Molecular Microbiology (2012) 86(4), 921–936 ᭿ doi:10.1111/mmi.12028 First published online 27 September 2012 New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens Grant English,1† Katharina Trunk,1† into how pathogens utilize antibacterial T6SSs to Vincenzo A. Rao,2 Velupillai Srikannathasan,2 overcome competitors and succeed in polymicrobial William N. Hunter2 and Sarah J. Coulthurst1* niches. 1Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK. 2Division of Biological Chemistry and Drug Discovery, Introduction College of Life Sciences, University of Dundee, Dundee, Protein secretion systems and their substrates are central UK. to bacterial virulence and interaction with other organisms (Gerlach and Hensel, 2007). Six different secretion Summary systems (Types I–VI) are used by Gram-negative bacteria to transport specific proteins to the exterior of the bacterial Protein secretion systems are critical to bacterial viru- cell or further inject them into target cells. The most lence and interactions with other organisms. The Type recently described of these is the Type VI secretion VI secretion system (T6SS) is found in many bacterial system (T6SS) (Filloux et al., 2008). T6SSs are complex species and is used to target either eukaryotic cells or multi-protein assemblies that span both bacterial mem- competitor bacteria. However, T6SS-secreted proteins branes and inject effector proteins directly from the bac- have proven surprisingly elusive. Here, we identified terial cytoplasm into target cells (Bonemann et al., 2010; two secreted substrates of the antibacterial T6SS from Cascales and Cambillau, 2012). T6SSs are encoded by the opportunistic human pathogen, Serratia marces- large, variable gene clusters that contain 13 ‘core’ essen- cens. Ssp1 and Ssp2, both encoded within the T6SS tial components, believed to make up the basic secretion gene cluster, were confirmed as antibacterial toxins apparatus. Two core proteins, Hcp and VgrG, form the delivered by the T6SS. Four related proteins encoded extracellular part of the secretion machinery and depend around the Ssp proteins (‘Rap’ proteins) included two on a functional T6SS apparatus for their movement to the specifically conferring self-resistance (‘immunity’) outside of the bacterial cell (indeed, the presence of Hcp against T6SS-dependent Ssp1 or Ssp2 toxicity. Bio- in the secreted fraction has provided a useful assay for chemical characterization revealed specific, tight basic T6SS assembly and activity; Pukatzki et al., 2009). binding between cognate Ssp–Rap pairs, forming Hcp and VgrG most likely form a needle-like membrane- complexes of 2:2 stoichiometry. The atomic structures puncturing device related to the bacteriophage tail spike; of two Rap proteins were solved, revealing a novel this structure is believed to be pushed to the outside of the helical fold, dependent on a structural disulphide secreting cell and likely into target cells upon contraction bond, a structural feature consistent with their func- of a tail sheath-like structure (Leiman et al., 2009; Bone- tional localization. Homologues of the Serratia Ssp mann et al., 2010; Basler et al., 2012). T6SSs occur in and Rap proteins are found encoded together within many pathogenic bacteria and are implicated in virulence other T6SS gene clusters, thus they represent founder in important pathogens, including Burkholderia mallei, members of new families of T6SS-secreted and Burkholderia pseudomallei, Burkholderia cenocepacia, cognate immunity proteins. We suggest that Ssp pro- Vibrio cholerae, Aeromonas hydrophila, Edwardsiella teins are the original substrates of the S. marcescens tarda and Pseudomonas aeruginosa (Zheng and Leung, T6SS, before horizontal acquisition of other T6SS- 2007; Cascales, 2008; Jani and Cotter, 2010; de Pace secreted toxins. Molecular insight has been provided et al., 2010; Burtnick et al., 2011; Rosales-Reyes et al., 2012). In several cases, the action of such ‘anti- Accepted 3 September, 2012. *For correspondence. E-mail s.j. eukaryotic’ T6SSs appears to result in disruption of the [email protected]; Tel. (+44) 1382 386208; Fax (+44) 1382 388216. †These authors contributed equally and are listed actin cytoskeleton (Pukatzki et al., 2007; Aubert et al., alphabetically. 