteins and lipids that are produced at varying timepoints throughout the inoculation process. Bioinformatic tools can then be used to understand the potential functions of each protein or lipid and potential functions or applications of the bacteriophage in general, including the pathogenicity of each bacteriophage.</p> <p><br></p> <p> Determined from proteomic and lipidomic analysis, a list of all proteins and lipids found within each phage infected sample was made. An important trend discovered is that more phage proteins were expressed at later times during the phage infection –Hour 7 and Hour 10, whereas more bacterial proteins were expressed initially –Hour 0 and Hour 3. A case study to investigate the usage of different intensity types produced from mass spectrometry was completed. Overall, it was determined that both the number of phageproteins and bacterial proteins can differ depending on if LFQ or iBAQ intensity type data was used. Correlation between proteins and lipid ontology classes was performed and shows whether groups of lipids are upregulated or downregulated at 14each time point. Understanding the function of lipid ontology groups and the type of regulation provides insight into how the phage or bacteria are potentially using the lipids produced. Some of the main findings include lipids that are involved in bacterial defense mechanisms/energy usage increase over time. Some correlation trends were not consistent across the different bacteriophages, which can be contributed to the different phage life cycles  and therefore different phage-host interactions. Further investigation should be performed to determine the specific biological function of proteins and lipids to confidently make claims about potential applications for each phage. Also, further investigation should be performed to understand if the differences in results between bacteriophage PotatoSplit and Zalkecks are due to the varying life cycles.</p>

<p>Bacteriophages (phages) have broad applications in diverse areas including phage therapy, agriculture, food safety, and environmental protection. In order to fully realize the potential for phage applications, it is critical to understand phage-bacteria interactions and characterize bacterial responses/targets to phage infection. Previous studies have largely focused on other classes of phages other than mycobacteriophages. This research provides the first global proteome investigation of the dynamic relationship between a mycobacteriophage and a mycobacterial host. Mycobacteriophages are viruses that infect mycobacteria. They have been reported to have vital potential uses in various fields, especially as an alternative in the prevention and treatment of mycobacterial diseases such as tuberculosis. Despite their potential, not much is known about the molecular interaction with mycobacteria during a mycobacteriophage infection, especially at the translational level. To better understand this, a novel mycobacteriophage, Ochi17 was first isolated and characterized based on the genome and structure. I then applied label-free quantitative proteomics using the model host, <i>Mycobacteria smegmatis</i>,which<i> </i>was infected with Ochi17<i> </i>at different infection time points. Phage Ochi17 was found to be a temperate phage and classified as a Siphoviridae. The proteome changes occurring at the mid-lytic stage of Phage Ochi17 infection was first examined followed by a temporal study of the global changes. More than 2,000 <i>M. smegmatis</i> proteins and at least 50 Ochi17 proteins were identified across all time points. Homologous recombination and host macromolecular synthetic processes were significantly upregulated, while lipid metabolism was significantly downregulated. The results suggested that Ochi17 suppressed the growth of Mycobacterium smegmatis not just by utilizing the macromolecular synthesis of the host, but also by suppressing host transcription, and fatty acid biosynthesis, in addition to the degradation of fatty acids irrespective of infection time. The two-component system was a target at only 24 h post infection. I also showed that phage Ochi17 proteome expression is time-dependent and the proteins typically cluster based on functional relatedness. The results presented here may contribute in the development of mycobacteriophages as antimicrobial therapies that can overcome various defense strategies employed by host mycobacteria.</p>

