Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

RfaH is a unique bacterial protein that enhances transcription of a select group of long operons in many Gram-negative bacteria. Operons regulated by RfaH possess an upstream operon polarity suppressor sequence, which recruits the RfaH protein to the RNA polymerase during transcription of genes, most of which are involved in the synthesis of cell-surface features. These include synthesis of the lipopolysaccharide (LPS) core and O-antigen in Salmonella and Escherichia coli, as well as F-plasmid conjugation pilus and capsule in E. coli. LPS is an important virulence factor in many Gram-negative bacteria, and protects Y. pseudotuberculosis against host antimicrobial chemokines. Recently published high-throughput transposon mutant screens have also suggested a role for RfaH in the ability of Y. pseudotuberculosis to colonize mice. However, the role of RfaH in Y. pseudotuberculosis and its descendent Yersinia pestis has not been carefully examined. In these studies we investigated the effect RfaH has on the structure of the LPS in both species at different temperatures. We also identified LPS-synthesis related genes that are regulated by RfaH. We determined the effect of RfaH on bacterial resistance to host defense peptides, and the ability of Y. pseudotuberculosis to colonize mice. We found that the loss of the rfaH gene had different effects in Y. pseudotuberculosis and Y. pestis. Loss of rfaH caused a truncation in the core region in Y. pseudotuberculosis strain IP32953 at both 21°C and 37°C, but only at 37°C in Y. pestis strain KIM6+. Similarly, we found that transcription of individual genes that are predicted to function in core or O-antigen synthesis were downregulated in the rfaH mutant strains in both species, but the impact of rfaH deletion was greater in Y. pseudotuberculosis. When tested for their ability to survive in the presence of antimicrobial peptides, the Y. pseudotuberculosis rfaH deficient bacteria were much more susceptible than wild-type to killing by polymyxin and by the antimicrobial chemokine CCL28. However, the Y. pestis rfaH mutant strain was equally susceptible to CCL28 as the wild-type strain. Infection of mice with Y. pseudotuberculosis show that rfaH deficient bacteria were able to survive as effectively as the wild-type following oral or intravenous inoculation, with or without the pYV virulence plasmid. Overall, our results show that while RfaH controls LPS gene expression in both Y. pseudotuberculosis and Y. pestis, its impact is much greater in Y. pseudotuberculosis. Furthermore, although loss of rfaH greatly reduces the ability of Y. pseudotuberculosis to resist antimicrobial peptides, it is not required for virulence in this species.

Yersinia pseudotuberculosis is a foodborne pathogen that is the ancestral strain to Yersinia pestis, the causative agent of Plague. Y. pseudotuberculosis invades a host through the intestinal epithelium. The bacteria resist mucosal innate immune defenses including antimicrobial chemokines and phagocytic cells, and replicate in local lymph nodes. They cause Tuberculosis-like symptoms, including necrosis of local tissue and granuloma formation. Like all bacteria, Y. pseudotuberculosis has a net negative charge, which contributes to its susceptibility to some cationic antimicrobial peptides. Y. pseudotuberculosis is able to reduce this negative charge by adding 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A portion of lipopolysaccharide. The production and addition of the L-Ara4N is coded for by the pmrHFIJKLM (pmrF) operon. A previous study has shown that the Y. pseudotuberculosis pmrF operon is important for resistance against polymyxin, but is not important for virulence in mice. Several previous reports have shown a strong influence of growth temperature on resistance to antimicrobial peptides and pmrF expression in pathogenic Yersinia species, but these studies also suggest significant variability between species, and even between strains of individual species. In particular, the regulation of the Y. pseudotuberculosis pmrF operon and its effect on bacterial interactions with mucosa-associated antimicrobial chemokines and neutrophils is not understood. In these studies, we investigated the environmental influences on pmrF expression in Y. pseudotuberculosis. We found that the promoter activity of the pmrHFIJKLM operon is increased at lower temperatures (21ºC) and in the presence of human serum. A ΔpmrI mutant strain of Y. pseudotuberculosis defective for addition of L-Ara4N was found to be more susceptible to killing by the antimicrobial chemokine CCL28 compared to wild-type. This suggests that this gene is important in the bacterial defense against antimicrobial chemokines. However, when the ΔpmrI mutant strain was exposed to human neutrophils, there was a decrease in phagocytosis as compared to wild-type bacteria. Our results suggest that the regulation of L-Ara4N modifications in Yersinia is more complex than previously appreciated and varies between species. Addition of L-Ara4N to Y. pseudotuberculosis appears to enhance resistance to some antimicrobial peptides like CCL28 and promote greater phagocytic engulfment by neutrophils. These opposing effects may partly explain why there is no net apparent survival defect in mutants lacking the pmrF operon during infection.

