Lack of evidence that Pseudomonas aeruginosa AmpDh3-PA0808 constitute a type VI secretion system effector–immunity pair

Type VI secretion systems (T6SSs) are cell envelope-spanning protein complexes that Gram-negative bacteria use to inject a diverse arsenal of antibacterial toxins into competitor cells. Recently, Wang et al. reported that the H2-T6SS of Pseudomonas aeruginosa delivers the peptidoglycan recycling amidase, AmpDh3, into the periplasm of recipient cells where it is proposed to act as a peptidoglycan degrading toxin. They further reported that PA0808, the open reading frame downstream of AmpDh3, encodes an immunity protein that localizes to the periplasm where it binds to and inactivates intercellularly delivered AmpDh3, thus protecting against its toxic activity. Given that AmpDh3 has an established role in cell wall homeostasis and that no precedent exists for cytosolic enzymes moonlighting as T6SS effectors, we attempted to replicate these findings. We found that cells lacking PA0808 are not susceptible to bacterial killing by AmpDh3 and that PA0808 and AmpDh3 do not physically interact in vitro or in vivo. Additionally, we found no evidence that AmpDh3 is exported from cells, including by strains with a constitutively active H2-T6SS. Finally, subcellular fractionation experiments and a 1.97 Å crystal structure reveal that PA0808 does not contain a canonical signal peptide or localize to the correct cellular compartment to confer protection against a cell wall targeting toxin. Taken together, these results cast doubt on the assertion that AmpDh3-PA0808 constitutes an H2-T6SS effector–immunity pair.     

铜绿假单胞菌T6SS的组装、分泌、功能和调控

作为一种对抗真核细胞和原核细胞的强有力细菌武器,Ⅵ型分泌系统(type Ⅵ secretion system,T6SS)广泛存在于革兰氏阴性菌中。铜绿假单胞菌是一种对多种抗生素具有耐药性并能够在人体引发急性和慢性感染的条件致病菌,它编码3套独立的T6SS,分别为H1-、H2-和H3-T6SS。T6SS通过介导细菌间竞争、生物被膜的形成、金属离子的摄取以及与真核宿主细胞之间的相互作用,对铜绿假单胞菌在毒力和适应环境方面发挥重要作用。本文主要对铜绿假单胞菌T6SS的组装、效应蛋白的分泌、功能及调控机制展开综述,旨在为T6SS的研究提供一定的参考,并为铜绿假单胞菌感染的预防和治疗提供一定的指导。     

Quorum sensing and alternative sigma factor RpoN regulate type VI secretion system I (T6SSVA1) in fish pathogen <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Type VI secretion system (T6SS) is a highly conserved bacterial protein secretion system and is precisely regulated in Gram-negative pathogens. In Vibrio alginolyticus, an important fish pathogen, two complete T6SS gene clusters (T6SSVA1 and T6SSVA2) were identified. In this study, expression of a hemolysin coregulated protein (Hcp1), which is one of the hallmarks of T6SS, was found to be strictly regulated in this bacterium. We showed that the expression of Hcp1 was growth phase-dependent and the production of Hcp1 reached a maximum in the exponential phase. The expression of Hcp1 was positively and negatively regulated by quorum sensing regulators LuxO and LuxR, respectively. In addition, we observed that Hcp1 expression required the alternative sigma factor RpoN and the enhancer-binding protein VasH, which is encoded in T6SSVA1 gene cluster. Moreover, LuxR, RpoN, and VasH could positively regulate the expression of other T6SS genes. Taken together, we demonstrated that the expression of T6SS in V. alginolyticus was under the regulation of quorum sensing and alternative sigma factor.     

The VgrG Proteins Are “à la Carte” Delivery Systems for Bacterial Type VI Effectors

The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1–3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier.     

