SummaryCell-permeable proteins, also called cell-penetrating peptides (CPPs), have the ability to cross cellular membranes, either alone or in association with bioactive cargo. We identified the Yersinia protein YopM as a novel bacterial cell-permeable protein. Here, we describe the ability of isolated recombinant YopM to enter host cells without a requirement for additional factors. This autonomous translocation of YopM was confirmed in several cell types, indicating that it is an intrinsic property of YopM. Using truncated versions of YopM, we show that either of the two N-terminal -helices of YopM mediates translocation into the cells. Furthermore, the two -helices are also able to deliver heterologous cargo, such as GFP or YopE. In addition, we found that, after entering the cells, YopM is functional and efficiently downregulates the transcription of pro-inflammatory cytokines (such as tumor necrosis factor- and interleukins 12, 15 and 18). This finding suggests the potential use of YopM as a tool for protein delivery. Furthermore, it can lead to important advances in understanding and evaluating the intracellular and molecular function of YopM without the need for infection with Yersinia.
SummaryEnteropathogenic Escherichia coli (EPEC), atypical enteropathogenic Escherichia coli (ATEC) and enterohemorrhagic Escherichia coli (EHEC) belong to the family of attaching and effacing (A/E) pathogens. Pathogenicity is mediated by subversion of host cell functions involving type III secretion system (TTSS)-dependent effector proteins. In this study, we have identified and characterized a novel TTSS-dependent effector protein encoded at the 5Ј-end of the locus of enterocyte effacement (LEE) pathogenicity island (PAI) of ATEC strain 3431-4/86 (O8:H -). Using affinity purification we identified IQGAP1, a scaffolding protein involved in the regulation of the actin cytoskeleton, as a putative host cell target. Accordingly, we termed the novel effector protein 'Ibe' for IQGAP1-binding effector. The interaction of Ibe and IQGAP1 was confirmed by co-immunoprecipitation from ATEC-infected cells and immunofluorescence analysis, which revealed colocalization of Ibe and IQGAP1 in ATEC-induced pedestals and actin-rich membrane ruffles. This suggests that the putative effector function of Ibe is mediated via IQGAP1. The Ibe-independent recruitment of IQGAP1 to ATEC-induced pedestals implies a general role for IQGAP1 in the subversion of host cell functions during infection. Homologues of the novel effector Ibe are widely distributed among EPEC, ATEC and EHEC strains but are not necessarily genetically linked to the LEE as they have occasionally also been found to be encoded within lambdoid prophages.
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