<p dir="ltr">Tuberculosis remains a global health challenge, ranking as the second leading cause of mortality worldwide in 2022. The resilience of <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, is enhanced by the high expression of efflux pumps that confer antibiotic tolerance and the formation of biofilms that confer resistance to antibiotics. Carbon nanomaterials (CNMs) exhibit a broad-spectrum of antibacterial efficacy, making them promising candidates for combating drug-resistant bacterial strains. The effects of the novel carbon allotropes called fullertubes (C₉₀) on any living cell have not been studied. In our study, we employed <i>Mycobacterium smegmatis</i> as a model organism for <i>M. tuberculosis</i> and exposed it to fullertubes and fullerenes. We explored the impact of these CNMs on efflux activity and biofilm formation through biochemical assays like ethidium bromide transport assay and crystal violet assay. We also investigated their impact on lipid biosynthesis associated with log-phase growth and biofilm formation using metabolic radiolabeling studies. We also investigated the effects of the efflux pump inhibitors (EPIs) piperine, berberine, 1-(1-naphthylmethyl)-piperazine and thioridazine on efflux activity, biofilm formation, and lipid biosynthesis associated with log-phase growth and biofilm formation in <i>M. smegmatis.</i> We utilized metabolic radiolabeling methods using ¹⁴C-palmitic acid and ¹⁴C-acetic acid which are precursors of lipid biosynthesis and analyzed the lipids by silica-thin layer chromatography and autoradiography. Our studies revealed that CNMs do not influence efflux activity. However, efflux pump inhibitors effectively block efflux activity in <i>M. smegmatis</i>. Biofilm formation was decreased by CNMs and EPIs. In biofilm cells, fullertubes increased the incorporation of radiolabeled ¹⁴C-palmitic acid into glycopeptidolipids on the cell surface as well as inside the cell. Piperine and berberine affected the incorporation of the radiolabels into lipids such as trehalose monomycolate, phosphatidylethanolamine and cardiolipin in planktonic and biofilm cells. Our study provides insights into the diverse effects of CNMs and efflux pump inhibitors on <i>M. smegmatis</i>.</p>

Homologous genetic recombination, because of its fundamental roles in the maintenance of genome stability and evolution, is an essential cellular function common to all organisms. This process also plays important roles in the repair of damaged DNA molecules, generation of genetic diversity and proper segregation of chromosomes. The genetic exchange is a highly orchestrated process that entails a plethora of control mechanisms and a large number of proteins, of which RecA and SSB are two proteins that have been chosen for further investigation(s) in the present study. In addition, we have also investigated the interaction between SSB and UvrD1, which plays an important role in DNA repair pathways, especially nucleotide excision repair (NER) and mismatch repair as well as DNA replication and recombination. Chapter 1 reviews the literature regarding various aspects of homologous recombination, with an emphasis on the biochemical and the biophysical aspects of RecA and SSB proteins. In addition, it provides an overview of the study of DNA repair and recombination in mycobacteria. RecA protein is ubiquitous and well conserved among bacterial species. Many archaeal species possess two RecA homologues (RadA and RadB) and eukarya possess multiple homologues of RecA including, Rad51, Rad51B, Rad51C, Rad51D, DMC1, XRCC2, or XRCC3. RecA or its homologues function as polymers, consisting of hundreds of monomers that cooperatively polymerize on single-stranded DNA to form a nucleoprotein filament. E. coli RecA protein participates in Trans Lesion Synthesis (TLS) of DNA and forms the minimal mutasome in association with DNA polymerase V (UmuD’2C). The fundamental mechanism underlying HR, i.e. DNA strand exchange, is one of the most fascinating examples of molecular recognition and exchange between biological macromolecules. Since the isolation of E. coli recA gene and the subsequent purification of its gene product and also from other organisms, RecA protein has been studied extensively for more than three decades. E. coli RecA protein has pivotal roles in DNA recombination and repair, and binding to DNA in the presence of ATP, is a fundamental property of RecA protein resulting in the formation of a nucleoprotein filament. This is the slow step of the HR process, and is considerably faster on ssDNA than on duplex DNA. Binding of RecA to dsDNA is slower at physiological pH, is accelerated at acidic pH, and the lag in binding at the higher pH values is due to slow nucleation. The ATP and the DNA binding functions of RecA display allosteric interaction such that ATP- binding leads to an increase in affinity to ssDNA-binding and vice-versa. X-ray structures of E. coli RecA complexed with nucleotide cofactors have implicated a highly conserved Gln196 in Mycobacterium smegmatis RecA in the coupling of ATP and the DNA