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The genus Salmonella includes facultative intracellular pathogens. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans killing about 2,00,000 people globally every year. Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) cause food poisoning in humans. Salmonellae also cause disease in animals of economic importance like poultry and cattle. Treatment of diseases caused by these notorious pathogens is becoming more and more difficult because of the emergence of drug resistant strains. Thus, it is vital to understand the virulence mechanisms of Salmonella which can lead us to potential drug targets and also help us design effective vaccines. Salmonella has evolved many strategies to enter the host, to evade intracellular and extracellular antimicrobial activities of the host and to extract nutrition in the stringent and hostile environment of the host. These strategies have enabled Salmonella to survive and multiply in the host making it a successful pathogen. Present study deals with four such survival strategies of Salmonella. S. Typhimurium causes a systemic disease in mice that is similar to typhoid fever caused by serovar Typhi in humans. This serves as a good model system to study and understand the pathogenesis of Salmonellae. This model system has been used throughout this study. In the present thesis attempts have been made to identify some novel survival strategies of Salmonella. The thesis is divided into five chapters. Chapter 1 gives an introduction into the basic biology of these notorious pathogens. The diseases caused by Salmonellae are introduced in this chapter. Typhoid fever is discussed in detail covering its epidemiology, clinical features, diagnosis, treatment and prevention. Next section covers the virulence determinants of Salmonella. In this section, Salmonella pathogenicity islands are discussed in detail. This chapter concludes with an overview of molecular pathogenesis of Salmonella covering its invasion strategy and its dangerous life inside the host cell. Salmonella stays and multiplies inside a specialized endosomal compartment of the host cell known as Salmonella-containing vacuole (SCV). It is believed that Salmonella multiplies inside SCV resulting in single big vacuole containing multiple bacteria. The results of Chapter 2 challenge this notion. Using transmission electron microscopy and confocal laser scanning microscopy we show that SCV also divides along with the division of Salmonella resulting in multiple SCVs containing single bacterium per vacuole. We also show that this division is mediated by the molecular motor dynein. This chapter concludes with a discussion on the advantages of SCV division with respect to Salmonella. Successful intracellular pathogens must have some strategy either to avoid lysosomal fusion or to endure the toxic molecules of lysosomes. In case of Salmonella, it is well accepted that SCV-lysosome fusion is blocked. However, the exact mechanism of this process is still unclear. The results of Chapter 3 enhance our understanding of this issue. This chapter explores an interesting possibility of Salmonella reducing the lysosomal number and thereby reducing the chances of SCV-lysosome fusion. Using flowcytometry and confocal laser scanning microscopy, we show that Salmonella decreases the number of acidic lysosomes in murine macrophages. Thus, our results suggest that there is an imbalance in the ratio of vacuoles to acidic lysosomes which decreases the probability of SCV-lysosome fusion thereby helping Salmonella avoid lysosomes. Multicellular organisms use various defense strategies to protect themselves from microbial infections; production of antimicrobial peptides (AMPs) is one of them. Being cationic in nature, AMPs interact and cause pores in the bacterial membrane eventually killing the bacteria. Pathogenic micro-organisms like Salmonella have evolved many strategies to counteract the AMPs they encounter upon their entry into the host systems. S Typhimurium genome has a gene cluster consisting of yejA, yejB, yejE and yejF genes which encode a putative ABC transporter. Chapter 4 deals with the detailed characterization of these genes. Our study shows that these genes constitute an operon. We have deleted the yejF gene which encodes the ATPase component of this putative ABC transporter. The ΔyejF strain showed increased sensitivity to AMPs like protamine, melittin, polymyxin B and human defensins and was compromised to proliferate inside activated macrophages and epithelial cells. In murine typhoid model, the ΔyejF strain displayed decreased virulence when infected intragastrically. These findings suggest that the putative transporter encoded by the yejABEF operon is involved in counteracting AMPs and contributes to the virulence of Salmonella. An important biochemical property of Salmonella that distinguishes it from the closely related E. coli is its inability to ferment lactose. In E. coli, lactose fermentation is carried out by the products of lac operon which is regulated by a repressor encoded by lacI. Salmonella does not have the lac operon and lacI. It has been proposed that S.enterica has lost lac region (lacI and lacZYA) during its evolution. Chapter 5 deals with the evolutionary and physiological significance behind the loss of lac region by S.enterica. We show that expression of LacI in S. enterica suppresses its virulence by interfering with the expression of SPI-2 virulence genes. We also observed that the genome of S. bongori which does not have the virulence genes of SPI-2 has a homologue of LacI. Our results suggest that presence of lacI has probably hindered the acquisition of virulence genes of SPI-2 in S. bongori, whereas absence of lacI has facilitated the same in S. enterica making it a successful systemic pathogen. Thus, lacI has played a remarkable role in the evolution of Salmonella virulence. Brief summary of four studies that are not directly related to survival strategies of Salmonella are included in Appendix. First two studies analyze molecular evolution of SPIs to understand the mechanism of host specificity in Salmonella and the last two studies explore the signaling of lipopolysaccharide (LPS) derived from Salmonella.

