Immunglobulin M-degrading enzyme of Streptococcus suis (IdeSsuis): modulation of B-cell function and consequences for mucosal colonization

猪链球菌是一种重要的猪源性致病菌，也是一种新兴的人类致病菌。猪S.猪非常有效地定殖上呼吸道粘膜，包括鼻咽、扁桃体（假定的进入口）和咽后淋巴结。S.在细菌病原体中，suis在表达免疫球蛋白（IG）M降解酶方面是独特的，命名为IdeSsuis，我们将其鉴定为半胱氨酸蛋白酶。在之前的项目中，我们证明了IdeSsuis介导的IgM裂解是一种新的补体逃避机制，导致调理作用减少和细菌存活增加。然而，我们发现，IgM裂解的IdeSsuis是不重要的侵袭性感染导致脑膜炎。然而，IdeSsuis对IgM的切割可能与S.猪在IgM浓度低于血清的粘膜表面定殖。因此，我们假设IdeSsuis通过定植链球菌介导的IgM蛋白水解足以减少该隔室中的IgM依赖性效应子功能。S.用野生型和突变型S.将通过实验诱导suis菌株，以定量测定粘膜表面上的定殖效力和IgM降解。我们将检验S. suis表达的IdeSsuis对表达膜IgM（即B细胞受体，BCR）的B细胞有效，尤其是在粘膜定殖部位。最近，我们发现IdeSsuis不仅切割可溶性IgM，而且切割在常规B细胞（即B2细胞）以及非常规B1样细胞上表达的IgM BCR。预期IdeSsuis的IgM和IgM-BCR裂解会导致粘膜B细胞应答缺陷和抗链球菌抗体产生减少。猪缺乏IgD，情况更是如此。详细地说，IdeSsuis可以破坏S.猪的IgM-BCR。随后，我们预期S的内化减少。这将导致减少的抗原呈递MHC-II和减少的相互作用与滤泡T辅助细胞（TfH）在淋巴结中。实验上呼吸道定植的S。无猪仔猪与包囊S. suis或其未包封的等基因突变体将用于分析被荚膜多糖激活的B1样细胞。在进一步的动物实验中，我们专门研究了S。IdeSsuis定植通过在粘膜部位处由IdeSsuis切割IgM来调节获得性免疫，导致攻击时保护性免疫受损。该实验将包括对表达野生型IdeSsuis或IgM切割缺陷的IdeSsuis的点突变变体的现有技术互补的猪链球菌突变体的比较。在这个提议中产生的数据预计将导致更好地理解IgM降解在定殖过程中的作用，并解释为什么IgM蛋白水解是一个高度保守的表型在所有调查的S。猪血清型。

Streptococcus suis is one of the most important porcine pathogens and an emerging human pathogen. In pigs, S. suis colonizes very efficiently the upper respiratory mucosa including nasopharynx, tonsils (the putative entry port), and retropharyngeal lymph nodes. S. suis is unique among bacterial pathogens in expressing an immunoglobulin (Ig) M degrading enzyme, designated IdeSsuis which we identified as a cysteine protease. In the preceding Project we demonstrated that IdeSsuis–mediated IgM cleavage is a novel complement evasion mechanism leading to reduced opsonisation and enhanced bacterial survival. We found, however, that IgM cleavage by IdeSsuis is not crucial during invasive infection leading to meningitis. However, cleavage of IgM by IdeSsuis might be relevant for S. suis colonization of mucosal surfaces where IgM concentrations are lower than in serum. Thus, we hypothesize that IdeSsuis–mediated IgM proteolysis by colonizing streptococci is sufficient to diminish IgM-dependent effector functions in this compartment. Colonization of S. suis-free piglets with wild-type and mutant S. suis strains will be induced experimentally to quantitatively determine colonization efficacy and IgM degradation on mucosal surfaces. We will test the hypothesis that S. suis-expressed IdeSsuis is effective on B cells expressing membrane IgM (i.e. B cell receptor, BCR) especially at mucosal sites of colonization. Recently we have generated evidence that IdeSsuis not only cleaves soluble IgM but also the IgM BCR expressed on conventional B cells (i.e. B2 cells) as well as on non-conventional B1-like cells. IgM and IgM-BCR cleavage by IdeSsuis is expected to result in a defective mucosal B cell response and reduced antibody production to S. suis, even more so as pigs lack IgD. In detail, IdeSsuis may disrupt the binding of S. suis to the IgM-BCR. Subsequently we expect reduced internalization of S. suis by B cells which should result in reduced antigen presentation on MHC-II and reduced interaction with follicular T helper (TfH) cells in the lymph node. Experimental colonization of the upper respiratory tract of S. suis-free piglets with encapsulated S. suis or its unencapsulated isogenic mutant will be conducted to analyze B1-like cells activated by capsular polysaccharides. In a further animal experiment we specifically investigate the hypothesis that S. suis colonization modulates adaptive immunity through IgM cleavage by IdeSsuis at mucosal sites leading to impaired protective immunity upon challenge. This experiment will include comparison of state-of-the-art complemented S.°suis mutants expressing either wild-type IdeSsuis or a point-mutated variant of IdeSsuis deficient in IgM cleavage. The data generated in this proposal is expected to lead to a better understanding of the role of IgM degradation during colonization and to explain why IgM proteolysis is a highly conserved phenotype in all investigated S. suis serotypes.