binding domains. The carboxyamide group of Gln196 makes an H-bond with the γ-phosphate group of ATP and the side chain of this residue is observed to move by approximately 2Å towards the ATP, relative to the other residues involved in ATP binding. In addition, a highly conserved Arg198 has also been postulated to interact with the γ-phosphate group of bound ATP and position it for a nucleophilic attack by a conserved residue-Glu96 leading to ATP hydrolyses. To elucidate the role of Gln196 and Arg198 in the allosteric modulation of RecA functions, we generated MsRecA variant proteins, where in Gln196 was substituted with alanine, asparagine or glutamate; Arg198 was mutated to a lysine. The biochemical characterization of MsRecA and its variant proteins with the objective of defining the allosteric interaction between the ATP- and the DNA-binding sites has been described with in Chapter 2. We observed that while the mutant MsRecA proteins were proficient in ATP-binding they were deficient in ATP hydrolyses. We assayed for the ability of these proteins to bind ssDNA using either nitrocellulose filter binding or Surface Plasmon Resonance (SPR). While we did not detect any ssDNA-binding by the mutant MsRecA proteins in the filter binding assay, we observed only ten-fold reduction in the affinity for ssDNA as compared to wild type MsRecA protein in MsRecAQ196A, Q196N and R198K in the SPR assay. MsRecA Q196E did not show any binding to ssDNA, in both nitrocellulose filter-binding as well as SPR assays. We assayed for the ability of the mutant RecA proteins for their ability to promote DNA-pairing as well as DNA strand exchange. While we observed limited pairing promoted by the mutant proteins relative to the wild-type MsRecA, we observed a complete abrogation of strand exchange in the case of mutant proteins. In addition, we assayed for the co-protease function of MsRecA, by monitoring the cleavage of MtLexA. We observed that only the wild-type MsRecA protein was able to cleave MtLexA, while none of the mutant RecA proteins were able to do so. In order to understand the differences observed between the wild -type and the mutant MsRecA proteins, we analyzed the conformational state of MsRecA and its variant proteins by circular dichroism spectroscopy upon ATP-binding. We observed that while MsRecA and MsRecAQ196N displayed a reduction in the absorbance at 220 nm upon ATP binding, we did not observe any such structural transitions in the other mutant MsRecA proteins that we tested. Based on our observations and the crystal structure of E. coli RecA bound to ssDNA, in Chapter 2, we propose a dual role for the Gln196 and Arg198 in modulating RecA activities. In the presynaptic filament Gln196 and Arg198 sense the presence of the nucleotide in the nucleotide binding pocket and initiate a series of conformation changes that culminate in the transition to an active RecA nucleoprotein filament. In the active RecA nucleoprotein filament these residues are repositioned such that they now form a part of the protomer-protomer interface. As such they perform two vital functions; they stabilize the protomer-protomer interface by participating in the formation of hydrogen bonds that span the interface as well transmit the wave of ATP hydrolysis across the interface leading to a coordinated hydrolyses of ATP essential for the heteroduplex extension phase of strand exchange reaction. The members of the super family of single stranded DNA binding proteins (SSB) play an important role in all aspects of DNA metabolism including DNA replication, repair, transcription and recombination. Prokaryotic SSBs bind ssDNA with high affinity and generally with positive cooperativity. Several lines of evidence suggest that prokaryotic SSBs are modularly organized into three distinct domains: the N-terminal DNA binding domain and acidic C-terminal domain are linked by a flexible spacer. Studies from our laboratory have revealed that M. smegmatis SSB plays a concerted role in recombination-like activities promoted by the cognate RecA. The C- terminal of SSB is known to be involved in its ability to interact with other proteins. We have previously reported that the C-terminal domain of M. smegmatis SSB, which is not essential for interaction with DNA, is the site for the binding of cognate RecA. The data in Chapter 3 describes the characterization of the SSB C-terminus with the objective of delineating the elements responsible for mediating protein-protein interaction, as well as to define the mechanism by which SSB is able to modulate the activities of RecA. To map the RecA interaction domain of SSB we created deletion mutants in MsSSB lacking 5, 10, 15 or 20 residues from the C-terminal. The truncated SSB proteins were expressed with a His- tag