Unusual, virulence plasmid-dependent growth behavior of Yersinia enterocolitica in three-dimensional collagen gels

Abstract

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37 degrees C. Motility at 37 degrees C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVDelta yadA-carrying yersiniae formed loosely packed microcolonies at 37 degrees C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca(2+)-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions.

FIG. 1.

Growth characteristics of Yersina in 3D-CoG. Imaging of the growth characteristics of Yersinia WA(pYVO8) and its plasmidless derivative WA-C in a 3D-CoG environment at 37°C after 20 h by different microscopic techniques. (A) Conventional phase contrast microscopy. Scale bar, 10 μm. (B) Confocal reflection contrast microscopy (an illustration of collagen fibers is shown in green) combined with fluorescence microscopy (an illustration of Yersinia cells expressing RFP is shown in red). Scale bar, 10 μm. (C) Electron microscopy. (C1a) Original magnification, ×1,650. Scale bar, 1 μm. (C1b) Original magnification, ×21,000. Scale bar, 100 nm. (C1c) Original magnification, ×66,740. Scale bar, 100 nm. (C1d) Excerpt of panel C1c. Scale bar, 100 nm. (C2a) Original magnification, ×20,080. Scale bar, 1 μm. (C2b) Excerpt of panel C2a. Scale bar, 100 nm. (C2c) Original magnification, ×37,000. Scale bar, 500 nm. (C2d and C2e) Excerpts of panel C2c. Scale bars, 100 nm. The plasmidless strain WA-C (A1, B1, and C1) grows singly or in small clusters of only a few cells, while the pYV-positive strain WA(pYVO8) (A2, B2, and C2) forms spherical dp microcolonies. An analysis of the cellular ultrastructure by electron microscopy reveals a fibrillar layer (red arrows) on top of WA(pYVO8) (C2), which forms dp microcolonies, in contrast to the case for WA-C (C1).

FIG. 2.

Growth progress of Yersinia in a 3D-CoG environment at 37°C. WA(pYVO8) and its plasmidless derivative WA-C were studied in 3D-CoG by time-lapse video microscopy (phase contrast) for 20 h, and growth characteristics were compared. WA-C (A to C) grows in small clusters of only a few cells or singly, while WA(pYVO8) forms chains of rods (which start to convolute) and establishes dp spherical microcolonies within 20 h (D to F). Scale bars, 10 μm.

FIG. 3.

Growth curve of WA(pYVO8) (A) and WA-C (B) within the 3D-CoG at 37°C. For the determination of the growth rate, the 3D-CoG was liquefied with collagenase at fixed time points to release yersiniae for plating on solid medium. From colony counts, doubling times were calculated. *, because of microcolony formation of WA(pYVO8), which did not allow disintegration into single bacterial cells, later time points were canceled. Data were collected from three independent experiments. Error bars indicate standard deviations.

FIG. 4.

Growth of Yersinia in three-dimensional motility agar. For comparison purposes, WA-C (A) and WA(pYVO8) (B) were analyzed in an agar environment instead of a collagen environment. After growing for 20 h at 37°C, both strains formed tiny aggregates. Scale bars, 10 μm.

FIG. 5.

Growth behavior of yadA as well as inv mutant strains in 3D-CoG after 20 h at 37°C. The growth phenotype of different yadA mutant strains (A to F) was studied with respect to the diverse functions of YadA. YadA-negative yersiniae (A) formed net-like, lp microcolonies, while collagen nonbinding YadA yersiniae (B) as well as neutrophil nonbinding YadA yersiniae (C) and stalk-deleted YadA yersiniae (E) featured spherical dp microcolonies. Only the deletion of the head domain of YadA (D) mediates lp microcolonies comparable to those of the yadA-negative strain (A). Complementation of the yadA-negative strain with wild-type YadA (F) restores the YadA-positive phenotype of dp microcolonies. In contrast to the pYV- encoded adhesin YadA, the chromosomally encoded invasin Inv of Yersinia has no impact on the growth behavior in the 3D-CoG (G to H): WAΔinv(pYVO8) and WA-CΔinv show no differences in the growth behavior within the 3D-CoG model compared to WA(pYVO8) and WA-C, respectively. Scale bars, 10 μm.

