Examinando por Autor "Mora, Guido C."
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Ítem Characterization of MgtC, a virulence factor of Salmonella enterica serovar typhi(Public Library of Science, 2009-04-14) Retamal, Patricio; Castillo-Ruiz, Mario; Mora, Guido C.The MgtC is a virulence factor in Salmonella Typhimurium that is required for growth at low-Mg2+ concentrations and intramacrophage survival. This gene is codified in a conserved region of the Salmonella pathogenicity island 3 (SPI-3), and is also present in the chromosome of other Salmonella serovars. In this study we characterized the MgtC factor in S. Typhi, a human specific pathogen, by using mgtC and SPI-3 mutant strains. We found that MgtC is the most important factor codified in the SPI-3 of S. Typhi for growth in low-Mg2+ media and survival within human cells. In addition, by using reporter genes we determined that the low-Mg2+ concentration, acidic media and PhoP regulator induce mgtC expression in S. Typhi. We suggest that MgtC is the most important virulence factor codified in the SPI-3 of S. Typhi. © 2009 Retamal et al.Ítem Modified intracellular-associated phenotypes in a recombinant Salmonella typhi expressing S. typhimurium SPI-3 sequences(Public Library of Science, 2010-02-24) Retamal, Patricio; Castillo-Ruiz, Mario; Villagra, Nicolás A.; Morgado, Juan; Mora, Guido C.A bioinformatics comparison of Salmonella Pathogenicity Island 3 sequences from S. Typhi and S. Typhimurium serovars showed that ten genes are highly conserved. However three of them are pseudogenes in S. Typhi. Our aim was to understand what functions are lost in S. Typhi due to pseudogenes by constructing a S. Typhi genetic hybrid carrying the SPI-3 region of S. Typhimurium instead of its own SPI-3. We observed that under stressful conditions the hybrid strain showed a clear impairment in resistance to hydrogen peroxide and decreased survival within U937 culture monocytes. We hypothesized that the marT-fidL operon, encoded in SPI-3, was responsible for the new phenotypes because marT is a pseudogen in S. Typhi and has a demonstrated role as a transcriptional regulator in S. Typhimurium. Therefore we cloned and transferred the S. Typhimurium marT-fidL operon into S. Typhi and confirmed that invasion of monocytes was dramatically decreased. Finally, our findings suggest that the genomic and functional differences between SPI-3 sequences have implications in the host specificity of Typhi and Typhimurium serovars. Copyright: © 2010 Retamal et al.Ítem RpoS integrates CRP, Fis, and PhoP signaling pathways to control Salmonella Typhi hlyE expression(BioMed Central Ltd., 2014-05) Jofré, Matías R.; Rodríguez, Leonardo M.; Villagra, Nicolás A.; Hidalgo, Alejandro A.; Mora, Guido C.; Fuentes, Juan A.Abstract Background: SPI-18 is a pathogenicity island found in some Salmonella enterica serovars, including S. Typhi. SPI-18 harbors two ORFs organized into an operon, hlyE and taiA genes, both implicated in virulence. Regarding the hlyE regulation in S. Typhi, it has been reported that RpoS participates as transcriptional up-regulator under low pH and high osmolarity. In addition, CRP down-regulates hlyE expression during exponential growth. Previously, it has been suggested that there is another factor related to catabolite repression, different from CRP, involved in the down-regulation of hlyE. Moreover, PhoP-dependent hlyE up-regulation has been reported in bacteria cultured simultaneously under low pH and low concentration of Mg2+. Nevertheless, the relative contribution of each environmental signal is not completely clear. In this work we aimed to better understand the regulation of hlyE in S. Typhi and the integration of different environmental signals through global regulators. Results: We found that Fis participates as a CRP-independent glucose-dependent down-regulator of hlyE. Also, Fis and CRP seem to exert the repression over hlyE through down-regulating rpoS. Moreover, PhoP up-regulates hlyE expression via rpoS under low pH and low Mg2+ conditions. Conclusions: All these results together show that, at least under the tested conditions, RpoS is the central regulator in the hlyE regulatory network, integrating multiple environmental signals and global regulators.