Salmonella enterica sérovar Typhi (Typhi) est une bactérie pathogène spécifique à l’homme. Typhi est l’agent étiologique de la fièvre typhoïde chez l’humain, causant plus de 16 millions de nouveaux cas par année et plus de 600 000 morts. Il a été démontré que pour causer une infection systémique, Salmonella doit nécessairement survivre dans les macrophages de l'hôte. Paradoxalement, S. enterica sérovar Typhimurium, très apparenté à Typhi (près de 90 % d’homologie), n’a pas la capacité de se disséminer dans l’organisme humain et peut infecter plusieurs espèces animales. Nous avons antérieurement identifié 36 gènes uniques à Typhi (absents chez Typhimurium) situés sur 15 régions différentes et exprimés sélectivement lors de l’infection de macrophages humains. Ainsi, l’une de ces régions a suscité notre attention, soit la région sty4217-4222 et plus particulièrement le produit du gène sty4221, une aminotransférase hypothétique. Ce dernier gène est d’intérêt dû à l’homologie qu’il détient avec une hémolysine connue (Hly) produite par Treponema denticola, possédant elle-même une activité d’aminotransférase. Chez T. denticola, Hly dégrade la cystéine et produit du H2S qui est toxique pour l’hôte. Notre hypothèse est que la spécificité d’hôte et la capacité de produire une infection systémique de Typhi sont dues à l’expression de gènes qui ne se retrouvent pas chez d’autres salmonelles. Le but de cette étude était donc de caractériser le gène sty4221 quant à son activité hémolytique, cytotoxique et tenter de déterminer son rôle dans la virulence de cette bactérie. Le gène sty4221 a été cloné sous le contrôle d’un promoteur inductible à l’arabinose et exprimé par E. coli. L’activité hémolytique du clone a été déterminée par simple observation sur gélose sang. Ce clone a également permis d’observer l’effet cytotoxique du surnageant de culture sur différentes lignées cellulaires, par quantification de la relâche de LDH. Le gène sty4221 a été muté chez la souche sauvage de Typhi, ISP1820, l’implication pathogénique du gène a ainsi pu être étudiée. Des tests de phagocytose, d’invasion et de survie dans des macrophages humains ont été effectués, ainsi que des tests d’adhésion et d’invasion sur des cellules HeLa. Par ailleurs, une première tentative de purification de la protéine a été entreprise. En somme, nous savons maintenant que STY4221 a des propriétés hémolytiques, augmentées par la présence de cystéine. De plus, STY4221 a un effet cytotoxique sur les macrophages THP-I, mais aucun effet sur les HeLa. Or, sty4221 ne semble pas impliqué dans les étapes d’adhésion, d’invasion, de phagocytose ou de survie. La caractérisation de sty4221 permettra sans doute d’approfondir nos connaissances sur les toxines trouvées uniquement chez Typhi. / Salmonella enterica serovar Typhi (Typhi) is a human restricted pathogen causing typhoid fever, a systemic infection. Annually, at least 16 million new cases with 600, 000 associated deaths are reported. It has been demonstrated that Salmonella has to survive in the macrophages of its host, in order to produce a systemic disease. This ability to cause a disseminated infection in human is unique to Typhi. Our laboratory had isolated 36 genes that were unique to Typhi (absent from Typhimurium’s genome), and that were expressed during human macrophages infection. One of these genes, sty4221, a putative aminotransferase, was of high interest since it shares sequence similarities with a known hemolysin (Hly), which also possesses an aminotransferase activity. That hemolysin is produced by Treponema denticola, it catabolizes cysteine and produces H2S, a toxic metabolite for the host. Our hypothesis is that host specificity and the ability to cause a systemic infection might be explained by the expression of genes that are not found in other salmonellas. The goal of this study was to characterize the gene sty4221, in terms of hemolytic and cytotoxic activity and to determine its role in virulence. The sty4221gene has been cloned in a vector under an arabinose inducible promoter and transformed in a strain of E. coli. The hemolytic activity has been investigated on blood-agar medium. To evaluate the cytotoxicity of the STY4221 protein on human cultured cells, direct observation by photonic microscopy was done. The cytotoxicity activity on human cultured cells has been quantitatively measured with a lactate dehydrogenase release assay. Moreover, the sty4221 gene has been deleted in order to study its implication in the infection and the survival within human macrophages and for adhesion/invasion on epithelial. Protein purification was also attempted. We now know that protein STY4221 has a hemolytic activity that is enhanced by cysteine. Also, we proved that the expression of sty4221 has a cytotoxic effect on THP-I macrophages, but not on epithelial HeLa cells. Meanwhile, sty4221 does not seem to be important during adhesion, invasion, infection nor survival. The characterization of protein STY4221 might extend the list of known exotoxin of Typhi.

