Made available in DSpace on 2016-06-02T19:02:40Z (GMT). No. of bitstreams: 1 3950.pdf: 2965296 bytes, checksum: 1e00d517bd464f272b8c1a2c9f67ca9c (MD5) Previous issue date: 2011-10-11 / Valée S.A. / Swine erysipelas is among the diseases that causes great economic losses in swine cultures worldwide. The disease is caused by the bacterium Erysipelothrix rhusiopathiae, and the surface protein SpaA is one of its main antigens. Herein, we report studies concerning the development of recombinant vaccines against swine erysipelas based on the SpaA antigen. Protein production for a subunit vaccine formulation was studied in shaken flasks and 5.0 L bioreactors. For this propose, a 1026 bp fragment of the spaA gene was cloned in Escherichia coli cells under the lac promoter control. The recombinant organism (E. coli BL21(DE3) pET28a_spaA) was cultivated in fed batch using complex medium with glycerol as carbon source. Nonconventional induction strategies were evaluated and high protein yield (198 mgprot/gDCW) and productivity values (0.4 gprot/L.h) were reached. The same antigen was cloned for expression and secretion in attenuated Salmonella typhimurium cells to obtain a live bacterial vector for the SpaA antigen. The recombinant lineage was able to express and secrete the SpaA fragment fused to the alpha-hemolysin secretion signal both in vitro and in vivo. High plasmid maintenance was observed in both conditions. The vaccinal vehicle showed to be able to colonize the Peyer patches and to invade the gut epithelial barrier in the inoculated animals. Immunization tests in murine model showed that the recombinant antigen delivered by Salmonella cells inoculated by oral route induced the production of seric IgG antibodies anti-SpaA. According to the literature, these antibodies must be able to promote pathogen opsonization in case of infection, contributing to confer a protective immunity against swine erysipelas to the vaccinated animals. In summary, this work presents contributions to development of subunit vaccines against swine erysipelas, in the form of recombinant protein formulations, or SpaA antigen delivery by attenuated S. typhimurium cells. / A erisipela suína é uma das enfermidades que causam grandes prejuízos na suinocultura em todo o mundo. A doença é causada pela bactéria Erysipelothrix rhusiopathiae, e a proteína de superfície SpaA desse microrganismo é um de seus principais antígenos. Neste trabalho, estudou-se o desenvolvimento de vacinas recombinantes contra a erisipela suína a partir do antígeno SpaA. Avaliou-se a produção de uma vacina de subunidade composta pelo antígeno recombinante, a qual foi estudada em frascos agitados e em biorretores de bancada de 5,0 L. Para isso, um fragmento de 1026 pb do gene spaA foi clonado em células de Escherichia coli sob controle do promotor lac e o organismo recombinante (E. coli BL21(DE3) pET28a_spaA) foi cultivado em batelada alimentada, utilizando-se meio complexo contendo glicerol como fonte de carbono. Estratégias não convencionais de indução foram avaliadas e altos valores de rendimento (198 mgprot/gDCW) e produtividade (0,4 gprot/L.h) da proteína recombinante foram alcançados. O mesmo antígeno foi clonado em um plasmídeo que possibilita a expressão e secreção da proteína recombinante em Salmonella typhimurium atenuada, a fim de se obter um vetor bacteriano vivo para o antígeno em questão. A linhagem recombinante foi capaz de expressar e secretar o fragmento da proteína SpaA fusionado ao sinal de secreção da alfa-hemolisina tanto in vitro quanto in vivo, apresentando alta taxa de manutenção plasmidial nas duas condições. Além disso, o veículo vacinal se mostrou capaz de colonizar as placas de Peyer e de invadir a barreira epitelial do intestino dos animais inoculados. Ensaios de imunização em modelo murino mostraram que a veiculação do antígeno pelas células de Salmonella inoculadas por via oral induziu a produção de anticorpos IgG séricos anti-SpaA, que de acordo com a literatura, devem ser capazes de promover a opsonização do patógeno em caso de infecção, contribuindo para conferir uma imunidade protetora contra a erisipela suína aos animais vacinados. Em suma, este trabalho apresenta contribuições para o desenvolvimento de vacinas de subunidade contra a erisipela suína na forma de uma vacina de proteína recombinante, ou por veiculação do antígeno SpaA por linhagens atenuadas de S. typhimurium.

Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes 1,367 serovars, some of which are commonly isolated from infected birds and mammals. The other subspecies mainly colonize cold blooded animals. Salmonella typhimurium, Salmonella typhiandSalmonella enteritidis are some of the serovars, which belong to s.enterica species. S. typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice. In immuno compromised patients the infection is often fatal if it is not treated with antibiotics. Clinical features of food poisoning include abdominal pain, vomiting, nausea, abdominal cramps, dehydration etc. S. typhi causes typhoid fever in humans. No other host has been identified for this serovar. Main source of infection is contaminated food and water. No age is exempted but it is less common before2 years. Incubation period is 360 days. Clinical features include stepladder type fever, malaise, headache, hepato splenomegaly, coated tongue, Neutrogena etc. It may be fatal if untreated. Among the serovars of Salmonella infecting humans S. typhimurium and S. typhi are the most important. While S. typhimurium infects many host species including birds and mammals, S. typhi is single host adapted and infects only human. The single host adaptation of S. typhi presents it with the need for establishing are servoir of infection in the community which can serve as a source of fresh infection. Also the single host adaptation of S. typhi has made it a highly specialized pathogen which has evolved certain unique genes needed for human colonization at the same time has lost a set of genes which are needed for survival in other hosts and in the highly variable external environment. This has led to the accumulation of a vast number of pseudo genesin S. Typhi. A comparative study of the two serovars is useful in many ways. Due to varied host defense systems encountered by the two serovars owing to different niche of infection the bacterial counter defense mechanisms are also different. By focusing on the differences between genes involved in the bacterial defense of host immune response we can decipher the role played by various genes in combating the antibacterial host response. Chapter 2 The role of TolA and peptidoglycan modification in detergent resistance of pathogenic Salmonella The major Salmonella serovars that infect human are Salmonella enterica serovar Typhi (S.typhi) which cause systemic typhoid and Salmonella enterica serovar Typhimurium (S. typhimurium) which cause gastro enteritis. S. typhi resides in the gall bladder during chronic infection and S .typhimurium infects intestine .Thus both pathogens encounter high concentrations of bile and have developed mechanisms to counter it. The Tol Pal complex spanning the outermembrane and the inner cytoplasmic membrane plays an important role in maintaining the stability of the outer membrane and providing detergent resistance. The tolA gene of S. Typhi Is shorter by 27 aminoacid than S. typhimurium. The tolA gene knockout of S. typhimurium and S. typhi differed in their tritonX resistance behavoiur, morphology and low osmolality tolerance. S. typhi tolA was unable to complement the tolA defect in S. typhimurium which could probably due to the difference in the peptidoglycan layer. An analys is of the peptidoglycan modifying genes of both the serovars revealed that dacD, pbgP, ynhG are different. dacD, pbgP genes are pseudogenes in S. typhi and ynhG has a major deletion in S. typhi. Further studies reveal that a double knockout of dacD and pbpG in S. typhimurium makes it sensitive to low osmolality similar to S. typhi. Based on these results we propose a mechanism, where shortening of TolA increases detergent resistance by bringing the outer membrane into closer contact with the peptidoglycan layer, but this is achieved at the cost of reduced Lpp (Bruan’slipoprotein) peptidoglycan linkage which plays a major role in low osmolality tolerance. The pathogen S. typhi is highly adapted to the human host and cannot infect any other host. The single host adaptation and the need to survive in high concentrations of bile have made S. typhi to acquire higher bile resistance at the cost of lowered osmotic tolerance through shortening TolA and reduced Lpp and peptidoglycan binding. Chapter 3 Development of a DNA vaccine against Salmonella The immune response against Salmonella is multifaceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify aprotective Tcellepitope (s) of Salmonella, as cell mediated immunity conferred by CD8+T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the GenBank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of S. typhimurium and S. typhi. They were subjected to BIMAS and SYFPEITHI analysis to map MHCI and MHC II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire Sop Band SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5fold on day4 and day8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone. Chapter 4 PCR based diagnosis and Serovar Determination of Blood Borne Salmonella Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhiand S. paratyphiA, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphiA. An additional set of primers amplify another region in S. typhi CT18 and S. typhiTy2 corresponding to the region between the genes STY0313 toSTY0316 but which is absent in S.paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying them from clinical is olates of patients from various geographical locations in India, there by showing that this region is potentially stable. These set of primers can also differentiate between S. typhiCT18, S. typhiTy2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivityof95%ascompared to the Widal test which had only 63%. As observed, in certain cases the PCR assay was more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A mutagenicidade do material particulado é atribuída primeiramente aos hidrocarbonetos policíclicos aromáticos (HPA). Investigamos a atividade mutagênica do material particulado (MP2,5) em amostras coletadas em três pontos da cidade do Rio de Janeiro. As coletas foram realizadas com auxílio de um amostrador de grande volume na Avenida Brasil, no campus da Universidade do Estado do Rio de Janeiro e no Túnel Rebouças em filtros de fibra de vidro. Metade de cada filtro foi submetido à extração por sonicação com o solvente diclorometano. Seis HPA foram identificados e quantificados por cromatografia gasosa com espectrometria de massa (GC/MS). Após a análise química as concentrações dos HPA obtidos foram correlacionados ao fatores físicos, além de ser realizado avaliação de risco para cada HPA estudado. Linhagens de Salmonella typhimurium (TA98 e derivadas TA98/1.8-DNP6, YG1021 e YG1024) foram utilizadas no ensaio de mutagenicidade e tratadas (10-50 g/placa) com extrato orgânico na presença e na ausência de metabolização exógena. Células de raiz de cebola foram tratadas com extratos orgânicos nas concentrações (5-25g/mL). A alta umidade encontrada no Túnel Rebouças pode ter influenciado na deposição de cinco dos seis HPA estudados em material particulado. Além disso, em diferentes condições de tráfego, motoristas de ônibus que cruzam a Avenida Brasil e o Rebouças túnel estão expostos ao risco induzidos por HPA na ordem de 10-6. Mutagenicidade foi detectada tanto na presença