We developed a new strategy for the analysis of correlation between genotype profile at baseline and virologic response.

Wall teichoic acid (WTA) are major constituents of Staphylococcus aureus ( S . aureus ) cell envelopes with important roles in the bacteria’s physiology, resistance to antimicrobial molecules, host interaction, virulence and biofilm formation. They consist of ribitol phosphate repeat units in which the ribitol residue is substituted with D-alanine (D-Ala) and N-acetyl-D-glucosamine (GlcNAc). The complete S . aureus WTA biosynthesis pathways was recently revealed with the identification of the two glycosyltransferases, TarM and TarS, respectively responsible for the α- and β-GlcNAc anomeric substitutions. We performed structural analyses to characterize WTAs from a panel of 24 S . aureus strains responsible for invasive infections. A majority of the S . aureus strains produced the β-GlcNAc WTA form in accordance with the presence of the tarS gene in all strains assessed. The β-GlcNAc anomer was preferentially expressed at the expense of the α-GlcNAc anomer when grown on stress-inducing culture medium containing high NaCl concentration. Furthermore, WTA glycosylation of the prototype S . aureus Newman strain was characterized in vivo in two different animal models, namely peritonitis and deep wound infection. While the inoculum used to infect animals produced almost exclusively α-GlcNAc WTA, a complete switch to β-glycosylation was observed in infected kidneys, livers and muscles. Overall, our data demonstrate that S . aureus WTA glycosylation is strongly influenced by environmental conditions and suggest that β-GlcNAc WTA may bring competitive advantage in vivo .

BackgroundLactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is “Generally Recognized As Safe”. Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development.ResultsA recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development.ConclusionsIn this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0271-z) contains supplementary material, which is available to authorized users.