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Agradecemos a compreensão. on 2017-07-26T13:34:20Z (GMT) / Submitted by DIEGO FELIPE ALVES BATISTA null (diegofelipe_vet@hotmail.com) on 2017-07-26T14:07:28Z No. of bitstreams: 1 Tese_Diego_Felipe_Alves_Batista.pdf: 4004591 bytes, checksum: 1de74c2da3ba5ba3e56c6bcf6f9ba6f2 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-07-26T19:26:28Z (GMT) No. of bitstreams: 1 batista_dfa_dr_jabo.pdf: 4004591 bytes, checksum: 1de74c2da3ba5ba3e56c6bcf6f9ba6f2 (MD5) / Made available in DSpace on 2017-07-26T19:26:28Z (GMT). No. of bitstreams: 1 batista_dfa_dr_jabo.pdf: 4004591 bytes, checksum: 1de74c2da3ba5ba3e56c6bcf6f9ba6f2 (MD5) Previous issue date: 2017-07-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O tifo aviário, causado por Salmonella Gallinarum biotipo Gallinarum, é uma infecção caracterizada pela alta mortalidade nos lotes de aves suscetíveis acometidos, enquanto S. Gallinarum biotipo Pullorum, o agente da pulorose, infecta as aves de produção industrial com as quais desenvolve relação mais branda. Ainda é escasso o conhecimento sobre os mecanismos moleculares que sustentam essas diferentes interações patógeno-hospedeiro. Nesse estudo, objetivou-se investigar o efeito de deleção parcial das sequências codificantes dos genes idnT (transportador de L-idonato ou D-gluconato), idnO (5-cetogluconato redutase) e ccmH (heme liase necessária na montagem de citocromos do tipo C) sobre a patogenicidade de S. Gallinarum 287/91 (SG287/91), uma vez que seus ortólogos são pseudogenes conservados em S. Pullorum. Os clones mutantes SG∆idnTO, SG∆ccmH e SG∆ccmHidnTO foram obtidos por meio da técnica de mutação sítio-dirigida, denominada de recombinação Lambda-Red e testados em dois experimentos independentes com aves comerciais semipesadas de postura suscetíveis ao tifo aviário. No 1º experimento não se observou alteração da patogenicidade dos clones mutantes após inoculação oral, pois todos os animais infectados desenvolveram sinais clínicos típicos do tifo aviário e vieram a óbito ao longo de 12 dias pós-infecção (dpi). Apesar dos 100% de mortalidade, as infecções desenvolvidas pelos clones SG∆idnTO e SG∆ccmHidnTO levaram os animais a óbito dentro de 48 horas desde o aparecimento dos sinais clínicos, enquanto SG287/91 o fez em 6 dias, sugerindo aumento da virulência dos clones mutantes. No 2º experimento observou-se que as mutantes invadiram o hospedeiro a partir do intestino, embora as quantidades recuperadas de SG∆idnTO e SG∆ccmHidnTO nos fígados e de SG∆idnTO nos baços, no 5º dpi, foram superiores a de SG287/91, reforçando a hipótese de aumento da virulência dos clones contendo a alteração idnTO. Apesar disso, os níveis de transcrição das citocinas CXCLi2 e IL6 produzidos à infecção por SG∆idnTO e SG∆ccmHidnTO não diferiram nas tonsilas cecais nos 1º e 3º dpi e nos baços no 3º dpi em relação à infecção por SG287/91. Somente SG∆ccmH inclinou-se a estimular a transcrição de CXCLi2 e IL6 nas tonsilas cecais no 1° dpi em relação ao grupo controle, enquanto SG287/91 tendeu a suprimi-la. Porém, não houve suporte estatístico para essa observação. Os níveis de mRNA do IFNγ estavam aumentados para todas as estirpes de S. Gallinarum, mutantes ou não, porém sem diferença estatística entre eles. Os resultados do presente estudo indicam que a ruptura nos genes idnTO, e em menor grau do gene ccmH, poderiam levar a perda de “fitness” em S. Gallinarum, lhes justificando a permanência no genoma desse micro-organismo, ao contrário do que ocorre com S. Pullorum. O estudo da patogenicidade de estirpe de S. Pullorum tendo reconstituídos os genes idnTO e ccmH no seu genoma poderia esclarecer os motivos pelos quais esses foram negativamente selecionados por esse micro-organismo. / Fowl typhoid, caused by Salmonella Gallinarum biovar Gallinarum, is an infectious disease which elicits high mortality into a flock of susceptible birds whereas S. Gallinarum biovar Pullorum, the aetiological agent of pullorum disease, infects poultry of commercial importance with which such a bacterium sets off a more permissive host-pathogen interaction. Little is known about the molecular mechanisms driving these distinct interplays with the host. Herein, we aimed at investigating the effect of partial deletions in the idnT (L-idonate / D-gluconate transporter), idnO (5-ketogluconase reductase) and ccmH (heme liase involved in the c-type cytochrome maturation) coding sequences on S. Gallinarum 287/91 (SG287/91) pathogenicity since they are conserved pseudogenes in S. Pullorum genomes. SG∆idnTO, SG∆ccmH and SG∆ccmHidnTO mutant strains were constructed through a one-step inactivation technique, known as Lambda-Red-mediated recombination, and tested on two independent experiments by using a commercial brown egg-producing layer line susceptible to fowl typhoid. On the experiment 1, no changing was observed in the pathogenicity of the mutant strains upon oral inoculation as the infected animals developed typical fowl typhoid clinical signs and died along 12 days post-infection (dpi). In spite of causing 100% mortality, SG∆idnTO and