The alternative sigma factors,rpoN1 and rpoN2 are required for mycophagous activity of Burkholderia gladioli strain NGJ1

Bacteria utilize RpoN, an alternative sigma factor (σ54) to grow in diverse habitats, including nitrogen-limiting conditions. Here, we report that a rice-associated mycophagous bacterium Burkholderia gladioli strain NGJ1 encodes two paralogues of rpoN viz. rpoN1 and rpoN2. Both of them are upregulated during 24 h of mycophagous interaction with Rhizoctonia solani, a polyphagous fungal pathogen. Disruption of either one of rpoNs renders the mutant NGJ1 bacterium defective in mycophagy, whereas ectopic expression of respective rpoN genes restores mycophagy in the complementing strains. NGJ1 requires rpoN1 and rpoN2 for efficient biocontrol to prevent R. solani to establish disease in rice and tomato. Further, we have identified 17 genes having RpoN regulatory motif in NGJ1, majority of them encode potential type III secretion system (T3SS) effectors, nitrogen assimilation, and cellular transport-related functions. Several of these RpoN regulated genes as well as certain previously reported T3SS apparatus (hrcC and hrcN) and effector (Bg_9562 and endo-β-1,3-glucanase) encoding genes are upregulated in NGJ1 but not in ΔrpoN1 or ΔrpoN2 mutant bacterium, during mycophagous interaction with R. solani. This highlights that RpoN1 and RpoN2 modulate T3SS, nitrogen assimilation as well as cellular transport systems in NGJ1 and thereby promote bacterial mycophagy.     

Subinhibitory Concentration of Kanamycin Induces the Pseudomonas aeruginosa type VI Secretion System

Pseudomonas aeruginosa is a Gram-negative bacterium found in natural environments including plants, soils and warm moist surfaces. This organism is also in the top ten of nosocomial pathogens, and prevalent in cystic fibrosis (CF) lung infections. The ability of P. aeruginosa to colonize a wide variety of environments in a lasting manner is associated with the formation of a resistant biofilm and the capacity to efficiently outcompete other microorganisms. Here we demonstrate that sub-inhibitory concentration of kanamycin not only induces biofilm formation but also induces expression of the type VI secretion genes in the H1-T6SS cluster. The H1-T6SS is known for its role in toxin production and bacterial competition. We show that the antibiotic induction of the H1-T6SS only occurs when a functional Gac/Rsm pathway is present. These observations may contribute to understand how P. aeruginosa responds to antibiotic producing competitors. It also suggests that improper antibiotic therapy may enhance P. aeruginosa colonization, including in the airways of CF patients.     

Translocon-independent intracellular replication by Pseudomonas aeruginosa requires the ADP-ribosylation domain of ExoS

Pseudomonas aeruginosa, a significant cause of human morbidity and mortality, uses a type 3 secretion system (T3SS) to inject effector toxins into host cells. We previously reported that P. aeruginosa uses ADP-ribosyltransferase (ADPr) activity of the T3SS effector ExoS for intracellular replication. T3SS translocon (ΔpopB)-mutants, which can export, but not translocate effectors across host membranes, retained intracellular replication. We hypothesized that secreted effectors mediate translocon-independent intracellular replication. Translocon mutants of PAO1 lacking one or more of its three known effectors (ExoS, ExoT and ExoY) were used. All translocon mutants, irrespective of effectors expressed, localized to intracellular vacuoles. Translocon-effector null mutants and translocon-exoS mutants showed defective intracellular replication. Mutants in exoT, exoY or both replicated as efficiently as translocon mutants expressing all effectors. Complementation of translocon-effector null mutants with native exoS or a membrane localization domain mutant of exoS, but not the ADPr mutant exoS (pUCPexoSE381D), restored intracellular replication, correlating with increased bacteria per vacuole. Thus, P. aeruginosa is capable of intravacuolar replication that requires ExoS ADPr activity, but not the translocon. These data suggest that T3SS effectors can participate in pathogenesis without translocon-mediated translocation across host membranes, and that intracellular bacteria can contribute to P. aeruginosa pathogenesis within epithelial cells.     

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.     

Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae

The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen’s arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7^th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.     

A gacS Deletion in Pseudomonas aeruginosa Cystic Fibrosis Isolate CHA Shapes Its Virulence

Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 3′ end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1-T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor.     

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

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co‐regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone‐like quality of Hcp. Application of this approach to the Hcp secretion island I‐encoded T6SS (H1‐T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (t ype VI s ecretion e xported 4), subsequently shown to act as a potent intra‐specific H1‐T6SS‐delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1‐T6SS effectors, Tse5 and Tse6, which differ from Hcp‐stabilized substrates by the presence of toxin‐associated PAAR‐repeat motifs and genetic linkage to members of the valine‐glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp‐stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1‐T6SS‐exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.     

