Structure And Function Of Galactosylated Wall Teichoic Acids In Serotype 4h Listeria Monocytogenes

Listeria monocytogenes（Lm）,an important foodborne zoonotic pathogen,relies on diverse surface proteins for interacting with the environment,including infecting the host.Surface proteins are mainly divided into covalently bound proteins,non-covalently bound proteins and membrane-anchored proteins,which attach to the cell wall through specific interactions.Wall teichoic acids（WTAs）,the most abundant sugar polymer on cell wall surface,provide anchor sites for surface proteins and regulates bacterial morphology,autolysis,and virulence.In addition,WTAs structures are fundamental to bacterial somatic antigens.Two structural classes of WTAs exist in Lm that are mainly distinguished by their variable repeating units into type Ⅰ（including serovars<SVs>1/2,3,and 7）and type Ⅱ（including SVs 4,5,and 6）.Notably,recent studies have demonstrated that L-rhamnosylation of type Ⅰ WTAs contributes to autolytic activity and virulence by promoting efficient surface association of the InlB and Ami through glycine-tryptophan（GW）domains.Moreover,galactosylated type ⅡWTAs is equally essential for surface anchoring of the virulence protein InlB.The GW domain is present in various Gram-positive bacteria and is responsible for anchoring proteins to the cell wall.However,it remains poorly understood about the specific binding mechanism of proteins to glycosylated WTAs.Lm XYSN were isolated from severe ovine listeriosis outbreaks.Comparative genomics suggested that their phylogenomic isolation was in a unified but distinct branch adjacent to lineage Ⅱ.In order to reveal the molecular pathogenic mechanism of hypervirulence of Lm XYSN,a transposable mutation library was constructed and human colon carcinoma epithelial cells were used for high-throughput screening of virulence-related genes in vitro.LMxysn₁095::Tn mutant significantly reduced the invasion abilities to Caco-2 BBe cells and was 105-fold less virulent in mice than the wild strain.Further analysis revealed that the gene cluster where the LMxysn₁095 gene（galT）was located may be involved in WTAs glycosylation modification,while the molecular mechanism of galT in bacterial virulence remains to be elucidated.In this study,Lm XYSN was used as biomaterial to study the bacterial and flagellar antigens as well as virulence phenotypes.We revealed that galT encoded galactosyltransferase responsible for the galactosylation of type Ⅱ WTAs（Gal-WTAs）in Lm XYSN strain,which played crucial roles in bacterial virulence.In addition,we demonstrated the anchoring mechanism between Gal-WTAs and virulence proteins,as well as the biological function of autolysin LygA.Moreover,ΔgalT mutant as a live attenuated vaccine candidate provided the protective immune response in a murine model.This study explored the pathogenic mechanism of the unique WTAs structure in SV 4h strains,providing a theoretical basis for the prevention and treatment of listeriosis.1.Newly named SV 4h strains are highly virulentAgglutination studies with antisera to somatic antigens indicated that Lm XYSN belonged to the serogroup 4.Moreover,Lm XYSN reacted with antisera for flagellar H-A and H-B antigen but not with H-C antigen,indicating that it is the member of a novel SV that was designated as 4h.The invasion rates of SV 4h isolates,including Lm XYSN,Lm 16E,and Lm 15LG were significantly higher than that of all tested Lm strains in Caco-2 BBe cell line.In addition,the bacterial loads in organs showed that SV 4h strains were hypervirulent compared to reference isolates,causing mouse death within 3-5 days.Guinea pigs orogastrically inoculated with Lm XYSN at a dose of 1×109 CFU,progressively succumbed to infection,no deaths were recorded for Lm EGD-e tested using the same dose.These results suggested that SV 4h strains were highly virulent.We further examined the virulence potential of Lm XYSN by comparing it with strain Lm EGD-e in an orogastric sheep infection model.Lm XYSN-infected sheep exhibited a significantly higher body temperature and lower body weight as compared with infected sheep of Lm EGD-e.Meanwhile,Lm XYSN showed 158-426 folds higher colonization capacity in liver and intestine than Lm EGD-e.Moreover,Lm XYSN successively broke through the intestinal barrier and blood-brain barrier to induce gastroenteritis and meningitis neuropathic listeriosis,leading to the