SG∆ccmHidnTO killed all the animals within 48 hours since the clinical signs appearance while SG287/91 did so in 6 days, indicating an increased virulence by these mutant strains. On the experiment 2 every mutant strain were able to invade the host system from the intestine albeit SG∆idnTO and SG∆ccmHidnTO were recovered from livers and SG∆idnTO alone from spleens at higher numbers than was SG287/91, supporting the hypothesis of increased virulence for those clones harbouring the idnTO mutation. Despite the results above, CXCLi2 and IL6 transcription levels during infection by SG∆idnTO and SG∆ccmHidnTO were similar to that induced by SG287/91 in caecal tonsils at 1 and 3 dpi and in spleens at 3 dpi. In contrast, SG∆ccmH trended to stimulate CXCLi2 and IL6 transcription in caecal tonsils at 1 dpi when compared to the negative, control group whereas SG287/91 tended to suppress it, but no statistical significance was found for such an observation. IFNγ mRNA were augmented for all S. Gallinarum strains, mutant or not, but without statistical difference amongst them. These findings indicate that gene decay into idnTO, and at a lesser extent, into ccmH sequences might lead to the loss of fitness by S. Gallinarum, raising an explanation for their maintenance on this bacterium chromosome when the opposite happens to S. Pullorum. Studying the pathogenicity of a S. Pullorum strain possessing both the idnTO and ccmH genes in its genome could bring to light the reasons whereby such genes were negatively selected by this microorganism. / FAPESP: 2013/22920-4 / FAPESP: 2013/26127-7

X-ray crystallography is one of the most powerful tools for the elucidation of the structure of biological macromolecules such as proteins and viruses. Crystallographic techniques are extensively used for investigations on protein structure, ligand-binding, mechanisms of enzyme catalyzed reactions, protein-protein interactions, role of metal ions in protein structure and function, structure of multi-enzyme complexes and viruses, protein dynamics and for a myriad other problems in structural biology. Crystallographic studies are essential for understanding the intricate details of the mechanism of action of enzymes at molecular level. Understanding the subtle differences between the pathogenic enzymes and host enzymes is necessary for the design of inhibitor molecules that specifically inhibit parasite enzymes. The current thesis deals with the application of biochemical and crystallographic techniques for understanding the structure and function of proteins from two pathogenic organisms – a plant virus Physalis Mottle Virus (PhMV), and a pathogenic bacterium, Salmonella typhimurium and also stress induced proteins from Oryza sativa. The thesis has been divided into seven chapters, with the first four chapters describing the work carried out on PhMV, while the rest of the chapters deal with the studies on stress response proteins from Oryza sativa and Salmonella typhimurium. The first part of the thesis deals with studies on viral capsids. Viruses are obligate parasites that have proteinaceous capsids enclosing the genetic material, which, in the case of small plant viruses, is invariably ss-RNA. X-ray diffraction studies on single crystals of viruses enable visualization of the structures of intact virus particles at near-atomic resolution. These studies provide detailed information regarding the coat protein folding, molecular interactions between protein subunits, flexibility of the N-and C-terminal segments and their probable importance in viral assembly, role of RNA in capsid assembly, nucleic acid (RNA)-protein interactions, the capsid structure and mechanism of assembly and disassembly. The present thesis deals with the capsid structure and analysis of the coat protein (CP) recombinant mutants of PhMV. Virus assembly, one of the important steps in the life cycle of a virus, involves specific interactions between the structural protein and cognate viral genome. This is a complex process that requires precise protein-protein and protein nucleic acid interactions. In fact, most of the biological functional units such as ribosomes and proteosomes also require highly co-ordinated macromolecular interactions for their functional expression. Viruses being simple in their architecture, serve as excellent model systems to understand mechanism of macromolecular assembly and provide necessary information for the development of antiviral therapeutics, especially in animal viruses. PhMV is a plant virus infecting several members of Solanaceae family. It belongs to the tymoviridae group of single stranded RNA viruses. Its genome is encapsidated in a shell comprising of 180 (architecture based on T = 3 icosahedral lattice) chemically identical coat protein (CP) subunits (~ 20,000Da) arranged with icosahedral symmetry. In an earlier phase of work, PhMV purified from infected plant leaves was crystallized in the space group R3 (a = 294.56 Å,  = 59.86). X-ray diffraction data to 3.8 Å resolution were recorded on films by screenless oscillation photography. Using this data of