Contribution of the major secreted yops of Yersinia enterocolitica O:8 to pathogenicity in the mouse infection model

Abstract

Pathogenic yersiniae (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) harbor a 70-kb virulence plasmid (pYV) that encodes a type III secretion system and a set of at least six effector proteins (YopH, YopO, YopP, YopE, YopM, and YopT) that are injected into the host cell cytoplasm. Yops (Yersinia outer proteins) disturb the dynamics of the cytoskeleton, inhibit phagocytosis by macrophages, and downregulate the production of proinflammatory cytokines, which makes it possible for yersiniae to multiply extracellularly in lymphoid tissue. Y. enterocolitica serotype O:8 belongs to the highly mouse-pathogenic group of yersiniae in contrast to Y. enterocolitica serotype O:9. However, there has been no systematic study of the contribution of Yops to the pathogenicity of Y. enterocolitica O:8 in mice. We generated a set of yop gene deletion mutants of Y. enterocolitica O:8 by using the novel Red cloning procedure. We subsequently analyzed the contribution of yopH, -O, -P, -E, -M, -T, and -Q deletions to pathogenicity after oral and intravenous infection of mice. Here we showed for the first time that a DeltayopT deletion mutant colonizes mouse tissues to a greater extent than the parental strain. The DeltayopO, DeltayopP, and DeltayopE mutants were only slightly attenuated after oral infection since they were still able to colonize the spleen and liver and cause systemic infection. The DeltayopO mutant was lethal for mice, whereas DeltayopP and DeltayopE mutants were successfully eliminated from the spleen and liver 2 weeks after infection. In contrast the DeltayopH, DeltayopM, and DeltayopQ mutants were highly attenuated and not able to colonize the spleen and liver on any of the days tested. The DeltayopH, DeltayopO, DeltayopP, DeltayopE, DeltayopM, and DeltayopQ mutants had only modest defects in the colonization of the small intestine and Peyer's patches. The DeltayopE mutant was eliminated from the small intestine 3 weeks after infection, whereas the DeltayopH, DeltayopP, DeltayopM, and DeltayopQ mutants continued to colonize the small intestine at this time.

FIG. 1.

(A) Coomassie blue-stained SDS-PAGE gel showing secreted proteins of WA-C(pYV::CM) (wild type [WT], lane 1) and Δyop mutants (lanes 2 to 9) precipitated from the culture supernatant. (B) Western blot of secreted proteins from culture supernatant of WA-C(pYV::CM) [WT, lane 1] and WA-C(pYVΔT) (ΔT, lane 2) with a rabbit anti-YopT polyclonal antibody.

FIG. 2.

Time course of colonization and persistence of Y. enterocolitica Δyop mutants and WA-C(pYV::CM) after oral infection of C57BL/6 mice with 10⁸ CFU. Two (A), five (B), seven (C), twelve (D), and twenty-one (E) days after infection, surviving bacteria in the SI (░⃞), PP (□), spleen (▪), and liver (▨) were determined. Values represent the average log CFU per organ for six mice with the standard errors of the means indicated by error bars. The limits of detection were 10 CFU/organ for PP and spleen and 50 CFU/organ for liver and SI. A “+” indicates that the strain was lethal for mice. Asterisks indicate statistical significance (P ≤ 0.05) between colonization of WA-C(pYV::CM) and the mutants.

FIG. 3.

Colonization and persistence in spleens over 3 weeks by Y. enterocolitica Δyop mutants and WA-C(pYV::CM) after oral infection of C57BL/6 mice with 10⁸ CFU. Asterisks indicate death of mice between days 7 and 12 postinfection. ΔyopQ, -H, and -M mutants were below the limit of detection (10 CFU/organ). Values indicate the average log CFU per organ for six mice.

FIG. 4.

Colonization of spleens, livers, PP, and SIs of C57BL/6 mice after oral infection with 10⁸ CFU of WA-C(pYVΔYopH) (▪), WA-C(pYVΔYopH+H) (□), and WA-C(pYV::CM) (▨) on day 5 postinfection.

FIG. 5.

Colonization of spleen (▪) and liver (□) of C57BL/6 mice 2 (A) and 4 (B) days after intravenous infection with 4 × 10⁴ CFU of the indicated Y. enterocolitica Δyop mutants and WA-C(pYV::CM). Each bar represents the mean log CFU for five mice. Asterisks indicate statistical significance between colonization of the Δyop mutants and of the WA-C(pYV::CM) (P ≤ 0.05).