Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Magíster en Bioquímica en el área de especialización de Bioquímica Toxicológica y Diagnóstico Molecular / Memoria para optar al Título de Bioquímica / El género Salmonella comprende dos especies, S. enterica y S. bongori, que en conjunto agrupan a más de 2.500 serovares. De éstos, los pertenecientes a S. enterica subespecie enterica son responsables de aproximadamente el 99% de los casos de salmonelosis en animales de sangre caliente. A nivel mundial se producen anualmente millones de casos de salmonelosis en el ser humano y miles de muertes, principalmente en países subdesarrollados. En esta tesis se propuso identificar un conjunto de genes requeridos para la colonización sistémica de un hospedero murino por tres serovares de Salmonella: S. Typhi, S. Typhimurium y S. Enteritidis. Este estudio se realizó mediante un análisis masivo de mutantes bajo selección negativa in vivo. La detección de aquellas mutantes con defectos en la colonización sistémica aguda de ratones BALB/c se realizó mediante hibridaciones comparativas utilizando un microarray genómico de Salmonella. El posterior análisis comparativo de las mutantes bajo selección negativa in vivo en los tres serovares, nos permitió identificar que mutantes en 131 genes serían atenuadas in vivo. Dentro de este grupo identificamos genes codificados en islas de patogenicidad conservadas del género Salmonella, genes necesarios para la biosíntesis de purinas y compuestos aromáticos (aro, pur y gua), genes relacionados con la biosíntesis y modificación del LPS (rfa, rfb) y genes que codifican reguladores globales asociados a patogenicidad (phoP, envZ, rpoN, dam y rsd). Otros genes identificados corresponden a los que codifican el sistema transportador de proteínas Twin-Arginine (tatABC), genes que codifican las diferentes subunidades de una NADH deshidrogenasa (genes nuo); un locus que corresponde a un transportador de péptidos del tipo ABC (sapBF). También pudimos detectar que mutantes en genes involucrados en el transporte de solutos se encuentran bajo selección, como trkH que codifica un transportador de potasio. El sistema de transporte Twin-Arginine corresponde a una de las dos vías de translocación de proteínas hacia el espacio periplasmático en bacterias Gram negativo. La participación de este sistema en la patogenicidad de Salmonella se confirmó mediante ensayos de competencia in vivo entre mutantes definidas del operón y la respectiva cepa silvestre en los tres serovares estudiados. El análisis global de mutantes en tres serovares nos permitió determinar un conjunto de genes comunes necesarios para establecer la colonización sistémica aguda en un hospedero murino. Posteriormente, se confirmó la participación del sistema de transporte de proteínas Tat en la patogenicidad de Salmonella. Los resultados de los ensayos de competencia nos permitieron confirmar la predicción obtenida en el análisis de masivo de mutantes bajo selección negativa in vivo. / The Salmonella genus comprises two species: S. bongori and S. enterica, which can be grouped into more than 2,500 serotypes. Serovars within S. enterica subspecies enterica account for ~99% of all salmonellosis in warm-blooded animals. Worldwide, these organisms are responsible for hundreds of millions of salmonellosis cases and hundreds of thousands of deaths, mainly in underdeveloped countries. In this thesis, we aimed to identify a group of genes required for systemic colonization of a murine host by three Salmonella serotypes: S. Typhi, S. Typhimurium and S. Enteritidis. We used a high-throughput microarray-based screening for mutants with defects in systemic colonization of BALB/c mice. Subsequent comparative analysis of mutants under negative selection in vivo allowed us to identify that mutants in 131 genes are attenuated in the three serotypes under study. Within this group we found genes encoded in some of the pathogenicity islands conserved in the Salmonella genus, genes required for biosynthesis of purines and aromatic compounds (aro, pur and gua), genes related to LPS biosynthesis (rfa and rfb) and genes encoding regulators previously associated with virulence (phoP, envZ, rpoN, dam and rsd). Other genes identified are those encoding the Twin-Arginine transport system (tatABC), genes coding the different subunits of a NADH dehydrogenase (nuo genes) and a locus encoding an ABC peptide transporter (sapBF). We also identified that mutants in genes involved in solute transport (i.e: trkH, that encodes a potassium transporter) are under negative selection in vivo. The Twin-Arginine transport system corresponds to one of the two pathways used by Gram-negative bacteria to translocate proteins to the periplasmatic space. Participation of this system in Salmonella pathogenicity was confirmed in the three serotypes under study by means of in vivo competition assays between targeted mutants of the operon and the corresponding wild-type strains. Overall, the global analysis of mutants under negative selection in vivo in three serotypes of Salmonella allowed us to identify a common group of genes required to establish acute systemic colonization of a murine host. We confirmed the participation of the Tat transport system in the pathogenicity of Salmonella using in vivo competition assays. These results further support the predictions obtained in our global analysis. / Fondecyt

