A Type VI Secretion System Trans-Kingdom Effector Is Required for the Delivery of a Novel Antibacterial Toxin in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has evolved multiple strategies to disarm and take advantage of its host. For this purpose, this opportunist pathogen has particularly developed protein secretion in the surrounding medium or injection into host cells. Among this, the type VI secretion system (T6SS) is utilized to deliver effectors into eukaryotic host as well as target bacteria. It assembles into a contractile bacteriophage tail-like structure that functions like a crossbow, injecting an arrow loaded with effectors into the target cell. The repertoire of T6SS antibacterial effectors of P. aeruginosa is remarkably broad to promote environmental adaptation and survival in various bacterial communities, and presumably in the eukaryotic host too. Here, we report the discovery of a novel pair of antibacterial effector and immunity of P. aeruginosa, Tle3 and Tli3. Tli3 neutralizes the toxicity of Tle3 in the periplasm to protect from fratricide intoxication. The characterization of the secretion mechanism of Tle3 indicates that it requires a cytoplasmic adaptor, Tla3, to be targeted and loaded onto the VgrG2b spike and thus delivered by the H2-T6SS machinery. Tla3 is different from the other adaptors discovered so far and defines a novel family among T6SS with a DUF2875. Interestingly, this led us to discover that VgrG2b that we previously characterized as an anti-eukaryotic effector possesses an antibacterial activity as well, as it is toxic towards Escherichia coli. Excitingly Tli3 can counteract VgrG2b toxicity. VgrG2b is thus a novel trans-kingdom effector targeting both bacteria and eukaryotes. VgrG2b represents an interesting target for fighting against P. aeruginosa in the environment and in the context of host infection.

A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens.

Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way.

Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosamgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

Gene transfer: transformation/electroporation.

Since Pseudomonas aeruginosa is a non-naturally competent bacterium, various methods have been developed to transfer exogenous DNA. Alternatively to transduction and conjugation, electroporation can also be used to transfer exogenous DNA molecules into Pseudomonas. Electroporation uses an electric field which generates pores in bacterial membranes allowing the entry of the exogenous DNA molecule. In contrast to conjugation which is restricted to the transfer of DNA from one bacterial cell to another, electroporation can be used to transfer all types of DNA resuspended in water.

Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa.

Three Type VI Secretion System (T6SS) loci called H1- to H3-T6SS coexist in Pseudomonas aeruginosa. H1-T6SS targets prokaryotic cells whereas H2-T6SS mediates interactions with both eukaryotic and prokaryotic host cells. Little is known about the third system, except that it may be connected to H2-T6SS during the host infection. Here we show that H3-T6SS is required for P. aeruginosa PAO1 virulence in the worm model. We demonstrate that the two putative H3-T6SS operons, called "left" and "right", are coregulated with H2-T6SS by the Las and Rhl Quorum Sensing systems. Interestingly, the RpoN σ54 factor has divergent effects on the three operons. As for many T6SSs, RpoN activates the expression of H3-T6SS left. However, RpoN unexpectedly represses the expression of H3-T6SS right and also H2-T6SS. Sfa2 and Sfa3 are putative enhancer binding proteins encoded on H2-T6SS and H3-T6SS left. In other T6SSs EBPs can act as σ54 activators to promote T6SS transcription. Strikingly, we found that the RpoN effects of H3-T6SS are Sfa-independent while the RpoN mediated repression of H2-T6SS is Sfa2-dependent. This is the first example of RpoN repression of a T6SS being mediated by a T6SS-encoded EBP.

The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells.

