Streptococcus Suis Type 2: Cross-protection By A Type 5 Avirulent Strain And Roles Of Type Ⅳ-like Secretion System In Pathogenesis

Streptococcus suis(S.suis,SS)is an emerging zoonotic bacteria distributed worldwide and can cause septicemia,endocarditis,arthritis and meningitis in humans and pigs through the respiratory tract or mucous membranes.It poses considerable public health concerns as S.suis type 2(SS2)strains could infect humans from dis-eased pigs.Two large outbreaks of human SS2 infection occurred in China in 1998 and 2005 with fifty-two deaths.Symptoms in these two outbreaks were different from previous cases and characterized as streptococcal toxic shock syndrome(STSS)with high fever,systemic hypotension,disseminated intravascular coagulation and multiple organs failure,shock and high mortality with 96.2%.A previous study indicated that the 89K pathogenicity island specific for recent epidemic strains predominantly clas-sified as sequence type 7 may have contributed to the development of STSS.Vaccina-tion is generally recognized as one of the most effective means for controlling infec-tious diseases.However,a safe and effective vaccine to prevent S.suis infections is still not available and current vaccines could not provide cross protection against oth-er serotypes.This study was aimed to:(1)identify avirulent S.suis strains other than type 2 that are capable of conferring cross protection against epidemic infections by strains of type 2 and 9 serotypes with relative high prevalence;(2)investigate the key com-ponent of the putative type Ⅳ-like secretion system(T4SS)within the 89K patho-genicity island involved in microbe-host interaction;and(3)explore the mechanisms of S.suis PrsA,a Parvulin-like peptidyl-prolyl isomerase,in contributing to cell death.1.A live avirulent Streptococcus suis type 5 strain XS045 provides cross-protection against infection by strains of types 2 and 9A S.suis type 5 strain XS045 with a relative high adhesive rate(26.7%)was identi-fied from a collection of isolates.This strain harbors most of the immunogens report-ed in S.suis and is deficient of virulence factors such as suilysin,MRP and DltA.It did not cause apparent pathogenicity in mice,zebrafish and piglets.The XS045 strain could persist in mouse blood or organs for at least 2 weeks without causing patholog-ical abnormalities and significant weight loss.Subcutaneous administration of the live XS045 strain to mice induced high antibody responses with serum tilter as 1:25600 after the boosting immunization and was able to provide cross-protection against challenges by a type 2 strain HA9801(100%protection)and a type 9(SS9)strain JX13(85%protection).The XS045 possess some common surface antigens in SS2 and SS9 as the anti-XS045 sera had good reactivity to the surface proteins of SS2 and SS9 strains and bound to the whole bacterial cells.Pretreatment of the SS2 and SS9 bacterial cells with the XS045 hyper-immune sera reduced their colonization in epi-thelium HEp-2 cells or survival in fresh whole blood,respectively(P<0.05).The hy-per-immune sera also facilitated the phagocytic activity of macrophages against SS2 and SS9(P<0.05).Efficient induction of antibodies with opsonizing activity as well as their cross-reactivity to surface proteins of the types 2 and 9 strains could be the mechanisms of cross-protection.2.The key component VirD4 of the putative T4SS in S.suis type 2 induced pro-files of differentially secreted proteins in response to oxidative stressTwo isogenic mutants with deletion of virB4-ORF or virD4-ORF were constructed from the wild-type strain HA9801 with a STSS feature.The VirD4 was confirmed as the key component of the putative T4SS with functions in virulence and an-ti-phagocytic effect.The AVirD4 mutant was more easily phagocytosed and had lower expression levels of inflammatory cytokines in mice and cell lines as compared with its parent strain.Expression of virD4 factor was significantly enhanced upon oxida-tive stress either directly with hydrogen peroxide stimulation or during bacte-ria-macrophage interaction by 55.3 and 9.8 folds respectively.There were significant differences of secreted protein profiles between AVirD4 and its parent strain when exposed to H2O2 as shown by two dimensional gel electrophoresis and mass spec-trometry analysis.A total of 148 protein spots were upregulated in the AVirD4 strain at 1.5-fold or higher as compared with only 33 protein spots up-regulated in the parent strain in response to hydrogen peroxide stress.Mass-spectrometry was used to identi-fy three spots in the parent strain(including PrsA)and nine spots in the AVirD4 mu-tant with high abundance and more than 3-fold increase.These proteins are involved in DNA damage repair,nucleotide biosynthesis,carbohydrate metabolism,stress modulation and infectivity.These up-regulated proteins were confirmed by qPCR.These data provide novel insights into possible effectors contributing to the patho-genicity of S.suis type 2 infection.3.PrsA protein of S.suis type 2 could induce cell death and release of inflam-matory cytokinesThe PrsA protein expressed in E.coli could cause a dose-or time-dependent cell death and increased expression of pro-inflammatory cytokines in murine macrophage cells.Cell death process induced by PrsA did not act in inducing pore formation like suilysin.Pyroptosis was the major cell death modality and necroptosis might play a role.Cell death was alleviated by the pyroptosis inhibitor Ac-YVAD-cmk(100μM).The N-and C-terminal parts of PrsA,but not the enzymatic PPIase domains,were re-sponsible for cell death and release of inflammatory cytokines.Knockdown of trl2 and myd88 by RNA interference did not affect cell death of mouse macrophage in-duced by PrsA,suggesting that TLR2/MyD88 pathway did not seem to be involved.These results provide important information regarding the role of PrsA in SS2 patho-genesis.In summary,the present study demonstrates that S.suis type 5 strain XS045 could be as a good live vaccine candidate which could induce cross-protection against strains of types 2 and 9.VirD4 in the putative T4SS is involved in SS2 pathogenecity,up-regulated upon oxidative stress and related to secretion of PrsA.PrsA could induce pyroptosis and release of pro-inflammatory cytokines.These findings contribute to better understanding of S.suis type 2 pathogenesis.