The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems

Abstract

The 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 Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3-4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4 O1 ) genes caused by a terminationcodon 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. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors.

Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system

“…Members of Rhs proteins include the antibacterial WapA in Gram-positive Bacillus subtilis 13 , the ABC insecticidal Tc-toxins 7,14,15 , and secreted effectors by the type VI secretion system (T6SS) in Gram-negative species 10,11 . Known T6SS Rhs effectors have been shown to be frequently associated with an Nterminal PAAR domain or downstream of VgrG and PAAR encoding genes 8,10,11,[16][17][18][19] .…”

The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors

“…Nucleases have either DNase [e.g., EsaD (T7SS), CdiA-CT O11 EC869 (CDI), colicin E2] or RNase activity [e.g., CdiA-CTII Bp1026b (CDI), colicin E6], targeting tRNA or rRNA molecules (6,7,12,(32)(33)(34). A variety of T6SS nucleases have been identified, including the Toxin_43 domain Tde family from Agrobacterium tumefaciens (12), the colicin/pyocin-related families (e.g., Usp, Hcp_ET3/ET4, VPA1263) (35,36), the endonuclease NS_2 family (e.g., RhsA) (33), the Tox-REase1 family (e.g., Tke10) (37), and effectors with an HNH endonuclease motif (e.g., RhsB, Rhs2, Hcp_ET1, and Tke2/4) (33,35,37,38) (SI Appendix, Table S2). Tse7 is the first characterized T6SS Tox-GHH2 member from the HNH family as well as the first T6SS nuclease identified in P. aeruginosa.…”

“…For example, Usp of APEC has a moderately active pyocin/colicin DNase domain and is an evolved Hcp (36). This is also the case for Hcp_ET1 of STEC, which has a Cterminal HNH-DNase domain, and Hcp_ET3 of ETEC with its C-terminal pyocin S3 DNase domain (35). However, in many instances the T6SS-dependent secretion of these toxins has not been confirmed.…”

Structure and Activity of the Type VI Secretion System

“…When the effector module is on the same polypeptide as the needle component, the T6SS subunit is called "specialized" or "evolved". Although effectors fused to Hcp or PAAR have been described (36,90,111), the best-characterized examples are C-terminal extensions of specialized VgrGs such as V. cholerae VgrG1 that cross-links actin and VgrG3 that has peptidoglycan glycoside hydrolase activity, A. hydrophila VgrG1 that ADP-ribosylates actin,…”