Table_5_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.

肠杆菌科大肠杆菌复合群（ECC）已成为医院获得性感染日益重要的原因，并且普遍表现出多重抗生素耐药性。为了识别可能参与其定植宿主能力的基因，我们采用了转座子测序（Tn-seq）方法。为此，我们从大肠杆菌亚种cloacae ATCC 13047中获得了高密度随机转座子插入文库，用于分析约30万个突变体在Galleria mellonella定植模型中的适应性。经过大规模并行测序后，我们鉴定出624个基因，这些基因对于在宿主体内最优生长和/或适应性至关重要。此外，发现63个基因的突变导致了正向选择，而576个基因可能参与了体内的适应性。这些发现突出了某些转录调控因子、VI型分泌系统和表面相关蛋白在大肠杆菌ATCC 13047的体内适应性中的作用。随后，我们根据其高正负折叠变化（FCs）选择了八个基因，并对相应的删除突变体进行了致病性和应对压力能力的测试。从而，我们证明了ECL_02247（编码NAD依赖性表异构酶/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，而调节因子ECL_00056参与了体内适应性。此外，缺乏鞭毛特异性ATP合酶FliI（ECL_03223）和假定的蛋白质ECL_01421的细菌细胞在移动性和对H2O2的抵抗力方面受到了影响。所有这些结果为E. cloacae ATCC 13047感染过程和应激反应中重要基因的信息提供了宝贵见解，并有助于更深入地理解这种细菌病原体的机会性特征。