The genome of Pseudomonas aeruginosa PAO1 contains three type VI secretion systems (T6SSs) called H1-, H2-, and H3-T6SS. The H1-T6SS secretes three identified toxins that target other bacteria, providing a fitness advantage for P. aeruginosa, and likely contributes to bacterial pathogenesis in chronic infections. However, no specific substrates or defined roles have been described for the two other systems. Here, we demonstrate that the expression of H2-T6SS genes of strain PAO1 is up-regulated during the transition from exponential to stationary phase growth and regulated by the Las and Rhl quorum sensing systems. In addition, we identify two putative Fur boxes in the promoter region and find that H2-T6SS transcription is negatively regulated by iron. We also show that the H2-T6SS system enhances bacterial uptake into HeLa cells (75% decrease in internalization with a H2-T6SS mutant) and into lung epithelial cells through a phosphatidylinositol 3-kinase-dependent pathway that induces Akt activation in the host cell (50% decrease in Akt phosphorylation). Finally, we show that H2-T6SS plays a role in P. aeruginosa virulence in the worm model. Thus, in contrast to H1-T6SS, H2-T6SS modulates interaction with eukaryotic host cells. Together, T6SS can carry out different functions that may be important in establishing chronic P. aeruginosa infections in the human host.

Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium.

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

The C-terminal amphipathic alpha-helix of Pseudomonas aeruginosa PelC outer membrane protein is required for its function.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes numerous infections and can adopt a versatile lifestyle. During chronic infection, P. aeruginosa becomes established as a bacterial community known as a biofilm. Biofilm formation results from the production of a matrix mainly comprised of exopolysaccharides. P. aeruginosa possesses several gene clusters which contribute to the formation of the matrix, including the pel genes. Among the pel genes, pelC encodes an outer membrane protein, which may serve as a transporter of polysaccharide to the bacterial cell surface. Whereas outer membrane proteins usually display an amphipathic beta-barrel fold, we show that PelC requires a C-terminal amphipathic alpha-helix for outer membrane insertion and function. Such a structural feature has only previously been reported for the Wza outer membrane protein of Escherichia coli, and our data suggest that this characteristic may be found in a large family of proteins, particularly outer membrane proteins specialized in polysaccharide transport.

PelC is a Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of proteins involved in exopolysaccharide transport.

Pseudomonas aeruginosa is a gram-negative bacterium, opportunistic pathogen, which causes severe acute or chronic infections, as is the case with cystic fibrosis patients. Chronic infections are frequently accompanied by the development of the bacterial population into a specialized community called biofilm. The pelA-G gene cluster of P. aeruginosa has been shown to be involved in pellicle production and biofilm formation. The pel genes have been proposed to contribute to the formation of the exopolysaccharide-containing pellicle. However, the function and the subcellular localization of the seven different Pel proteins are poorly understood. Based on bioinformatics analysis, we have previously considered that PelF is a putative glycosyltransferase (GT4 family), whereas PelG is a Wzx-like polysaccharide transporter from the PST family. In this study we have further characterized the PelC protein. We have shown that PelC is an outer membrane lipoprotein. The N-terminal signal peptide of the PelC lipoprotein is sufficient to target the protein into the membranes. However, by constructing various PelC hybrid proteins we also proposed that efficient and functional outer membrane insertion of PelC requires not only the signal peptide and the lipid modification, but also requires the C-terminal domain of PelC. Because the gene encoding the outer membrane lipoprotein PelC is part of a putative gene cluster involved in exopolysaccharide biogenesis, we suggest that PelC is a new member of the outer membrane auxiliary (OMA) family of lipoprotein whose Wza, involved in Escherichia coli capsular polysaccharide transport, is an archetype.

Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa.

Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (DeltafliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (DeltamotAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.

Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes.

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a wide range of acute and chronic infections. The transition to chronic infections is accompanied by physiological changes in the bacteria favoring formation of biofilm communities. Here we report the identification of LadS, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharides. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection by transcriptional and posttranscriptional mechanisms.

Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1.

A systematic analysis of the type III secretion (T3S) genes of Pseudomonas aeruginosa strain PAO1 revealed that they are under quorum-sensing control. This observation was supported by the down-regulation of the T3S regulon in the presence of RhlR-C4HSL and the corresponding advanced secretion of ExoS in a rhlI mutant.