Zugl.: Berlin, Humboldt-Univ., Diss., 2009

No description available.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2001. / Spine title: Yersinia uptake by mammalian cells. Includes bibliographical references (leaves 214-240). Also available online through Digital Dissertations.

Made available in DSpace on 2014-06-11T19:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-22Bitstream added on 2014-06-13T20:04:04Z : No. of bitstreams: 1 monnazzi_lgs_dr_arafcf.pdf: 1628105 bytes, checksum: 439e61c2131f392d13c659297a95020d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / As três espécies patogênicas do gênero Yersinia, Y. pestis, Y. enterocolitica e Y. pseudotuberculosis, compartilham um tropismo pelos tecidos linfóides e um plasmídeo de 70-kb que é essencial para a virulência. O plasmídeo codifica um sistema de secreção do tipo III e proteínas efetoras chamadas Yops (Yersinia outer proteins). Este sistema de secreção é responsável por translocar as Yops para dentro das células do hospedeiro, onde elas interagem com alvos específicos e alteram as funções destas células. As Yops são capazes de modular a resposta imune do hospedeiro, permitindo à bactéria se replicar extracelularmente nos tecidos e órgãos linfóides. Embora haja muita informação sobre os mecanismos usados pela Yersinia para evadir do sistema imune inato de defesa, pouco se sabe sobre como ela afeta a resposta imune adaptativa in vivo. O objetivo deste trabalho foi analisar a influência das Yops E, H e M, translocadas pela Y. pseudotuberculosis, na colonização e persistência da bactéria no baço e fígado dos animais infectados, nas quantidades de LT-CD4 e LT-CD8 durante a infecção e na produção das principais citocinas Th1 e Th2 por estas subpopulações de linfócitos. Além disso, foi verificado o papel destas Yops sobre a ativação do fator nuclear κB (NF-κB) e sobre a atividade citotóxica dos LT-CD8. Para isso, camundongos BALB/c fêmeas foram infectados intravenosamente com a amostra selvagem de Y. pseudotuberculosis (WT), ou com amostras mutantes incapazes de secretar as Yops E, H e M (YopE-, YopH- e YopM-), ou ainda com a amostra curada do plasmídeo de virulência (YpIII). No 5°, 7°, 14° e 21° dia pós-infecção (pi), os animais foram sacrificados e as células esplênicas foram obtidas de camundongos infectados e de camundongos não infectados (grupo controle). Os níveis de colonização no baço e no fígado foram determinados por... / The three pathogenic species of the genus Yersinia, Y. pestis, Y. enterocolitica and Y. pseudotuberculosis, share a tropism for lymphoid tissues and a 70-kb plasmid essential for virulence. The plasmid encodes a type III secretion system and effector proteins called Yops (Yersinia outer proteins). This secretion system is responsible for translocating the Yops into the host cells, where they interact with specific host targets and alter the functions of these cells. Yops are able to modulate the host immune defenses allowing the bacteria to replicate extracellularly in lymphoid tissues and organs. Although there is ample information on the mechanisms used by Yersinia to evade the innate immune system, very little is known about how it affects the adaptive immune response in vivo. The aim of this research was to analyze the influence of translocated Yops E, H and M of Y. pseudotuberculosis on the colonization and persistence of the bacterium in the spleen and liver of infected animals, on the quantities of CD4 and CD8 T cells during the infection and on the production of the main Th1 and Th2 cytokines by these lymphocyte subpopulations. In addition, it was verified the role of these same Yops on the activation of nuclear factor κB (NF- κB) and on the CD8 T cells cytotoxic activity. To this end, female BALB/c mice were infected intravenously with the wild type Y. pseudotuberculosis (WT), or with the mutant strains unable to secrete the Yops E, H and M (YopE-, YopH- and YopM-) or with the plasmid-cured strain (YpIII). On the 5th, 7th, 14th and 21st days post-infection (pi), the animals were sacrificed and the spleen cells were isolated from infected and uninfected mice (control group). The levels of colonization in the spleen and liver were determined by counting the number of colony-forming units. Both the phenotypic analysis of lymphocytes and the intracellular... (Complete abstract click electronic access below)

