Formate acts as a diffusible signal to induce Salmonella invasion

Abstract

To infect an animal host, Salmonella enterica serovar Typhimurium must penetrate the intestinal epithelial barrier. This process of invasion requires a type III secretion system encoded within Salmonella pathogenicity island I (SPI1). We found that a mutant with deletions of the acetate kinase and phosphotransacetylase genes (ackA-pta) was deficient in invasion and SPI1 expression but that invasion gene expression was completely restored by supplying medium conditioned by growth of the wild-type strain, suggesting that a signal produced by the wild type, but not by the ackA-pta mutant, was required for invasion. This mutant also excreted 68-fold-less formate into the culture medium, and the addition of sodium formate to cultures restored both the expression of SPI1 and the invasion of cultured epithelial cells by the mutant. The effect of formate was pH dependent, requiring a pH below neutrality, and studies in mice showed that the distal ileum, the preferred site of Salmonella invasion in this species, had the appropriate formate concentration and pH to elicit invasion, while the cecum contained no detectable formate. Furthermore, we found that formate affected the major regulators of SPI1, hilA and hilD, but that the primary routes of formate metabolism played no role in its activity as a signal.

FIG. 1.

Effects of conditioned medium on sipC expression. The wild type and barA and ackA-pta mutants with a sipC::lacZY fusion were grown standing overnight in 50% 2× MOPS minimal medium with 1% glucose and 50% of either fresh or conditioned medium. Fresh medium (gray bars) consisted of 1× MOPS minimal medium with 0.5% glucose and amino acids. Conditioned medium was made by growing the wild type (striped bars), sirA mutant (white bars), or ackA-pta mutant (black bars) standing overnight in this same medium and then filter sterilizing the culture supernatant. Expression of sipC was assessed by using β-galactosidase assays. Values represent the mean for each condition tested in triplicate. Asterisks show a significant difference (P ≤ 0.05) for the strain with the genotype shown when grown with the addition of conditioned medium compared to the fresh-medium control. Error bars show standard deviations.

FIG. 2.

Effects of formate on sipC expression. (A) Strains were grown as standing overnight cultures in LB with 100 mM MOPS, pH 6.7, and with 10 mM sodium chloride (gray bars) or 10 mM sodium formate (black bars). Triplicate cultures of each strain were assayed for sipC::lacZY expression by using β-galactosidase assays. (B) Overnight standing cultures of the wild type (gray bars) and ackA-pta mutant (black bars) with the sipC::lacZY fusion were grown in LB with 100 mM MOPS, pH 6.7, and with 0, 1, 5, 10, 20, or 30 mM sodium formate. Triplicate cultures for each condition were assayed for sipC::lacZY expression. Single asterisks show a significant difference (P ≤ 0.05) for the strain with the genotype shown when grown with the addition of formate compared to the NaCl control. Double asterisks show a significant difference for the mutant strain compared to the wild type when both were grown without formate. Error bars show standard deviations.

FIG. 3.

Invasion of epithelial cells. Bacteria were grown overnight as standing cultures in LB broth buffered with 100 mM MOPS, pH 6.7, and with either 10 mM sodium chloride (gray bars) or 10 mM sodium formate (black bars). Bacteria were added to HEp-2 cells, and the level of invasion was assessed by using a gentamicin protection assay. Invasion is shown in comparison to that of the wild-type strain set to 100. Each strain and condition was tested in quadruplicate. Single asterisks show a significant difference (P ≤ 0.05) for the strain with the genotype shown when grown with the addition of formate compared to the NaCl control. Double asterisks show a significant difference for the mutant strain compared to the wild type when both were grown without formate. Error bars show standard deviations.

FIG. 4.

Effects of medium pH on sipC expression in response to formate. The wild type and ackA-pta mutants with a sipC::lacZY fusion were grown standing overnight in LB with 100 mM HEPES, pH 8.0, or 100 mM MOPS, pH 6.7, as indicated, and with either 10 mM sodium chloride (gray bars) or 10 mM sodium formate (black bars). Expression of sipC was assessed by using β-galactosidase assays. Values represent the mean for each condition tested in duplicate. Single asterisks show a significant difference (P ≤ 0.05) for the strain with the genotype shown when grown with the addition of formate compared to the NaCl control. Double asterisks show a significant difference for the mutant strain compared to the wild type when both were grown without formate. Error bars show standard deviations.

FIG. 5.

Effects of formate metabolism on sipC expression. (A) Pathways for the integration of formate into central carbon metabolism. (B) Strains with the genotype shown and with the sipC::lacZY fusion were grown as standing overnight cultures in LB with 100 mM MOPS, pH 6.7. Triplicate cultures of each strain were assayed for sipC::lacZY expression by using β-galactosidase assays. Double asterisks show a significant difference for the mutant strain compared to the wild type. Error bars show standard deviations.

FIG. 6.

Effects of pflB on invasion gene expression in response to formate and pyruvate. Strains were grown as standing overnight cultures in LB with 100 mM MOPS, pH 6.7, and with 10 mM sodium chloride (gray bars) or 10 mM sodium formate (black bars) (A) or 50 mM sodium chloride (gray bars) or 50 mM sodium pyruvate (black bars) (B). Triplicate cultures of each strain were assayed for sipC::lacZY expression by using β-galactosidase assays. Single asterisks show a significant difference (P ≤ 0.05) for the strain with the genotype shown when grown with the addition of formate (A) or pyruvate (B) compared to the NaCl control. Double asterisks show a significant difference for the mutant strain compared to the wild type when both were grown without additions to the medium. Error bars show standard deviations.

FIG. 7.

Effects of formate on SPI1 gene expression. The ackA-pta mutant strain was grown in LB broth buffered with 100 mM MOPS, pH 6.7, and with either 10 mM sodium chloride (gray bars) or 10 mM sodium formate (black bars). Total RNA was used to create cDNA and quantified by real-time PCR. Values were normalized by using the housekeeping gene icdA, and levels found in the control cultures were set to 1. Data are shown as the mean expression of two independent cultures with each culture tested in triplicate. Asterisks show a significant difference (P ≤ 0.05) for expression of the gene shown when bacteria were grown with the addition of formate compared to that of the NaCl control. Error bars show standard deviations.