VE-Cadherin Cleavage by LasB Protease from Pseudomonas aeruginosa Facilitates Type III Secretion System Toxicity in Endothelial Cells

Infection of the vascular system by Pseudomonas aeruginosa (Pa) occurs during bacterial dissemination in the body or in blood-borne infections. Type 3 secretion system (T3SS) toxins from Pa induce a massive retraction when injected into endothelial cells. Here, we addressed the role of type 2 secretion system (T2SS) effectors in this process. Mutants with an inactive T2SS were much less effective than wild-type strains at inducing cell retraction. Furthermore, secretomes from wild-typeswere sufficient to trigger cell-cell junction opening when applied to cells, while T2SS-inactivated mutants had minimal activity. Intoxication was associated with decreased levels of vascular endothelial (VE)-cadherin, a homophilic adhesive protein located at endothelial cell-cell junctions. During the process, the protein was cleaved in the middle of its extracellular domain (positions 335 and 349). VE-cadherin attrition was T3SS-independent but T2SS-dependent. Interestingly, the epithelial (E)-cadherin was unaffected by T2SS effectors, indicating that this mechanism is specific to endothelial cells. We showed that one of the T2SS effectors, the protease LasB, directly affected VE-cadherin proteolysis, hence promoting cell-cell junction disruption. Furthermore, mouse infection with Pa to induce acute pneumonia lead to significant decreases in lung VE-cadherin levels, whereas the decrease was minimal with T2SS-inactivated or LasB-deleted mutant strains. We conclude that the T2SS plays a pivotal role during Pa infection of the vascular system by breaching the endothelial barrier, and propose a model in which the T2SS and the T3SS cooperate to intoxicate endothelial cells.     

The role of type III secretion System 2 in Vibrio parahaemolyticus pathogenicity

Vibrio parahaemolyticus, a Gram-negative marine bacterial pathogen, is emerging as a major cause of food-borne illnesses worldwide due to the consumption of raw seafood leading to diseases including gastroenteritis, wound infection, and septicemia. The bacteria utilize toxins and type III secretion system (T3SS) to trigger virulence. T3SS is a multi-subunit needle-like apparatus used to deliver bacterial proteins, termed effectors, into the host cytoplasm which then target various eukaryotic signaling pathways. V. parahaemolyticus carries two T3SSs in each of its two chromosomes, named T3SS1 and T3SS2, both of which play crucial yet distinct roles during infection: T3SS1 causes cytotoxicity whereas T3SS2 is mainly associated with enterotoxicity. Each T3SS secretes a unique set of effectors that contribute to virulence by acting on different host targets and serving different functions. Emerging studies on T3SS2 of V. parahaemolyticus, reveal its regulation, translocation, discovery, characterization of its effectors, and development of animal models to understand the enterotoxicity. This review on recent findings for T3SS2 of V. parahaemolyticus highlights a novel mechanism of invasion that appears to be conserved by other marine bacteria.     

Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation

Productive intercellular delivery of cargo by secretory systems requires exquisite temporal and spatial choreography. Our laboratory has demonstrated that the haemolysin co-regulated secretion island I (HSI-I)-encoded type VI secretion system (H1-T6SS) of Pseudomonas aeruginosa transfers effector proteins to other bacterial cells. The activity of these effectors requires cell contact-dependent delivery by the secretion apparatus, and thus their export is highly repressed under planktonic growth conditions. Here we define regulatory pathways that orchestrate efficient secretion by this system. We identified a T6S-associated protein, TagF, as a posttranslational repressor of the H1-T6SS. Strains activated by TagF derepression or stimulated through a previously identified threonine phosphorylation pathway (TPP) share the property of secretory ATPase recruitment to the T6S apparatus, yet display different effector output levels and genetic requirements for their export. We also found that these two pathways respond to distinct stimuli; we identified surface growth as a physiological cue that activates the H1-T6SS exclusively through the TPP. Coordination of posttranslational triggering with cell contact-promoting growth conditions provides a mechanism for the T6SS to avoid wasteful release of effectors.