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We demonstrated that infection of HeLa cells, which are non-responsive to flagellin, with wild type Salmonella enterica serotype Typhimurium (S. typhimurium) activated chemokine expression at higher level than S. typhimurium lacking sipAsopABDE2, indicating that the corresponding effector proteins (SipA, SopA, SopB, SopD and SopE2) are required to induce chemokines independent of flagellin. The S. typhimurium sipAsopABDE2 mutant complemented with sipA activated IL-8 expression at significantly higher level than a S. typhimurium sipAsopABDE2 mutant. However, extracellular addition of recombinant SipA failed to induce IL-8. Phosphorylation analyses demonstrated that S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant) induced phosphorylation of CREB1, JUN and p38MAPK, which are proteins involved in IL-8 expression. The contribution of effector proteins to S. typhimurium-induced intracellular Ca2+ mobilization and its role in IL-8 expression and bacterial internalization were also investigated. Our results demonstrated that wild type S. typhimurium significantly increased the amplitude of intracellular Ca2+ beginning 30 sec after infection. However, further analyses of intracellular Ca2+ changes in HeLa cells infected with S. typhimurium mutants indicated no correlation between increased intracellular Ca2+ and IL-8 expression or bacterial internalization. To analyze specific cell populations targeted by wild type S. typhimurium or S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant), laser capture microdissection was performed. Our data indicated that in wild type S. typhimurium-infected bovine Peyer’s patches, high levels of IL-8 were expressed in enterocytes of crypts, whereas Gro-α was expressed in enterocytes of both crypts and absorptive villi. A strain carrying a chromosomal copy of sipA colonized the same cell population as wild type, but induced IL8 and Gro-α in enterocytes of both crypts and absorptive villi. In conclusion, we demonstrated that in vitro S. typhimurium effector proteins induce chemokine expression independent of Ca2+ changes through phosphorylation of proteins related to IL-8 pathway. In vivo, we found higher levels of IL-8 expression in enterocytes of crypts than enterocytes of absorptive villi, although both cell populations contributed to Gro-α expression. These data extend the knowledge of the molecular mechanism by which S. typhimurium induces inflammatory genes by identifying pathogen and host molecules involved in inflammation.

ABSTRACT There are more than 2000 serotypes in the typhoid, septic and enteritidis Salmonella strains. This has long been a perplexity for the differentiation of Salmonella and discrimination from other food poisoning bacteria. In this research, we used polymerase chain reaction method ¡]PCR¡^ to develop a molecular technique for the detection of Salmonella. An invasion factor invA gene had been used to design the PCR primers. The results showed that under specific PCR conditions, many related bacterias such as Shigella, Yersinia, Serratia, Enterobacter, Vibrio and E. coli could be detected only with the primer annealing temperature ranging from 56¢J~66¢J. However, all tested Salmonella strains could be detected above 69¢J. This is potential of being developed into a rapid method for detecting Salmonella strains in food or clinical specimens. In the future, it can be further increased the accuracy of detection in coordination with multi-primer PCR technique or developed into a biochip for the detection of Salmonella.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 30-33).

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xv, 170 p.; also includes graphics. Includes abstract and vita. Advisor: Ahmed E. Yousef, Dept. of Food Science and Nutrition. Includes bibliographical references (p. 145-170).

published_or_final_version / Microbiology / Master / Master of Philosophy