The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype

Aim. To estimate WaaL ligase contribution in the lipopolysaccharide (LPS) phenotype profile formation of Y. enterocolitica O:3 (YeO3) bacteria. Methods. The waaL-knock-out mutants were created by an allelic exchange strategy. The LPS phenotypes of created mutants were visualized by silver-stained DOC-PAGE and immunoblotting with specific outer core (core oligosaccharide, hexasaccharide, OC) and O-polysaccharide (OPS or O-Ag) monoclonal antibodies. Results. Deletion of waaL OS gene from YeO3 genome has a marked effect on OC ligation in either single or double mutants. The waaL PS deletion has an opposite effect on the OPS ligation – barely detected increasing of OPS bands. Conclusions. The LPS ligases of YeO3 exhibit relaxed donor substrate specificity. Under given conditions the effect of WaaL OS ligase is more significant for OC and OPS ligation onto lipid A than that of WaaL PS .


Introduction.
In Europe, yersiniosis is the third most common bacterial zoonosis after campylobacteriosis and salmonellosis [1].Y. enterocolitica is a well known human and animal pathogen.Among humans, the pathway of Y. enterocolitica is associated with intestinal disease, such as enterocolitis, with inflammatory diarrhea, ileitis, mesenteric appendicitis and gastroenteritis.A diarrheal disease is sometimes followed by post-infectious reactive arthritis.
It is well known that all Gram-negative bacteria contain an outer leaflet with a large amount of lipopolysaccharide (LPS).LPS is a glycolipid consisting of three domains: the lipid A moiety, the core and the distal Opolysaccharide (OPS or O-Ag).The homopolymeric O-Ag is composed of b1,2-linked 6-deoxy-L-altrose residues.Together with the hexasaccharide core, the O-Ag is linked to the inner core (IC) of LPS to form a branched structure [2].The antigenic variations of OPS in the Y. enterocolitica isolates are distinguished serologi-cally.Nowadays, more than 50 serotypes are known, of which O:3, O:5, 27, O:8 and O:9 are pathogenic [3].
The LPS biosynthesis is a complex process that includes the stepwise transformation of the primary substrate under enzymatic treatment.The WaaL proteins are involved in the ligation of OC and O-Ag onto the lipid A core.According to the in silico investigations, the Y. enterocolitica O:3 (YeO3) genome contains at least three genes responsible for the WaaL proteins expression.Yersinia pestis and Yersinia pseudotuberculosis, however, carry only the waaL PS gene, whereas either waaL OS or waaL XS or both are additionally present in other Yersinia species.
It was shown that deletion of waaL OS and waaL PS genes correlate with the OPS and the OC expression.For this purpose LPS ligases were named as WaaL PS and WaaL OS respectively.The third ligase named as WaaL XS was not involved in the LPS or ESA biosynthesis [4].
The current study is aimed at the estimation of a role of the ligases in the Y. enterocolitica' LPS phenotype profile formation.The waaL-knock-out mutants of YeO3 were created by the allelic exchange strategy.Phenotypes of created mutants were visualized by silver-stained DOC-PAGE and immunoblotting with specific OC and O-Ag monoclonal antibodies.
Materials and methods.Bacterial strains and culture conditions.Bacterial strains are listed in Table .Yersinia strains were grown at 22-25 o C (RT) and Escherichia coli strains at 37 o C in Luria Broth (LB) media.LB supplemented with 1.5 % Bacto Agar was used for all solid cultures.As a selective medium CIN agar supplemented with appropriate antibiotics was used.When appropriate, the antibiotics were added to the media at the following concentrations: kanamycin (Km), 100 µg/ml in agar plates and 20 µg/ml in broth; chloramphenicol (Clm), 20 µg/ml.
General DNA techniques.Isolation of plasmids and genomic DNA was done with kits.All enzymes were used according to the supplier's specifications.Smallscale plasmid DNA preparations were carried out using plasmid mini prep kits.Plasmid DNA was moved by electroporation into Y. enterocolitica or heat shock transformation.Recombinant plasmids were mobilized from E. coli strains to Y. enterocolitica by conjugation.
Mutants construction.The waaL OS and waaL PS genes were amplified by PCR with primer pairs O3lig YE1727F5 & O3ligYE1727R5 and O3ligYE532F2 & O3ligYE532R2 with DyNAzyme DNA-polymerase («Thermo Scientific», USA) from isolated genomical DNA of YeO3.Amplified DNA was purified with Kit method and digested with NsiI (Mph 1103I) for the waaL OS gene and PstI for waaL PS .The digested and purified fragments were cloned into the PstI digested suicide vector pSW23T and the constructed plasmids were named as pSW23T-waaL OS and pSW23T-waaL PS respectively.The constructions were mobilized from E. coli w7249 into YeO3 strains by conjugation as described earlier [5].For elimination of suicide vector and the wildtype genes, the optimized cycloserine enrichment method was used [5].
For large-scale screening of knock-out mutants among Clm sensitive bacteria (Clm S ) colonies we used Colony hybridization kit method («Roche», France).Isolated genomical DNA from negative colonies were diluted and used as a template for PCR with different primer pairs.DNA of wild-type strain YeO3 was used as a control.