death of all sheep within 21-39 days post-infection.In the early phase of infection,Lm XYSN suppressed the transcript levels of IFN-γ,TNFα,and IL-6 in spleen,liver,and mesenteric lymph nodes（MLN）.In addition,IFN-γ expressionin peripheral blood was quantified using ELISA.The results showed that Lm XYSN significantly inhibited IFN-γ secretion in sheep at days 1 and 2 post-infection compared with infected sheep of Lm EGD-e.These results indicated that SV 4h strains significantly inhibit the expression of pro-inflammatory factors,thereby promoting the survival and pathogenesis of bacteria in vivo.2.galT mediates virulence in Lm XYSNLMxysn₁095 gene（galT）was predicted to be a glycosyltransferase and may be involved in WTAs glycosylation modification.Therefore,the structures of WTAs were determined using electrospray mass spectrometry.The results showed that Lm XYSN produced a type ⅡWTA with only galactose decoration（Gal-WTAs）.Deletion of galT abrogated galactose decoration on WTA,indicating that it encoded a galactosyl transferase.Agglutination reaction with serogroup 4-specific antiserum showed galactose decoration on WTAs was associated with integrity of O-antigen.Transmission electron microscopy（TEM）of Lm XYSN and theΔ1095 revealed ultrastructural changes in the surface of the cell wall of the mutant strain.The mutant was also more sensitive to LL-37 and CRAMP than the wild type strain,indicating that galactosylated WTAs increased bacterial resistance toward antimicrobial peptides by maintaining the integrity of the cell wall structure.We examined the influence of galactose-based glycosylation on the location of specific virulence factors to the bacterial surface by Western blot.Deletion of galT showed a strong reduction in the levels of surface-bound InlB,Ami and ActA proteins.In addition,confocal images of Lm stained by anti-ActA monoclonal antibody showed a loss of "actin tail"formation and a defect in intracytoplasmic motility of ΔgalT mutant during infection of Caco2 BBe cells.Taken together,these data indicated that the galactose-based decoration of WTAs was required for stable localization of important well-characterized virulence genes on the surface of the bacteria.The results of Caco-2 BBe cells infection model showed that deletion of galT significantly reduced bacterial invasion and intracellular replication,while the adhesion levels were similar among the three strains.In addition,we examined the contribution of galT to the virulence potential of Lm XYSN in vivo using the mouse oral infection model.The ability of the mutant to colonize deeper organs was severely damaged.Moreover,the LD50 showed a 105-fold increase in the ΔgalT mutant compared to Lm XYSN.These results strongly confirmed that galT played a key role in intestinal barrier translocation and successful organ colonization.3.Galactosylated WTAs interacts with autolysin LygA to mediate bacterial homeostasis and virulenceTo identify novel surface proteins potentially associated to the cell wall surface through WTAs galactosylation,label-free quantitative proteomic analysis of surface proteins from the Lm XYSN and ΔgalT strains was performed.In addition to InlB and Ami,a previously uncharacterized GW domain protein,LMxysn₁092（LygA）,was identified to have the most significant change in abundance（24.42-fold）.Moreover,Western blot analysis showed that the redistribution of LygA in ΔgalT mutant from the cell surface to the secreted compartment,while the transcript levels of lygA did not change.These results indicated that WTAs galactosylation was crucial for the efficient association of LygA to the bacterial surface.In order to determine the specific interaction between LygA and WTAs with surface plasmon resonance（SPR）,we successfully purified different types of WTAs and produced recombinant LygA.The results revealed that the binding affinity of Gal-WTAs to LygA（KD=1.54 μM）was stronger.than that of galactosylation-deficient WTAs（KD=7.61 μM）.Meanwhile,the interaction of LygAGw with Gal-WTAs showed the same trend as that of LygA.In addition,analysis of the effect of GW motif numbers on LygA anchoring showed LygA was gradually shed from the cell wall surface as the number of GW domains continued to decrease,especially LygA almost lost its anchoring ability when the four GW domains were deleted.These data indicated that the non-covalent binding of LygA to WTAs is coordinately modulated by a