| Plague, the causative agent of which is believed to be Yersinia pestis, is a deadly infectious disease that accounts for three pandemics throughout history. It is transmitted between animals and humans by the bite of infected fleas, direct contact, inhalation and rarely, ingestion of infective materials. Clinical plague infection manifests itself in three forms depending on the route of infection:bubonic, septicemic and pneumonic.Type III secretion system (T3SS), harboring a plasmid70kb pCD1, is one of the most important mechanisms accounting for the pathogenesis of Yersinia pestis. T3SS encodes a syringe-like structure consisting of more than20proteins, and can inject multiple protein effectors into host cells to manipulate the cellular functions. Based on complete chromosome sequence analysis of Y. pestis, there are90ORFs in plasmid pCD1, which includ pseudogenes, transferase genes, plasmid replication-relating genes and57other genes. A total of57genes, including all the pCD1encoded genes except those involved in plasmid replication and partition, pseudogenes, and the putative transposase genes, were subjected to yeast mating analysis.19pairs of interaction proteins were identified, among which8pairs had been previously reported and11novel pairs were identified.5pairs of11novel pairs were studied in this thesis, that were YpCD1.09-YopN, YpCD1.09-YpCD1.08, YpCD1.16-TyeA, YpCD1.15-1.16, YscF-YscI. These genes and proteins of Y. pestis T3SS were studied in order to reveal the relation between these genes and T3SS and the YscI-YscF interactions domain of T3SS, which provides valuable hints for further studies.Part1Study on the function of potential new genes of Yersinia pestis type three secretion systemYopN and TyeA plays a negtive effect in the Yops secretion, and YscF assembles to form the needle of the injectisome, while the YscI connects the needle to the basal side of the base body, but the function of YpCD1.09, YpCD1.08, YpCD1.16are still unknown. The YopN and TyeA played a negtive effect in Yops secretion, and YopN, the SycN/YscB chaperon and TyeA could form a YopN/SycN/YscB/TyeA complex that is required to block the Yops secretions under the non-secretion permission conditions.The interactions between YpCD1.09, YpCD1.08, YpCD1.16and YopN, TyeA indicated that they may be new members of T3SS family and participate in the regulation of effectors secretion by some ways. Therefore, the transcription level, low calcium response and influence on effectors transportation of YpCD1.09,YpCD1.08, YpCD1.16were studied to reveal the relation between these genes and T3SS function. The expressions of YpCD1.08, YpCD1.09, YpCD1.16of Y. pestis and their dependence on temperature were determined by quantitative PCR. Quantitative real time PCR assay showed that transcriptions of thses genes at37℃was higher than those at26℃, which was consistent with the well established temperature dependence of Yersinia T3SS genes. Mutants of Y. pestis defective in three genes were constructed using λ-Red recombinant system. At37℃, the low calcium response of all the mutants was normal. The YopN stability of YpCDl.09mutant was analysed, and our results suggested that YpCD1.09may not act as a chaperon. A TEM P-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS, and the results showed that ΔYpCD1.08could translocate much higher level of YopE into HeLa cells. The results above showed that all the presumed genes were expressed in Y.pestis, and YpCD1.08, YpCD1.09, YpCD1.16genes at37℃were expressed higher than those at26℃, which is consistent with the well established temperature dependence of T3SS genes. The low calcium response of mutants at37℃was normal, however the translocation of YopE of YpCD1.08mutant increased by30%, indicating the YpCD1.08may involve in the secretion and regulation of the effector YopE.The results above show that, although YpCD1.09, YpCD1.08, YpCD1.16genes don’t affect the secretion of T3SS effector protein, but these three genes are closely related with the function of T3SS.Part2YscF and YscI interactions within Type III Secretion System of Y. pestisThe central proteins of type III secretion system injectome which are conserved in all bacteria isn’t more than nine, including YscF and YscI. The YscF assembles to form the needle of the injectisome, while YscI connects the needle to the basal side of the base body. After Yscl mutated, Yersinia type Ⅲ secretion system can secrete effector proteins but not secreted needle protein as YscF. The YscI-YscF interactions domain was studied in order to reveal the structure and function of T3SS, which provides a lot of valuable informations about the functional mechanism of Y.pestis T3SS.GST-pull down approach was used to study the interaction regions of YscI which is important for the binding to YscF, GST-tagged truncates of YscI were expressed in E. coli and determined for their abilities to bind with YscF. Further work was also conducted focusing on the possible functions of these proteins on the secretion of the effector proteins of T3SS. YscF and YscI were both known as important structural proteins of T3SS, but no interaction between them had been reported. In previous study, the interaction between them were validated by GST pull down experiment, while in the present work we identified that the C-terminal domain spanning83-91amino acids of YscI is crucial for the YscI-YscF interactions, which provides valuable hints for further studies. |
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