Study On The Interaction Of YscI-YscF Protein Of The Type III Secretion System Of Yersinia Pestis And The Identification Of New Virulence Factors In The Protein Secretion Group

The paper covers two parts,“An Interaction between the Inner Rod Protein YscI and the Needle Protein YscF Is Required to Assemble the NeedleStructureofthe Yersinia Type Three Secretion System” and “Secretome and Comparative Proteome of Yersinia pestis Grown Under Conditions Mimicking the Two Typical Niches in Its Life cycle”.The abstract and the paper will be divided into two parts.Abstract 1Type III secretion system?T3SS?is a virulence mechanism widely distributed in Gram-negative bacterium,which can translocate effector proteins into host cells during infection.Three kinds of pathogenic Yersinia pestis species share a common T3 SS encoded by a 70kb-plasmid called pCD1.The virulence effector proteins are injected via a needle-like structure called Ysc injectisome lead to cell cytosol.Injectisome is composed of more than 20 necessary proteins,most of which are structure parts while the rest are assistant parts during assembly,such as chaperonin.The core components of injesctisome of all the bacterium are so conservative.The needle tip protein LcrV and Yersinia outer protein?Yops?can interfere with the normal physiological function of host cells.The pCD1 plasmid of the Yersinia pestis encodes more than 90 genes,of which 57 genes encode T3SS-related proteins.In a yeast two-hybrid screening that aimed to identify interactions among potential components of the T3 SS,we found that YscI interacted with YscF.GST pull-down assay using GST-tagged Ysc I truncants found that the C-terminal domain of YscI,which is highly conserved among different inner rod protein family members,was essential for binding Ysc F.And the amino acid residues 83–102 and 93–102 in the C-terminal domain of YscI are critical for the in vitro interaction between YscF and Ysc I,whereas the other tested regions had limited effects on binding.Firstly,the construction of ?yscI strain was based on the ?-Red recombination system.And we created site-directed mutagenesis of yscI with FLAG--encoding sequence by introducing the recombinant plasmids into the?35?yscI strain.The site-directed mutagenesis of ysc I were ?yscI-FLAG-YscI,?yscI-C?73-85,?yscI-C?83-92,?yscI-C?93-102 and ?ysc I-C?93-102.Then,we found that YscF was successfully co-immunoprecipitated by FLAG-Ysc I,as well as by the Ysc I?73–82 and YscI?103–115 mutants.However,no YscF band was detected when using Y.pestis strains expressing the YscI?83–92 or YscI?93–102 proteins to perform the co-IP assay.These results indicate that YscI can bind to YscF,and that amino acid residues 83–92 and 93–103 of YscI are essential for binding Ysc F inside bacteria.To observe the influence of Ysc I mutations on needle assembly,we used BS3 treatment to detect the polymerization of YscF in?35?yscI strains that were trans-complemented with plasmids expressing different YscI truncated mutants.Nevertheless,we observed a ladder of bands of polymerized YscF in the wild-type Y.pestis strain.YscF polymerization was basically normal in the ?yscI-C?73–82 and ?yscI-C?103–115 strains.In contrast,no ladders of polymerized YscF could be observed in BS3-treated ?yscI-C?83–92 and ?yscI-C?93–102 strains.These results imply that the interaction between Ysc I and YscF might be important for the assembly of the YscF needle.Then,we detected the secretion of several T3 SS substrates in the culture supernatants of yscI null mutants that were trans-complemented with plasmids expressing different YscI mutants.As expected,the secretion of T3 SS substrates was only detected in the ?yscI-C?73–82 and ?yscI-C?103–115 strains,but it was completely abolished in the ?yscI-C?83–92 strain.Surprisingly,the amino acids 93–102 are essential for the YscF binding and the needle assembly,Yop secretion is not abolished in the ?yscI-C?93–10 2strain,suggesting that Ysc I?93–102 likely forms an assembled inner rod structure and is able to switch substrate,while YscI?83–92 could not assemble rod structure or is unable to switch substrate.A sequence alignment of YscI and YscF showed that they shared high amino acid identity at residues 85–92,and amino acid residues 85,86,88,and 92 of YscI are identical to those of YscF.First,we created single amino acid mutations of yscI,they were ?yscI-CW85 A,?yscI-CS86 A,?ysc I-CI88 A and ?yscI-CI92 A.The binding of the YscI mutants to YscF was analyzed by co-IP.The results showed that the ?yscI-CW85 A and ?yscI-CS86 A mutants did not co-immunoprecipitate YscF,while the ?yscI-CI88 A and ?yscI-CI92 A mutants did not exhibit any obvious defects in binding.Then,we sought to determine the YscF binding affinity of purified YscI point mutants in in vitro assay.GST pull-down assay using GST-tagged YscI point mutants found that I88 A or I92 A YscI mutants exhibit no defect in YscF binding,but mutation of 85 or 86 amino acids of YscI resulted in the disruption of Ysc F binding.Taken together,these results indicate that YscI can bind directly to YscF and mutations of W85 or S86 of YscI totally abolishes YscF binding.To investigate whether the YscI mutations that disrupt YscF binding also affect needle assembly,YscF polymerization was analyzed by a cross-linking assay using Y.pestis strains expressing various YscI mutants.The results showed the ?yscI-CW85 A and ?yscI-CS86 A strains no longer polymerized Ysc F while normal YscF polymerization was found in the?yscI-CI88 Aand ?yscI-CI92 A strain.These results clearly demonstrate that mutation of W85 or S86 not only disrupts the binding of YscI to YscF,but also results in the failure of needle assembly.Next,we analyzed substrate secretion by Y.pestis strains expressing YscI single amino acidmutants.The secretion was completely abrogated in the ?yscI-CW85 A and ?yscI-CS86 A strains,except that the ?yscI-CW85 A strain still secreted a small amount of YopE.The above results showing that YscII88 A and YscII92 A bind YscF and that strains expressing YscII88 Aor YscII92Acan assemble the YscF needle.In this study,we tested the cytotoxicity to HeLa cell of YscI mutants by observeing morphologies of cells and detecting their virulence attenuated by RTCA.The ?yscI-CI88 Aand ?yscI-CI92 A strains caused cytotoxic effects to HeLa cells similar to the wild-type strain,while the morphologies of cells that were infected with the ?yscI strain was similar to that of the mock-infected control.T he ?yscI-C?83–92,?yscI-C?93–102,?yscI-CW85 A and ?yscI-CS86 A mutants exhibited greatly reduced cytotoxicity to HeLa cells,which is in line with the above results showing that YscI?82–93,YscI-C?93–102,YscIS86 A and YscIW85 A do not bind Ysc F,and that those strains do not form the polymerized YscF needle either.Then,proteins from the soluble fractions of the infected HeLa cells were analyzed for the presence of YopE,YopM and LcrV.The results showed ?yscI-CW85 A and ?yscI-CS86 A could not translocated YopE,YopM and LcrV into the cytoplasm of HeLa cells.Abstract 2Plague is a potent infectious disease caused by Yersinia pestis,which is mainly transmitted from fleas to the rodents by biting and may be transmitted to people,leading to gland plague,pneumonia plague and sepsis.In this study,on the basis of the whole genome sequencing of Yersinia pestis,we further studied the proteome under different physiological conditions,namely,26 °C calcium?simulated flea environment?and 37 °C low calcium?simulating the mammalian environment?in order to discover new virulence factors related to pathogenicity,which will be helpful for the understanding of Y.pestis pathogenesis as well as the development of novel measurements for treatment and prevention of plague.Y.pestis 201 strain was grown in chemically defined TMH medium,with2.5 m Mcalcium at 26°C or without calcium at 37°C,as indicated for the specific experiments.Bacterial cultures were collected at 8h after the temperature shift from 26 °C to 37 °C.The secreted proteins present in the culture supernatant were concentrated bycentrifugation through an 3k D Ultra-15 centrifugal filter device.The bacterial pelletswere re-suspended in lysis buffer and lysed by sonication in ice bath.Theconcentrations of protein samples from the supernatants and the cell pellets weredetermined by using Pierce BCA Protein Assay Kit.For sample preparations to studythe dynamics of substrates secretion of Y.pestis T3 SS,Y.pestisstrain 201 was grownas described above,except that both the bacterial cultures were collected at 80,160,240,400 and 480 min after the temperature shift from 26 °C to 37 °C.Protein samplesform the culture supernatant and the cell pellets were extracted and the proteinconcentrations were determined as described above.Here,comparative proteomics analysis of Y.pestis cultured under conditions mimicking the two typical niches were performed using TMT?tandem mass tag?labeling quantitative proteomics technology.After removing of the redundant proteins,expressions of 2141 proteins were identified,representing 52.2 %of the genome,among which 1081 proteins could be quantitatively measured in both of the two types of samples.Thus,the criterions fordefinition of a protein to be a secreted protein in this study were set as Score ? 5 and the matched Unique Peptides ? 2.According to these criterions,total of 767 proteins were identified to be the secreted proteins of Y.pestis Among the proteins in the list of secreted proteins,Yp-2058,Yp-3657 and Yp₃415-Yp₃418 were remarkable and may be closely related to the virulence of Yersinia pestis.In the study of dynamics of substrates secretion of T3 SS,we did not observe any specific changes in T3 SS proteins over time.It may be due to the proteins have been basically stabilized at the minimum secretion time of 80 min.This study established a new method to identify the secreted protein of T3 SS,that is,S/P PSM> 1 as a diagnostic criteria for secreted protein.T3 SS secreted proteins which were identified by the criteria are all reported in the literature.Among the proteins in the list of secreted proteins,several proteins encoded by an operon Yp₃416^Yp₃418 were remarkable.We constructed the deletion mutant of YP₃416,YP₃418 and YP₃416-3418 and analyzed their virulence.The ability to survival in the mouse macrophages were analyzed the deletion mutants.The results showed the survival curves of yp₃416-3418 and yp₃416 mutant were significantly different from that of wild-type strain otherwise there was no significant difference between wild-type strain and yp₃418 mutant after infection with tail vein.And after the nasal infection,the survival curves of the wild-type strain and the above three mutants were statistically significant.We further cloned yp₃416,yp₃417,yp₃418,yp₂058 and yp₃657 gene into ?-lactamase reporter plasmidp BBR1-TEM to analyze weather those proteins could be translocated into eukaryotic cells.The lipophilic substrate CCF2-AM readily enters eukaryotic cells,where endogenous cytoplasmicesterasesconvertit into CCF2 that consists of a cephalosporin core linking a 7-hydroxycoumarin to a fluorescein.Translocation of proteins fused with ?-lactamase will introduces ?-lactamase into host cytosol,which cleavages the substrates of CCF2 andspatially separates the two dyesand disrupts fluorescent energy resonance transfer?FRET?,leading to the increased ratio of blue to green fluorescence?447/520 nm?with excitation at 409 nm.The results showed Yp₃416,Yp₃417,Yp₃418,Yp₂058 and Yp₃657 can be translocated into He La cells during infection,which did not only confirm that these 5 proteins could be secreted by Y.pestis,but also showed that they could be translocated into host cytosol,which hints that those proteins could contribute to the pathogenesis of Y.pestis.