Type ? Secretion Systems In Pathogenic Escherichia Coli Are Involved In Full Virulence And Antibacterial Mechanism

Escherichia coli typically colonize in the intestinal tract and other mucosal surface of human and animals.Most E.coli are commensal,while several clonal complexes have evoloped into virulent strains with specific pathogenic characteristics;and could divided into two different groups:Intestinal Pathogenic and Extraintestinal Pathogenic E.coli(ExPEC).Avian Pathogenic E.coli(APEC)is an important member of ExPEC,it cause acute diseases and great economic loss in the poultry industry.Moreover,it shares the same virulent factors and pathogenic characteristics with human ExPEC with a great threaten to the public health.The type VI secretion system(T6SS)is a widespread macromolecular machine that delivers diverse effectors as molecular weapons thereby participating in interbacterial competition and virulence.Many Gram-negative bacteria use T6SS to compete for specific niches by the contact-dependent killing of competitors.However,the functions of T6SS in the pathogenic process of E.coli are still unclear,and identification of antibacterial effectors and their interbacterail competitive machenisms are in urgent need of further exploring.1 Two type VI secretion systems in APEC present different functionsThe type VI secretion systems(T6SSs)make contributions for the pathogenesis of avian pathogenic Escherichia coli(APEC),one of the leading causative agents of sepsis and meningitis in poultry.T6SSs facilitate APEC to colonize in host epithelium or blood,to escape from inflammatory response,and to enhance bacterial virulence.Haemolysin coregulated protein(Hcp)is both a core component of T6SS tail tube,and also acts as an exported receptor and chaperone of effectors.Here,four dissimilar Hcp types were designated in Gram-negative bacteria,and three of them were distributed widely in APEC.However,previous studies and our data indicated that these Hcps play different roles in pathogenic processes of APEC and immunoprotection assays.Thus,we managed to explore the potential mechanism of this functional differentiation.Notably,our results exhibited the divergence in transcription levels of three hcps under 50%duck serum,and demonstrated the transcriptional activation of hcpl by relieving Fur's repression.Further analyses revealed that host serum could up-regulate hcp2B operon to transcribe downstream xmtU/xmtV gene pair for interbacterial antagonism.Otherwise,delicate modifications of Hcp in protein structure were also required for its excellent properties.The comparison of Hcp structures based on their genetic classification exhibited diverse extended loop regions.In particular,the variant region Vs2(Loop L2,3)in Hcpl and Hcp2B was demonstrated to be essential for delivery of antibacterial effectors and inhibition of microphage phagocytosis.All above results indicated that the functional differentiation of Hcp homologs and their T6SSs is greatly drove by specific transcriptional regulations and diverse extended loop regions.2 Diverse antibacterial effectors are harbored by vgrG islands in T6SSHere,an overlooked approach based on the vgrG islands was established to search T6SS effectors.Previous studies reported that vgrG islands within T6SS clusters represent rearrangement hot spots to acquire potential effector genes,suggesting the evolutionary implications of horizontal transfer of T6SS-related toxins.Indeed,the vgrG islands are widespread and variegated among interspecies or distant species,while the flanking genes,vgrG and icmF,always remain conserved commendably.Using this conservatism to perform BlastN search,a large number of putative effectors with diverse predicted toxin domains were found in vgrG islands of E.coli,and classified into six clans(designated as VT1-VT6).Our experimental results validated that VT4 mediated cell-wall digestion by its losozyme_like activity,while VT6 was identified as a novel amidase to hydrolyze D-lactyl-L-Ala crosslinks between N-acetylmuramoyl and L-Ala in peptidoglycan.Based on the above discovery,a large-scale identification was performed in various bacteria,and more than 270 predicted VT effectors from 21 bacterial species were found and classified into 15 identified or unknown toxins.Briefly,this work significantly expands our current understanding of effector encoding patterns,and diversify antibacterial effectors of T6SS.3 The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in T6SSsThe type VI secretion system(T6SS)is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria.Using a dynamic injection mechanism,diverse effectors can be delivered by T6SS directly into recipient cells.Here,we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains.Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins,designated as Hcp-ET,exist widely in the Enterobacteriaceae.To verify our findings,Hcp-ET1 was tested for its antibacterial effect,and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery.Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tlel phospholipase(encoded by DUF2235 domain)activity.Notably,comprehensive analyses of protein homology and genomic neighbourhoods revealed that Hcp-ET3-4 is fused with two toxin domains(Pyocin S3 and Colicin-DNase)C-terminally,and its encoding gene is followed three duplications of the cognate immunity genes.However,some bacteria encode a separated hcp-et3 and an orphan et4(et4O1)genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments.Our results demonstrated that both of these toxins had antibacterial effects.Further,all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin,which has not been reported previously.4 PAAR-Rhs proteins harbor various C-terminal toxins to diversify the antibacterial pathways of T6SSsRecently,Rhs proteins with polymorphic C-terminal toxin-domains that inhibit or kill neighboring cells were identified.In this report,we identified a novel Rhs with an MPTase4(Metallopeptidase-4)domain(designated as Rhs-CT1)that showed an antibacterial effect via T6SS in Escherichia coli.We managed to develop a specific strategy by matching the diagnostic domain-architecture of Rhs-CT1(Rhs with an N-terminal PAAR-motif and a C-terminal toxin domain)for effector retrieval and discovered a series of Rhs-CTs in E.coli.Indeed,the screened Rhs-CT3 with a REase-3(Restriction endonuclease-3)domain also mediated interbacterial antagonism.Further analysis revealed that vgrGO1 and eagR/DUF1795(upstream of rhs-ct)were required for the delivery of Rhs-CTs,suggesting eagR as a potential T6SS chaperone.In addition to chaperoned Rhs-CTs,neighborless Rhs-CTs could be classified into a distinct family(Rhs-Nb)sharing close evolutionary relationship with T6SS2-Rhs(encoded in the T6SS2 cluster of E.coli).Notably,the Rhs-Nb-CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B-VgrG2 module.In a further retrieval analysis,we discovered various Effector/Immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans,suggesting that Rhs-CTs greatly diversify the antibacterial pathways of T6SS.