severely limited quality and poor completion (40%), the structure PhMV was determined by molecular replacement using the related turnip yellow mosaic virus (TYMV) structure as the phasing model. There was therefore a need to re-determine and improve the structure, which could be useful for understanding the earlier detailed studies on its biophysical properties. As a continuation of these studies, the present investigations were conceived with the goal of determining the natural top and bottom component capsid structures of PhMV. Investigations were also carried out to examine the possibility of enhancing the diffraction quality of PhMV crystals. The thesis begins with a review of the current literature on the available crystal structures of viruses and their implications for capsid assembly (chapter I). All experimental and computational methods used during the course of investigations are described in chapter II, as most of these are applicable to all the structure determinations and analyses. The experimental procedures described 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 analysis. Chapter III describes structural studies on top and bottom components of PhMV. Purified tymoviruses including PhMV are found to contain two classes of particles that sediment at different velocities through sucrose gradients and are called the top (sedimentation coefficient 54 Svedberg units(S)) and the bottom (115S) components. The top component particles are either devoid of RNA or contain only a small subgenomic RNA (5%) while the bottom component particles contain the full length genomic RNA. Only the bottom component is infectious. The top and bottom components were separately crystallized in P1 and R3 space groups, respectively. It is of interest to note that crystals of the bottom component obtained earlier belonged to R3 space group while recombinant capsids that lack of full length RNA as in natural top component crystallized in the P1 space group. A polyalanine model of the homologous TYMV was used as the phasing model to determine the structures of these particles by molecular replacement using the program AMoRe. The refinement of top and bottom component capsid structures were carried out using CNS version 1.1 and the polypeptide models were built into the final electron-density map using the interactive graphics program O. The quality of the map was sufficient for building the model and unambiguous positioning of the side chains. There is a significant difference in the radius of the top and bottom component capsids, the top component being 5 Å larger in radius. Thus, RNA makes the capsid more compact, even though RNA is not a pre-requisite for capsid assembly. Partially ordered RNA was observed in the bottom component. The refined models could form the basis for understanding the architecture, protein-protein interactions, protein-nucleic acid interactions, stability and assembly of PhMV. Chapter IV provides a detailed description of the mutations carried out on PhMV coat protein towards enhancing the diffraction quality of crystals. The gene coding for PhMV coat protein (PhMVCP) and several of its deletion and substitution mutants were originally cloned in pRSETC and pET-21 vectors by Mira Sastri and Uma Shankar in Prof. Savithri’s laboratory at the Department of Biochemistry. It was observed that the recombinant intact coat protein and several mutants lacking up to 30 amino acids from the N-terminal end could assemble into empty shells resembling the natural top component. None of these deletion mutants crystallized in forms that diffracted to high resolution. Based on the intersubunit contacts observed, three more site-specific mutants were designed. These three mutants were expressed in BL21 (DE3), purified and crystallized. Even these mutant crystals did not diffract to high resolution. The polypeptide fold of PhMV coat protein therefore was carefully examined for probable reasons. It was found that PhMV subunit has three major cavities. Three cavities are likely to increase the flexibility of protein subunits, which in turn may result in crystals of poor quality. Mutations V52W, S158Q and A160L were shown to fill up these cavities and with the view of obtaining better crystals. These site specific mutations were carried out the mutant proteins were purified. It was shown that the recombinant capsids are stable and possess T=3 architecture. Two mutants were crystallized and a data set for V52W extending to 6.0 Å resolution could be collected. Due to the limited resolution, further work was not pursued. It is plausible that the triple mutant will diffract to higher resolution. The second part of the thesis deals with stress response proteins from Oryza sativa and Salmonella typhimurium. It is known that viral infection and abiotic and biotic stresses induce a network of proteins in plants. Chapter V presents a review of the current literature on stress proteins, focusing mainly on Oryza sativa and S. typhimurium stress response proteins. Chapter VI describes the over expression of stress proteins SAP1 and SAP2 from rice. These