2008; Suarez et al., 2010). Exciting recent work has dem- © 2012 Blackwell Publishing Ltd 922 G. English et al. ᭿ onstrated that some T6SSs are used to target other bac- toxins. We have also identified and characterized the Rap teria, efficiently killing or inhibiting competitors. This has proteins, which include the cognate immunity proteins to been reported for T6SSs in P. aeruginosa, Burkholderia these toxins. Biochemical analyses demonstrated a tight thailandensis, V. cholerae and Serratia marcescens and specific interaction between secreted and immunity (Hood et al., 2010; MacIntyre et al., 2010; Schwarz et al., proteins. These secreted toxins and immunity proteins 2010; Murdoch et al., 2011). The discovery that certain represent two new protein families, co-occurring within T6SSs may be ‘antibacterial’ rather than, or in addition to, T6SS gene clusters of many other organisms. Addition- ‘anti-eukaryotic’ is highly relevant to the competitive ally, determination of high-resolution crystal structures of fitness and success of pathogens, particularly within pol- two members of the Rap protein family revealed that this ymicrobial infection sites. Such a system could provide family possesses a previously undescribed protein fold the pathogen with a large competitive advantage against that is dependent on formation of a disulphide bond. other bacteria in the host or the environment, enabling it to proliferate and mount a successful infection. Identifying the proteins secreted by T6SSs is a priority, Results as they will be the ‘effectors’ that directly act on target The T6SS gene cluster harbours self-resistance eukaryotic or bacterial cells. A special case of T6-secreted determinants and candidate secreted effectors effector is the class of ‘evolved’ VgrG proteins found in a minority of T6SSs which have extra C-terminal effector The T6SS gene cluster of S. marcescens Db10, domains, e.g. the actin cross-linking domain of V. chol- SMA2244–2281, contains 38 genes, including many with erae VgrG1 that is translocated into mammalian cells no known function (Murdoch et al., 2011). We speculated (Pukatzki et al., 2007; Jani and Cotter, 2010). However, that encoded within this cluster might be T6-secreted excluding the structural components VgrG and Hcp, very effectors and/or self-resistance determinants, the latter few ‘true’ T6SS-secreted proteins have been confirmed so preventing the T6SS-expressing cell from harming itself or far. Best characterized are three effector proteins, anti- being harmed by its isogenic (sibling) neighbours. In order bacterial toxins named Tse1–3, secreted by the antibac- to determine whether the cluster did indeed contain self- terial HSI-1 T6SS of P. aeruginosa. Tse1 and 3 are resistance determinants (such as specific immunity pro- peptidoglycan hydrolases that attack the cell wall of target teins analogous to the Tsi proteins), we generated a bacteria (Hood et al., 2010; Russell et al., 2011). Tse2 is mutant lacking the entire T6SS gene cluster (DT6SS) and active in the cytoplasm of target cells, where it efficiently examined whether it was fully resistant to the T6SS of the induces quiescence (Li et al., 2012). All three have adja- wild type strain. Co-culture of two target strains, wild type cently encoded cognate ‘immunity’ proteins (Tsi1–Tsi3) Db10 and the DT6SS mutant, each with a wild type and a which protect the secreting cell from harming itself or DclpV attacker, showed that the DT6SS mutant had lost being harmed by its sibling neighbours (Hood et al., 2010; resistance to T6SS-mediated inhibition or killing by the Russell et al., 2011). Significantly, obvious homologues of wild type strain (Fig. 1A, left). Recovery of DT6SS was the Tse and Tsi proteins are not detectable outside of decreased 100-fold when it was co-cultured with the wild P. aeruginosa (Hood et al., 2010). A recent report has also type strain, compared with when the wild type strain was identified a number of candidate T6SS substrates in co-cultured with itself. This effect was dependent on a B. thailandensis, one of which was confirmed as a new functional T6SS in the attacker as there was no loss of T6-secreted peptidoglycan amidase (Russell et al., 2012). DT6SS when it was co-cultured with a DclpV mutant. The Opportunistic Gram-negative bacteria cause a large ATPase ClpV is one of the core, structural components of proportion of problematic and antibiotic-resistant hospital- the T6SS and we have shown previously that it is essen- acquired infections. Enterobacteria (especially extended- tial for Hcp secretion and T6-mediated antibacterial killing spectrum b-lactamase producing isolates) are among the activity of S. marcescens Db10 (Murdoch et al., 2011). To leading culprits, including S. marcescens (Choi et al., simplify the analysis of multiple mutants, we defined the 2007; Lockhart et al., 2007). We previously reported that ‘resistance index’ of a strain as the difference between S. marcescens Db10 possesses a T6SS with potent anti- recovery when co-cultured with the wild type strain and bacterial activity (Murdoch et al., 2011). How this activity recovery when co-cultured with the DclpV mutant, specifi- is mediated,