The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL.

Lihi Shaulov,Jenia Gershberg,B Brett Finlay,Wanyin Deng,Neta Sal-Man

doi:10.1128/mbio.01733-16

Abstract

ABSTRACTThe type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins.

Highlights

Gram-negative bacterial pathogens, such as pathogenic Escherichia coli and Salmonella, Shigella, Yersinia, and Pseudomonas spp., are causative agents of serious human diseases ranging from lethal diarrhea to the plague that account for millions of deaths annually worldwide
The T3SS is a syringe-like structure that comprises about 20 different proteins. This large protein complex consists of three major substructures: a basal body embedded within the bacterial membranes, an extracellular needle that bridges the extracellular space between the bacteria and the host cell, and a pore-forming complex, termed the translocon, which forms a channel in the membrane of the host cell
We found that the enteropathogenic E. coli (EPEC) protein EscP, which was previously suggested to function as the ruler protein and is involved in the first substrate specificity switch [42], is involved in regulation of the second substrate specificity switch

Summary

RESULTS

Western blot analysis with the anti-Flag antibody detected the EscP protein in the WCL but not in the secreted fraction of either the WT or the ⌬escP mutant EPEC strain (Fig. 4B). These results suggest that EscP contains a secretion signal, it is not secreted through the T3SS, and its function occurs inside the bacteria. Co-IP experiments were done with cell lysates of BL21 strain expressing EscP-Flag and SepL-His. Samples that contained EDTA or BAPTA showed significantly lower levels than the calcium-containing sample (Fig. 6B), confirming that the calcium dependence of the EscPSepL interaction is not dependent on any additional T3SS components. In line with a previous report, we found that calcium does not affect the interaction between SepL and SepD under conditions similar to those used to examine the SepL-EscP interaction (see Fig. S4 in the supplemental material) [34]

DISCUSSION

MATERIALS AND METHODS

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