The bacterial cytoskeleton contains structural and functional homologues of actin, tubulin and intermediate filament-like proteins. In Escherichia.coli (E.coli) and Bacillus.subtilis (B.subtilis), the bacterial actin homologue mreB, along with mreC and mreD are all involved in cell shape determination. In silico searches confirmed the presence of actin gene J |: homologues in Salmonella. The putative cytoskeletal genes of Salmonella Typhimurium mreB, mreC, and mreD, he in close proximity to four additional genes named; maf, cafA, yhdP, and tldD. Evidence was provided which to suggest they are part of the mreB operon.

No description available.

No description available.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an intracellular pathogen, causes gastroenteritis in humans and a systemic disease in mice. The ability of Salmonella to replicate inside host cells requires translocation of effector proteins across the vacuolar membrane, mediated by the Salmonella pathogenicity island‐2 (SPI‐2) type three secretion system (T3SS). However, the repertoire of effectors involved in this process has not been defined. The first part of this PhD work focused on SrfJ, a putative effector of the SPI‐2 T3SS with similarity to human lysosomal glucosylceramidase. Expression of its gene was dependent on SsrA/B, a two‐component regulatory system required for expression of most SPI‐2 effector genes. Expression of srfJ was also shown to occur under SPI‐2 T3SS activation conditions. However, there was no detectable secretion or translocation of the protein, although a srfJ mutant strain had an intracellular replication defect in primary bone marrow‐derived macrophages. Using a dual‐fluorescence reporter system that allows direct measurement of intracellular replication, the contribution to replication of individual SPI‐2 T3SS effectors was investigated. The replication kinetics of S. Typhimurium deletion mutants for all known SPI‐2 effectors were measured and compared in mouse bone marrow‐derived macrophages. Several mutant strains with replication defects were identified, thereby revealing that intracellular replication is the result of the contribution of numerous effectors. Two S. Typhimurium polymutant strains were generated whose replication defects closely resemble that of a SPI‐2 T3SS null mutant and are severely attenuated in virulence in vivo. These strains retained an intact T3SS and delivered a CD8+ T cell epitope via the SPI‐2 T3SS into the cytoplasm of infected cells. Since an S. Typhi mutant strain lacking the SPI‐2 T3SS has been shown to be safe and immunogenic in humans, these polymutant strains could have applications in vaccine design, as carrier strains for the delivery of heterologous antigens.

Salmonella species can cause wide-ranging disease from mild food-poisoning enteritis to a systemic, sometimes fatal typhoid infection. These bacteria have evolved to survive in different environments within and outside the host and do so through the regulation of differential gene expression following activation of certain stress response systems. In gram negative bacteria such as Salmonella, envelope stress responses (ESR) are response systems that target stresses affecting components of the cell envelope such as the periplasm and outer membrane proteins. The two best characterised ESRs are the RpoE stress response system and the CpxAR two-component signal transduction system. Two further ESRs, the BaeSR response and the phage shock response have also recently been identified. The intention of this thesis was to characterise the ESR systems of S. Typhimurium to widen our current knowledge of genes involved in these systems and their role in the pathogenesis of S. Typhimurium with the ultimate aim of identifying possible candidate vaccine genes that may be used in future therapeutics against Salmonella infection. Firstly, extensive mutagenesis and phenotypic analysis studies were undertaken to characterise genes thought to be members of the RpoE regulon. Study of the phage shock response was initiated through mutagenesis, characterisation and regulation studies. A microarray experiment was designed in collaboration with colleagues at the Sanger Centre to identify members of the S. Typhimurium CpxAR regulon, with several members of this regulon being characterised further. The structural components of HtrA, an important ESR protein in S. Typhimurium, were analysed and finally work within this thesis was involved in the investigation of potential overlaps between both the RpoE and CpxAR systems. This led to the establishment of preliminary studies to investigate the vaccine potential of the tol - pal genes in S. Typhimurium.