at the N- terminus and purified to homogeneity using a Ni-NTA affinity matrix. We observed unlike MsSSB, MsSSB∆C5 and MsSSB∆C10, MsSSB∆C15 and MsSSB∆C20 were unable to support three-strand exchange catalyzed by MsRecA. Based on the observation that interaction with SSB is essential for MsRecA to catalyze the strand Exchange reaction, we postulate that the RecA interacting domain of SSB is situated between the 15th and the 20th residue from the C-terminal. Further, the C-terminal of MsSSB modulates the transitions between DNA binding modes. Unlike the case with EcSSB where deletion of the last 8 residues from the C-terminal stabilizes the (SSB)35 mode of ssDNA binding, we observe that in case of MsSSB the deletion of C-terminal seems to destabilize the (SSB)35. In addition, the transition from the low density binding mode to a high density mode involves the formation of several intermediates when the C-terminal residues are deleted. With the objective of understanding the functions to the C-terminal of SSB independent of its DNA-binding domain in modulating RecA functions, we employed a peptide corresponding to the 35 residues from the C-terminal of the MsSSB. We observed that the C-terminal region alone is capable of interacting with RecA. In addition we also observed that the C-terminal domain of SSB stimulates RecA functions independent of its DNA binding domain. To address the question, whether the stimulatory effect of the C-terminal domain of SSB in the absence of its DNA-binding domain is restricted to RecA or is a generalized phenomenon associated with all SSB interacting proteins; we tested the effect of C-terminal domain of SSB on UvrD which is known to interact with SSB. UvrD participates in several pathways of DNA metabolism, which include the nucleotide excision repair (NER) and mismatch repair pathway, replication and recombination. Genetic evidence suggests that UvrD and SSB interact in vivo. We tested the effect of mycobacterial SSB on M. tuberculosis UvrD1 (MtUvrD1) functions in vitro. We observe that MtUvrd1 physically interacts with SSB. Further, presence of SSB has an inhibitory effect on the helicase activity of MtUvrD1 and that this effect is dependent on the C-terminal region as the deletion of residues from the C-terminal of SSB abrogates the inhibitory effect of SSB. However, unlike RecA, the C-terminal region of SSB alone had no effect on the helicase activity of UvrD1. We also observed that MsSSB has opposing effects on the ATPase activity of MtUvrD1. In the presence of low concentrations of SSB the ATPase activity is enhanced, while we observed an inhibition when the concentration of MsSSB is high. The precise mechanistic details of how SSB is able to act as an accessory protein to RecA, in context of homologous recombination and stimulates its biochemical activities have been a subject of debate. Whereas research from some groups has shown that the stimulatory effect SSB is mediated through its ability to melt DNA secondary structure, thereby allowing RecA to overcome the kinetic barrier imposed by the presence of secondary structure in ssDNA, others postulate that SSB plays a direct role in the stabilization of RecA nucleoprotein filament and prevents its dissociation. Chapter 3 discusses the experimental evidence in favor of the aforesaid models and based on the results of our experiments; we propose that the accessory functions of SSB may be mediated by a mechanism that involves elements of both models. While interaction with SSB can bring about a conformational change in RecA that is reflected in the enhanced levels of strand exchange and co-protease activity, the helix destabilizing function of SSB is essential during heteroduplex extension and to sequester the displaced strand such that it does not participate in any further pairing reactions. The novel finding that we present in Chapter 3 is that the interaction of SSB C-terminal alone has a stimulatory effect upon RecA activities. Furthermore, we observed that M. tuberculosis UvrD1 is a weak interaction partner of SSB. The physical and functional interactions between MsSSB with RecA on the one hand, and MsSSB and UvrD1 on the other highlight different types of cross-talk between the components of HR and DNA repair pathways. In contrast to the results of earlier studies, our results indicate that protein-protein interactions alone between SSB and RecA may modulate the RecA mediated processes of presynapsis, homologous pairing and strand exchange between homologous DNA molecules as well as modulate its co-protease activity. In addition, our studies indicate that a direct protein-protein interaction is responsible for the modulation of UvrD1 activities by SSB.

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