Table_6_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.

肠杆菌科克洛埃复合体（ECC）物种已成为医院获得性感染的日益重要原因，并且通常表现出多重抗生素耐药性。为了识别可能在其定植宿主能力中发挥作用的基因，我们采用了转座子测序（Tn-seq）方法。为此，我们从克洛埃杆菌亚种克洛埃（E. cloacae subsp. cloacae ATCC 13047）获得了高密度随机转座子插入文库，用以分析在大黄蜂（Galleria mellonella）定植模型中约30万个突变体的适应性。经过大规模并行测序，我们鉴定出624个基因，这些基因对于在宿主体内的最佳生长和/或适应性似乎是必需的。此外，还发现了63个基因，突变导致其发生正选择，而576个基因可能参与体内的适应性。这些发现指出了一些转录调控因子、VI型分泌系统和表面相关蛋白在克洛埃杆菌ATCC 13047的体内适应性中的作用。随后，我们根据其高正或负变化倍数（FCs）选择了八个基因，并测试了相应的缺失突变体在致病性和应对压力方面的能力。从而，我们证明了ECL_02247（编码NAD依赖性赤藓糖异构酶/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与体内适应性相关。此外，缺乏鞭毛特异性ATP合酶FliI（ECL_03223）的细菌细胞在运动能力上受到影响，而假定的蛋白质ECL_01421在抵抗H2O2方面受到影响。所有这些结果为克洛埃杆菌ATCC 13047感染过程和压力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该细菌病原体的机会性特征。