肺炎链球菌寄居和感染中的唾液酸酶NanC-NanA双向调节机制研究

The sialidase NanA specifically recognizes and hydrolyzes the terminal sialic acid (Sia) from cell surface glycans to promote pneumococcal colonization and infection,which has been proved to be a key virulence factor of Streptococcus pneumoniae (S. pneumoniae). We have previously reported that S. pneumoniae also expresses a third different sialidase NanC, catalyzing a unique reaction that releases DANA(Neu5Ac2en). The sialidase activity of NanA could be suppressed by DANA as confirmed in our experiments. Further study revealled that DANA could be steadily hydrated by NanC when it accumulates to a certain concentration. Therefore, we propose that S. pneumoniae might employ NanC as a dual regulator for Sia signalling and NanA activities in vitro and in vivo, thereby form a novel NanC-NanA cross-talk syetsm that might adjust the virulence status of this bacterium at different infection stages. In this project, we will test TIGR4,D39 and the sialidase knock-out strains of S. pneumoniae in some cell, tissue and animal infection models, to investigate the cross-talk between NanC and NanA at different conditions;and confirm the potential findings by using specific inhibitors. We will aslo use the fluoresence-based imaging system to monitor the mobility of S. pneumoniae in vivo and the pathological changes of the BALB/C mice. Meanwhile,the role of Sia and its related Sia binding lectins SiglecG, as well as their interactions with NanC will be studied. We speculate that this study will elucidate the precise molecular mechanisms of NanC-NanA mutual regulating system in the S. pneumoniae colonization, infections and tissue damages. This project will probably provide meaningful insights towards the discovery of novel diagnostic and therapeutic measures based on the sialidases systems against S. pneumoniae infectons.

研究表明：肺炎链球菌以唾液酸酶NanA特异性结合和水解宿主细胞表面的唾液酸（Sia）分子，促进细菌寄居及感染。我们已证实：该菌还表达另一种唾液酸酶NanC，其酶学反应释放的产物DANA，能有效拮抗NanA活性；进一步研究发现：DANA富集时又会被NanC水化为游离Sia，失去抑制效能，但可能是刺激细菌移行的信号分子。本项目据此提出：肺炎链球菌或利用NanC独特的酶学性质,组成NanC-NanA双酶双向调节系统，调校该菌在不同感染时相的状态。我们拟用TIGR4、D39及唾液酸酶敲除菌株，分别感染细胞、组织和小鼠模型，研究不同条件下NanC/A的相互作用模式，并通过双靶向抑制进行验证；同时深入探讨NanC对宿主Sia分子及Sia结合性凝集素G（SiglecG）功能的影响，以期揭示NanC/A在肺炎链球菌寄居与感染切换及炎性损伤中的作用和机制，为发掘基于机制的肺炎链球菌感染诊治新策略提供科学依据

肺炎链球菌以唾液酸酶NanA特异性结合和水解宿主细胞表面的唾液酸（Sia）分子，促进细菌寄居及感染, 该菌还表达另一种唾液酸酶NanC。本项目提出：肺炎链球菌或利用NanC独特的酶学性质,组成NanC-NanA双酶双向调节系统，调校该菌在不同感染时相的状态。在本研究周期内，我们研究发现：NanC与NanA识别唾液酸衍生物，但其酶学反应释放的产物不同，NanC催化的中间产物DANA能有效拮抗NanA活性；而DANA富集时又会被NanC水化为游离Sia，失去抑制效能。我们检测感染模型小鼠肺泡灌洗液IL-4，IL-5，IL-13和IL-37治疗组与MSC组比较显示，肺泡灌洗液中IL-4，IL-5，IL-1的浓度明显下降。NanC蛋白体外对人急性早幼粒白血病细胞株HL60细胞和人急性单核白血病细胞株THP-1细胞有明显的生长抑制作用，且抑制作用呈剂量依赖性。NanC蛋白作用48h后能明显影响NF-κB 信号通路中P65蛋白的表达，其中P65蛋白表达降低。本研究为发掘基于机制的肺炎链球菌感染诊治新策略提供科学依据。