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. 2013 Aug 27;4(5):e00565-13.
doi: 10.1128/mBio.00565-13.

Genome and Transcriptome Adaptation Accompanying Emergence of the Definitive Type 2 Host-Restricted Salmonella Enterica Serovar Typhimurium Pathovar

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Free PMC article

Genome and Transcriptome Adaptation Accompanying Emergence of the Definitive Type 2 Host-Restricted Salmonella Enterica Serovar Typhimurium Pathovar

Robert A Kingsley et al. mBio. .
Free PMC article

Abstract

Salmonella enterica serovar Typhimurium definitive type 2 (DT2) is host restricted to Columba livia (rock or feral pigeon) but is also closely related to S. Typhimurium isolates that circulate in livestock and cause a zoonosis characterized by gastroenteritis in humans. DT2 isolates formed a distinct phylogenetic cluster within S. Typhimurium based on whole-genome-sequence polymorphisms. Comparative genome analysis of DT2 94-213 and S. Typhimurium SL1344, DT104, and D23580 identified few differences in gene content with the exception of variations within prophages. However, DT2 94-213 harbored 22 pseudogenes that were intact in other closely related S. Typhimurium strains. We report a novel in silico approach to identify single amino acid substitutions in proteins that have a high probability of a functional impact. One polymorphism identified using this method, a single-residue deletion in the Tar protein, abrogated chemotaxis to aspartate in vitro. DT2 94-213 also exhibited an altered transcriptional profile in response to culture at 42°C compared to that of SL1344. Such differentially regulated genes included a number involved in flagellum biosynthesis and motility. IMPORTANCE Whereas Salmonella enterica serovar Typhimurium can infect a wide range of animal species, some variants within this serovar exhibit a more limited host range and altered disease potential. Phylogenetic analysis based on whole-genome sequences can identify lineages associated with specific virulence traits, including host adaptation. This study represents one of the first to link pathogen-specific genetic signatures, including coding capacity, genome degradation, and transcriptional responses to host adaptation within a Salmonella serovar. We performed comparative genome analysis of reference and pigeon-adapted definitive type 2 (DT2) S. Typhimurium isolates alongside phenotypic and transcriptome analyses, to identify genetic signatures linked to host adaptation within the DT2 lineage.