stress related proteins confer tolerance to cold, dehydration and salt stress in rice. These proteins have been cloned in the expression vector pEt-28(a) and expressed in E. coli strain BL21 CodonPlus(DE3)RIL. The proteins were purified and crystallization trials were made. However, there were no hits. In an attempt to get crystals, nine deletion constructs of SAP1 were designed eliminating potentially disordered and unfolded regions based on a bioinformatics analysis. Crystallization trails are being carried out on three of the constructs. Structural studies on a universal stress protein from Salmonella typhimurium, which shares homology with the rice universal stress proteins, was initiated. Apart from this, several other stress related proteins of Salmonella typhimurium have also been selected for structural and functional studies. These include YdaA, YbdQ, Yic, Ynaf, Yec, Spy and Usb. All these were cloned and expressed in E. coli. Out of seven proteins, Ynaf, YdaA and YbdQ were found in the soluble fraction and were expressed in quantities suitable for structural studies. I could crystallize YdaA and Ynaf. X-ray diffraction data to resolutions of 3.6 Å and 2.3 Å were collected on crystals of YdaA and YnaF, respectively. A tentative structure of YnaF has been obtained. Further attempts to determine these structures are in progress. Biophysical, Biochemical functional characterization of YdaA and YnaF proteins are described. Structural studies on mannose-6-phosphate isomerase, an enzyme related to stress regulatory proteins from S. typhimurium are dealt with in Chapter VII. Mannose 6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The structure could be solved in its apo and holo forms (with two different metal atoms, Y3+ and Zn2+), and complexed with the cyclic form of the substrate fructose 6-phosphate (F6P) and Zn2+. Isomerization involves acid/base catalysis with proton transfer between C1 and C2 atoms of the substrate. Lys 132, His 131, His 99 and Asp 270 are close to the substrate and are likely to be the residues involved in proton transfer. Interactions observed at the active site suggest that the ring opening step is catalyzed by His 99 and Asp 270. An active site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. The metal ion is not close to the substrate atoms involved in proton transfer. Binding of the metal induces structural order in the loop consisting of residues 50-54. Hence, the metal atom does not appear to play a direct role in catalysis, but is probably important for maintaining the architecture of the active site. Based on these structures and earlier biochemical work, a probable isomerization mechanism has been proposed. The thesis concludes with a brief discussion on the future prospects of the work. The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis:

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I performed two randomised controlled trials (RCTs) to determine the best treatments for enteric fever in Kathmandu, Nepal, an area with a high proportion of nalidixic acid resistant S. Typhi and S. Paratyphi A isolates. I recruited 844 patients with suspected enteric fever to compare chloramphenicol versus gatifloxacin. 352 patients were culture confirmed. 14/175 patients treated with chloramphenicol and 12/177 patients treated with gatifloxacin experienced treatment failure (HR=0.86 (95% CI 0.40 to 1.86), p=0.70). The median times to fever clearance were 3.95 and 3.90 days, respectively (HR=1.06 [CI 0.86 to 1.32], p=0.59). The second RCT compared ofloxacin versus gatifloxacin and recruited 627 patients. Of the 170 patients infected with nalidixic acid resistant strains, the number of patients with treatment failure was 6/83 in the ofloxacin group and 5/87 in the gatifloxacin group (Hazard Ratio, HR=0.81, 95% CI 0.25 to 2.65; p=0.73); the median times to fever clearance were 4.7 and 3.3 days respectively (HR=1.59 [CI 1.16 to 2.18], p=0.004). I compared conventional blood culture against an electricity free culture approach. 66 of 304 patients with suspected enteric fever were positive for S. Typhi or S. Paratyphi A, 55 (85%) isolates were identified by the conventional blood culture and 60 (92%) isolates were identified by the experimental method. The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% and 96.0%, respectively. This electricity free blood culture system may have utility in resource-limited settings or potentially in disaster relief and refugee camps. I performed a literature review of RCTs of enteric fever which showed that trial design varied greatly. I was interested in the perspective of patients and what they regarded as cure. 1,481 patients were interviewed at the start of treatment, 860 (58%) reported that the resolution of fever would mean cure to them. At the completion of treatment, 877/1,448 (60.6%) reported that they felt cured when fever was completely gone. We suggest that fever clearance time is the best surrogate for clinical cure in patients with enteric fever and should be used as the primary outcome in future RCTs for the treatment of enteric fever.

