Enterohemorrhagic E. coli Requires N-WASP for Efficient Type III Translocation but Not for EspFU-Mediated Actin Pedestal Formation

Abstract

Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspFU (aka TccP) that trigger the formation of F-actin-rich ‘pedestals’ beneath bound bacteria. EspFU is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspFU into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspFU, we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspFU were the only bacterial effectors required for pedestal formation, and the EspFU sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspFU, presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization.

Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenicEscherichia coliTir effector

“…We propose that Nck is required for the translocation of Tir EPEC and Map EPEC , in a similar way that efficient translocation of Tir EHEC and EspFu EHEC requires N-WASP and actin assembly. 53 In our studies, EPEC-infected Nck-deficient cells presented a reduction of Tir and pedestals of 88 % and 94 % respectively ( Fig. 1; Fig.…”

“…S3) might be a consequence of the low levels of Tir found in these cells; in agreement with the reported decrease adhesion of EHEC to N-WASP-deficient cells that present decrease levels of Tir EHEC . 53 However, the major EPEC adherence factor for HeLa cells is the bundle forming pilus followed by Tir-Intimin interaction. Thus, it is likely that the difference in binding reflects the absence of a receptor for this human-specific pathogen on murine cells.…”

Human Endothelial Cells Internalize Candida parapsilosis via N-WASP-Mediated Endocytosis

“…Although Als3 is present preferentially on hyphae, other adhesins are present on both yeast and hyphae (16) and may serve to trigger N-WASP activation. Other pathogens, such as Shigella (40) and enterohemorrhagic Escherichia coli (41), have been shown to require N-WASP-mediated actin polymerization, but the current study is the first report showing its role in internalization for Candida. These findings suggest that N-WASP plays a central role in internalization of pathogenic microorganisms.…”

A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion

“…It has been demonstrated by several research groups that Tir translocation appears to be a hierarchical process (37,39,47), occurring before other effectors are injected into host cells. Our data indicate Tir secretion even with alteration of CesT at the C-terminal region, whereas in stark contrast, NleA secretion was impaired with CesT alteration.…”

“…A quantitative assay for Tir-mediated actin reorganization (binding index) was performed on HeLa or HT29 cells using the method described by Vingadassalom et al (47), with detection by fluorescent phalloidin staining as previously described (42). To detect Tir and NleA protein translocation resulting from EPEC infection, HeLa cells were mechanically fractionated and immunoblotted with appropriate antibodies, as previously demonstrated (48).…”