Salmonella is a Gram-negative bacterium that consists of two species; S. enterica and S. bongori. The species S. enterica can be further divided into 6 subspecies and subspecies I is predominantly associated with disease in warm blooded animals and contains over 2,500 antigenically distinct serovars. Each serovar is >90% identical at the DNA level but can infect a different range of hosts and cause different diseases. Poultry are an important reservoir of entry of Salmonella into the human food chain owing to the contamination of their eggs and meat. The molecular mechanisms underlying colonisation of food producing animals with Salmonella are unknown. Fimbrial genes encode proteinaceous surface exposed appendages which have been shown to mediate adhesion of bacterial cells but the precise role for fimbriae in the carriage and virulence of Salmonella is poorly defined. The purpose of this study was to annotate and characterise the fimbrial genes of the poultry-associated S. enterica serovars Enteritidis and Gallinarum and relate this role to host-specificity. The availability of the genome sequences of several strains of S. enterica allowed a comparison of the sequence, location and repertoire of fimbrial genes and although no unique fimbrial genes were identified all serovars possessed a unique repertoire. The host-specific serovars contain a higher number of pseudogenes within fimbrial operons than the ubiquitous serovars and the rate of attrition of fimbrial genes was 3-4 fold higher than the genomic mean. Such gene decay may partially explain the narrowing of host-range of the host-restricted and host-specific serovars. Polymorphisms that may alter transcription were identified along with targets that may be associated with phase variation of the fimbrial genes. Lambda red-mediated homologous recombination was used to construct a panel of S. Enteritidis P125109 and S. Gallinarum 287/91 strains lacking major fimbrial subunit genes which were examined in vitro and in vivo. Several fimbrial subunits played a role in the adherence to and invasion of different cell lines in different growth conditions and the role appeared to be serovar-specific. A mutation in the steA gene impaired interactions with different cell lines in vitro but this phenotype was found to be due to a polar effect on genes downstream of steA. The majority of fimbrial subunits played no significant role in the colonisation of the alimentary tract in an established chicken model. Mutation of the stcA gene resulted in the greatest degree of attenuation in vivo of all of the fimbrial mutants examined. This phenotype was trans-complemented and was not the result of a polar or second-site defect thereby fulfilling molecular Koch’s postulates. The stcA genes therefore play a significant role in the colonisation of the chicken caeca.

A chemically defined medium for Salmonella growth was developed and optimised using supplements of amino acids, nucleosides, vitamins and different carbon sources. The medium developed was compared to commercially available pre-enrichment media BPW and Salmosyst. Growth of Salmonella was significantly higher in BPW and Salmosyst than the medium developed. The amino acid and nucleoside supplement was directly compared with peptone. The results show peptone to be nutritionally superior and promoting better growth of Salmonella. Three different ELISA assays were used to detect Salmonella growing in four different media. The ELISA assay sensitivity was determined and a degree of media interference with the immunoassays was established. Salmonella culture viability was investigated using three different procedures: differential culturing on selective and non-selective media; fluorescence microscopy with BACLIGHT stained cells and flow cytometry analysis of BACLIGHT and BEP stained cells. Flow cytometry was found to be the most consistent, sensitive and rapid procedure for cell viability measurement. Clusters of viable cells unable to grow on solid media and therefore remaining undetectable by cultural methods were identified using flow cytometry. Severely heat injured Salmonella was used to determine media recoverability. The results indicate that media which contain peptone recover injured Salmonella better than chemically defined or other media. Detection of Salmonella was performed using PCR assay after sample pre-enrichment. The amplification of Salmonella DNA extracted using a crude method resulted in an assay sensitivity of 20 Salmonella cells in pure cultures. The specificity of the oligonucleotide primers employed in the PCR assay was confirmed. Non-salmonella organisms present in high numbers interfered with PCR detection of Salmonella. Food components also interfered with PCR amplification and reduced the assay sensitivity. Interference by food components and non-salmonella DNA was eliminated by the use of a 24 hour pre-enrichment followed by a 3 hour secondary enrichment, a rapid DNA extraction and template preparation. Using this system it was possible to detect 3 Salmonella cells per gram of food in the presence of 106 non-salmonella cells within 28 hours.

