Characterization of a novel porin involved in systemic Yersinia enterocolitica infection

Abstract

Yersinia enterocolitica is an enteric pathogen capable of causing systemic disease in a murine model. We have identified a novel protein, systemic factor protein A (SfpA), conserved in other pathogenic bacteria that is involved in systemic disease. Analysis of bacterial colonization revealed that a DeltasfpA strain is defective in mesenteric lymph node colonization. Bioinformatics and functional studies suggest that SfpA is a porin.

FIG. 1.

Bacterial colonization of the mouse by the sfpA mutant. (A) C57BL/6j mice were orally infected with 10⁸ CFU wild-type Y. enterocolitica (JB580v) (▪) or the sfpA mutant (YVM1051) (□). Mice were sacrificed and organs harvested on day 3 and day 7. The bacterial load for each organ was determined by counting the number of viable bacteria after plating serial dilutions. Results are expressed as CFU per gram of tissue. Each symbol represents an individual mouse, and symbols on the x axis were below the limits of detection (limits of detection were as follows: for Peyer's patches, 240; for MLN, 100; and for spleens, 65). The Mann-Whitney test was used to calculate the P value. (B) C57BL/6j mice were infected intraperitoneally with 10⁴ CFU of wild-type Y. enterocolitica (JB580v) (▪) or the sfpA mutant (YVM1051) (□). The Mann-Whitney test was used to calculate the P value. Bacterial loads for mesenteric lymph nodes were determined as described above. (C) C57BL/6j mice were orally infected with 10⁸ CFU wild-type Y. enterocolitica (JB580v) (▪), the sfpA mutant (YVM1051) (□), or the complemented sfpA mutant strain (YVM1220) (▴). Every “X” on the x axis represents one dead mouse; symbols on the x axis indicate that the CFU were below the limits of detection. P, Peyer's patch; M, mesenteric lymph node; S, spleen.

FIG. 2.

Levels of SfpA^HA in Y. enterocolitica during in vitro growth. Cultures of Y. enterocolitica sfpA^HA (YVM1219) were grown in Luria-Bertani broth at 26°C or 37°C, samples were collected at various time points, and aliquots with equivalent OD₆₀₀ values were loaded into the lanes. Monoclonal anti-HA antibodies (Sigma, St. Louis, MO) were used in immunoblot analysis to determine the levels of SfpA.

FIG. 3.

Localization and heat modifiability of SfpA^HA in Y. enterocolitica. (A) Y. enterocolitica sfpA^HA cultures (YVM1219) were grown in Luria-Bertani broth at 37°C to an OD₆₀₀ of 0.5 and fractionated as described previously (17). Monoclonal anti-HA antibodies were used in immunoblot analysis. (B) Y. enterocolitica sfpA^HA cultures (YVM1219) were grown as described above. Whole-cell lysates were boiled for 10 min in β-mercaptoethanol (BME) or Laemmli buffer (no BME) to rule out any effects of the BME. All samples were run in sodium dodecyl sulfate-polyacrylamide gels and subjected to immunoblot analysis with monoclonal anti-HA antibodies. The 35-kDa molecular mass marker is labeled.

FIG. 4.

Sugar diffusion through the SfpA porin. sfpA (pSM1) was expressed in the E. coli MCR106 lamB mutant or in lamB⁺ E. coli strain MC4100 and tested for growth on minimal medium in the presence of differently sized sugars. Overnight cultures grown in Luria-Bertani broth were pelleted, washed with M63 salts, and plated on minimal medium containing M63 salts, 10 μM IPTG, and 0.2% (wt/vol) of the indicated sugars. Growth was assessed after 24 to 48 h at 37°C.