Table_7_Landscape of in vivo Fitness-Associated Genes of Enterobacter cloacae Complex.xls

Species of the Enterobacter cloacae complex (ECC) represent an increasing cause of hospital-acquired infections and commonly exhibit multiple antibiotic resistances. In order to identify genes that may play a role in its ability to colonize the host, we used the transposon-sequencing (Tn-seq) approach. To this end, a high-density random transposon insertion library was obtained from E. cloacae subsp. cloacae ATCC 13047, which was used to analyze the fitness of ca. 300,000 mutants in Galleria mellonella colonization model. Following massively parallel sequencing, we identified 624 genes that seemed essential for the optimal growth and/or the fitness within the host. Moreover, 63 genes where mutations resulted in positive selection were found, while 576 genes potentially involved in the in vivo fitness were observed. These findings pointed out the role of some transcriptional regulators, type VI secretion system, and surface-associated proteins in the in vivo fitness of E. cloacae ATCC 13047. We then selected eight genes based on their high positive or negative fold changes (FCs) and tested the corresponding deletion mutants for their virulence and ability to cope with stresses. Thereby, we showed that ECL_02247 (encoding the NAD-dependent epimerase/dehydratase) and ECL_04444 (coding for a surface antigen-like protein) may correspond to new virulence factors, and that the regulator ECL_00056 was involved in in vivo fitness. In addition, bacterial cells lacking the flagellum-specific ATP synthase FliI (ECL_03223) and the hypothetical protein ECL_01421 were affected for mobility and resistance to H2O2, respectively. All these results yield valuable information regarding genes important for infection process and stress response of E. cloacae ATCC 13047 and participate to a better understanding of the opportunistic traits in this bacterial pathogen.

肠杆菌科克洛阿菌（Enterobacter cloacae complex，简称ECC）已成为医院获得性感染的日益重要的原因，且普遍表现出多重抗生素耐药性。为探究可能影响其宿主定植能力的基因，本研究采用了转座子测序（Tn-seq）方法。为此，从克洛阿菌亚种克洛阿菌（E. cloacae subsp. cloacae ATCC 13047）中构建了一个高密度随机转座子插入文库，用于分析约30万突变体在蜜蜂幼虫定植模型中的适应性。经过大规模并行测序，我们鉴定出624个基因，这些基因对于细菌在宿主体内的最佳生长及适应性至关重要。此外，发现63个基因的突变导致了正向选择，而576个基因可能与体内的适应性有关。这些发现凸显了某些转录调控因子、VI型分泌系统和表面相关蛋白在E. cloacae ATCC 13047体内适应性中的作用。基于这些基因在正负变化率（FC）上的显著差异，我们选取了八个基因，并对其对应的缺失突变体进行了致病力和抗应激能力的测试。结果表明，ECL_02247（编码NAD依赖性异构酶/脱水酶）和ECL_04444（编码表面抗原样蛋白）可能对应新的致病因子，而调控因子ECL_00056则参与了体内的适应性。此外，缺乏鞭毛特异性ATP合酶FliI（ECL_03223）和假定的蛋白ECL_01421的细菌细胞在运动性和抵抗H2O2的能力上受到了影响。所有这些结果均提供了有关克洛阿菌ATCC 13047感染过程和应激反应中重要基因的有价值信息，并有助于更深入地理解该条件致病菌的侵袭性特征。