Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype typhimurium: identification of intestinal colonization and persistence determinants - PubMed (original) (raw)

Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype typhimurium: identification of intestinal colonization and persistence determinants

Robert A Kingsley et al. Infect Immun. 2003 Feb.

Abstract

The shdA gene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of the Salmonella enterica serotype Typhimurium chromosome. In addition to shdA, the CS54 island of Salmonella serotype Typhimurium strain LT2 contains four open reading frames designated ratA, ratB, sivI, and sivH. DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution within the genus Salmonella. Homologues of shdA and ratB were detected only in serotypes of Salmonella enterica subsp. I. In contrast, sequences hybridizing with ratA, sivI, and sivH were present in S. enterica subsp. II and S. bongori in addition to S. enterica subsp. I. Deletion of the ratA and sivI genes did not alter the ability of Salmonella serotype Typhimurium to colonize the organs of mice. Insertional inactivation of the sivH gene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum, mesenteric lymph nodes, and spleen. Deletion of the shdA gene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in the ratB gene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen. The shdA and ratB deletion strains exhibited a shedding defect in mice, whereas the sivH deletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is required for efficient fecal shedding in mice.

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Figures

FIG. 1.

ORFs of the CS54 island of Salmonella serotype Typhimurium strain ATCC 14028 and deletion or insertion mutations constructed for phenotypic analysis. ORFs are indicated by arrows. The position of a dispersed repeat (cross-hatched bar) and remnant of an IS_1_ element (open bar) are indicated. The lengths (in base pairs) of the ORFs are indicated from the ATG codon (+1) to the stop codon. The position of the deletion or insertion in shdA, ratB, ratA, sivI, or sivH in strains AH9, AH12, AH8, AH10, and RAK19 are indicated.

FIG. 2.

Position and alignment of the imperfect repeats of RatA and RatB. In the Clustal alignment of the imperfect repeats (top), dark shaded boxes indicate identical residues, light shaded boxes indicate residues with similar biochemical properties, and dashes indicate gaps in alignment. The positions of the repeats in the RatA and RatB proteins (arrows) are indicated (filled bars) (bottom).

FIG. 3.

Phylogenetic distribution of the ratB, ratA, sivI, and sivH genes within the genus Salmonella. Southern blot analysis with representative serotypes of S. enterica (subspecies are indicated in roman numerals) and S. bongori (S.b.) is shown. Genomic DNA prepared from serotypes indicated on the left (strain designations are indicated in parentheses) was hybridized with DNA probes pRA59, pRA64, pRA71, and pRA73. The locations of these DNA probes (closed bars) relative to the ORFs of the CS54 island (arrows) are indicated on the map shown at the top.

FIG. 4.

Recovery of bacteria from the cecum, Peyer's patch, mesenteric lymph node (MLN), and spleen of BALB/c mice 5 days post-oral inoculation with an equal mixture of AH9 (Δ_shdA_::Kmr, open bars) or RAK60 (AH9 complemented, filled bars) and IR715 (wild type [wt]) (A), AH12 (Δ_ratB_, open bars) or RAK58 (AH12 complemented, filled bars) and AJB715 (wt) (B), AH8 (Δ_ratA_::Kmr) and IR715 (wt) (C), AH10 (Δ_sivI_::Kmr) and IR715 (wt) (D), or RAK19 (sivH::Kmr, open bars) or RAK59 (RAK19 complemented, filled bars) and IR715 (wt) (E). The ratio of the two strains present is given as the mean ± standard error. An asterisk indicates that the output ratio was significantly different (P < 0.05) from that present in the inoculum.

FIG. 5.

Recovery of bacteria from fecal pellets collected after inoculation of CBA/J mice with an equal mixture of AH9 (Δ_shdA_::Kmr) (A), RAK19 (sivH::Kmr) (B), or AH12 (Δ_ratB_) and IR715 (wild type) (C) Salmonella serotype Typhimurium strains. The ratio of the two strains present in the fecal pellets is given as the mean ± standard error. An asterisk indicates that the output ratio was significantly different (P < 0.05) from that present in the inoculum.

FIG. 6.

Recovery of bacteria from fecal pellets, the cecum, and the Peyer's patches of BALB/c mice following oral inoculation with an equal mixture of AH9 (Δ_shdA_::Kmr) and IR715 (wild type [wt]) (A), AH12 (Δ_ratB_) and AJB715 (wt) (B), or RAK19 (sivH::Kmr) and IR715 (wt) (C). The ratio of the two strains present is given as the mean ± standard error. An asterisk indicates that the output ratio was significantly different (P < 0.05) from that present in the inoculum.

FIG. 7.

References

1. Aleksic, S., F. Heinzerling, and J. Bockemühl. 1996. Human infection caused by salmonellae of subspecies II to VI in Germany, 1977-1992. Zentbl. Bakteriol. 283:391-398. - PubMed
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1994. Current protocols in molecular biology. John Wiley & Sons, New York, N.Y.
1. Bäumler, A. J., R. M. Tsolis, P. J. Valentine, T. A. Ficht, and F. Heffron. 1997. Synergistic effect of mutations in invA and lpfC on the ability of Salmonella typhimurium to cause murine typhoid. Infect. Immun. 65:2254-2259. - PMC - PubMed
1. Bäumler, A. J., R. M. Tsolis, A. W. M. van der Velden, I. Stojiljkovic, S. Anic, and F. Heffron. 1996. Identification of a new iron regulated locus of Salmonella typhi. Gene 193:207-213. - PubMed
1. Blanc-Potard, A.-B., and E. A. Groisman. 1997. The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival. EMBO J. 16:5376-5385. - PMC - PubMed

Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype typhimurium: identification of intestinal colonization and persistence determinants - PubMed (original) (raw)