Functional Characteristics Of The Type ? Secretion System Effector Hcp In Aeromonas Hydrophila

Aeromonas hydrvphila is a common opportunistic pathogen which is widely spread in aquatic environments.This bacterium can cause hemorrhagic septicaemia in various freshwater fishes,and can also cause food poisoning infectious diarrhea,and septicaemia in humain The type ? secretion system?T6SS?is a versatile weapon used by many Gram-negative bacteria to target eukaryotic host cells or other bacteria,and plays an important role in the bacterial virulence and environmental fitness.Hemolysin co-regulatory protein?Hep?is a central structural component and effector of T6SS,and its secretion is considered as an indicator of a functional T6SS.However,the role of T6SS in A.hydrophila still remains unclear.The bioinformatics analys:is performed on 14 whole-genome sequenced A.hydrophila showed that the T6SS gene clusters are diverse in different isolates of A.hydrophila.On this basis,the role of the T6SS effector,Hcp,was investigated in the A.hydrophila Chinese strain NJ-35 and American strain ML09-119;the immunogenicity and protective efficacy of the recombinant Hcp protein were evaluated in common carp?Cyprinus carpio?;and a new effector-immunity pair TseP/TsiP was identified and investigated in A.hydrophila NJ-35.1.Identification of the T6SS gene cluster and analysis of Hep expression diversity in A.hydrophila Chinese strain NJ-35Bioinformatics analysis was performed on 14 whole genome sequenced A.hydrophila strains.Except for GYK1,all the other six A.hydrophila Chinese strains contained one T6SS gene cluster.However,in the seven A.hydrophila American strains,no T6SS gene cluster was found except for ATCC 7966 and AHNIH1.The presence of Hcp in the culture supernatant of A.hydrophila NJ-35 suggested a functional T6SS in this strain Hcp secretion was found to be strongly induced by low temperature in NJ-35.In this study,three copies of the hcp gene were identified in the genome of strain NJ-35.We targeted these Hcp family proteins for generating deletion mutants by homologous recombination method.Futher,the corresponding complemented strains of these mutants were constructed.In addition,the cross complemented strains of the hcp genes were also constructed.These mutants showed varying levels in Hcp production The hcpl gene was shown to play a primary role in Hcp expression and secretion of NJ-35;hcp3 might play a complementary role when hcp1 was knocked out;whereas hcp2 was not expressed in vitro.These findings suggest that Hcpl may be involved in the assembly and secretion function of T6SS.2.Diverse roles of Hcp family proteins in the pathogenicity and environmental fitness of A.hydrophila Chinese strain NJ-35The biological characteristics of the wild-type,the hcp gene-deleted and their complemented strains of A.hydrophila NJ-35 were investigated.Hcpl was found necessary for T6SS assembly and played a predominant role in the bacterial competition;Hcp2 negatively functioned in the biofilm formation and bacterial adhesion and was more involved in the A.hydrophila virulence in zebrafish and survival against the predation of Tetrahymena;and Hcp3 positively influenced the biofilm formation and bacterial adhesion.These findings illustrate that the T6SS of A.hydrophila NJ-35 is active,and the three Hcp family proteins take part in different processes in the virulence and environmental adaptation of this bacterium3.Diverse roles of Hep family proteins in the pathogenicity and environmental fitness of A.hydrophila American strain ML09-119The bioinformatics analysis of the whole genome sequence of A.hydrophila ML09-119 showed that T6SS gene cluster was absent in its genome,whereas two hcp genes present To investigate the function ofthe hcp genes in ML09-119,single and double gene-deleted strains of hcp-1 and hcp-2 were constructed.The hcp-2 gene was transcribed while hcp-1 was not in ML09-119 under standard culture conditions in vitro.In this American strain ML09-119,neither hcp-1 nor hcp-2 was involved in bacterial motility,biofilm formation,or bacterial competition.In the animal infection experiment,the catfish infected by the wild-type strain and Ahcp-2 all died within 30 h and 36 h,respectively.In contrast,the catfish infected by?hcp-1 and ?hcp-1/2 showed survival rates of 40%and 