假基因在伤寒沙门菌中的生物学意义研究

Salmonella enterica serovar Typhi is distinct from other Salmonella serovars due to its single host range and systemic infection symptom, with the molecular mechanism unknown. Genome comparison has revealed that the Typhi genome harbors much more pseudogenes than the non-typhoidal genomes. A well-recognized hypothesis for the formation of pseudogenes is that, during the restriction of host range, many genes become functionally unnecessary and thus are inactivated by the accumulation of frame-shift and non-sense mutations. Based on our preliminary research, however, a few pseudogenes are able to translate into functional proteins through Recoding. A few other pseudogenes, though inactivated indeed, enhance the bacterial virulence against the host defense. These findings suggest that pseudogenes present a much broader biological significance other than that in host restriction. This proposal mainly focuses on two tasks. First, we plan to observe the expression of pseudogenes under different culturing conditions by proteomic approach, with the aim to verify if recoding could regulate the expression of pseudogenes. Second, we plan to repair the open reading frame of pseudogenes to restore their biological functions and observe if any changes occur in regard to host range or virulence in comparison with the wild strain. We hope this study would help gain a better understanding of pathogenecity of Salmonella Typhi, and lay a basis for developing novel methods of diagnosis and treatment for typhoid infections.

伤寒沙门菌因其单一的宿主范围及独特的全身性感染而区别于众多非伤寒沙门菌，其分子机制目前尚未阐明。比较基因组学研究发现，伤寒沙门菌的假基因数量明显多于非伤寒沙门菌。学术界对此现象的共识为：随着宿主范围的减小，部分广宿主感染相关基因失去其存在价值从而失活变为假基因。我们前期研究却发现，若干假基因可通过重编码机制克服移码突变及无义突变并最终正常表达蛋白；另有个别假基因虽确实无法表达蛋白，但其失活反而增强了伤寒沙门菌的毒力。上述发现提示假基因除参与宿主选择外还有其他丰富的生物学意义，故引出了本项目的两大研究内容：①利用蛋白质组学手段研究不同环境下假基因的表达，明确重编码对假基因表达的调控作用；②构建突变株恢复假基因在伤寒沙门菌中的正常表达，比较突变株与野生株在宿主范围及毒力表现上的差异。我们希望通过对假基因的系统研究来增进对伤寒沙门菌致病机制的了解，并为未来开发新诊疗手段提供科学依据。

假基因由于携带移码突变或无义突变往往被认为无法正常表达蛋白。伤寒、甲型副伤寒等单一人宿主沙门菌相比于广宿主沙门菌拥有明显更多的假基因，这可能是因细菌随宿主范围减小，部分环境应答基因失去其存在价值从而失活变为假基因。在本项目中，我们利用液相色谱-串联质谱技术，发现在伤寒、甲型副伤寒沙门菌中超过半数的假基因有肽段表达。其中又有超过2/3的基因在失活位点下游可检测到肽段表达，暗示这些假基因能够表达完整蛋白。这些可表达假基因或因移码突变，或因无义突变失活，且其蛋白表白水平显著低于同源正常基因。我们将这些假基因克隆至大肠杆菌内，用MALDI-TOF同样可检测到他们的表达。RNA-seq转录组测序表明，所有可表达假基因在RNA水平仍保留失活突变状态，表明它们的正常表达依赖翻译水平的纠错机制完成。我们在失活突变位点及其上下游并未发现高度保守的序列特征，表明这种纠错表达机制可能具备高度多样化的特点。利用质粒表达载体高表达若干假基因的正常同源基因后，可观察到伤寒沙门菌的毒力发生显著变化。根据以上发现，我们提出“补偿理论”来解释假基因的表达：沙门菌在由广宿主向窄宿主进化的过程中，由于生态位逐渐狭窄，原本用于应对多样环境的一些基因由于功能冗余逐渐假基因化。但在某些情况下沙门菌仍可能偶尔需要从单宿主环境返回广宿主环境，因而需要通过重编码暂时恢复已失活假基因的原本功能来补偿这种需求。我们相信，细菌假基因的广泛表达是一项重要的基础研究发现，是对现有中心法则标准编码机制的重要补充。