quanto na ausência de metabolização, para as linhagens YG1021 e YG1024 nos três pontos, sugerindo a presença de nitro e amino derivados de HPA. As amostras do Túnel Rebouças apresentaram os maiores valores para rev/g e rev/m3. Estes resultados podem estar relacionados ao longo trajeto e a restrita ventilação. Efeito citotóxico foi detectado pelo ensaio Allium cepa nos três pontos de monitoramento. Além disso os extratos orgânicos provenientes das coletas da Avenida Brasil, UERJ e do Túnel Rebouças induziram efeito clastogênico em células de raiz de Allium cepa / The mutagenicity of airborne particles from combustion is attributed primarily to polycyclic aromatic hydrocarbons (PAHs). We investigated the mutagenic activity of particulate matter (PM2.5) samples collected from three sites in Rio de Janeiro. Samples were collected using a high-volume sampler at Avenida Brasil, the campus of the Rio de Janeiro State University, and Rebouças tunnel. Half of each filter was submitted to sequential extraction by sonication with dichloromethane. Six PAHs were quantified by gas chromatography/mass spectrometry (GC/MS). Salmonella typhimurium TA98 and the derivative strains TA98/1.8-DNP6, YG1021 and YG1024 used in mutagenicity assays were treated (10-50 g/plate) with and without exogenous metabolization. Onion root cells were treated with organic extracts concentrations (5-25g/mL). The high humidity detected in the Rebouças Tunnel may have influenced the deposition of five of the six PAHs studied in particulate matter. Moreover, in different traffic conditions can put the bus drivers that cross the Brasil Avenue and Rebouças tunnel at the risk of exposure induced by HPA in the order of 10-6. Independently of exogenous metabolization, mutagenicity was detected for strains YG1021 and YG1024 at all the sites, suggesting the presence of nitro and amino derivatives of PAHs. Rebouças tunnel presented the highest values for rev/g and rev/m3. These could be related to the fact that this long, enclosed passageway and restricts ventilation. Cytotoxic effect was detected by the Allium cepa test in the three monitoring sites. Also organic extracts from Brazil Avenue, UERJ and Rebouças Tunnel showed clastogenic effect in Allium cepa roots

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A mutagenicidade do material particulado é atribuída primeiramente aos hidrocarbonetos policíclicos aromáticos (HPA). Investigamos a atividade mutagênica do material particulado (MP2,5) em amostras coletadas em três pontos da cidade do Rio de Janeiro. As coletas foram realizadas com auxílio de um amostrador de grande volume na Avenida Brasil, no campus da Universidade do Estado do Rio de Janeiro e no Túnel Rebouças em filtros de fibra de vidro. Metade de cada filtro foi submetido à extração por sonicação com o solvente diclorometano. Seis HPA foram identificados e quantificados por cromatografia gasosa com espectrometria de massa (GC/MS). Após a análise química as concentrações dos HPA obtidos foram correlacionados ao fatores físicos, além de ser realizado avaliação de risco para cada HPA estudado. Linhagens de Salmonella typhimurium (TA98 e derivadas TA98/1.8-DNP6, YG1021 e YG1024) foram utilizadas no ensaio de mutagenicidade e tratadas (10-50 g/placa) com extrato orgânico na presença e na ausência de metabolização exógena. Células de raiz de cebola foram tratadas com extratos orgânicos nas concentrações (5-25g/mL). A alta umidade encontrada no Túnel Rebouças pode ter influenciado na deposição de cinco dos seis HPA estudados em material particulado. Além disso, em diferentes condições de tráfego, motoristas de ônibus que cruzam a Avenida Brasil e o Rebouças túnel estão expostos ao risco induzidos por HPA na ordem de 10-6. Mutagenicidade foi detectada tanto na presença quanto na ausência de metabolização, para as linhagens YG1021 e YG1024 nos três pontos, sugerindo a presença de nitro e amino derivados de HPA. As amostras do Túnel Rebouças apresentaram os maiores valores para rev/g e rev/m3. Estes resultados podem estar relacionados ao longo trajeto e a restrita ventilação. Efeito citotóxico foi detectado pelo ensaio Allium cepa nos três pontos de monitoramento. Além disso os extratos orgânicos provenientes das coletas da Avenida Brasil, UERJ e do Túnel Rebouças induziram efeito clastogênico em células de raiz de Allium cepa / The mutagenicity of airborne particles from combustion is attributed primarily to polycyclic aromatic hydrocarbons (PAHs). We investigated the mutagenic activity of particulate matter (PM2.5) samples collected from three sites in Rio de Janeiro. Samples were collected using a high-volume sampler at Avenida Brasil, the campus of the Rio de Janeiro State University, and Rebouças tunnel. Half of each filter was submitted to sequential extraction by sonication with dichloromethane. Six PAHs were quantified by gas chromatography/mass spectrometry (GC/MS). Salmonella typhimurium TA98 and the derivative strains TA98/1.8-DNP6, YG1021 and YG1024 used in mutagenicity assays were treated (10-50 g/plate) with and without exogenous metabolization. Onion root cells were treated with organic extracts concentrations (5-25g/mL). The high humidity detected in the Rebouças Tunnel may have influenced the deposition of five of the six PAHs studied in particulate matter. Moreover, in different traffic conditions can put the bus drivers that cross the Brasil Avenue and Rebouças tunnel at the risk of exposure induced by HPA in the order of 10-6. Independently of exogenous metabolization, mutagenicity was detected for strains YG1021 and YG1024 at all the sites, suggesting the presence of nitro and amino derivatives of PAHs. Rebouças tunnel presented the highest values for rev/g and rev/m3. These could be related to the fact that this long, enclosed passageway and restricts ventilation. Cytotoxic effect was detected by the Allium cepa test in the three monitoring sites. Also organic extracts from Brazil Avenue, UERJ and Rebouças Tunnel showed clastogenic effect in Allium cepa roots

Chapter -I Introduction Salmonella is a pathogen well-known for its ability to infect a wide variety of hosts and causes disease ranging from mild gastroenteritis to typhoid fever. During infection, it is exposed to a myriad of conditions; from the aquatic environment, the gut lumen to the phagolysosome. The success of Salmonella as a pathogen lies in its ability to sense each of these environments and adapt itself for survival and proliferation accordingly. This is done mainly via the action of specific two-component systems (TCSs) which sense cues specific to each of these niches and trigger the appropriate transcriptional reprogramming. This reprogramming is best studied for the genes directly known to be involved in virulence. In the case of Salmonella, most of these genes are a part of specific clusters, acquired through horizontal gene transfer, known as Salmonella Pathogenicity Islands (SPIs). Of the various SPIs, the two most important are SPI-1 and SPI-2. SPI-1 is classically involved in orchestrating bacterial invasion of non-phagocytic cells in the gut, allowing the pathogen to invade the host. Furthermore, its role is well characterized in the classic inflammation associated with gastroenteritis. On the other hand, SPI-2 is specialized for