FIG. 6.

YadA expression in the 3D-CoG. (A) Verification of the presence of YadA in Yersinia WA(pYVO8) microcolonies by immunofluorescence using a YadA-specific rabbit antiserum. The left panel shows phase contrast; the right panel shows fluorescence. Scale bars, 10 μm. (B) Verification of the presence of YadA in Yersinia WA(pYVO8) microcolonies in cell lysates by SDS-PAGE. The arrow indicates multimeric YadA (180 to 200 kDa). Lane 1, WA(pYVO8); lane 2, WA(pYVO8ΔvirF).

FIG. 7.

Growth behavior and motility of different mutant strains in the 3D-CoG after 20 h at 37°C. Studies with motAB mutant strains [WAΔmotAB(pYVO8) and WA-CΔmotAB] revealed immotile yersiniae and the growth of WAΔmotAB(pYVO8) yersiniae in spherical dp microcolonies (A) and that of WA-CΔmotAB yersiniae in large irregular clusters (B), dissimilar to dp microcolonies. WA(pYVO8ΔvirF) showed the same growth behavior as that of the parent strain WA(pYVO8), with dp spherical microcolonies (C) and no motile cells. Strain WA-C(pTTSS), carrying a minivirulence plasmid with the genes of the TTSS and yadA, formed spherical dp microcolonies and no motile cells could be detected (D). WA-C(pBR322 EH-5), expressing only YadA, was also able to form dp microcolonies with immotile yersiniae (E). Furthermore, strain WA-C(pLCR), with a 30-kb SalI/XbaI fragment of pYVO8, which harbors the genes of the TTSS needle complex, but neither yadA nor genes encoding one of the six Yop effector proteins nor YscM2, showed a growth behavior similar to that of WA-C (that is, the formation of small clusters and single motile bacteria) (F). Scale bars, 10 μm.

FIG. 8.

Documentation of the motility phenotype of strain WA-C within the 3D-CoG at 37°C. (A) Strains WA-C and WA(pYVO8) were grown with 1% anti-flagellum antiserum within the 3D-CoG and checked microscopically in regard to the growth phenotype. WA-C yersiniae were found to be immotile and formed large irregular clusters (A1a). Control antiserum did not inhibit the motile phenotype of strain WA-C (A1b). Furthermore, dp microcolony formation of WA(pYVO8) was not affected by the addition of anti-flagellum (A2a) or control antisera (A2b). Scale bars, 10 μm. (B) Documentation of the motility of strain WA-C (B1) in contrast to that of WA-CΔmotAB (B2) by tracking experiments.

FIG. 9.

Expression of yop effector genes of Yersinia in the 3D-CoG. To check the influence of Ca²⁺ on growth behavior and on yopE expression, WA(pYVO8, pYopE₁₃₈-GFP) was grown in normal 3D-CoG medium (A and C1) and in parallel in Ca²⁺-depleted 3D-CoG medium (B and C2). (A) By using a yopE-gfp reporter plasmid [WA(pYVO8, pYopE₁₃₈-GFP)], we demonstrated that yopE is expressed in the 3D-CoG at 37°C. Scale bars, 10 μm. The left panel shows fluorescence; the right panel shows phase contrast. (B) Ca²⁺ depletion in the 3D-CoG medium led to a drastic growth inhibition. Scale bars, 10 μm. The left panel shows fluorescence; the right panel shows phase contrast. (C) Intensity plot of GFP fluorescence of WA(pYVO8, pYopE₁₃₈-GFP) in 3D-CoG medium (C1) and in Ca²⁺-depleted 3D-CoG medium (C2) after 20 h at 37°C. Images were recorded with the same parameter settings to allow quantitative evaluation.