Comparative analysis of genes required for the intracellular survival of Salmonella enterica serovar Typhimurium in murine macrophages and Dictyostelium discoideum
Salmonella is an intracellular pathogen that causes a variety of illnesses ranging from self-limiting gastroenteritis to severe systemic infections that can cause the death of the host. Once inside the organism, these bacteria can cross the epithelial barrier and interact with professional phagocyti...
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| Tipo de recurso: | tesis de maestría |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | Chile |
| OAI Identifier: | oai:repositorio.anid.cl:10533/210999 |
| Acceso en línea: | https://hdl.handle.net/10533/210999 |
| Access Level: | acceso abierto |
| Palabra clave: | Ciencias Naturales Otras Ciencias Naturales |
| Sumario: | Salmonella is an intracellular pathogen that causes a variety of illnesses ranging from self-limiting gastroenteritis to severe systemic infections that can cause the death of the host. Once inside the organism, these bacteria can cross the epithelial barrier and interact with professional phagocytic cells of the innate immune system, causing a local inflammatory response which culminates in the excretion of the pathogen to the environment. The pathogenicity of Salmonella is associated with its ability to survive in macrophages and dendritic cells, which can act as dissemination vectors inside the host. The molecular mechanisms used for these bacteria to survive and replicate in macrophages have been widely studied. However, no in-depth study has been conducted in order to understand the molecular mechanisms required for Salmonella survival in other stages of its life cycle. For instance, in the environment Salmonella interacts with other phagocytic cells that feed on bacteria and fungus. Among these, the amoebae use similar endocytic and degradation mechanisms to those described in innate immune cells. In this thesis, we aimed to identify a common group of genes required for the intracellular survival of Salmonella Typhimurium in murine macrophages and the amoeba Dictyostelium discoideum. To this end, we performed a high-throughput analysis of mutants under negative selection using different mutant libraries. The identification of mutants unable to survive intracellularly in both phagocytic cells was carried out by deep-sequencing. First, we identified 719 mutants of S. Typhimurium under negative selection in murine macrophages. These mutants included genes encoded in pathogenicity islands conserved in the Salmonella genus, genes involved in transport and biosynthesis of amino acids and carbohydrates, genes encoding regulators associated with response to external signals, genes linked to biosynthesis and modification of lipopolysaccharide (LPS) and genes associated to nutritional and oxidative stress, among other. The comparative analysis between the data of this thesis and data obtained in our laboratory that identified mutants with defects in intracellular survival in D. discoideum, allow us the identification of mutants in 213 genes of S. Typhimurium required to survive intracellularly in both phagocytic cells. Within this group, we found genes encoded in Salmonella pathogenicity islands (SPI-1 and SPI-3), genes involved in iron uptake (iroC, iroN and feoB), genes related with response to starvation and acid pH (spoT and adiY) and genes associated to LPS biosynthesis and modification (waaB, waaI, waaJ, waaL, waaZ, wbaC, wbaK, wbaM, wbaN, wbaD, oafA, wzzfepE and genes in the arn operon), among other. To confirm predictions from our comparative analysis, we choose mutants involved in LPS biosynthesis and evaluated their intracellular survival in both infection models. We demonstrated that mutants ΔwaaL, ΔwzzST and ΔarnBCADTEF are deficient in intracellular survival in murine macrophages and D. discoideum. Hence, a complete LPS containing 16 to 35 AgO units (L-AgO) would be necessary for survival of this pathogen in murine macrophages and D. discoideum. Similarly, a modified LPS containing 4-deoxy-aminoarabinose bound to lipid A would contribute to the intracellular survival of S. Typhimurium in both phagocytic cells. Overall, our results constitute a first step towards understanding the molecular mechanisms exploited by S. Typhimurium in order to survive in strikingly different niches such as mammalians and environmental protozoa. |
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