Genetically distinct pathways guide effector export through the type VI secretion system

Self Cite

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI + bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT EC536 deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT EC536 binary complex and the neutralized ternary complex of CysK/CdiA-CT/ CdiI EC536 . CdiA-CT EC536 inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT EC536 exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT EC536 thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT EC536 , and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.bacterial competition | structural biology | toxin chaperone | tRNase activity

Section: Discussionmentioning

confidence: 99%

Unraveling the essential role of CysK in CDI toxin activation

Johnson

Beck

Morse

et al. 2016

Self Cite

“…The striking conservation of genetic modules encoding homologous VgrG, a distinct set of potential adaptor/chaperone, and a specific effector in various Proteobacteria strongly suggest a conserved mechanism in type VI effector delivery. (30,31). Recent studies further identified a DUF4123-domaincontaining protein, Tap-1/Tec, that is required for loading a specific effector onto cognate VgrG for delivery in V. cholerae (32,33).…”

Section: Significancementioning

confidence: 99%

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

Bondage

Lin

et al. 2016

144

273

Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity.type VI secretion system | VgrG | DNase effector | interbacterial competition | Agrobacterium tumefaciens

“…The T6 apparatus is a multiprotein, cell envelope spanning complex comprised of core Tss proteins. A key component of the machinery is a needle-like structure, similar to the T4 contractile bacteriophage tail, which is assembled in the cytoplasm where it is loaded with toxic effectors (8)(9)(10). Contraction of the sheath surrounding the needle apparatus drives expulsion of the needle from the cell, delivering the needle and associated effectors either into the supernatant of in vitro grown bacteria, or across the membrane of prey cells.…”

mentioning

confidence: 99%

Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species

Chatzidaki‐Livanis

Geva‐Zatorsky

Comstock

2016

181

176

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis. Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses, trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.Bacteroides | T6SS | gut microbiota | bacterial competition | type VI secretion