Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis

Abstract

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10(10) CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.

FIG. 1

Results from the STM screen in mice and calves. A total of 260 mutants were screened for virulence in both mice and calves. Mutants not recovered from output pools of mice are shown in the left circle. Mutants not recovered from output pools of calves are shown in the right circle. The genotypes of these mutants are shown in Table 1.

FIG. 2

Percent identity determined by pairwise alignment of amino acid sequences of SlrP, IpaH 60.0, IpaH 65.4, and YopM with the CLUSTAL program.

FIG. 3

Pustell alignment of the SlrP amino acid sequence against itself (window size = 8; minimum % identity = 60%; hush value = 2) (left). Lines parallel to the diagonal identify direct amino acid repeats. The SlrP primary structure is shown as an arrow on the bottom (left). The positions of 10 copies of a 21-amino-acid repeat are indicated. A CLUSTAL alignment of the repeats is shown on the right (top). A comparison of the SlrP repeat consensus sequence with the consensus of 20-amino-acid repeats found in IpaH 60.0, IpaH 65.4, and YopM is shown on the right (bottom) (59). Capital letters in the consensus sequence indicate amino acids, which are conserved at this position in at least 50% of the repeats.

FIG. 4

Invasiveness of different S. typhimurium strains for HEp-2 cells. The number of gentamicin-protected bacteria recovered after the lysis of tissue culture cells is given as a fraction of the total number of bacteria added to each well at the beginning of the assay. Each bar represents the mean from six wells ± standard deviation.

FIG. 5

Comparison of the uvrB-yphK intergenic region of E. coli K-12 and S. typhimurium ATCC 14028 (top). Arrows indicate the positions of genes. Comparisons of nucleotide sequences from the left and right boundaries of the pathogenicity islet are shown on the bottom. E.c., E. coli; S.t., S. typhimurium.

FIG. 6

Distribution of genes (slrP, sspH1, and sspH2) encoding leucine-rich repeat proteins among 16 SARC collection strains (right) and 72 SARB collection strains (left and center). The SARC collection contains serotypes of Salmonella bongori (s3041 and s3044) and of S. enterica subsp. I (s4194 and s3333), II (s2985 and s2993), IIIa (s2980 and s2983), IIIb (s2978 and s2979), IV (s3015 and s3027), VI (s2995 and s3057), and VII (s3013 and s3014). The SARB collection consists of 72 strains of S. enterica subsp. I. Ag, S. agona, An, S. anatum, Ba, S. brandenburg, Cs, S. choleraesuis, De, S. derby, Di, S. duisburg, Dt, S. decatur, Du, S. dublin, Em, S. emek, En, S. enteritidis, Ga, S. gallinarum, Ha, S. haifa, He, S. heidelberg, Id, S. indiana, In, S. infantis, Mi, S. miami, Mo, S. montevideo, Mu, S. muenchen, Np, S. newport, Pa, S. paratyphi A, Pb, S. paratyphi B, Pc, S. paratyphi C, Pn, S. panama, Pu, S. pullorum, Re, S. reading, Ru, S. rubislaw, Se, S. sendai, Sf, S. senftenberg, Sp, S. saintpaul, St, S. stanley, Sv, S. stanleyville, Sw, S. schwarzengrund, Th, S. thompson, Tm, S. typhimurium, Tp, S. typhi, Ts, S. typhisuis, Wi, S. wien.