O lançamento de esgotos domésticos in natura, efluentes das estações de tratamento de esgoto e escoamento superficial, são relatados como importantes causas de poluição das águas superficiais. Sabe-se que a alteração da qualidade das águas dos rios restringe seus múltiplos usos e contribui para o aumento de doenças de veiculação hídrica, em decorrência da exposição oral a bioagentes patogênicos. Neste contexto, o objetivo do presente estudo foi identificar e quantificar bioagentes presentes na água do rio Pardo, Brasil, e estimar o risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. para a população, devido ao uso do rio como fonte de abastecimento público e recreação de contato primário, por meio da abordagem da Avaliação Quantitativa de Risco Microbiológico (AQRM). Durante os anos de 2015 e 2016, foram realizadas seis coletas de amostras da água do rio Pardo (período chuvoso e período seco) em seis pontos, totalizando 36 amostras. Foram realizadas análises de identificação e quantificação de E. coli, Salmonella Não Tifóide, Cryptosporidium spp. e Giardia spp. Para estimativa de risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. (AQRM), foram considerados diferentes populações (crianças e adultos), volumes de água ingerido, concentração de (oo)cistos e duração e frequência da exposição, de acordo com o cenário estabelecido. Os valores médios para E. coli variaram de 6,57 x 101 UFC/100 mL a 6,07 x 103 UFC/100 mL, apresentando diferenças estatisticamente significantes (p < 0,05) entre os períodos chuvoso e seco. As densidades de Salmonella Não Tifóide foram baixas (<0,6473 a 1,55 NMP/100 mL), com frequência de 13,9% das amostras positivas, evidenciando a circulação desse patógeno no ambiente. A concentração de (oo)cistos de Cryptosporidium spp. e Giardia spp. variou de <0,1 a 0,4 oocistos/L e <0,1 a 4,4 cistos/L, respectivamente. Para abordagem da AQRM devido a ingestão da água do rio Pardo usada para abastecimento público, a probabilidade anual de infecção por Cryptosporidium spp. e Giardia spp. foi maior para adultos do que para crianças, sendo que na maioria dos pontos apresentou resultados superiores ao risco anual tolerável pela USEPA (1 x 10-4). No que diz respeito ao uso da água do rio Pardo para recreação de contato primário, a probabilidade diária e anual de infecção, bem como a probabilidade de doenças, foi maior para crianças, seguida de adultos/homens e adultos/mulheres. A probabilidade de criptosporidiose e giardíase esteve abaixo do limite tolerável pela USEPA (3,6 x 10-2), exceto no Ponto 4, em que a estimativa de risco de doença por Giardia spp. para crianças esteve acima deste valor. A presença de bioagentes em amostras de água do rio Pardo pode estar relacionada à poluição das águas por fontes pontuais e difusas. Esses achados refletem a importância de priorizar os recursos para implantação e complementação das Estações de Tratamento de Esgoto na UGRHI 4, a fim de prevenir as doenças de veiculação hídrica em populações que utilizam a água do rio Pardo para abastecimento público e recreação de contato primário / The discharge of domestic sewage, effluents of wastewater treatment plants and surface runoff, are reported as important causes of surface water pollution. It is known that the alteration of river water quality restricts its multiple uses and contributes to the increase of waterborne diseases, due to oral exposure to pathogenic bioagents. In this context, the aim of the present study was to identify and quantify bioagents present in Pardo river water, Brazil, and to estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. for the population, due to the use of the river as source of public supply and primary contact recreation, through the approach of Quantitative Microbial Risk Assessment (QMRA). During the years of 2015 and 2016, six samples of water from the Pardo river (rainy and dry season) were collected at six points, totaling 36 samples. Identification and quantification analyzes of E. coli, Non-typhoid Salmonella, Cryptosporidium spp. and Giardia spp. To estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. (QMRA), different populations (children and adults), volumes of ingested water, concentration of (oo) cysts, duration and frequency of exposure were considered according to the established scenario. Mean values for E. coli varied from 6.57 x 101 CFU / 100 mL to 6.07 x 103 CFU / 100 mL, showing statistically significant differences (p <0.05) between the rainy and dry season. Non-typhoid Salmonella densities were low (<0.6473 at 1.55 MPN / 100 mL), with a frequency of 13.9% of the positive samples, evidencing the circulation of this pathogen in the environment. Cryptosporidium spp. and Giardia spp. concentration ranged from <0.1 to 0.4 oocysts / L and <0.1 to 4.4 cysts / L, respectively. In order to approach the QMRA due to the ingestion of Pardo river water used for public supply, the probability of annual infection by Cryptosporidium spp. and Giardia spp. was higher for adults than for children, and in most points presented results higher than the risk tolerable by USEPA (1 x 10-4). Regarding the use of Pardo river water for primary contact recreation, the daily and annual probability of infection, as well as the probability of illness, was higher for children, followed by adults / men and adults / women. The probability of cryptosporidiosis and giardiasis was below the limit tolerable by USEPA (3.6 x 10-2), except in Point 4, where the estimated risk of disease by Giardia spp. for children was above this value. The presence of bioagents in Pardo river water may be related to water pollution by point and diffuse sources. These findings reflect the importance of prioritizing the resources for implementation and complementation of wastewater treatment plants at UGRHI 4, in order to prevent waterborne diseases in populations that use Pardo river water for public supply and primary contact recreation

