阴沟肠杆菌复合体中介导多粘菌素异质性耐药的机制研究

Enterobacter cloacae complex (ECC) comprises multiple genetically closely related bacterial species and is one of the major pathogens of human infection. Colistin is the last line antimicrobial agent to treat infections caused by ECC. However, it is common that ECC displays heteroresistance to colistin, which refers to the situation that some colonies of an ECC strain are resistance to colistin while the major of colonies of the same strain are susceptible. The exact mechanisms responsible for heteroresistance to colistin in ECC remain unclear. During our previous studies, we identified that hp_01194, a gene encoding a small protein, mediated heteroresistance to colistin in six non-duplicate ECC strains. Based on our findings and those in literature, we hypothesize that hp_01194 encodes a transmembrane protein, which is able to act on the PhoP-PhoQ regulatory system and the AcrAB-TolC efflux system and then confers resistance to colistin. The unstable gene amplification of hp_01194 is likely to explain the heterogeneity of colistin resistance. In addition, we have also found that there are other mechanisms conferring heteroresistance to colistin in ECC. In this study, we will employ methods including Nanopore long-read sequencing, transcriptome sequencing and analysis, two-hybrid assay, Western blot and sequential peptide affinity purification to investigate the mechanisms of hp_01194 to mediate colistin heteroresistance at the gene, transcription and protein three tiers. We will also use Illumina whole genome sequencing, transcriptome sequencing, two-direction shotgun cloning, cloning and complementary experiment of target genes to investigate the mechanisms mediating colistin heteroresistance in 13 ECC strains that had no hp_01194. Combination of all above studies will help us to reveal the overall mechanisms of colistin heteroresistance in ECC and the generated information will help to improve clinical examination and to provide critical theoretical support for optimizing therapeutic schemes and finding targets for drug improvement.

阴沟肠杆菌复合体（ECC）包含多种紧密相关菌种，是重要致病菌，常对多粘菌素这一底线药物异质性耐药，但相关机制尚未被阐明。前期实验中发现编码小分子蛋白的基因hp_01194在6株不同ECC菌株中介导了多粘菌素异质性耐药。结合文献推测该基因编码跨膜蛋白，作用于PhoP-PhoQ双因子调节系统和AcrAB-TolC外排泵而对多粘菌素耐药；其不稳定基因拷贝数增加则可能导致异质性。前期实验提示ECC中还有其他机制介导多粘菌素异质性耐药。本项目将（1）通过Nanopore三代测序、转录组测序分析、双杂交实验、Western blot、顺序肽亲和纯化实验等从基因、转录和蛋白三层面揭示hp_01194作用机制；（2）通过基因组和转录组测序分析、双向鸟枪克隆、基因克隆/补回等揭示无hp_01194基因的13株ECC中多粘菌素异质性耐药机制。两者结合将为改进临床检测、优化治疗和药物改造等提供重要理论支持。

本研究围绕肠杆菌对多粘菌素异质性耐药机制开展。通过PAP菌群剖析法证实肠杆菌临床菌株中多粘菌素异质性耐药常见（可达27.1%）。对前期发现的介导多粘菌素耐药的跨膜蛋白编码基因ecr（Enterobacter colistin resistance，曾为hp_01194），我们通过转录谱测序，发现ecr通过正向调节PhoP-PhoQ双组分调节系统，激活arnBCADTEF操作子将L-Ara4N 氨基酸转移至脂质A，而对多粘菌素耐药；这是全新的机制。不过，大多数多粘菌素异质性耐药肠杆菌没有ecr，提示有其他机制。我们选取了多粘菌素异质性耐药的肠杆菌临床菌株120027为代表深入研究。我们随机挑取3个单克隆做为3个生物学重复（代表初始菌群），并从3个单克隆PAP实验中多粘菌素浓度最高平板上随机挑取三个单菌落做为3个技术重复（3×3重复），得到9个多粘菌素耐药克隆（代表耐药亚群）。通过PAP，我们发现在没有多粘菌素时，这些克隆均同时有敏感和耐药两种亚群，但耐药亚群占比低（仅为1.67-6.67×10-4），菌群总体表现为多粘菌素敏感；但在有多粘菌素时，耐药亚群占比显著上升到66.7%，使菌群总体表现为多粘菌素耐药。进行基因组测序发现9个耐药克隆中7个的phoP-phoQ基因有错义突变，1个的mgrB有错义突变；通过克隆/补回验证了这些错义突变可导致多粘菌素耐药。剩下1个克隆无错义突变，但q-RT-PCR发现arnBCADTEF表达水平显著提高。为了揭示异质性机制，我们选取了phoQ突变的克隆，高保真PCR后进行100万×超深度测序。我们发现在同一菌群中phoQ的同一个位点存在碱基杂合，但介导多粘菌素耐药的碱基在同一位点所有碱基中占比低（1.15×10-5到6×10-4）。总体上，肠杆菌对多粘菌素的耐药主要围绕PhoP-PhoQ双组分系统及其调节基因，而异质性的机制为预先存在的碱基杂合和抗菌药物筛选。复杂的异质性耐药机制反映了细菌对生存压力的“对赌式”应对机制，通过数量优势和菌群多样性而使生存可能最大化。我们还发现了新的质粒介导多粘菌素耐药基因mcr-10、发现了一些肠杆菌新菌种，并系统梳理了肠杆菌属分类。以上的发现，对于理解肠杆菌这一重要临床病原菌起到了重要推动作用，并有利于设计和制定相应的干预措施。..