RafX-mediated Attachment Of Teichoic Acids To The Cell Wall In Streptococcus Pneumoniae: Implication For Bacterial Virulence

Objective: Streptococcus pneumoniae teichoic acid （TA） is one of themajor virulence factors which is important for bacteria to survive in thehost. Pneumococcal teichoic acids play a crucial role in the maintenance ofthe bacterial surface cation environment. In addition, TAs provide dockingstation for choline-binding proteins. A previous bioinformatic analysisrevealed a complex biosynthesis process for pneumococcal teichoic acid.Attachment of the teichoic acid precursors to the cell wall represents animportant step of pneumococcal TA biosynthesis, but the involved enzymesare not known. Our previous studies have shown that the RafX （SPD₁672）protein may be required for teichoic acid metabolism; however, the featuresof the RafX protein, the enzymatic activity of RafX in teichoic acidmetabolism and role of RafX-mediated TA biosynthesis in bacterialvirulence need to be further clarified.Methods: We first analyzed by bioinformatic analysis the localization inthe genome of pneumococcal D39strain and function of the RafX protein. Next, the rafX gene was deleted from the genome of Streptococcuspneumoniae strains. RT-PCR method was used to characterize thetranscription of rafX gene. Phenotypes of rafX mutants were characterizedby in vitro culture. By using optical microscopy and electron microscopy,the morphology of rafX mutant were assessed. Using laser scanningconfocal and Western blotting analysis, we characterized the subcellularlocalization of the RafX protein.In the second part of the experiments, we used several analyticalmethods, including2%choline elution, SDS treatment, proteinase Kdigestion, lgt gene mutation, lysozyme plus mutanolysin hydrolysis,deoxycholate lysis to prepare and analyze the teichoic acid. Based on thesefindings, deoxycholate solution was therefore used to generate teichoicacids. FACS analysis and ELISA techniques were used to revealed theamount of surface exposed teichoic acid. Bioinformatic analysis was usedto reveal the enzymatic activity of the extracellular loop （EL4） of the RafXprotein. Furthermore, site-directed mutagenesis was used to determineenzymatic roles of the extracellular loops and key amino acids inpneumococcal TA biosynthesis. PCR was used to amplify rafX gene withthe genome DNA from20different ST types of clinical Streptococcuspneumoniae strains. By using Western blotting analysis, the TA bandingpattern of clinical isolates were assessed to address whether RafX-mediatedteichoic acid metabolism is a conserved phenomenon. Finally, the contribution of teichoic acids in bacterial virulence were determined byusing the in vitro adhesion assays and in vivo challenging models.Results: Bioinformatic analysis predicted that RafX harbors O-antigenligase activity. rafX gene was co-transcribed with its upstream gtfA gene.Compared to the wild type D39strain, D39ΔrafX mutant grown on theblood agar plates displayed a smaller colony. In addition, D39ΔrafXmutant was impaired in bacteria growth in comparison with D39wild typewhen grown in the C+Y medium, displaying premature autolysis. By usingoptical microscopy and transmission electron microscopy D39ΔrafX wasshown to harbor a shorter chain length; in addition, D39ΔrafX mutantdisplayed an abnormal cell division and morphology under transmissionelectron microscopy. Using GFP-RafX fusion structures, we showed thatRafX protein was located on the cell membrane, and both of the N-terminaland C-terminal of the protein were located in the cytoplasm.In contrast to wrongly assigned LTA, analytical methods including2%choline washing, SDS treatment, lgt deletion mutation, mutanolysin pluslysozyme hydrolysis, sodium deoxycholate lysis actually generated andanalyzed the wall teichoic acid （WTA） and peptidoglycan （PG） complex（WTA-PG）. Western blotting analysis showed that RafX mutants wereimpaired in WTA-PG banding patterns and were reduced in the amount ofcell wall associated teichoic acids. FACS and ELISA confirmed that RafXmutants were reduced in the total amount of surface exposed teichoic acids. rafX gene could be PCR amplified from all of the20different types ofclinical isolates and DNA sequencing data revealed a sequence identityover99.9%. In addition, all clinical isolates displayed typical teichoic acidbanding pattern without obviously shifting to larger molecular weights.Molecular docking simulations using Molegro Virtual Docker softwareindicated that the amino acid residues of Arg260, His304and His306ofEL4domain was able to fix and bind the truncated teichoic acid precursormolecule （C14H26O7P2） through the hydrogen bonds, suggesting theligase activity of RafX protein. Furthermore, alanine substitutionmutagenesis suggested that H151, R154, R266, W270, G284, P287, H304Aand H306were key amino acid residues for RafX protein function.In vitro adhesion assays showed that R6ΔrafX mutant was only10-15%adhesive of the wild type R6strain. Furthermore, the addition ofanti-CWPS antiserum significantly inhibited the adhesion of wild type R6to MLE12and HUVEC cells, suggesting that teichoic acid itself is requiredfor bacterial adhesion. In addition, RafX ΔEL4, H306A mutant strains wereas adhesive as the R6ΔrafX mutant, while RafX H106A complementedmutant was as adhesive as wild type R6strain, indicating that RafX proteinitself seems not required for pneumococcal adhesion. Flow cytometryanalysis showed that the surface protein CbpA was reduced on the surfaceof R6and TIGR4ΔrafX mutants when compared to the wild type strains,suggesting that the decreased expression of the CbpA protein is partially responsible for the impaired adhesion of rafX mutants.Mice infected with wild type strains, RafX H106A and RafX WLcomplemented strains eventually proceed to the death, whereas miceinfected with ΔrafX mutant and RafX H306A complemented strainsurvived significantly longer than the wild type strain infected mice.Histopathological examination of lung tissue revealed that mice infectedwith wild type D39strain displayed a typical hemorrhage, necrosis,inflammatory cell infiltration, whereas lungs from D39ΔrafX mutantinfected mice only demonstrated a mild inflammation, without bleedingand necrosis lesions being observed, suggesting teichoic acids is closelyrelated to bacterial virulence. In addition, the bacterial loads in nasalwashes, lung, brain and blood following challenge with D39ΔrafX mutantwere significantly lower than those following challenge with the wild typestrain, indicating that teichoic acid is necessary for bacterial colonizationand invasive diseases. To reveal the causes of the virulence changes, D39and D39ΔrafX mutant were grown in the presence of rabbit serum, rabbitwhole blood or neutrophils and macrophages. The data revealed that ΔrafXmutant was impaired in the growth in the presence of rabbit serum andcould be killed by the normal rabbit blood. However, compared to the wildtype D39, ΔrafX mutant was as sensitive to wild type strain to the killing bythe innate immune cells, indicating that the growth defect of ΔrafX mutantis the main contributor to the virulence attenuation. Conclusion: ΔrafX mutants were impaired in bacterial growth, atuolysisas well as bacterial shape and division. The RafX membrane protein islikely a ligase involved in the attachment of pneumococcal teichoic acidprecursors to the cell wall. RafX-mediated teichoic acids biosynthesis ishighly conserved in Streptococci. Teichoic acid itself is an importantnon-protein adhesin which influences the bacterial adhesion also involvingthe contribution of choline binding protein （eg. CbpA）. rafX mutants weresignificantly reduced in bacterial virulence which was primarily attributedto the growth defects and the reduced surface retention of choline bindingproteins.