Comprehensive identification of <i>Vibrio vulnificus</i> genes required for growth in human serum

<i>Vibrio vulnificus</i> can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent <i>V. vulnificus</i> lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the <i>V. vulnificus</i> essential gene set; comparisons of these genes with similar TIS-based classification of underrepresented genes in other vibrios enabled the compilation of a common <i>Vibrio</i> essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum suggested that genes involved in capsule biogenesis are critical for YJ016 complement resistance. Notably, homologues of two genes required for YJ016 serum-resistance and capsule biogenesis were not previously linked to capsule biogenesis and are largely absent from other <i>V. vulnificus</i> strains. The relative abundance of mutants after exposure to heat inactivated serum was compared with the findings from the serum screen. These comparisons suggest that in both conditions the pathogen relies on its Na⁺ transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive/proliferate within the metabolic constraints of serum. Collectively, our findings reveal the potency of comparative TIS screens to provide knowledge of how a pathogen overcomes the diverse limitations to growth imposed by serum.创伤弧菌（Vibrio vulnificus）是一种高侵袭性病原体，可从感染部位播散至血流，引发败血症乃至死亡。为在血液中存活并增殖，该病原体需要相关机制以克服宿主微环境的先天免疫防御与代谢限制。我们在YJ016菌株——即代表最具毒力的创伤弧菌谱系（或系统发育群）的模式菌株——中构建了高密度转座子突变体文库，并借助转座子插入测序（TIS）筛选，鉴定出可帮助该病原体在人血清中存活与增殖的基因位点。最初，我们通过统计插入位点缺失的基因，估算出创伤弧菌的必需基因集；将这些基因与其他弧菌中基于TIS鉴定的插入缺失基因进行比较，最终汇编得到弧菌属（Vibrio）通用必需基因集。对血清暴露后文库中插入突变体的相对丰度进行分析，结果表明参与荚膜生物合成的基因对YJ016的补体抗性至关重要。值得注意的是，两种与YJ016血清抗性及荚膜生物合成相关的基因同源物，此前从未被关联至荚膜生物合成过程，且在其他创伤弧菌菌株中基本不存在。我们将热灭活血清暴露后的突变体相对丰度，与血清筛选的结果进行了对比。对比结果显示，在两种条件下，病原体均依赖其钠泵型NADH-泛醌还原酶（NQR）复合物与II型分泌系统，以在血清的代谢限制环境中存活/增殖。综上，我们的研究结果证实了比较型TIS筛选的有效性，可为解析病原体如何克服血清所施加的多重生长限制提供新认知。