RcsB contributes to the distinct stress fitness between Escherichia coli O157:H7 curli variants of 1993 Hamburger-associated outbreak strains

Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation and biofilm formation. We previously reported that natural curli variants of E. coli O157:H7 (EcO157) displayed distinct acid resistance; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence between the curli variants. Among curli-producing (C+) variants isolated from the 1993 U.S. hamburger-associated outbreaks strains, we identified large deletions in the rcsB gene that encodes the response regulator of RcsCDB two-component signal transduction system of rcsB ,. Further comparison of stress fitness revealed that C+ variants were also significantly more sensitive to heat shock, but remained similar resistance to osmotic stress and oxidative damage as curli-deficient (C-) variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by the enhanced expression of genes related to biofilm formation, virulence, catabolic activity and nutrients uptake, but marked decrease in transcription of genes related to various stress resistance in C+ variants. Supplying C+ variants with a functional rcsB restored cells resistance to heat shock and acid challenge, but blocked the curli production, confirming that inactivation of RcsB in C+ variants was the basis of fitness segregation within the EcO157population. This study provides an example of how genome instability of EcO157promotes the intra-population diversification, generating sub-populations carrying an array of distinct phenotypes that may confer the pathogen survival advantages in host and nonhost environments. Overall design: Three replicates for isolates RM6607R, RM6607W, RM6608R, RM6608W independently grown from single colonies in LB broth and incubated at 28°C overnight on a shaker (150 rpm). The cells were collected by centrifugation, washed once in LBNS broth, and inoculated in 25ml of LBNS broth with a concentration equivalent to 0.001OD600 ml-1. The cultures were incubated at 28°C for 24 h with aeration (150 rpm). At the end of incubation, ice-cold phenol-ethanol (5%:95%) solution was immediately added to culture at a final concentration of 20% (volume), and the mixture was incubated on ice for 30 min. The cells were collected by centrifugation at 4°C and the pellets were stored at -80°C until RNA extraction. The cells were collected by centrifugation, washed once in LBNS broth, and inoculated in 25ml of LBNS broth with a concentration equivalent to 0.001OD600 ml-1. The cultures were incubated at 28°C for 24 h with aeration (150 rpm). At the end of incubation, ice-cold phenol-ethanol (5%:95%) solution was immediately added to culture at a final concentration of 20% (volume), and the mixture was incubated on ice for 30 min. The cells were collected by centrifugation at 4°C and the pellets were stored at -80°C until RNA extraction. RNA was isolated using Promega SV Total RNA kit. A type 2 gene expression experimental design was used, with fluorescently labeled genomic DNA as a reference channel in each experiment as described by Lucchini, S., et al. 2005. Infect Immun 73:88-102.

Curli（粘附性菌毛）是肠杆菌科细菌的表面附着、细胞聚集和生物膜形成的关键因素。我们先前报道，大肠杆菌O157:H7（EcO157）的自然Curli变体表现出独特的耐酸性；然而，这种差异并非直接与Curli菌毛本身相关。在本研究中，我们探讨了Curli变体之间这种表型差异的潜在分子基础。在1993年美国汉堡相关爆发菌株中分离得到的产生Curli（C+）变体中，我们发现了编码RcsCDB双组分信号转导系统的响应调节因子rcsB基因的较大缺失。进一步的应激适应性比较显示，C+变体对热冲击的敏感性显著增加，但与Curli缺陷（C-）变体相比，在渗透压应激和氧化损伤方面的抗性保持相似。转录组学分析揭示了Curli变体之间大量差异表达基因，其中与生物膜形成、致病性、代谢活性和营养物质摄取相关的基因表达增强，而在C+变体中，与多种应激抗性相关的基因转录显著降低。向C+变体提供功能性的rcsB基因恢复了细胞对热冲击和酸挑战的抵抗力，但阻断了Curli的产生，证实了RcsB在C+变体中的失活是EcO157种群内适应性分化的基础。本研究提供了一个实例，说明了EcO157的基因组不稳定性如何促进种群内的多样性，产生携带一系列不同表型的亚种群，这些表型可能在宿主和非宿主环境中赋予病原体生存优势。总体设计：对RM6607R、RM6607W、RM6608R、RM6608W四个菌株分别从单个菌落中独立培养于LB培养基中，在28°C的摇床上过夜培养（150 rpm）。通过离心收集细胞，用LBNS培养基洗涤一次，然后接种于25ml的LBNS培养基中，浓度相当于0.001OD600 ml-1。在28°C下培养24小时，充气（150 rpm）。培养结束时，立即向培养物中加入冰冷的苯酚-乙醇（5%:95%）溶液，最终浓度为20%（体积），混合物在冰上孵育30分钟。在4°C下离心收集细胞，沉淀在-80°C下储存，直至提取RNA。细胞通过离心收集，用LBNS培养基洗涤一次，然后接种于25ml的LBNS培养基中，浓度相当于0.001OD600 ml-1。在28°C下培养24小时，充气（150 rpm）。培养结束时，立即向培养物中加入冰冷的苯酚-乙醇（5%:95%）溶液，最终浓度为20%（体积），混合物在冰上孵育30分钟。在4°C下离心收集细胞，沉淀在-80°C下储存，直至提取RNA。RNA使用Promega SV Total RNA试剂盒进行提取。采用Lucchini, S.等人（2005）所描述的II型基因表达实验设计，在每个实验中，使用荧光标记的基因组DNA作为参考通道，如Infect Immun 73:88-102所述。