survival within the harsh intracellular environment of host cells such as macrophages and epithelial cells. Other important virulence determinants include motility, chemotaxis as well as adhesins. The transcription of these virulence genes is under tight regulation and responsive to environmental conditions. Many small molecules such as short chain fatty acids, pp(p)Gpp, bile and acyl homoserine lactones among others are known to be potent regulators of virulence in Salmonella. Furthermore, the metabolic products of the normal flora in the gut also affect its virulence. Thus the metabolic status, of both the host as well as the pathogen, plays an important role in determining the outcome of the infection. Many metabolic enzymes and their products are now known to directly or indirectly affect virulence gene expression. In this study, we explore one such class of metabolic enzymes viz amino acid racemases. They catalyze the chiral conversion of L-amino acids to D-amino acids and vice versa. We have studied the biochemical properties of two such non-canonical racemases as well as their role in bacterial survival and pathogenesis. Chapter-II Identification and characterization of putative aspartate racemases in Salmonella Amino acid racemases, such as alanine and glutamate racemases, are ubiquitously found in all bacteria and they play an essential role in cell wall biosynthesis. Recently it has been found, that bacteria possess other amino acid racemases which produce non-canonical D-amino acids. These D-amino acids, upon secretion, further orchestrate various phenotypes such as cell wall remodeling and biofilm dispersal. In this study, we have explored the ability of Salmonella to produce such non-canonical D-amino acids. The genome of S. Typhimurium possesses genes encoding two putative aspartate racemases; ygeA and aspR. These genes were maximally expressed in mid-log phase of bacterial growth and their corresponding proteins ar localized in the outer membrane of the bacterium. The biochemical characterization of the proteins YgeA and AspR revealed that only the latter is catalytically active under in vitro conditions. AspR could catalyze the conversion of L-Aspartate to D-Aspartate and vice versa, however was unable to use any other amino acid as its substrate. With atleast one of the racemases showing catalytic activity, the profiling of the secreted D-amino acids in Salmonella conditioned medium was undertaken using LC-MS. It was observed that the bacterium actively secreted specific D-amino acids such as D-Ala and D-Met into the culture medium in a growth-phase dependent manner. Furthermore, analysis of the secreted D-amino acid profile of the strains lacking either one or both the racemases revealed that atleast a subset of the secreted D-amino acids were dependent on the activity of YgeA and AspR. Thus, D-amino acids secreted by S. Typhimurium might represent a novel class of signaling molecules. Chapter – III Role of aspartate racemases in growth and survival of S. Typhimurium In order to understand the role of ygeA and aspR in vivo, we created knockouts of these genes (both single as well as double knockout) in S. Typhimurium using λ Red recombinase strategy. These knockouts were then assessed for their growth and morphology. The aspartate racemase knockouts behave similar to the wild type during growth in LB as well as M9 minimal medium. While their gross morphology remained the same as the wild type, the size distribution of the racemase knockouts was slightly different in the stationary phase. Unlike the wild type bacteria, the mutants did not exhibit the characteristic reduction in cell size upon entry into stationary phase. In addition, the survival of the mutants in the presence of cell wall damaging agents such as bile and Triton-X 100 was compromised as compared to the wild type. This can be ascribed to changes in the cell wall of the bacterium, wherein the mutants accumulated peptidoglycan in the stationary phase of growth. This suggests that aspartate racemases might have an effect on cell wall biosynthesis in Salmonella in the stationary phase. Another important strategy employed by bacteria to survive in stress conditions is biofilm formation. It was seen that the mutants were compromised in their ability to form a biofilm at the liquid-air interface in vitro. This defect is due to a transcriptional downregulation of the genes required for biofilm formation. These results demonstrate that, contrary to the established inhibitory effects of D-amino acids on biofilms of various bacteria, the aspartate racemases appear to act as positive regulators of biofilm formation in Salmonella. Chapter – IV Involvement of aspartate racemases in the regulation of Salmonella pathogenesis Salmonella’s success as a pathogen can be broadly assessed by its ability to invade and replicate within two major cell types: epithelial cells and macrophage-like cells. We have studied the fate of the aspartate racemase knockout strains in both these cell types. While the mutants replicate as well as the wild type in macrophage cell lines, their ability to invade epithelial cell lines is highly compromised. This defect can be ascribed to the downregulation of the Salmonella Pathogenicity Island-1 (SPI-1) in the racemase knockouts at the transcriptional level. One of the major pathways that regulate SPI-1 activation is the flagellar pathway. It was observed that in addition to SPI-1, the motility of the racemase mutants was also highly compromised. The mutants did not possess any flagella and showed a high transcriptional downregulation of all the three classes of flagellar genes. Transcriptome analysis revealed a global reprogramming in the aspartate racemase mutants, resulting in the differential regulation of motility, adhesion, amino acid transport, cell wall biosynthesis and other pathways. Of the genes upregulated in the knockouts, FimZ is known for its negative effect on motility and might be responsible for the observed downregulation of the flagellar regulon. This suggests that ygeA and aspR might be repressors of fimbrial gene expression. In totality, the racemases affected the pathogenesis of Salmonella, where the double knockout was severely compromised in the colitis model of infection. Overall the study is the first to identify secretion of non-canonical D-amino acids by Salmonella and suggests that YgeA and AspR might be the source of the same. This is supported in part by in vitro studies with the purified proteins. Studies in vivo further highlight the possible substrates that might be utilized by these enzymes. Physiologically, the aspartate racemases appear to regulate cell wall remodeling and biofilm formation. In contrast to the established literature, aspartate racemases (and their possible D-amino acid products) seem to be essential for formation of biofilms and regulate this phenotype at the transcriptional level. Furthermore, our studies put forth aspartate racemases as novel positive regulators of Flagella and SPI-1, affecting the success of Salmonella in the colitis model of infection in mice. Transcriptome analysis hints at the pleiotropic effects of aspartate racemases in Salmonella, bringing forth hitherto unexplored roles for this class of enzymes in the biology of this pathogen.

