Requirement of the Yersinia pseudotuberculosis effectors YopH and YopE in colonization and persistence in intestinal and lymph tissues

Abstract

The gram-negative enteric pathogen Yersinia pseudotuberculosis employs a type III secretion system and effector Yop proteins that are required for virulence. Mutations in the type III secretion-translocation apparatus have been shown to cause defects in colonization of the murine cecum, suggesting roles for one or more effector Yops in the intestinal tract. To investigate this possibility, isogenic yop mutant strains were tested for their ability to colonize and persist in intestinal and associated lymph tissues of the mouse following orogastric inoculation. In single-strain infections, a yopHEMOJ mutant strain was unable to colonize, replicate, or persist in intestinal and lymph tissues. A yopH mutant strain specifically fails to colonize the mesenteric lymph nodes, but yopE and yopO mutant strains showed only minor defects in persistence in intestinal and lymph tissues. While no single Yop was found to be essential for colonization or persistence in intestinal tissues in single-strain infections, the absence of both YopH and YopE together almost eliminated colonization of all tissues, indicating either that these two Yops have some redundant functions or that Y. pseudotuberculosis employs multiple strategies for colonization. In competition infections with wild-type Y. pseudotuberculosis, the presence of wild-type bacteria severely hindered the ability of the yopH, yopE, and yopO mutants to persist in many tissues, suggesting that the wild-type bacteria either fills colonization niches or elicits host responses that the yop mutants are unable to withstand.

FIG. 1.

Colonization of intestinal and lymph tissues at day 1 and day 2 postinfection with 2 × 10⁹ CFU of wild-type Y. pseudotuberculosis (filled diamonds), yopHEMOJ (open circles), or yopBHEMOJ (open triangles). Data from four to six mice from two experiments were pooled. Each symbol represents the log₁₀ CFU/gram of tissue from one mouse colonized with the appropriate strain. All points were above the limit of detection of 10 CFU/gram of tissue. At day 1, colonization by yopHEMOJ and yopBHEMOJ was statistically different (**; P < 0.01) from that by the wild-type strain in the ileum, colon, and PP. At day 2, colonization by yopHEMOJ and yopBHEMOJ are statistically different from the wild-type strain (P < 0.01) in all tissues.

FIG. 2.

Colonization of ileum, PP, and MLN at day 2 and day 5 postinfection with either 2 × 10⁹ CFU or 2 × 10⁸ CFU of wild-type (wt), yopB (ΔB), yopH (ΔH), yopE (ΔE), or yopO (ΔO) Y. pseudotuberculosis. Data from four to eight mice from two to four experiments were pooled. Each symbol represents the log₁₀ CFU/gram of tissue for the tissue sample from one mouse. Shaded bars represent the average log₁₀ CFU/gram of tissue. Open symbols indicate that less than 10 CFU were recovered/tissue. Asterisks indicate statistical significance of difference between colonization levels of yop mutants and wild-type bacteria as follows: *, P = 0.1 to 0.01; **, P < 0.01.

FIG. 3.

Colonization of the spleen at 2 and 5 days postinfection with either 2 × 10⁹ CFU or 2 × 10⁸ CFU of wild-type (wt), yopB (ΔB), yopH (ΔH), yopE (ΔE), yopO (ΔO) or ΔHEMOJ Y. pseudotuberculosis. Data from four to eight mice from two to four experiments were pooled. Each symbol represents the log₁₀ CFU/gram of tissue for the spleen from one mouse. Open symbols indicate that less than 10 CFU/tissue were recovered.

FIG. 4.

Colonization of intestinal and lymph tissues at day 2 and day 5 postinfection with 2 × 10⁸ CFU of wild-type, yopHE (ΔHE), yopHO (ΔHO), yopEO (ΔEO), yopHEO (ΔHEO), or yopHEMOJ (ΔHEMOJ) Y. pseudotuberculosis. Data from four to six mice from two to three experiments were pooled. Each symbol represents the log₁₀ CFU/gram of tissue for the tissue sample from one mouse. Shaded bars represent the average log₁₀ CFU/gram of tissue. Open symbols indicate that less than 10 CFU were recovered/tissue. Asterisks indicate statistical significance of difference between colonization levels of yop mutants and wild-type bacteria. *, P = 0.1 to 0.01; **, P < 0.01.

FIG. 5.

Competition of yopHE versus yopHEMOJ (closed circles) and yopHEO versus yopHEMOJ (open circles), 1 day postinfection with 2 × 10⁹ CFU of an equal mixture of yopHE and yopHEMOJ or yopHEO and yopHEMOJ. Data from three to six mice from two experiments were pooled. Data are graphed as C.I. {[yopHE(O)/yopHEMOJ output ratio]/[yopHE(O)/yopHEMOJ input ratio]} values for the tissue samples from one mouse. Shaded bars represent geometric means of the C.I. values. All points were above the limit of detection.

FIG. 6.

Competition between wild-type Y. pseudotuberculosis and yop mutants at 5 days postinfection with 2 × 10⁹ CFU of a mixture of equal amounts of the wild-type strain and either yopB, yopH, yopE, yopO, or yopHEMOJ. Data from six to eight mice from three to four experiments were pooled. Each point represents the C.I. [(mutant/wild-type output ratio)/(mutant/wild-type input ratio)] value for the tissue sample from one mouse. Shaded bars represent the geometric means of the C.I. values. Open symbols indicate that the yop mutant was below the limit of detection, which was usually 200 bacteria. Asterisks indicate the yop mutants that were significantly outcompeted by wild-type bacteria (*, P = 0.1 to 0.01; **, P < 0.01).