Complementation.The waaL OS and waaL PS genes were amplified with Phusion DNA polymerase from YeO3-c with O3ligYe1727f & O3ligYe1727r, O3lig Ye532f & O3ligYe532r primer pairs.PCR fragments were phosphorylated with polynucleotide kinase in the presence of 10 mM ATP, digested with EcoRI and ligated with EcoRI and ScaI digested, SAP-treated pTM100.The constructed plasmids were named pEPlig1727 & pEPlig532 and electroporated into S17-1l pir with further mobilization into YeO3 ligase mutants by conjugation.Obtained colonies were screened on appropriate antibiotic plates with CIN agar [4].DOC-PAGE analysis.The bacteria were grown 16-20 h at RT in 5 ml of LB medium with appropriate antibiotics.The exact optical density of the cultures was measured at 600 nm (OD 600 ), 3 ml of the cultures were centrifuged and the pellets were resuspended in deoxycholate lysis buffer (2 % DOC, 4 % 2-mercaptoethanol, 10 % glycerol and 0.002 % bromophenol blue in 1 M Tris-HCl buffer, pH 6.8) in a volume adjusted according to density of the culture (100 ml/OD 600 ~1).Lypopolysaccaride phenotypes were analyzed by silver stained DOC-PAGE with previous proteinase K treated whole cell lysates [4].The Western blotting O-polysaccharide, outer core and inner core expression were detected by O:3 specific mAbs 2B5 and TomA6.
Results and discussion.Mutants construction.The waaL OS and waaL PS mutants of YeO3 were constructed from fully virulent serotype O:3 patient isolate expressing complete LPS.With help of the allelic exchange strategy we managed to inactivate waaL OS and waaL PS encoding regions in the YeO3 genome.The constructed single and double mutants were complemented with the pEPlig1727 & pEPlig532 plasmids, which were supplemented with the functional waaL gene.
Traditional cycloserine enrichment method was additionally optimized [5].The possibility was conside-red that constructed merodiploids (MD) are not fully resistant to Clm, as it should be (weak operon, etc.).To examine this possibility we tested several lines of conditions (concentration of Clm, incubation time, density of bacteria, etc.).According to these experimental data, the next improvements were done: reducing the Clm concentration in media to 2.5 ml v/v (instead of 10 ml v/v) and prolongation of incubation to 4-5 h (instead of 2-3 h) before D-cycloserition solution was added.A current modification of the method allowed us to pick up only bacteria after second crossing over (Fig. 1).
The colony hybridization method was used for specific detection of deletion in the waaL gene among Clm S .Further justifications of deletion were performed by PCR (Fig. 2).
Phenotype analysis.We used two approaches to analyze the difference in the expression of OC and O-Ag: 1) Silver staining of DOC-PAGE; 2) Immunoblotting with OC and O-Ag specific antibodies.
It is noticeable from silver staining of DOC-PAGE that deletion in the waaL OS gene leads to a dramatic decrease in the OC expression and appearance of strong IC band (Fig. 3).However, the level of O-Ag expression reduced, as well, compared to the mutant with deletion in the waaL PS gene.The deletion in the waaL PS gene Fig. 2. Amplification of waaL OS gene by PCR with pair of primers O3lig Ye1727f & O3ligYe1727r: 1-11checking colonies; 12 -gene ruler; 13 -waaL OS -knock-out mutant; 14 -YeO3 wild type control seems to be insignificant for the OC expression and even to stimulate the OPS formation.In case of the double YeO3_Dos_Dps mutants, it is noticeable a strong expression of IC bands, the absence of OC in both variants and the absence of O-Ag expression in one variant.
Western blotting analysis of LPS samples of the YeO3 ligase mutants and their complementation of single and double mutants were performed with the OCspecific mAb 2B5 and O-Ag-specific mAb TomA6 (Fig. 4).The deletion in the waaL OS gene resulted in reduction of the OC expression, as it was shown with silver staining of DOC-PAGE.Also, the waaL OS -knockout mutant complemented with the functional waaL OS gene showed full recovery of OC and the decreasing of O-Ag expression at the same time.
Similar results were obtained with the double mutant and complementation variants.The LPS profile of the double YeO3_Dos_Dps mutant in immunoblotting was similar to that with silver staining where only the O-Ag expression took place.The complementation with the functional waaL OS and waaL PS genes showed inhibition of O-Ag expression and full recovery of OC in both cases.Disruption in the waaL PS gene as a single mutation leads to hardly noticeable stimulation of the O-Ag expression.
Thereby, the data obtained from silver staining and immunoblotting of DOC-PAGE evidence the participation of WaaL ligase in the LPS phenotype creation.However, strict substrate specificity of the LPS ligase in YeO3 was not detected.
Conclusions.Summarizing the obtained data we can conclude that the LPS ligases of YeO3 exhibit relaxed donor substrate specificity.It has been established that under given conditions the effect of the WaaL OS ligase is more significant for the OC and OPS ligation onto lipid A than for the WaaL PS ligase.It is possible that deletion of the waaL OS and waaL PS genes and changes in the OC or O-Ag moieties of LPS enhance the ability of pathogen to evade host defenses.Further work is required to elucidate the biological significance of these different settings.
Funding.This work was supported by grants from Center for International Mobility (CIMO), Finland TM-12-8286.