The genus Mycobacterium comprises some of the most devastating pathogens that infect humans. Mycobacterium tuberculosis causes tuberculosis in humans leading to high morbidity and mortality. The disease is especially prevalent in the under-developed and developing countries of the tropics. Diseases like AIDS and cancer compromise the immune system of an individual leaving him/her susceptible to secondary infections, particularly of tuberculosis. Thus, tuberculosis is making reappearance even in the well-developed countries of the west. The emergence of multi drug resistant strains of tuberculosis makes this deadly disease difficult to cure. A vaccine against tuberculosis is therefore the need of the hour. Mycobacterium smegmatis is a non-pathogenic member of the same family. It has a relatively fast multiplication time when compared to M. tuberculosis and shares the same unique features of the family that make pathogenic members extremely resistant to chemicals and drugs. Proteins of M. smegmatis and M. tuberculosis share high sequence identities, making M. smegmatis the microorganism of choice to study its more deadly counterpart from the same family. A striking feature of all mycobacterial genomes is the abundance of genes coding for enzymes involved in fatty acid and lipid metabolism; more than 250 in Mycobacterium tuberculosis compared to only 50 in Escherichia coli. The mycobacterial genome codes for over a hundred enzymes involved in fatty acid degradation. Apart from providing energy, lipids and fatty acids also form an integral part of the cell wall and cell membrane of Mycobacteria. The abundance and importance of lipid metabolizing enzymes in Mycobacteria make them attractive targets for drug discovery. It is therefore of interest to biochemically and structurally characterize these enzymes. Thiolases are a group of enzymes that are involved in lipid metabolism. In the last step of the β-oxidation pathway, degradative thiolases catalyze the shortening of fatty acid chains by degrading 3-keto acyl CoA to acetyl CoA and a shortened acyl CoA molecule. Thiolases are a subfamily of the thiolase superfamily. This superfamily also includes the Ketoacyl-(Acyl-carrier-protein)-Synthase (KAS) enzymes, polyketide synthases and chalcone synthases. Most members of this superfamily are dimers and while only a few have been found to be tetramers. The tetramers are loosely held dimers of tight dimers. Examination of the Mycobacterium smegmatis genome revealed the presence of several putative thiolase genes. These genes have been annotated as thiolases on the basis of sequence analysis. However, none of them has been biochemically or structurally characterized. The sequence identity between some of these proteins and the other well-characterized thiolases is rather low. The work described in this thesis attempts to characterize two such enzymes from M. smegmatis structurally and functionally. Chapter 1 begins with a brief introduction to the genus Mycobacteria and the role of fatty acid metabolism in mycobacterial virulence. This is followed by a review of the current literature on the enzymes of the thiolase superfamily and their role in fatty acid metabolism. The chapter concludes with a brief summary on the aims and objectives of the work. Chapter 2 describes all the common experimental procedures and computational methods used during the course of these investigations, as most of them are applicable to all the structure determinations and analyses presented in later chapters. The experimental procedures described include overexpression, purification, site directed mutagenesis, isolation of plasmids, crystallization of proteins and X-ray diffraction data collection. Computational methods include structure determination protocols along with details of various programs used during data processing, structure determination, refinement, model building, structure validation and analysis. Chapter 3 describes the cloning, expression, purification, crystallization and structure determination of a thiolase-like protein (TLP1) from M. smegmatis. All enzymes of the thiolase superfamily that have been structurally characterized so far share four features: 1) conservation of the core α/β/α/β/α-layered structure of the thiolase domain, 2) conservation of the extensive dimerization interface, 3) the location of the active site pocket and conservation of key active site residues and 4) the use of a nucleophilic cysteine residue in catalysis. The crystal structure of MsTLP1 revealed some interesting differences when compared to classical thiolases. Of the four characteristic features of thiolases, MsTLP1 has the conserved thiolase fold. The location of its putative active site is similar to that in classical thiolases. However, the dimerization is not a conserved feature in MsTLP1, which appears to be a monomer in solution as well as in the crystal structure. The ligand binding groove of MsTLP1, identified by structural superposition with Z. ramigera thiolase, is larger than that of Z. ramigera. The absence of the catalytic cysteine suggested that though the protein has the strictly conserved thiolase fold, it might perform an entirely different function. A unique extra C-terminal domain of unknown function present only in MsTLP1 has been described towards the end of the chapter. A thorough sequence and structural analysis suggested that MsTLP1 might belong to a new subfamily in the thiolase superfamily. Chapter 4 describes the attempts made towards the biochemical characterization of MsTLP1. Thiolase assays carried out for the synthetic and degradative reactions revealed that the enzyme is inactive in both the directions. However, surface plasmon resonance binding studies revealed that the protein could bind to Coenzyme A, a feature it shares with other enzymes of the thiolase superfamily. Thorough bioinformatics analyses of the structure to determine the residues involved in CoA binding have also been described. The chapter ends with a discussion on the probable function of TLPs in Mycobacteria. Chapter 5 describes the cloning, expression, purification and X-ray structural studies on MsT1-L thiolase. This is the first structural report of a probable T1-thiolase. The protein crystallized in three different space