Découverte il y a plus d’un siècle, Salmonella n’a cessé d’intriguer les chercheurs. Sa capacité à résister à de nombreux antibiotiques est de plus en plus préoccupante. La surveillance de ce pathogène repose sur un typage rapide et discriminant de façon à identifier le plus précocement possible les sources alimentaires contaminées. Les méthodes classiques sont longues, lourdes et non automatisables. Comprendre l’émergence et l’évolution des Salmonella est la clé pour éradiquer ce pathogène resté l’une des premières causes de diarrhées bactériennes d’origine alimentaire dans le monde. Au cours des dernières décennies, des progrès spectaculaires ont été menés dans le monde de la microbiologie avec l’arrivée des séquenceurs de paillasse, passant du traitement d’une dizaine à des centaines de millions de séquences. L’accès facilité aux séquences génomiques et aux outils qui leurs sont dédiés sont devenus une nécessité. Les outils actuellement disponibles ne sont pas assez discriminants pour sous-typer S. enterica sérotype Typhimurium (STM), sérotype prédominant de Salmonella. Nous avons voulu lors de ce travail, montrer l’intérêt du séquençage entier du génome, pour l’étude génomique de Salmonella. (1) Après avoir séquencé plus de 300 génomes de STM, nous avons mis au point un outil de sous-typage in silico de ce sérotype, basé sur le polymorphisme des CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). La surveillance à haut débit des salmonelloses a été validée en routine sur plus de 800 génomes. L’étude de la coévolution entre le chromosome (SNPs) et les régions CRISPR ont permis d’établir une nomenclature définissant les différentes populations de STM. (2) L’analyse génomique de 280 souches historiques de STM a montré que les gènes de bêta-lactamase conférant une résistance à l’ampicilline et portés par des plasmides étaient répandus chez STM à la fin des années 1950, bien avant l’utilisation de cet antibiotique. La présence de la pénicilline G dans le milieu agricole où ces composés ont été utilisés en tant que promoteurs de croissance ont pu conduire à la sélection des premières souches résistantes à l’ampicilline. (3) L’étude phylogénétique d’un génome issu du cadavre d’une femme décédée il y a plus de 800 ans, probablement à cause de la fièvre entérique et de 219 génomes historiques et récents des sérotypes Paratyphi C, Choleraesuis et Typhisuis ont montré que leurs génomes étaient très similaires au cours des 4000 dernières années. Ainsi, la combinaison des approches génotypique et phylogénétique ont accru nos connaissances sur l’évolution de ce pathogène.Mots clés : Séquençage entier du génome, surveillance épidémiologique, CRISPR, SNP, résistance antibiotique, phylogénie, évolution / Over a century has passed since the discovery of Salmonella and yet, this pathogen still intrigues researchers. Its ability to withstand many antibiotics is of increasing concern. The monitoring of this pathogen is based on a rapid and discriminatory typing to identify the sources of contaminated food as early as possible. The conventional methods are long, heavy and non-automatable. Understanding the emergence and evolution of Salmonella is the key to eradicate this pathogen, which has remained one of the leading causes of foodborne bacterial diarrhea in the world. During the last decades, spectacular progress has been made in the world of microbiology with the arrival of workbench sequencers, passing from a dozen to hundreds of millions of sequences processed. Facilitated access to numerous genome sequences and dedicated tools are mandatory. Tools currently available are not sufficiently discriminating for the subtype of S. enterica serotype Typhimurium, a predominant serotype of Salmonella. Throughout this study, we showed the interest of whole genome sequencing, a multidisciplinary tool, for the genomic study of Salmonella. (1) After sequencing over 300 S. enterica serotype Typhimurium genomes, we have developed an in silico subtyping tool for this serotype, based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) polymorphism. High-throughput microbiological monitoring of salmonellosis has been routinely validated on over 800 genomes. The study of coevolution between the chromosome (SNPs of the core genome) and the two CRISPR regions made it possible to establish a nomenclature defining the different populations of this serotype. (2) Genomic analysis of 280 historical strains of S. enterica serotype Typhimurium showed that plasmids carrying beta-lactamase genes, which confer resistance to ampicillin, were widespread within this serotype in the late 1950s, years before ampicillin was first used for clinical purposes. The presence of penicillin G in the farming environment where these compounds were used as growth promoters, may have led to the selection of the first ampicillin-resistant strains. (3) The phylogenetic study of a genome from the corpse of a young woman who died over 800 years ago, probably due to enteric fever, and 219 historical and recent genomes of the serotypes Paratyphi C, Choleraesuis and Typhisuis have shown, despite the differences in host specificity, that their genomes were very similar over the past 4000 years. Thus, the combination of genotypic and phylogenetic approaches has increased our knowledge of the evolution of this pathogen.Key words: Whole genome sequencing, epidemiological monitoring, CRISPR, SNP, antibiotic resistance, phylogeny, evolution