Differences in Host Cell Invasion and Salmonella Pathogenicity Island 1 Expression between Salmonella enterica Serovar Paratyphi A and Nontyphoidal S. Typhimurium

Abstract

Active invasion into nonphagocytic host cells is central to Salmonella enterica pathogenicity and dependent on multiple genes within Salmonella pathogenicity island 1 (SPI-1). Here, we explored the invasion phenotype and the expression of SPI-1 in the typhoidal serovarS Paratyphi A compared to that of the nontyphoidal serovarS Typhimurium. We demonstrate that while S. Typhimurium is equally invasive under both aerobic and microaerobic conditions, S. Paratyphi A invades only following growth under microaerobic conditions. Transcriptome sequencing (RNA-Seq), reverse transcription-PCR (RT-PCR), Western blot, and secretome analyses established that S. Paratyphi A expresses much lower levels of SPI-1 genes and secretes lesser amounts of SPI-1 effector proteins than S. Typhimurium, especially under aerobic growth. Bypassing the native SPI-1 regulation by inducible expression of the SPI-1 activator, HilA, considerably elevated SPI-1 gene expression, host cell invasion, disruption of epithelial integrity, and induction of proinflammatory cytokine secretion by S. Paratyphi A but not by S. Typhimurium, suggesting that SPI-1 expression is naturally downregulated inS Paratyphi A. Using streptomycin-treated mice, we were able to establish substantial intestinal colonization byS Paratyphi A and showed moderately higher pathology and intestinal inflammation in mice infected with S. Paratyphi A overexpressing hilA Collectively, our results reveal unexpected differences in SPI-1 expression between S. Paratyphi A andS Typhimurium, indicate that S. Paratyphi A host cell invasion is suppressed under aerobic conditions, and suggest that lower invasion in aerobic sites and suppressed expression of immunogenic SPI-1 components contributes to the restrained inflammatory infection elicited by S. Paratyphi A.

FIG 1

Effect of environmental conditions on S. Paratyphi A and S. Typhimurium invasion. Salmonella strains were grown at 37°C in Lennox LB supplemented with 0, 0.17, and 0.3 M NaCl. Cultures were grown for 16 h aerobically to an OD₆₀₀ of 4.5 (stationary aerobic), diluted 1:100, grown for 2.5 h under aerobic conditions to an OD₆₀₀ of 1.2 to 1.4 (late logarithmic aerobic) or for 16 h statically in capped tubes to an OD₆₀₀ of 0.6 (stationary microaerobic), and used to infect HeLa cells (A) or Caco-2 cells (B). Data represent the means and standard errors of the means (SEM) from at least three biological replicates under each condition. (C) Overnight S. Typhimurium SL1344 and S. Paratyphi A 45157 cultures were diluted 1:100 into fresh LB broth and grown for 2 h to early logarithmic phase under aerobic conditions. At 2 h postinoculation, these cultures were split in two. One portion of the culture continued growth under aerobic conditions for an additional 1 h (growing for 3 h in total to an OD₆₀₀ of 1.2), and the other portion was transferred to microaerobic conditions for 3 more hours (growing for 5 h in total to an OD₆₀₀ of 0.6). All four cultures were used to infect HeLa cells. Data represent the means and SEM from 11 biological replicates. In all experiments, invasion was determined at 2 h postinfection using the gentamicin protection assay and is shown as the percentage of intracellular bacteria (CFU) from the infection inoculum. An unpaired t test with two tails was used to determine the significance of the differences between S. Typhimurium (STM) and S. Paratyphi A (SPA) invasion. **, P < 0.01; ***, P < 0.0001; ns, not significant.

FIG 2

Invasion of S. Paratyphi A is impaired at the logarithmic phase under aerobic conditions. Sixteen S. Typhimurium strains (A) and 16 S. Paratyphi A strains (B) were grown in LB aerobically to the late logarithmic phase (OD₆₀₀ of 1.2 to 1.4). Invasion was determined at 2 h p.i. using the gentamicin protection assay. (C) Adhesion was determined in the presence of cytochalasin D and is shown as the percentage of cell-associated bacteria from the total number of CFU used to infect the cells. Bars represent the mean adhesion and SEM from at least three biological replicates. *, P < 0.05; ***, P < 0.0001; ns, not significant.

FIG 3

T3SS-1 is not functional in S. Paratyphi A grown to the late logarithmic phase aerobically. S. Typhimurium SL1344 (STM) and S. Paratyphi A 45157 (SPA) and their isogenic invA and invG null mutant strains were grown to the late logarithmic phase aerobically (A and B) or in LB supplemented with 0.17 M NaCl under microaerobic conditions (C and D), as described for Fig. 1, and used to infect Caco-2 cells. Under these conditions, the invasion rates of S. Paratyphi A were 0.06% ± 0.02% and 5.1% ± 1.7% (from the infection inoculum), respectively. Salmonella invasion was determined at 2 h p.i. using the gentamicin protection assay and is shown relative to the invasion of the wild-type background. One-way analysis of variance (ANOVA) with Dunnett's multiple-comparison test was implemented to compare the invasion of the different strains to that of the wild type. ***, P < 0.0001; ns, not significant.