Ítem S. Typhimurium sseJ gene decreases the S. Typhi cytotoxicity toward cultured epithelial cells(BMC, 2010) Trombert, A Nicole; Berrocal, Liliana; Fuentes, Juan A.; Mora, Guido C.Background. Salmonella enterica serovar Typhi and Typhimurium are closely related serovars as indicated by >96% DNA sequence identity between shared genes. Nevertheless, S. Typhi is a strictly human-specific pathogen causing a systemic disease, typhoid fever. In contrast, S. Typhimurium is a broad host range pathogen causing only a self-limited gastroenteritis in immunocompetent humans. We hypothesize that these differences have arisen because some genes are unique to each serovar either gained by horizontal gene transfer or by the loss of gene activity due to mutation, such as pseudogenes. S. Typhi has 5% of genes as pseudogenes, much more than S. Typhimurium which contains 1%. As a consequence, S. Typhi lacks several protein effectors implicated in invasion, proliferation and/or translocation by the type III secretion system that are fully functional proteins in S. Typhimurium. SseJ, one of these effectors, corresponds to an acyltransferase/lipase that participates in SCV biogenesis in human epithelial cell lines and is needed for full virulence of S. Typhimurium. In S. Typhi, sseJ is a pseudogene. Therefore, we suggest that sseJ inactivation in S. Typhi has an important role in the development of the systemic infection. Results. We investigated whether the S. Typhi trans-complemented with the functional sseJ gene from S. Typhimurium (STM) affects the cytotoxicity toward cultured cell lines. It was found that S. Typhi harbouring sseJ STMpresents a similar cytotoxicity level and intracellular retention/proliferation of cultured epithelial cells (HT-29 or HEp-2) as wild type S. Typhimurium. These phenotypes are significantly different from wild type S. Typhi. Conclusions. Based on our results we conclude that the mutation that inactivate the sseJ gene in S. Typhi resulted in evident changes in the behaviour of bacteria in contact with eukaryotic cells, plausibly contributing to the S. Typhi adaptation to the systemic infection in humans. © 2010 Trombert et al; licensee BioMed Central Ltd.Ítem SPI-9 of Salmonella enterica serovar typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture(Microbiology Society, 2016-08) Velásquez, Juan C.; Hidalgo, Alejandro A.; Villagra, Nicolás; Santiviago, Carlos A.; Mora, Guido C.; Fuentes, Juan A.The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.Ítem The Salmonella enterica serovar Typhi tsx gene, encoding a nucleoside-specific porin, is essential for prototrophic growth in the absence of nucleosides(American Society for Microbiology, 2005-10) Bucarey, Sergio A.; Villagra, Nicolás A.; Martinic, Mara P.; Trombert, A. Nicole; Santiviago, Carlos A.; Maulén, Nancy P.; Youderian, Philip; Mora, Guido C.The Salmonella enterica serovar Typhi tsx gene encodes a porin that facilitates the import of nucleosides. When serovar Typhi is grown under anaerobic conditions, Tsx is among the outer membrane proteins whose expression increases dramatically. This increase in expression is due, at least in part, to increased transcription and is dependent on Fnr but not on ArcA. A mutant derivative of serovar Typhi strain STH2370 with a deletion of the tsx gene is an auxotroph that requires either adenosine or thymidine for growth on minimal medium. In contrast, an otherwise isogenic nupG nupC double mutant, defective in the inner membrane nucleoside permeases, is a prototroph. Because anaerobic growth enhances the virulence of serovar Typhi in vitro, we assessed the role that the tsx gene plays in pathogenicity and found that the serovar Typhi STH2370 Δtsx mutant is defective in survival within human macrophage-like U937 cells. To understand why the Δtsx mutant is an auxotroph, we selected for insertions of minitransposon T-POP in the Δtsx genetic background that restored prototrophy. One T-POP insertion that suppressed the Δtsx mutation in the presence of the inducer tetracycline was located upstream of the pyrD gene. The results of reverse transcription-PCR analysis showed that addition of the inducer decreased the rate of pyrD transcription. These results suggest that the Tsx porin and the balance of products of the tsx and pyrD genes play critical roles in membrane assembly and integrity and thus in the virulence of serovar Typhi. Copyright © 2005, American Society for Microbiology. All Rights Reserved.