groups, in all of which the enzyme was found to be in a tetrameric form. Analysis of the tetramer structures from the three different crystal forms revealed that MsT1-L exhibits some rotational flexibility about the central tetramerization loop. A qualitative and quantitative analysis of this movement has been described. Structural comparisons revealed that the overall structure of MsT1-L is very similar to that of the well-characterized biosynthetic thiolase form Z. ramigera. However, a detailed analysis of the ordered waters near the active site cavity revealed interesting differences between the two. The probable functional relevance of this observation has been discussed. The crystal structure of MsT1-L complexed with CoA has also been described in detail. Structural comparisons with classical thiolases also revealed significant differences in the organization of the loop domain that harbors most of the residues required for catalysis. These differences cause the active site cavity of MsT1-L to be larger than that of biosynthetic thiolase suggesting that MsT1-L thiolase could probably bind larger substrates. This cavity is large enough to accommodate a medium chain length fatty acyl CoA as substrate. Co-crystallization experiments with hexanoyl CoA revealed a novel binding site for the fatty acyl chain in MsT1-L and this has been described in detail. Contributions made towards the cloning and expression of other thiolases from S. typhimurium and P. falciparum have been described in Chapters 6 and 7. The thesis concludes with a brief discussion on the future prospects of the investigations presented here.

Three-dimensional structures of proteins provide insights into the mechanisms of macromolecular assembly, enzyme catalysis and mode of activation, substrate-specificity, ligand-binding properties, stability and dynamical features. X-ray crystallography has become the method of choice in structural biology due to the remarkable methodological advances made in the generation of intense X-ray beams with very low divergence, cryocooling methods to prolong useful life of irradiated crystals, sensitive methods of Xray diffraction data collection, automated and fast methods for data processing, advances and automation in methods of computational crystallography, comparative analysis of macromolecular structures along with parallel advances in biochemical and molecular biology methods that allow production of the desired biomolecule in quantities sufficient for X-ray diffraction studies. Advances in molecular biology techniques and genomic data have helped in identifying metabolic pathways responsible for metabolism of short-chain fatty acids (SCFAs). The primary objective of this thesis is application of crystallographic techniques for understanding the structure and function of enzymes involved in the metabolism of SCFAs in S. typhimurium. Pathways chosen for the present study are (i) propionate degradation to pyruvate and succinate by 2-methylcitrate pathway involving gene products of the prp operon, (ii) acetate activation to acetyl-CoA by AckA-Pta pathway involving gene products of the ack-pta operon, (iii) threonine degradation to propionate involving gene products of the tdc operon, (iv) 1,2-propanediol (1,2-PD) degradation to propionate involving gene products of the pdu operon. These metabolic pathways utilize a large number of enzymes with diverse catalytic mechanisms. The main objectives of the work include structural and functional studies on 2-methycitrate synthase (PrpC), acetate kinase (AckA), propionate kinase isoforms (PduW and TdcD) and propanol dehydrogenase (PduQ) from S. typhimurium. In the present work, these proteins were cloned, expressed, purified and characterized. The purified proteins were crystallized using standard methods. The crystals were placed in an X-ray beam and diffraction data were collected and used for the elucidation of structure of the proteins. The structures were subjected to rigorous comparative analysis and the results were complemented with suitable biochemical and biophysical experiments. The thesis begins with a review of the current literature on SCFAs metabolism in bacteria, emphasizing studies carried out on S. typhimurium and the closely related E. coli as well as organisms for which the structure of a homologue has been determined (Chapter 1). Metabolic pathways involving acetate utilization by activation to acetyl- CoA, propionate degradation to pyruvate and succinate, anaerobic degradation of Lthreonine to propionate and, 1,2-PD degradation to propionate are described in this chapter. Common experimental and computational methods used during the course of investigations are described in Chapter 2, as most of these are applicable to all structure determinations and analyses. Experimental procedures described here include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and structural analysis. In Chapter 3, X-ray crystal structure of S. typhimurium 2-methylcitrate synthase (StPrpC; EC 2.3.3.5) determined at 2.4 Å resolution and its functional characterization is reported. StPrpC catalyzes aldol-condensation of oxaloacetate and propionyl-CoA to 2- methylcitrate and CoA in the second step of 2-methylcitrate pathway. StPrpC forms a dimer in solution and utilizes propionyl-CoA more efficiently than acetyl-CoA or butyryl- CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might be responsible for the specificities for acyl-CoAs of these enzymes. Structural comparison with the ligand free (open) and bound (closed) states of CSs showed that StPrpC represents the first apo structure among xvi CS homologs in a nearly closed conformation. StPrpC molecules were organized as decamers, composed of five identical dimer units, in the P1 crystal cell. Higher order oligomerization of StPrpC is likely to be due to high pH (9.0) of the crystallization condition. In gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural and functional studies on S. typhimurium acetate kinase (StAckA; EC 2.7.2.1) are described in Chapter 4. Acetate kinase, an enzyme widely distributed in the bacteria and archaea domains, catalyzes the reversible phosphoryl transfer from ATP to acetate in the presence of a metal ion during acetate metabolism. StAckA catalyzes Mg2+ dependent phosphate transfer from ATP to acetate 10 times more efficiently when compared to propionate. Butyrate was found to inhibit the activity of the enzyme. Kinetic analysis showed that ATP and Mg2+ could be effectively substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. The X-ray crystal structure of StAckA was determined in two different forms at 2.70 Å (Form-I) and 1.90 Å (Form-II) resolutions, respectively. StAckA contains a fold with the topology βββαβαβα, similar to those of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin. StAckA consists of two domains with an active site cleft at the domain interface. Comparison of StAckA structure with those of ligand complexes of other acetokinase family proteins permitted the identification of residues essential for substrate binding and catalysis. Conservation of most of these residues points to both structural and mechanistic similarities between enzymes of this family. