Introduction

The major virulence system of pathogenic Yersinia spp. is a type III secretion system that mediates the translocation of effector proteins (Yersinia outer proteins; Yops) into infected cells. These Yops interact with host proteins and exhibit manifold functions in modulating host cell immune responses. YopP is an acetyltransferase, which, via acetylation of NF-kB and MAPK pathway-related kinases, disrupts central innate immunity pathways within the host cell. Phosphorylations are common post-translational modifications, which may alter the function and activity of the modified protein. Our results indicate that YopP is phosphorylated by the host cell.

Materials & Methods

A fusion construct between a peptide tag (ALFA-Tag) and YopP was created and introduced into a knockout Yersinia lineage. J774 macrophages were infected and cell lysates were analyzed by immunoblotting. ALFA-tagged YopP was immunoprecipitated and analyzed via Mass spectrometry (MS) to identify YopP phosphorylation sites. Point mutations of the identified sites were inserted in YopP in order to study the relevance of YopP phosphorylation in host cell infection.

Results

Yersinia infection of J774 macrophages followed by immunoblotting of cell lysis revealed the appearance of additional YopP bands with slower electrophoretic mobility. These additional YopP bands disappeared by phosphatase treatment, but were stabilized by the phosphatase inhibitor Calyculin A, indicating being a result of a phosphorylation. After SDS-PAGE, a band corresponding to phosphorylated YopP was analyzed via MS, which revealed Serin-6 and -10 of YopP as phosphorylated by the host cell, starting as early as 15 mins post infection. Then, phospho-mimicking (S6/10D) and phospho-nil (S6/10A) YopP mutants were created, which resulted in expectedly altered electrophoretic mobility patterns in westernblot experiments.

Summary

Our results show that Y. enterocolitica YopP is phosphorylated at S6 and S10 by yet unknown host cell kinases. Studies are ongoing in order to assess the physiological relevance of these phosphorylation events on YopP functions and their consequences for the host cell immune response.