Humanpatogena stammar av bakterien Yersinia enterocolitica kan orsaka akut gastroenterit. För identifiering av bakterien odlas fecesprover ut på CIN-agar. På senare år har en kromogen agarplatta framtagits som differentierar mellan patogena och apatogena stammar av Y. enterocolitica. Syftet med studien är att jämföra och utvärdera två CIN-agar, med agarbaser och supplement från två olika företag (Liofilchem och Oxoid), och CHROMagar Y. enterocolitica (CHROMagar). Odling av fecesprover samt seriespädning av sex Y. enterocolitica stammar och en Y. pseudotuberculosis utfördes. Vid utodlade fecesprover jämfördes växt och hämning av övriga bakterier. Vid seriespädning räknades antal kolonier på plattorna för respektive spädning, samt utseende av kolonier på plattor bedömdes. Resultatet tyder på att skillnad av hämningseffekt av Y. enterocolitica och utseende på kolonierna finns mellan de två CIN-agarplattorna. Oxoid’s CIN-agar erhöll större kolonier, lägre hämningseffekt av Y. enterocolitica och detektionsgräns än Liofilchem’s CIN-agar. På CHROMagar-plattan växte de patogena stammarna med bleklila kolonier och de apatogena stammarna med blåa kolonier. Hämningseffekt av Y. enterocolitica hos CHROMagar-plattan är densamma som Oxoid’s CIN-agar. Slutsatsen är således att Oxoid’s CIN-agar och CHROMagar har samma hämningseffekt av Y. enterocolitica men CHROMagar differentierar mellan patogena och apatogena stammar. Liofilchem’s CIN-agar har högre hämningseffekt än CHROMagar och Oxoid’s CIN-agar. / Pathogenic strains of Yersinia enterocolitica can cause acute gastroenteritis in humans. To identify the bacterium, cultivation of stool samples on CIN-agar are performed. A chromogenic medium has been developed that differentiate between pathogenic and nonpathogenic strains of Y. enterocolitica. The purpose is to compare and evaluate two CIN-agar, with agar bases and supplements from two companies (Liofilchem and Oxoid), and CHROMagar Y. enterocolitica (CHROMagar). Growth of stool samples and serial dilutions of six Y. enterocolitica strains and one strain of Y. pseudotuberculosis were performed. Comparisons of the growth and inhibition of other bacteria were done for the stool samples. Colonies for each dilution were counted and appearance of the colonies was evaluated. The result indicates that a difference in inhibitory effect on Y. enterocolitica and appearance of colonies exist between the two CIN-agar. All strains grew with larger colonies on Oxoid CIN-agar than on Liofilchem’s. Oxoid CIN-agar and CHROMagar have a lower inhibitory effect on Y. enterocolitica than Liofilchem’s. On CHROMagar, the pathogenic strains grew with mauve colonies, whilst the nonpathogenic strains grew with blue colonies. Thus, the conclusion is that CHROMagar and Oxoid CIN-agar have less inhibitory effect on Y. enterocolitica than Liofilchem’s. CHROMagar can differentiate between pathogenic and nonpathogenic strains.

Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compounds