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards acetate than propionate. Intriguingly, a major conformational reorganization and partial disorder in a large segment consisting of residues 230-297 of the polypeptide was observed in Form-II. Electron density corresponding to a plausible xvii citrate was observed at a novel binding pocket present at the dimeric interface. Citrate bound at this site might be responsible for the observed disorder in the Form-II structure. A similar ligand binding pocket and residues lining the pocket were also found to be conserved in other structurally known enzymes of acetokinase family. These observations and examination of enzymatic reaction in the presence of citrate and succinate (tricarboxylic acid cycle intermediates) suggested that binding of ligands at this pocket might be important for allosteric regulation in this family of enzymes. Propionate kinase (EC 2.7.2.15) catalyzes reversible conversion of propionylphosphate and ADP to propionate and ATP. S. typhimurium possess two isoforms of propionate kinase, PduW and TdcD, involved in 1,2-propanediol degradation to propionate and in L-threonine degradation to propionate, respectively. In Chapter 5, structural and functional analyses of PduW and TdcD, carried out to gain insights into the substrate-binding pocket and catalytic mechanism of these enzymes, are described. Both isoforms showed broad specificity for utilization of SCFAs (propionate > acetate), nucleotides (ATP ≈ GTP > UTP > CTP) and metal ions (Mg2+ ≈ Mn2+). Molecular modeling of StPduW indicated that the enzyme is likely to adopt a fold similar to other members of acetokinase family. The residues at the active site are well conserved. Differences in the size of hydrophobic pocket where the substrate binds, particularly the replacement of a valine residue in acetate kinases (StAckA: Val93) by an alanine in propionate kinases (StPduW: Ala92; StTdcD: Ala88), could account for the observed greater affinity towards their cognate SCFAs. Crystal structures of TdcD from S. typhimurium in complex with various nucleotides were determined using native StTdcD as the phasing model. Nucleotide complexes of StTdcD provide a structural rationale for the broad specificity of the enzyme for its cofactor. Binding of ethylene glycol close to the γ-phosphate of GTP might suggest a direct in-line transfer mechanism. The thesis concludes with a brief discussion on the future prospects of the work. xviii Projects carried out as part of Master of Science projects and as additional activity during the course of the thesis work are described in three appendices. Analysis of the genomic sequences of E. coli and S. typhimurium has revealed the presence of hpa operon essential for 4-hydroxyphenylacetate (4-HPA) catabolism. S. typhimurium hpaE gene encodes for a 55 kDa polypeptide (StHpaE; EC 1.2.1.60) which catalyzes conversion of 5-carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) to 5-carboxymethyl-2-hydroxymuconic aldehyde (CHMA) in 4-HPA metabolism. Sequence analysis of StHpaE showed that it belongs to aldehyde dehydrogenase (ALDH) superfamily and possesses residues equivalent to the catalytic glutamate and cysteine residues of homologous enzymes (Appendix A). The gene was cloned in pRSET C expression vector and the recombinant protein was purified using Ni-NTA affinity chromatography. The enzyme forms a tetramer in solution and shows catalytic activity toward the substrate analog adipic semialdehyde. Crystal structure of StHpaE revealed that it contains three domains; two dinucleotide-binding domains, a Rossmann-fold type domain, and a small three-stranded β-sheet domain, which is involved in tetrameric interactions. NAD+-bound crystal of StHpaE permitted identification of active site pocket and residues important for ligand anchoring and catalysis. Mutarotases or aldose 1-epimerases (EC 5.1.3.3) play a key role in carbohydrate metabolism by catalyzing the interconversion of α- and β-anomers of sugars. S. typhimurium YeaD (StYeaD), annotated as aldose 1-epimerase, has very low sequence identity with other well characterized mutarotases. In Appendix B, the crystal structure of StYeaD determined in orthorhombic and monoclinic crystal forms at 1.9 Å and 2.5 Å resolutions, respectively are reported. StYeaD possesses a fold similar to those of galactose mutarotases (GalMs). Structural comparison of StYeaD with GalMs has permitted identification of residues involved in catalysis and substrate anchoring. In spite xix of the similar fold and conservation of catalytic residues, minor but significant differences in the substrate binding pocket were observed compared to GalMs. Therefore, the substrate specificity of YeaD like proteins seems to be distinct from those of GalMs. Pepper Vein Banding Virus (PVBV) is a member of the genus potyvirus and infects Solanaceae plants. PVBV is a single-stranded positive-sense RNA virus with a genome-linked viral protein (VPg) covalently attached at the 5'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV VPg was over-expressed in E. coli and purified using Ni-NTA affinity chromatography (Appendix C). PVBV NIb was found to uridylylate Tyr66 of VPg in a templateindependent manner. Studies on N- and C-terminal deletion mutants of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation by PVBV NIb.

