Yersinia enterocolitica Targets Cells of the Innate and Adaptive Immune System by Injection of Yops in a Mouse Infection Model

Abstract

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80+, 11% of CD11c+, 7% of CD49b+, 5% of Gr1+ cells, 2.3% of CD19+, and 2.6% of CD3+ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19+CD21+CD23+ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.

Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein

“…Rho GTPbinding proteins appear to constitute one way of control based on findings that inhibition of Rho and Rac in cells can block Yop translocation (1,42,44). Here we provide evidence that the Rho GTP-binding protein activator CNF-Y strongly increases Yop translocation.…”

“…Here we provide evidence that the Rho GTP-binding protein activator CNF-Y strongly increases Yop translocation. We found that the CNF-Y effect was almost entirely dependent on Rac and independent of RhoA, -B, and -C. This result was unexpected because first, CNF-Y was reported to prefer RhoA as a substrate over Rac and Cdc42, and second, inhibition of Rho had been shown to affect Yop translocation (42,44). As described by Hoffmann et al (49) and confirmed in this study, the CNF-Y effect on the actin cytoskeleton is dominated by stress fibers with only a few membrane ruffles and filopodia present, which suggested Rho activation but no prominent Rac and Cdc42 activation, respectively.…”

“…FRET-based Real-time Translocation Analysis-Methods for detection of translocated effectors by effector-␤-lactamase fusions and cephalosporin-derived FRET substrates were described previously and adapted here for real-time analysis by live cell fluorescence microscopy (44,54,55). Cells were seeded in 96-well plates (black with clear bottom, Greiner Bio-One) at a density of 2 ϫ 10 4 cells/well.…”

“…While Yop delivery progresses, the increasing amount of YopE in the host cell cytoplasm is thought to prevent further translocation by down-regulating Rho GTPbinding proteins. Inactivation of Rho (A, B, and C) with bacterial toxins as well as knockdown of Rac1 via siRNA were reported to inhibit Yop translocation (42)(43)(44); however, a more comprehensive analysis of the Rho family proteins involved in Yop translocation has not been performed.…”

Different Bacterial Pathogens, Different Strategies, Yet the Aim Is the Same: Evasion of Intestinal Dendritic Cell Recognition

“…Despite its predominantly extracellular lifestyle, the bacteria have a TTSS, allowing them to inject a number of effector proteins, including Yersinia outer proteins (Yops) into host cells. Notably, DCs are also targeted by Yops in vivo and injection of these bacterial effector molecules has been shown to inhibit Ag presentation by DCs (57). By interacting directly with the actin cytoskeleton, Yops inhibit the phagocytic activity of DCs (58) and, as proof of principle, transfection of DC with YopP has been shown to reduce uptake of OVA (59).…”