The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with nosocomial infections and cystic fibrosis. Chronic bacterial infections are increasingly associated with the biofilm lifestyle in which microcolonies are embedded in an extracellular matrix. Screening procedures for identifying biofilm-deficient strains have allowed the characterization of several key determinants involved in this process. Biofilm-deficient P. aeruginosa PAK strains affected in a seven-gene cluster called pel were characterized. The pel genes encode proteins with similarity to components involved in polysaccharide biogenesis, of which PelF is a putative glycosyltransferase. PelG was also identified as a putative component of the polysaccharide transporter (PST) family. The pel genes were previously identified in the P. aeruginosa PA14 strain as required for the production of a glucose-rich matrix material involved in the formation of a thick pellicle and resistant biofilm. However, in PA14, the pel mutants have no clear phenotype in the initiation phase of attachment. It was shown that pel mutations in the PAK strain had little influence on biofilm initiation but, as in PA14, appeared to generate the least robust and mature biofilms. Strikingly, by constructing pel mutants in a non-piliated P. aeruginosa PAK strain, an unexpected effect of the pel mutation in the early phase of biofilm formation was discovered, since it was observed that these mutants were severely defective in the attachment process on solid surfaces. The pel gene cluster is conserved in other Gram-negative bacteria, and mutation in a Ralstonia solanacearum pelG homologue, ragG, led to an adherence defect.

Global regulation in Pseudomonas aeruginosa: the regulatory protein AlgR2 (AlgQ) acts as a modulator of quorum sensing.

The Pseudomonas aeruginosa protein AlgR2 (AlgQ) was originally identified as a regulatory protein implicated in alginate production. It also regulates the synthesis of polyphosphate as well as of a variety of secretable virulence factors, upregulating neuraminidase and siderophore synthesis and downregulating rhamnolipid biosurfactant and extracellular protease synthesis. In this study, we show that the regulatory effect of AlgR2 on elastase protease synthesis is exerted at transcriptional level on the lasB gene. We also demonstrate that AlgR2 negatively modulates the expression of quorum sensing regulatory genes lasR and rhlR. Finally, results obtained from DNA retardation assays provide evidence that AlgR2 can bind specifically to the lasR and rhlR promoters. Altogether, these data provide strong support for the hypothesis that AlgR2 is a global transcriptional regulator in P. aeruginosa.

Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR.

Pseudomonas aeruginosa controls the production of many exoproteins and secondary metabolites via a hierarchical quorum sensing (QS) regulatory cascade involving the LuxR-like proteins LasR, RhlR and their cognate signal molecules N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-l-homoserine lactone (C4-HSL). The finding of a third LuxR-type protein in P. aeruginosa, QscR, adds further complexity to this regulatory network. It has been shown previously that QscR represses transcription of three QS-controlled gene clusters, phz (phenazine), hcn (hydrogen cyanide) and qsc105 (Chugani, Whiteley, Lee, D'Argenio, Manoil, and Greenberg, 2001, Proc Natl Acad Sci USA 98: 2752-2757). In this study, we identify two novel QscR targets these are lasB, encoding the extracellular elastase, and the second phenazine gene cluster, both of which are downregulated by QscR. In addition, we show that QscR synthesis is regulated by the two-component response regulator GacA. Taking advantage of the in vivo fluorescence anisotropy technology that we have developed, we show that QscR can be found in several different types of association. Indeed, we identify QscR multimers in the absence of any acyl-HSL, lower order QscR oligomers associated either with C4-HSL or 3O-C12-HSL and QscR-containing heterodimers with LasR or RhlR. The formation of heterodimers between QscR and LasR or RhlR, in the absence of acyl-HSLs, is a very exciting, new result that should improve our understanding of the QscR network and its relationship to the production of P. aeruginosa virulence factors.