O lançamento de esgotos domésticos in natura, efluentes das estações de tratamento de esgoto e escoamento superficial, são relatados como importantes causas de poluição das águas superficiais. Sabe-se que a alteração da qualidade das águas dos rios restringe seus múltiplos usos e contribui para o aumento de doenças de veiculação hídrica, em decorrência da exposição oral a bioagentes patogênicos. Neste contexto, o objetivo do presente estudo foi identificar e quantificar bioagentes presentes na água do rio Pardo, Brasil, e estimar o risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. para a população, devido ao uso do rio como fonte de abastecimento público e recreação de contato primário, por meio da abordagem da Avaliação Quantitativa de Risco Microbiológico (AQRM). Durante os anos de 2015 e 2016, foram realizadas seis coletas de amostras da água do rio Pardo (período chuvoso e período seco) em seis pontos, totalizando 36 amostras. Foram realizadas análises de identificação e quantificação de E. coli, Salmonella Não Tifóide, Cryptosporidium spp. e Giardia spp. Para estimativa de risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. (AQRM), foram considerados diferentes populações (crianças e adultos), volumes de água ingerido, concentração de (oo)cistos e duração e frequência da exposição, de acordo com o cenário estabelecido. Os valores médios para E. coli variaram de 6,57 x 101 UFC/100 mL a 6,07 x 103 UFC/100 mL, apresentando diferenças estatisticamente significantes (p < 0,05) entre os períodos chuvoso e seco. As densidades de Salmonella Não Tifóide foram baixas (<0,6473 a 1,55 NMP/100 mL), com frequência de 13,9% das amostras positivas, evidenciando a circulação desse patógeno no ambiente. A concentração de (oo)cistos de Cryptosporidium spp. e Giardia spp. variou de <0,1 a 0,4 oocistos/L e <0,1 a 4,4 cistos/L, respectivamente. Para abordagem da AQRM devido a ingestão da água do rio Pardo usada para abastecimento público, a probabilidade anual de infecção por Cryptosporidium spp. e Giardia spp. foi maior para adultos do que para crianças, sendo que na maioria dos pontos apresentou resultados superiores ao risco anual tolerável pela USEPA (1 x 10-4). No que diz respeito ao uso da água do rio Pardo para recreação de contato primário, a probabilidade diária e anual de infecção, bem como a probabilidade de doenças, foi maior para crianças, seguida de adultos/homens e adultos/mulheres. A probabilidade de criptosporidiose e giardíase esteve abaixo do limite tolerável pela USEPA (3,6 x 10-2), exceto no Ponto 4, em que a estimativa de risco de doença por Giardia spp. para crianças esteve acima deste valor. A presença de bioagentes em amostras de água do rio Pardo pode estar relacionada à poluição das águas por fontes pontuais e difusas. Esses achados refletem a importância de priorizar os recursos para implantação e complementação das Estações de Tratamento de Esgoto na UGRHI 4, a fim de prevenir as doenças de veiculação hídrica em populações que utilizam a água do rio Pardo para abastecimento público e recreação de contato primário / The discharge of domestic sewage, effluents of wastewater treatment plants and surface runoff, are reported as important causes of surface water pollution. It is known that the alteration of river water quality restricts its multiple uses and contributes to the increase of waterborne diseases, due to oral exposure to pathogenic bioagents. In this context, the aim of the present study was to identify and quantify bioagents present in Pardo river water, Brazil, and to estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. for the population, due to the use of the river as source of public supply and primary contact recreation, through the approach of Quantitative Microbial Risk Assessment (QMRA). During the years of 2015 and 2016, six samples of water from the Pardo river (rainy and dry season) were collected at six points, totaling 36 samples. Identification and quantification analyzes of E. coli, Non-typhoid Salmonella, Cryptosporidium spp. and Giardia spp. To estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. (QMRA), different populations (children and adults), volumes of ingested water, concentration of (oo) cysts, duration and frequency of exposure were considered according to the established scenario. Mean values for E. coli varied from 6.57 x 101 CFU / 100 mL to 6.07 x 103 CFU / 100 mL, showing statistically significant differences (p <0.05) between the rainy and dry season. Non-typhoid Salmonella densities were low (<0.6473 at 1.55 MPN / 100 mL), with a frequency of 13.9% of the positive samples, evidencing the circulation of this pathogen in the environment. Cryptosporidium spp. and Giardia spp. concentration ranged from <0.1 to 0.4 oocysts / L and <0.1 to 4.4 cysts / L, respectively. In order to approach the QMRA due to the ingestion of Pardo river water used for public supply, the probability of annual infection by Cryptosporidium spp. and Giardia spp. was higher for adults than for children, and in most points presented results higher than the risk tolerable by USEPA (1 x 10-4). Regarding the use of Pardo river water for primary contact recreation, the daily and annual probability of infection, as well as the probability of illness, was higher for children, followed by adults / men and adults / women. The probability of cryptosporidiosis and giardiasis was below the limit tolerable by USEPA (3.6 x 10-2), except in Point 4, where the estimated risk of disease by Giardia spp. for children was above this value. The presence of bioagents in Pardo river water may be related to water pollution by point and diffuse sources. These findings reflect the importance of prioritizing the resources for implementation and complementation of wastewater treatment plants at UGRHI 4, in order to prevent waterborne diseases in populations that use Pardo river water for public supply and primary contact recreation