Shevchenko I. The role of lipopolysaccharide WaaL ligase biological properties implementation in the Yersinia enterocolitica bacteria

The aim of current study was to estimate the influence of waaLOS and waaLPS genes deletion on lipopolysaccharide (LPS) synthesis, motility and stress resistance of bacteria Y. enterocolitica 6471/76 and 8081. An optimized expression system for obtaining recombinant WaaLOS ligase in quality and quantity required for crystallization has been designed. Single and double waaL mutants were created by replacing the wild-type alleles in bacterial chromosome for mutant ones. The phenotypes of mutants were visualized by DOC-PAGE gels stained with silver and immunoblot with specific to O-polysaccharide (OPS) and outer core (OC) monoclonal antibodies. Deletion of waaLOS gene from Y. enterocolitica genome has a marked effect on OC and SR-LPS ligation either in single or double mutants. The waaLPS deletion has an opposite effect on the OPS ligation - barely detected increasing of OPS bands among single mutants. On the other hand, clear OPS and OC ligation was not detected neither by immunoblotting nor by bacteriophage specificity assay with FWA1 and F80-18 phages. Through the lack of lysis zones one can testify the lack of full receptor for bacteriophage binding. The ligase structural and the ligase specificity studies will elucidate the functional mechanism of the LPS ligases. We have been able to design an optimized expression system for obtaining His6-tagged WaaLOS ligase in quality and quantity required for determining the 3D-structure. Recombinant WaaLOS ligase biological activity confirmation was provided with help waaL knock-out Escherichia coli CLM24 strain, which electroporated with pTTQ18-waaLos. The His6-tagged WaaLOS ligase activity was analyzed by visualization LPS phenotype in silver stained DOC-PAGE. Around 30% of transformed waaL knock-out E. coli bacteria produced weak OPS bands, which confirmed that pTTQ18_waaLos contain biologically active ligase and is able to reproduce it. To establish the role of WaaL ligase in Y. enterocolitica mutants' biology, we conducted a series of experiments, which included virulence research, motility and stress resistance evaluation. To assess the impact of WaaLOS, WaaLPS or both ligase deletion in virulence creation for pathogenic Y. enterocolitica strains we examined the sensitivity of mutant bacteria to influence of human serum killing by performing serum killing assay. The results of normal serum killing showed clear reduction of serum resistance among single and double ligase mutants for both serotypes. Y. enterocolitica 8081 ligase mutants, however, showed full resistance to alternative pathway mediated killing. Further work will be needed to clarify the role of WaaL LPS' ligases of Y. enterocolitica for virulence in vivo. The ability to move in and colonize the surface of substrates associates with biofilm formation and virulence of pathogenic bacteria. Swimming motility of mutants was analyzed by motility assay and flagellin production was detected by immunoblotting with flagellin-specific mAb 15D8. We found that deletion of waaL genes drastically affected the production of flagella and correlate with the significantly reduced swimming motility among double ligase mutant of Y. enterocolitica 6471/76. In Y. enterocolitica 8081 bacteria motility decreasing was associated with WaaLPS ligase deletion. Both waaL ligase genes deletion lead to a drastic reduction of bacterial stress resistance. There are hypertonic medium, surfactants, antimicrobial peptides and reactive oxygen species - stress agents in the growth media, which in minor concentrations caused Y. enterocolitica 6471/76 double ligase mutant growth decline. It is likely that the absence of polysaccharide chains at the surface of the double mutants leads to inhibition of their growth in the medium with stressors. However, for growth of Y. enterocolitica 8081 double mutants at the level of the wild-type SR-LPS was sufficient.