Yersinia pseudotuberculosis Membrane Vesicles Deliver Virulence Factors into Host Cells In vitro

Abstract

Yersinia pseudotuberculosis is a Gram-negative pathogenic bacterium that is believed to be an evolutionary ancestor of Yersinia pestis, a deadly pathogen causing bubonic plague. As a pathogen, Y. pseudotuberculosis causes a foodborne disease in humans and zoonotic infections in a variety of animal hosts, both wild and domestic. It is known that Yersinia pseudotuberculosis delivers various virulence factors into the host cells via type III secretion system (TTSS) during infection. Recent studies related to pathogenic bacteria showed most pathogenic bacteria uses an alternative mode of the delivery vehicle called membrane vesicles (MVs) in addition to their secretion systems. The current study focused on Yersinia pseudotuberculosis membrane vesicles and their impact on mammalian host cell in vitro. Transmission electron and super resolution microscopy analysis revealed 24 hours grown Yersinia pseudotuberculosis produced MVs. Further proteomic analysis by LC-MS/MS identified 887 proteins associated with membrane vesicles. MVs contained some secreted bacterial proteins, including chaperonin (GroEL), Attachment invasion locus proteins (AliA), urease, etc. Based on subcellular localization predictions, MVs were found to be a double membrane structure. Further studies on human airways epithelial cell NuLi1 revealed purified MVs induced morphological changes and were shown to be cytotoxic as they were internalized into the cells successfully delivering 60 kDa Chaperonin and cytotoxic necrotizing factor (CNFy). When purified MVs were tested on murine macrophage, RAW 264.7 cells severer actin rearrangement could be observed within 3 hours. Significant loss in raw cell viability could be observed after 48 hours in a dose dependent manner as determined by MTT assay while confocal microscopy revealed giant cell multinucleated phenotype due to MVs exposure. Flow cytometric analysis confirmed that apoptosis is not a major event in cytotoxicity with only 2% cells found to be apoptotic. Furthermore, MVs induced high levels of TNF-α in raw cells. Finally, as cytotoxic necrotizing factor toxin (CNFy) is one of the crucial virulence factor allied with Yersinia pseudotuberculosis YPIII (+), we also tried to characterize this CNFy in detail in this study. Some early proteomics study confirmed that there was some signature post translational modification (PTM) of the Y. pseudotuberculosis CNFy protein. For further confirmation, we cloned and expressed Y. pseudotuberculosis (termed YCNFy) in E.coli (termed ECNFy). Analyzes with both Yersinia and E.coli CNFy for multinucleation in NuLi1 cells confirmed that YCNFy was 8 times more active than ECNFy. Comparative proteomics unveiled a greater PTM value of 61 in the Yersinia CNFy compared to that expressed in E. coli with a modification of only 6 amino acids. Out of all, only Lysine 1003 is “methylation” was found to be common in both YCNFy and ECNFy whereas 17 glutamic acid methylation could only be found in YCNFy suggesting that PTM is required for the activity of CNFy. This study thus describe some novel and stirring characteristics of Y. pseudotuberculosis MVs and CNFy. Follow-up studies designed to utilize such unique findings should extend the scope of Y. pseudotuberculosis research regarding infection prevention and control.