combination of fixed GW domains in a Gal-dependent manner.Moreover,GW protein Auto,is not modulated by WTAs L-rhamnosylation,strongly associated with GalWTAs（KD=0.08 μM）.The homology of amino acids in GW domains was very low between LygA and Auto.These results indicated that the affinity of WTAs and GW proteins depended on the glycosylation pattern of WTAs and the specific amino acids of GW motif.To further investigate the function of LygA,SMART analysis showed that LygA consisted of LYZ2 and GW domains.We tested the hydrolase activity of LygA using M.lysodeikticus cells.The results showed that autolysin LygA with an activity of 211 U,may change the cell surface hydrophobicity of M.lysodeikticus,resulting in a large number of cell aggregation.WTAs L-rhamnosylation promoted surface association of Ami to regulate bacterial autolysis.Therefore,ΔlygA,Δami,and ΔlygAlami mutants were constructed to study the role of LygA in autolysis.Physiological levels of autolysis in ΔlygA mutant were dramatically decreased compared with Δami mutant,while Δami/lygA mutant exhibited a small lysis deficiency in comparison to ΔlygA strain within 34 h.In addition,LygA was far more abundant than Ami on the cell surface,as revealed by quantitative proteomic analysis.Altogether,these data indicated that autolysin LygA and Ami were related to the autolysis activity of Lm.In particular,LygA dominated the physiological process of auto lysis.To examine the role of LygA in infecting eukaryotic cells,Caco-2 and HepG-2 cells were used to assess the invasion capability of Lm.The ΔlygA mutant exhibited impaired adhesion and invasion abilities in comparison with the WT and complemented strains.Moreover,the deficiency of LygA in bacteria significantly reduced their invasion and colonization abilities in the colon,liver and brain in the mouse oral model infection.Altogether,these results confirmed that LygA was required for the bacterial virulence and played crucial roles in the early colonization of the intestine and further infection of the host.4.ΔgalT attenuated vaccine confers protective immunity against SV 4h LmC57BL/6 mice were inoculated subcutaneously with Lm XYSN and ΔgalT mutant to evaluate the safety of the candidate vaccine.The bacterial loads of ΔgalT mutant in the liver and spleen were significantly lower than those infected with Lm XYSN.Additionally,the mutant could be rapidly cleared by the body.In addition,histopathology did not observe any pathological changes in each organ of mice vaccinated with ΔgalT.These results indicated that the ΔgalT mutant had a certain potential safety as a live attenuated vaccine.ELISA was used to detect the presence of antibodies in the sera of vaccinated mice.After the 14th day post-immunization,anti-Lm XYSN antibodies were induced in mice immunized with ΔgalT mutant.Antibody levels increased successively and reached the highest levels at day 21 post-immunization.In addition,the transcription levels of cytokines in the spleens of mice were measured at day 21 post-immunization.qRT-PCR results showed that the ΔgalT attenuated vaccine candidate induced high expression levels of Thl-related cytokines IFN-γ,TNF-α,IL-6,and IL-17,Importantly,mice were challenged with a lethal dose of Lm XYSN after day 28 post-immunization,showing the ΔgalT vaccine candidate provided 100%immune protection in mice and 83.3%protection efficiency up to 105th day after immunization.These results suggested that the ΔgalT vaccine candidate stimulated a strong humoral and cellular immune responses,mediating high levels of protective immunity.In summary,newly named SV 4h strains,according to the unique combinations of somatic and flagellar antigens,were highly pathogenic in a variety of animal models.Lm XYSN produced a type Ⅱ WTA with only galactosylation.galT encoded a galactosyltransferase involved in galactose modification on WTAs and mediated surface anchoring of important virulence proteins InlB,Ami,and ActA.Meanwhile,the newly identified autolysin LygA directly interacted with Gal-WTAs through a fixed number of GW domains to modulate bacterial homeostasis and virulence.In addition,ΔgalT as an attenuated vaccine candidate was highly effective in protecting against challenge with homologous strains.This study revealed the mechanism of the unique type Ⅱ WTAs in the enhanced virulence of SV 4h strains,providing the theoretical basis for the prevention and control of listeriosis.