30%,respectively,within 48 h.At 12 h post infection,the colonization ability of Ahcp-1 and Ahcp-1/2 mutants in kidney of catfish significantly decreased compared to the wild-type strain,and the ?hcp-1 mutant also showed a significantly decreased colonization ability in liver of catfish.These findings indicated that hcp-1 was involved in the virulence of ML09-119 on catfish.4.Comparative proteomics analysis betmeen the hcpl gene-deleted and wild-type strains of A.hydrophilaThe adhesion ability of hcpl gene-deleted strain to HEp-2 cells was significantly lower than that of the wild-type strain?P<0.001?.Lable-free quantitative proteomics techniques were used to investigate the differentily expressed proteins in outer membrane protein samples between the ?hcpl mutant and wild-type strain of NJ-35.Among the differentially expressed proteins,23 were up-regulated and 30 were down-regulated.These proteins was involved in flagellar formation,pilus formation and outer membrane proteins,which may be related to the adhesion ability of A.hydrophila to HEp-2 cells.The results of qRT-PCR showed that the mRNA levels of fimC and fimD,which were involved in type L pilus formation,were significantly downregulated?P<0.001?in the ?hcpl mutant.It is speculated that the hcpl gene may regulate the expression of genes such as fimC and fimD to affect the bacterial adhesion ability.5.Protective efficacy of the recombinant Hcp protein of A.hydrophila in Common carp?Cyprinus carpio?Prophylactic vaccination against motile aeromonad septicemia?MAS?and other diseases is essential for continued growth of aquaculture.The distribution of the hcp gene in 240 isolates of Aeromonas was determined in this study The T6SS effector Hcp was expressed and characterized,and its immunogenicity and protective efficacy were evaluated in common carp?Cyprinus carpio?.PCRresults showed that the hcp gene was widely present in different species of Aeromonas,especially in A.hydrophila?39/39?.Immunoblot analysis demonstrated that Hcp is conserved among A.hydrophila strains of different origins.The vaccination with recombinant Hcp resulted in an increased survival?46.67%?in common carp during a 10-day challenge time compared to non-vaccinated fish?7.14%?.The vaccinated fish also showed the significantly increased levels of IgM antibody in serum and cytokines such as inerleukin-1??IL-1??and tumor necrosis factor-??TNF-??in kidney,spleen and gills.The recombinant Hcp shows promise as a vaccine candidate against A.hydrophila.6.Identification and functional analysis of a new effector-immunity pair TseP/TsiP in A.hydrophila NJ-35The T6SS of Gram-negative bacteria can be used to inhibit and kill other bacteria or eukaryotic cells through the delivery of effectors.In this study,bioinformatics analysis of the whole genome sequences of 14 A.hydrophila strains identified a variety pairs of potential efectors and their cognate immunity proteins.A new antibacterial effector-immunity pair TseP/TsiP was identified in A.hydrophila NJ-35 for the first time.TseP is encoded by U876₁1145 and carres a Pyocin_S domain.To investigate the role of TseP in NJ-35,the tseP or tsiP gene-deleted strains and their corresponding complemented strains were constructed.The results showed that in NJ-35,the deletion of tseP or tsiP gene resulted in a significant increase in bacterial biofilm formation while not affecting the bacterial motility and survival against the predation of Tetrahymena.The results of bacterial competition assay showed that the wild-type strain NJ-35 had a significant killing effect on ?tsePtsiP and Escherichia coli?P<0.001?;the deletion of tseP in NJ-35 obviously attenuated this killing effect of NJ-35 on AtsePtsiP,while not reduce the killing effect of NJ-35 on E.coli,which suggested that TseP might be involved in the intraspecies competition of A.hydrophila.Also,the antibacterial effect of TseP depended on the T6SS,and its cognate immunity protein TsiP could protect the bacteria itself against this effect.In summary,the T6SS gene clusters are diverse in different isolates of A.hydrophila;the T6SS effector Hcp family proteins play different roles in the pathogenicity and environmental fitness of A.hydrophila NJ-35.This study provided new insights into how the bacteria use the T6SS to mediate interactions between the microorganisms.