Modified Intracellular Associated Phenotypes

Modified Intracellular-Associated Phenotypes in a Recombinant Salmonella Typhi Expressing S. Typhimurium SPI-3 Sequences
´ Patricio Retamal1, Mario Castillo-Ruiz2, Nicolas A. Villagra2, Juan Morgado2, Guido C. Mora2*
´ 1 Departamento de Medicina Preventiva Animal, Universidad de Chile, Santiago, Chile, 2 Departamento de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chile

AbstractA 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 ownSPI-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.
Citation: Retamal P, Castillo-Ruiz M, Villagra NA, Morgado J, MoraGC (2010) Modified Intracellular-Associated Phenotypes in a Recombinant Salmonella Typhi Expressing S. Typhimurium SPI-3 Sequences. PLoS ONE 5(2): e9394. doi:10.1371/journal.pone.0009394 Editor: Martin G. Marinus, University of Massachusetts Medical School, United States of America Received September 29, 2009; Accepted February 5, 2010; Published February 24, 2010 Copyright: ß 2010 Retamal et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by FONDECYT grant 1060999, UNAB grant 0404 and predoctoral fellowships from CONICYT and PUC. The funders had no role in studydesign, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gmora@unab.cl

Introduction
The Salmonella enterica genome has five DNA regions that are related to pathogenicity and are shared by all serovars, referred to as the Salmonella pathogenicity islands (SPI). Theacquisition of these SPI regions seems to have played a major role in the adaptation of Salmonella to new niches in its vertebrate hosts, supporting many bacterial requirements during infection [1]. One such island, SPI-3, is located in the selC locus of S. enterica and contains ten ORFs [2], some of them have been experimentally associated with virulence functions in this pathogen. This is the case ofthe mgtCB operon, for which there is evidence of involvement in intramacrophage survival and virulence in mice [3–5]. This operon is encoded in all Salmonella serovars within a very conserved SPI-3 region [6]. The mgtC seems to be a pathogenic factor that has been repeatedly acquired by horizontal gene transfer throughout bacterial evolution, because it has also been related to virulence inMycobacterium tuberculosis and Brucella suis [7–9]. Other SPI-3 genes that have a role in pathogenicity are misL and marT. The misL gene encodes an autotransporter protein that is involved in the adhesion of S. enterica serovar Typhimurium (S. Typhimurium) to the extracellular matrix in mice and chicks, thereby acting as an intestinal colonization factor [10,11]. It has also been shown that marT encodes...