Identification of conditionally essential genes for Streptococcus suis infection in pigs

Streptococcus suis is a Gram-positive bacterium and zoonotic pathogen that causes meningitis and sepsis in pigs and humans. The aim of this study was to identify genes required for S. suis infection. We created Tn-Seq libraries in a virulent S. suis strain 10, which was used to inoculate pigs in an intrathecal experimental infection. Comparative analysis of the relative abundance of mutants recovered from different sites of infection (blood, cerebrospinal fluid, and meninges of the brain) identified 361 conditionally essential genes, i.e. required for infection, which is about 18% of the genome. The conditionally essential genes were primarily involved in metabolic and transport processes, regulation, ribosomal structure and biogenesis, transcription, and cell wall membrane and envelope biogenesis, stress defenses, and immune evasion. Directed mutants were created in a set of 10 genes of different genetic ontologies and their role was determined in ex vivo models. Mutants showed different levels of sensitivity to survival in whole blood, serum, cerebrospinal fluid, thermic shock, and stress conditions, as compared to the wild type. Additionally, the role of three selected mutants was validated in co-infection experiments in which pigs were infected with both wild type and isogenic mutant strains. The genetic determinants of infection identified in this work contribute to novel insights in S. suis pathogenesis and could serve as targets for novel vaccines or antimicrobial drugs.猪链球菌（Streptococcus suis）是一种革兰氏阳性菌（Gram-positive bacterium），同时也是人畜共患病原菌（zoonotic pathogen），可引发猪与人类的脑膜炎及败血症。本研究旨在鉴定猪链球菌感染所需的功能基因。我们在强毒猪链球菌10型菌株中构建了转座子测序（Tn-Seq）文库，并将其用于鞘内实验感染猪的接种。通过对不同感染部位（血液、脑脊液及脑膜）回收的突变株相对丰度进行比较分析，共鉴定出361个感染必需的条件必需基因（conditionally essential genes），约占基因组的18%。上述条件必需基因主要参与代谢与转运过程、基因调控、核糖体结构与生物发生、转录过程、细胞壁膜及包膜生物发生、应激防御以及免疫逃逸等功能。我们针对10个隶属于不同基因本体（genetic ontologies）的基因构建了定向突变株，并通过体外模型（ex vivo models）验证了其功能。与野生型菌株相比，各突变株在全血、血清、脑脊液中的存活能力以及对热休克（thermic shock）和各类应激条件的耐受性均呈现不同程度的差异。此外，我们通过共感染实验验证了3个选定突变株的功能：实验猪同时接种野生型菌株与同基因突变株（isogenic mutant strains）。本研究鉴定的感染相关遗传决定因子，为猪链球菌致病机制（pathogenesis）的研究提供了全新视角，同时可作为新型疫苗或抗菌药物（antimicrobial drugs）的研发靶点。