A type IVB secretion system adapted for bacterial killing, biofilm invasion and biocontrol

Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their environmental niches. Here, we show that Pseudomonas putida IsoF is able to outcompete a wide range of bacteria with the aid of a novel type IVB secretion system (T4BSS) that can deliver toxic effectors into bacterial competitors. This extends the host range of T4BSSs, which were so far thought to transfer effectors only into eukaryotic cells, to prokaryotes. Bioinformatic and genetic analyses showed that this killing machine is entirely encoded by a rare genomic island, which has been recently acquired by horizontal gene transfer. IsoF utilizes this secretion system not only as a defensive weapon to antagonize bacterial competitors but also as an offensive weapon to invade existing biofilms, allowing the strain to persist in its natural environment. Furthermore, we show that IsoF can protect tomato plants against the plant pathogen Ralstonia solanacearum in a T4BSS-dependent manner, suggesting that IsoF capabilities can be exploited for pest control and sustainable agriculture.

Contributions of lipopolysaccharide and the type IVB secretion system to Coxiella burnetii vaccine efficacy and reactogenicity

Coxiella burnetii is the bacterial causative agent of the zoonosis Q fever. The current human Q fever vaccine, Q-VAX®, is a fixed, whole cell vaccine (WCV) licensed solely for use in Australia. C. burnetii WCV administration is associated with a dermal hypersensitivity reaction in people with pre-existing immunity to C. burnetii, limiting wider use. Consequently, a less reactogenic vaccine is needed. Here, we investigated contributions of the C. burnetii Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of C. burnetii infection, vaccination-challenge, and post-vaccination hypersensitivity. The NMI ∆dot/icm strain was avirulent, protective as a WCV against a robust C. burnetii challenge, and displayed potentially altered reactogenicity compared to NMI. Nine Mile phase II (NMII) strains of C. burnetii that produce rough LPS, were similarly tested. NMI was significantly more protective than NMII as a WCV; however, both vaccines exhibited similar reactogenicity. Collectively, our results indicate that, like phase I LPS, the T4BSS is required for full virulence by C. burnetii. Conversely, unlike phase I LPS, the T4BSS is not required for vaccine-induced protection. LPS length does not appear to contribute to reactogenicity while the T4BSS may contribute to this response. NMI ∆dot/icm represents an avirulent phase I strain with full vaccine efficacy, illustrating the potential of genetically modified C. burnetii as improved WCVs.

Polar targeting and assembly of theLegionellaDot/Icm type IV secretion system (T4SS) by T6SS-related components

SummaryLegionella pneumophila, the causative agent of Legionnaires’ disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells’ cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of theLegionellacontaining vacuole (LCV). Here we show that polar targeting of the Dot/Icm core-transmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles ofL. pneumophilaby themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose thatLegionellaco-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of theLegionellaT4BSS apparatus is mediated by an innovative “outside-inside” mechanism.

Dot/Icm-Translocated Proteins Important for Biogenesis of theCoxiella burnetii-Containing Vacuole Identified by Screening of an Effector Mutant Sublibrary

ABSTRACTCoxiella burnetiiis an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary forC. burnetiivirulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identifyC. burnetiiNine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies inCoxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations incbu0414(coxH1),cbu0513,cbu0978(cem3),cbu1387(cem6),cbu1524(caeA),cbu1752, orcbu2028resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. Thecbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain ofC. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by thecbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire ofC. burnetiifactors that contribute to CCV biogenesis.

Structural insights into the roles of the IcmS–IcmW complex in the type IVb secretion system of Legionella pneumophila

The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers ∼300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS–IcmW recognizes effectors, and what the roles of IcmS–IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/β fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS–IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL–IcmS–IcmW complex reveals extensive and highly stable interactions between DotL and IcmS–IcmW. Moreover, IcmS–IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS–IcmW also functions as an inseparable integral component of the DotL–T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS–IcmW complex in T4BSSs.

Polar delivery ofLegionellatype IV secretion system substrates is essential for virulence

A recurrent emerging theme is the targeting of proteins to subcellular microdomains within bacterial cells, particularly to the poles. In most cases, it has been assumed that this localization is critical to the protein’s function.Legionella pneumophilauses a type IVB secretion system (T4BSS) to export a large number of protein substrates into the cytoplasm of host cells. Here we show that theLegionellaexport apparatus is localized to the bacterial poles, as is consistent with many T4SS substrates being retained on the phagosomal membrane adjacent to the poles of the bacterium. More significantly, we were able to demonstrate that polar secretion of substrates is critically required forLegionella’s alteration of the host endocytic pathway, an activity required for this pathogen’s virulence.