Figures

FIG 1
FIG 1
Phylogram of S. Typhimurium isolates based on SNPs determined from the whole-genome sequence. A maximum-likelihood tree showing the phylogenetic relationship of S. Typhimurium DT2 isolates and a collection of diverse strains from various sources. The length of the scale bar is the estimated number of SNPs determined from the rate of substitution per variable site. MLST groups are color-coded as indicated above the tree. All nodes have >80% bootstrap support unless stated. The sequence type (ST) is indicated: ST19 (red), ST128 (green), ST34 (blue), ST313 (yellow), ST568 (cyan), and ST98 (orange).
FIG 2
FIG 2
Colonization of the cecum and liver of C57BL/6 mice by S. Typhimurium SL1344 and DT104 and representatives of the DT2 lineages. Mice were inoculated with 5 × 107 CFU by the intragastric route with S. Typhimurium representing each of the three sublineages of DT2a (dark green), DT2b (light green), and DT2c (medium-green circles) or ST19 isolates, strains SL1344 and DT104 (red circles). Four days postinoculation, the CFU in organ homogenates was determined for the cecum (A) and liver (B). The numbers of CFU per organ in 3 to 9 mice are plotted (circles), and geometric means are indicated (horizontal lines). In order to determine lineage-specific colonization phenotypes, the colonization of the cecum (C) and the liver (D) for isolates from each lineage were plotted together (circles), and the geometric means are indicated. The probability that the colonization of isolates from each DT2 sublineage is significantly different from that of SL1344 and DT104 was calculated using an unpaired Mann-Whitney test. The P value is indicated above the plots for the DT2a, DT2b, and DT2c sublineages.
FIG 3
FIG 3
Mixed-inoculum competitive infection of C57BL/6 mice to compare virulence of S. Typhimurium RAK113 and SL1344 containing deletion of pseudogenes in 94-213. An equal mixture (log10 ratio = 0) containing approximately 1 × 107 CFU of RAK113 and test strain was inoculated orally. Filled bars indicate the log10 ratio ± standard errors (SE) of CFU (RAK113/test strain) on day five postinoculation in organ homogenates: mesenteric lymph nodes (MLN), cecum (Cec), ileum (Ile), spleen (Spl), and liver (Liv). *, Student’s t test was used to determine if the output log10 ratio was significantly different from the input one.
FIG 4
FIG 4
Frequency distribution of Δbit scores calculated from a Pfam scan of 94-213 and DT104 peptides compared with SL1344 orthologues that contain amino acid substitutions. The bit score of HMM domains determined using a Pfam scan were measured, and the differences between SL1344/94-213 and SL1344/DT104 were calculated. The frequency distributions for SL1344/94-213 (A) and SL1344/DT104 (B) are plotted as bars. The normal distribution, centered around a Δbit score of 0, was calculated using the variation observed in the calculated data.
FIG 5
FIG 5
Polymorphisms in the Tar protein of S. Typhimurium DT2 strain 94-213 results in loss of function of the methyl-accepting chemotaxis protein. The tar gene of strain 94-213 was transferred to strain SL1344 using bacteriophage P22-mediated transduction by selecting for the cotransfer of a cat gene inserted in the 5′ region of the tar promoter. The SW769 transductant, that cotransduced the ΔA98 and V400A polymorphisms, no longer responded by chemotaxis toward aspartate in the agar (dashed yellow line), while the SW767 transductant, in which the cat gene but not the tar polymorphisms were transferred, responded to aspartate in a similar fashion to SL1344.
FIG 6
FIG 6
Comparison of genes differentially expressed at 42°C and 37°C in S. Typhimurium DT2 strain 94-213 and SL1344. (A) Venn diagram summarizing the numbers of genes differentially expressed in 94-213 and SL1344, SL1344 only, and 94-213 only. The log2-fold changes in expression at 42°C compared to that at 37°C of genes that were significantly differentially expressed are plotted for strain 94-213 (y axis) and SL1344 (x axis). (A) Venn diagram summarizing the number of genes that were differentially expressed (DE): differentially expressed in 94-213 and SL1344 (B), SL1344 only (C), and 94-213 only (D). (E) Differentially expressed type associated with the SPI-1 (red triangles), SPI-2 (blue squares), or flagellum biosynthesis, motility, and chemotaxis (green circles).
FIG 7
FIG 7
Flagellum gene expression, elaboration of flagella, and invasion of enterocytes. (A) Relative expression of the fliD gene determined by quantitative RT-PCR of four strains from outside the DT2 clade and 10 DT2 isolates during culture at 42°C and 37°C. (B) Transmission electron micrograph of negatively stained S. Typhimurium cultured at 42°C or 37°C showing expression of flagella. (C) Enumeration of flagella associated with negatively stained S. Typhimurium in random TEM fields. (D) Invasion of cultured T84 enterocyte-like cells by S. Typhimurium strains following culture of the inoculum at 42°C or 37°C determined using a gentamicin protection assay. The number of CFU recovered 2 h postinoculation is expressed as the percentage of the initial inoculum. The mean percentages and standard errors from five biological replicates are indicated. Data from a representative of duplicate experiments are shown.
FIG 8
FIG 8
Impact of temperature of S. Typhimurium culture on interaction with host enterocytes in culture at 37°C and 42°C. Presence of the proinflammatory cytokine IL-8 in the supernatant of cultured T84 enterocyte-like cells after exposure to S. Typhimurium strains following culture of the inoculum at 37°C (A) or 42°C (B). The mean concentration of IL-8 (pg/ml) in the tissue culture medium supernatant ± standard deviation from three replicate wells infected with SL1344 (triangle), 94-213 (square), and 88-138 (circle).

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