The thesis describes structural and functional studies of two PLP-dependent enzymes, diaminopropionate (DAP) ammonia lyase (DAPAL) and N-acetylornithine aminotransferase (AcOAT). The main objective of this work was to understand the structural features that control and impart specificity for PLP-dependent catalysis. DAPAL is a prokaryotic enzyme that catalyzes the degradation of D and L forms of DAP to pyruvate and ammonia. The first crystal structure of DAPAL was determined from Escherichia coli (EcDAPAL) in holo and apo forms, and in complex with various ligands. The structure with a transient reaction intermediate (aminoacrylate-PLP azomethine) bound at the active site was obtained from crystals soaked with substrate, DL-DAP. Apo and holo structures revealed that the region around the active site undergoes transition from disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. Based on the crystal structures and biochemical studies, as well as studies on active site mutant enzymes, a two base mechanism of catalysis involving Asp120 and Lys77 is suggested. AcOAT is an enzyme of arginine biosynthesis pathway that catalyses the reversible conversion of N-acetylglutamate semialdehyde and glutamate to N-acetyl ornithine and α-ketoglutarate. It belongs to subgroup III of fold type I PLP dependent enzymes. Many clinically important aminotransferases belong to the same subgroup and share many structural similarities. We have carried out extensive comparative analysis of these enzymes to identify the unique features important for substrate specificity. Crystal structures of AcOAT from Salmonella typhimurium were determined in presence of two ligands, canaline and gabaculine, which are known to act as general inhibitors for most of the enzymes of this class. There structures provided important insights into the mode of binding of the substrates. The structures illustrated the switching of conformation of an active site glutamate side chain on binding of the two substrates. In addition to that, structural transitions involving three loop regions near the active site were observed in different ligand bound structures. Kinetics of single turnover fast reactions and multiple turnover steady state reactions indicated that N-AcOAT dimer might follow a mechanism involving sequential half site reactivity for efficient catalysis. The changes observed in loop conformation that resulted in asymmetric forms of the dimer enzyme might form the structural basis for half site reactivity. Single site mutants were designed to understand the significance of these structural transitions and the specific role of active site residues in determining substrate specificity and catalysis. Biochemical characterization of wild type and mutant enzymes by steady state and fast kinetic studies, along with their crystal structures provided detailed insights into subtlety of active site features that manifest substrate specificity and catalytic activity. The thesis also describes the investigations on fold type II enzymes directed towards analyses of polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance. Contributions made towards structural and functional studies of three other PLP-dependent enzymes, serine hydoxymethyltransferase (SHMT), D-serine deaminase (DSD) and D-cysteine desulfhydrase (DCyD) are described in an appendix.

La creciente demanda de alimentos con características lo más parecidas al producto fresco, está impulsando el desarrollo de nuevas tecnologías "no térmicas" de conservación. Dentro de las más prometedoras se encuentran el tratamiento por Pulsos Eléctricos de Alta Intensidad (PEF) y la tecnología de Altas Presiones Hidrostáticas (HHP). Estas tecnologías permiten conservar, en mayor medida que los tratamientos térmicos, la calidad (sabor, aroma, color y vitaminas) de determinados alimentos frescos e inactivar microorganismos y enzimas, incrementando su vida útil en refrigeración y facilitando su comercialización. El objetivo general de la presente tesis doctoral ha sido estudiar la posibilidad de procesar por PEF y HHP solos o combinados con calor de una nueva bebida mezcla de zumo de naranja y leche incluyendo aspectos microbiológicos y de calidad. El plan de trabajo comenzó con la elaboración y caracterización físico-química y sensorial del nuevo producto eligiendo la formulación adecuada para desarrollar los estudios cinéticos y de vida útil. Los parámetros de calidad más importantes en los zumos de fruta son la actividad enzimática y el contenido en aroma (concentración de compuestos volátiles). En el caso del zumo de naranja la pectin metil esterasa (PME) es una de las enzimas de mayor importancia. Se evaluó el efecto del tratamiento por PEF, HHP y calor en la inactivación de PME. Todas las tecnologías estudiadas lograron un nivel de inactivación enzimática del 90%. Se observó la aparición de dos fracciones con diferente resistencia al tratamiento, por ello, el modelo bifásico fue el que mejor describió las curvas de inactivación de PME mediante tratamiento combinado de HHP y calor en el producto. Posteriormente se estudió la variación en el contenido en aroma (concentración de compuestos volátiles) tras el tratamiento de HHP, PEF y calor en el producto siendo la tecnología por PEF la que mejor preservó el aroma original del producto fresco. Una vez establecidos / Sampedro Parra, F. (2008). Impacto del tratamiento por pulsos eléctricos de alta intensidad y altas presiones hidrostáticas sobre la calidad y seguridad microbiológica de un alimento mezcla de zumo de naranja y leche [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1992 / Palancia

Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea. Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity. In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation. Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.