Identification of potential adhesin receptors for Yersinia ruckeri: expression and purification of Yersinia ruckeri Invasin

Abstract

Yersinia ruckeri is a causative agent of yersiniosis or enteric redmouth disease (ERM) in salmonid fish. It leads to significant economic losses in fish aquaculture industry each year. The exact virulence mechanism and route of infection of this bacterium in not yet known. Therefore, it was very interesting to investigate if this bacterium uses the same virulence factors to cause disease in fish as the three human pathogenic Yersinia species use to cause disease in humans, Y. pestis, Y. enterocolitica and Y. pseudotuberculosis. Adhesins of the pathogenic Yersiniae are well described virulence factors mediating binding of the bacterial cells to the host cells. Their activity is essential for Yersinia virulence and establishment of infection. In this work two Y. ruckeri protein sequences were identified, namely Y. ruckeri Invasin (YrInv) and Invasin-like molecule (YrIlm), and YrInv was studied in more detail. The amino acid sequence of YrInv was analyzed with multiple bioinformatics tools, and its structure has been based on the comparison with Invasin (InvA) from Y. pseudotuberculosis and Y. enterocolitica, and E. coli Intimin (Int). The YrInv gene sequence was used for making multiple constructs for YrInv expression in E. coli BL21 (DE3) Gold. The expression of both full-length protein in the E. coli outer membrane (OM) and soluble YrInv fragments in E. coli cytoplasm and periplasm were tested. The optimization of soluble YrInv fragment expression was done for large scale expression and purification purposes. The results show high similarity in protein domain organization between the YrInv and compared sequences. In this work, we demonstrate that YrInv can be expressed in E. coli cells, and that the full-length YrInv in E. coli OM is surface exposed. Also, expression of soluble YrInv fragments is shown on both small and large scale. Both full-length YrInv and its fragments are the tools to be used in receptor identification studies on the fish cell lines.