| Streptococcus suis(S.suis)is an important zoonotic pathogen,which can not only cause disease to pigs,but also infect humans to cause meningitis and toxic shock syndrome.Therefore,the study of S.suis has a great significance of public health.In recent years,the study of pathogenic mechanism and vaccines of S.suis are mainly focus on S.suis serotype2.With the identification of more and more clinical isolates of S.suis with undetermined serotypes,the study on clinical isolates of S.suis with typical pathogenic characteristics becomes more and more urgent.The strain of S.suis serotype Chz CZ130302,isolated from piglet meningitis case,is showed typical meningitis symptoms in mouse infection model,and is an ideal model for bacterial meningitis research.This study mainly focused on the strains of S.suis serotype Chz(CZ130302,AH681 and HN136)to study the Zinc metalloproteinase(Zmp)and Integrative conjugative elements(ICE),aiming to provide a new idea for the reveal the mechanism of pathogenic and drug resistance of S.suis.1 Evolutionary analysis of zinc metalloproteinases in Streptococcus suisZmp is a kind of widely distributed proteolytic enzyme.It has been reported to be associated with bacterial pathogenicity in many bacteria.This study had analyzed the distribution of Zmp in 70 S.suis strains,and it is found that the Zmp is distributed in67.14%(47/70)S.suis strains.The phylogenetic analysis of S.suis Zmp showed obvious evolutionary groups,which are Zmp A,Zmp B,ZmpC,Zmp E and Zmp N,respectively.Among them,ZmpC of CZ130302 is closely related to ZmpC of Streptococcus pneumoniae(S.pneumoniae)TIGR4.And Zmp E was identified a new Zmp group in S.suis.2 The effect of ZmpC on the virulence of CZ130302The zmp deletion(Δzmp C,Δzmp E,Δzmp N and Δzmp B)strains were constructed with the CZ130302 background.Growth curve measurements showed that the deficiency of zmp didn’t affect the growth of S.suis.The ability of biofilm formation of S.suis were significantly reduced after deleting zmp(P < 0.0001).Virulence related assays showed that compared with the wild-type strain,the adhesion ability of Δzmp C to Hep-2 cell was significantly reduced(P < 0.01);the brain of mice infected with Δzmp C’ transcription levels of M-TNF-α,M-IL-8 and M-MMP-9 were significantly lower(P < 0.05;P < 0.001);the mice survival time of infected Δzmp C strain is prolonged,and the organ bacterial loading of mice infected with Δzmp C was significantly decreased(P < 0.05;P < 0.0001).All of this indicated that the ZmpC of CZ130302 promotes the pathogenic process of bacteria.3 Study on function of zinc metalloproteinase in S.suis serotype ChzThe 3D structural prediction of Zmp revealed that ZmpC,Zmp E and Zmp B of S.suis serotype Chz CZ130302 were highly similar to Ig A1 protease and ZmpC of S.pneumoniae TIGR4.The prokaryotic expression vectors of ZmpC-M26,Zmp E-M26 Zmp B-M26 were constructed to study the function of them.The gelatin enzyme assay revealed that the ZmpC of CZ130302 could cleavage the hinge region of human MMP-9.The polyclonal antibodies against ZmpC-M26,Zmp E-M26 and Zmp B-M26 were prepared by immunizing mice and the assays of subcellular localization of ZmpC-M26,Zmp E-M26 and Zmp B-M26 were confirmed that these proteins anchored the cell wall.Immune protection test in mice showed that ZmpC-M26,Zmp E-M26 and Zmp B-M26 have 75%,50% and 37.5%protective efficacy to mice against the infection of S.suis serotype Chz CZ130302,and62.5%,75% and 62.5% protective efficacy to mice against the infection of S.suis serotype2 ZY05719,respectively.Therefore,ZmpC,Zmp E and Zmp B all have the potential to as the candidate immugen for S.suis.4 Identification of ICESsu AH681 in AH681As a mobile genetic element,ICE is a main reason that contributes to the spread of drugresistant genes in bacteria.In this study,the genome sequence analysis of multi-drug resistance strains of S.suis serotype Chz AH681 and HN136 showed that the exogenous inserted gene islands(73 K and 115 K)in their genome have the typical characteristics of ICE and possess the integrase of ICESa2603 family.It is speculated that ICESsu AH681 and ICESsu HN136 could be an ICE.Cyclization assay showed that ICESsu AH681 could excised from genome and existed the integrate form and circular form,and the ICESsu HN136 only have the integrate form but doesn’t have circular form.It revealed that ICESsu AH681 is an activity ICE.However,when the mating assays was performed by using AH681 as the donor and P1/7 as the recipient,the P1/7 conjugate of ICESsu AH681 was not obtained.In summary,the protein evolutionary analysis of Zmp in S.suis showed that they have five distinct evolutionary groups(Zmp A,Zmp B,ZmpC,Zmp E and Zmp N).Virulence related assays found that ZmpC of S.suis serotype Chz CZ130302 have an effect on bacterial virulence.Subcellular localization confirmed that ZmpC,Zmp E and Zmp B are anchored to the cell wall surface.Immune protection test in mice showed ZmpC,Zmp E and Zmp B can protect mice against the infection of S.suis serotype Chz CZ130302 and serotype 2 ZY05719.Besides,it is confirmed that the ZmpC of S.suis serotype Chz CZ130302 can cleavage the hinge region of human MMP-9.At the same time,an activity ICE(ICESsu AH681),from the multi-drug resistant S.suis serotype Chz AH681,was identified in this study. |
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