体外传代选择对鼠衣原体质粒缺失株培养特性的影响

Effect of In vitro Passage with Selection Pressure on Fitness of Plasmid-free Chlamydia Muridarum Strain CMUT3

目的探讨传代选择对鼠衣原体质粒缺失株CMUT3菌株培养特性的影响及其相应机制。方法采用"无辅助感染"和"辅助感染"交替方式体外传代培养CMUT3菌株,而后观察传代菌株和亲代感染细胞时的吸附效率以及对离心因素的依赖性,并利用下一代测序技术对传代菌株CMUT3G40和亲代CMUT3G0进行全基因组测序分析。结果传代菌株CMUT3G40在感染He La细胞时对离心因素依赖性降低,吸附试验表明CMUT3G40菌株对细胞的吸附能力增强;比较基因组学分析结果表明CMUT3G40菌株与亲代CMUT3G0在tc0237基因存在差异,TC0237Q117E错义突变出现于CMUT3G40菌株基因组(CMUT3G40,99.7%;CMUT3G0,0%)。结论通过体外传代培养CMUT3菌株可获得吸附能力增强的菌株,该表型与传代选择所致的TC0237Q117E密切相关。

Objective To unravel the effect of passage selection pressure on fitness of plasmid-free Chlamydia muri- darnm strain CMUT3 and uncover the correlation between genetic and phenotypic signatures. Methods CMUT3 organisms were in vitro passaged for 40 generations under the alternating unassisted/assisted-infection cycle conditions and the infectivity of initial and final generation organisms in cell culture was measured by comparing their attachment efficiency phenotype in the presence or absence of centrifugation.Next-generation sequencing technology was then used to sequence the genomes of passaged CMUT3G40 and the parental CMUT3G0. Results Passaged CMUT3G40 organisms became less de- pendent on centrifugation and more efficient for infecting HeLa cells in the absence of centrifugation compared to the paren- tal CMUT3G0.Comparative genomes analysis of CMUT3G40 and CMUT3G0 revealed that a G to C substitution at position 277313,resulting in a glutamine to glutamic acid amino acid change in the protein product of TC0237 （99. 7% in CMUT3G40 versus 0% in CMUT3G0 genomes）. Conclusion The TC0237Q117E missense mutation under in vitro pas- sage selection pressure can be attributed to an in vitro attachment enhancement phenotype of plasmid-free C. muridarum CMUT3 organisms.