FIG 4

S. Paratyphi A grown aerobically to the late logarithmic phase expresses lower levels of SPI-1 genes than S. Typhimurium. (A) Total RNA was harvested from S. Typhimurium SL1344 and three S. Paratyphi A strain (45157, 118239, and 9150) cultures grown aerobically to the late logarithmic phase at 37°C, and extracted RNA was subjected to qRT-PCR. The fold change in the abundance of SPI-1 gene transcripts (normalized to rpoD) in S. Paratyphi A strains relative to their expression in S. Typhimurium is shown. The indicated values present the means and the SEM from three independent RT-PCR experiments from two independent RNA extractions. (B) SDS-PAGE Western blot analysis of bacterial cell lysate from S. Typhimurium SL1344 (STM) and S. Paratyphi A 45157 (SPA) strains grown aerobically to the late logarithmic phase. Protein fractions were probed using anti-HA antibody and anti-DnaK antibody as a control. (C) S. Typhimurium SL1344 and S. Paratyphi A 45157 cultures were grown in LB for 5.5 h and normalized to an OD₆₀₀ of 2.4 to 2.6. Exogenous human cytochrome c (8 mg) was added to the bacterial cultures as a precipitating and loading control. Supernatant (5 ml) cultures were precipitated by TCA, and equal amounts (25 μl) from the precipitated fractions were separated on an SDS-12% polyacrylamide gel and stained with Coomassie G-250.

FIG 5

Induced expression of SPI-1 increases S. Paratyphi A invasion. (A) Total RNA was harvested from S. Paratyphi A 45157 carrying pBAD18 or pBAD::hilA grown in LB supplemented with 100 μl/ml ampicillin and 50 mM arabinose to late logarithmic phase aerobically and was subjected to qRT-PCR. The fold change in the abundance of SPI-1 gene transcripts (normalized to rpoD) in S. Paratyphi A harboring pBAD::hilA is shown relative to their expression in S. Paratyphi A carrying pBAD18. The indicated values present the means and the SEM from three independent RT-PCR experiments from two independent RNA extractions. (B) S. Paratyphi A cultures were grown aerobically in LB to logarithmic phase and normalized to an OD₆₀₀ of 1.2. Secreted fractions from S. Paratyphi A carrying pBAD18 (lane 2) or pBAD::hilA grown in the presence of 1 M glucose (lane 3) or increasing arabinose concentrations (lanes 4 to 6) were separated on SDS-12% polyacrylamide gels and stained with Coomassie G-250. (C) S. Typhimurium SL1344 (wild type) and its isogenic strains carrying pBAD or pBAD::hilA were grown in LB, LB supplemented with 1 M glucose, or LB with increasing concentrations of arabinose under aerobic conditions and used to infect HeLa cells. Invasion is shown relative to that of the wild-type strain (grown in LB supplemented with 50 mM arabinose), presenting a 3.7% ± 0.76% invasion rate (from the infecting inoculum) under these conditions. (D) S. Paratyphi A 45157 and its isogenic strains carrying pBAD or pBAD::hilA were grown and used to infect HeLa cells as described for panel C. Invasion is shown relative to that of the S. Paratyphi A wild-type strain, presenting, on average, 0.05% ± 0.001% invasion. ANOVA with Dunnett's multiple-comparison test was implemented to compare the invasion of the different strains to that of the wild type. The results represent the means and SEM from at least three biological replicates. **, P < 0.01; ***, P < 0.0001; ns, not significant.

FIG 6

Induced expression of hilA increases epithelium disruption and secretion of proinflammatory cytokines by S. Paratyphi A. The integrity of the epithelial monolayer was determined for polarized Caco-2 cells infected at an MOI of 1:10 at 0, 3, 6, 8, 10, and 12 h p.i. and is shown as the change in transepithelial electrical resistance (TEER) from the time of infection (T0). S. Typhimurium SL1344 (A) and S. Paratyphi A 45157 (B) strains were grown under aerobic conditions to the late logarithmic phase in the presence of 100 μl/ml ampicillin and 50 mM arabinose. Data represent the means and SEM from three to five infections. An unpaired t test with two tails was used to determine the significance of the differences between S. Paratyphi A/pBAD18 and S. Paratyphi A/pBAD::hilA measurements. At 2 h p.i., supernatant was taken from the polarized Caco-2 cells and the concentration of secreted IL-8 (C) and Mip3α (D) was measured by ELISA. Data shown represent the means and standard deviations from three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ns, not significant.

FIG 7

S. Paratyphi A colonizes the intestine of streptomycin-treated mice. Groups of C57BL/6 mice were treated with streptomycin 1 day preinfection and inoculated orally with 1 × 10⁸ to 2 × 10⁸ CFU of S. Paratyphi A carrying pBAD18 (circles) or pBAD::hilA (closed squares) grown to the late logarithmic phase under aerobic conditions. Mice from day one (A) and day three (B) p.i. were sacrificed, and homogenized tissues were plated on XLD plates supplemented with ampicillin to determine bacterial load per organ. Geometric means from both groups are shown as horizontal lines.

FIG 8

hilA-overexpressing S. Paratyphi A induces greater pathology and increased host immune response in the mouse model. Cecal sections were taken at day one p.i. from streptomycin-pretreated mice that were infected with S. Paratyphi A carrying pBAD18 (A, C, and E) or pBAD::hilA (B, D, and F). (A and B) Immunofluorescence staining of cecal sections. Nuclei were stained with DAPI (blue), and S. Paratyphi A organisms were stained with anti-Salmonella serum (green). Original magnification, ×400. (C and D) Staining against CD68 (macrophages; red) and MPO (neutrophils; green) was conducted as explained in Materials and Methods, and nuclei were stained with DAPI (blue). The lumen, mucosa, and Peyer's patches (PP) are indicated. Original magnification, ×100. (E and F) Representative micrographs of hematoxylin and eosin staining of cecal sections (original magnification of ×100). Desquamations of the epithelium are indicated by arrows, ulceration is shown by an asterisk, and dead cells in lumen are indicated by arrowheads. (G) Histopathological scoring was assessed by the determination of the infiltration of inflammatory cells together with the evaluation of epithelial damage. *, P < 0.05.