Diversity of Virulence Phenotypes among Type III Secretion Negative Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease.     

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.     

Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P₂. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.     

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.     

Plant Immunity Directly or Indirectly Restricts the Injection of Type III Effectors by the Pseudomonas syringae Type III Secretion System

Plants perceive microorganisms by recognizing microbial molecules known as pathogen-associated molecular patterns (PAMPs) inducing PAMP-triggered immunity (PTI) or by recognizing pathogen effectors inducing effector-triggered immunity (ETI). The hypersensitive response (HR), a programmed cell death response associated with ETI, is known to be inhibited by PTI. Here, we show that PTI-induced HR inhibition is due to direct or indirect restriction of the type III protein secretion system''s (T3SS) ability to inject type III effectors (T3Es). We found that the Pseudomonas syringae T3SS was restricted in its ability to inject a T3E-adenylate cyclase (CyaA) injection reporter into PTI-induced tobacco (Nicotiana tabacum) cells. We confirmed this restriction with a direct injection assay that monitored the in planta processing of the AvrRpt2 T3E. Virulent P. syringae strains were able to overcome a PAMP pretreatment in tobacco or Arabidopsis (Arabidopsis thaliana) and continue to inject a T3E-CyaA reporter into host cells. In contrast, ETI-inducing P. syringae strains were unable to overcome PTI-induced injection restriction. A P. syringae pv tomato DC3000 mutant lacking about one-third of its T3E inventory was less capable of injecting into PTI-induced Arabidopsis plant cells, grew poorly in planta, and did not cause disease symptoms. PTI-induced transgenic Arabidopsis expressing the T3E HopAO1 or HopF2 allowed higher amounts of the T3E-CyaA reporter to be injected into plant cells compared to wild-type plants. Our results show that PTI-induced HR inhibition is due to direct or indirect restriction of T3E injection and that T3Es can relieve this restriction by suppressing PTI.Plants come into contact with a myriad of microorganisms and rely on their innate immune systems to perceive potential microbial infections and induce immune responses. Plant innate immunity can be broadly portrayed as consisting of two major branches, distinguished primarily by their mode of microbe detection. The first branch is activated by extracellular pattern recognition receptors (Boller and Felix, 2009; Nicaise et al., 2009) that perceive broadly conserved molecules called pathogen (microbe)-associated molecular patterns (PAMPs; Medzhitov and Janeway, 1997; Ausubel, 2005). The response induced by this recognition is termed PAMP-triggered immunity (PTI; Jones and Dangl, 2006). A well-characterized example of PTI in plants is the recognition of and subsequent immune response to a small N-terminal region of bacterial flagellin by the FLS2 receptor kinase of Arabidopsis (Arabidopsis thaliana; Felix et al., 1999; Zipfel et al., 2004). Plant resistance (R) proteins activate the second branch of the plant innate immune system by recognizing specific pathogen effector proteins. The response induced by this recognition is termed effector-triggered immunity (ETI; Jones and Dangl, 2006). ETI and PTI induce similar innate immune responses, including ion fluxes, reactive oxygen species (ROS), and callose (β-1,3-glucan) deposition in the cell wall (Tsuda et al., 2008; Boller and Felix, 2009); however, ETI generally also includes the induction of a programmed cell death called the hypersensitive response (HR; Heath, 2000).The induction of ETI in response to a bacterial plant pathogen is generally due to the recognition of bacterial type III effector (T3E) proteins injected into the plant cell by the pathogen''s type III protein secretion system (T3SS; Alfano and Collmer, 1997; Buttner and He, 2009). These recognized T3Es were classically known as avirulence (Avr) proteins because they induced ETI responses sufficient to prevent a normally virulent pathogen from causing disease, thereby rendering it avirulent (Leach and White, 1996). However, it has become increasingly apparent that many T3Es benefit their bacteria by suppressing PTI and ETI (Block et al., 2008; Cui et al., 2009; Guo et al., 2009). Under the current model, plants first developed PTI to reduce microbial colonization of the apoplast. Successful bacterial pathogens countered this by acquiring a T3SS and PTI-suppressing T3Es (Espinosa and Alfano, 2004; Chisholm et al., 2006; Jones and Dangl, 2006).The bacterial pathogen Pseudomonas syringae infects the aerial parts of many plant species. It displays host specificity, and its strains have been separated into more than 50 pathovars based on the host plants that they infect. For example, P. syringae pv tabaci is virulent in tobacco (Nicotiana tabacum), but it triggers nonhost resistance in Arabidopsis, a plant-microbe interaction referred to as a nonhost interaction. Nonhost resistance describes the resistance observed when all members of a plant species are resistant to a specific pathogen (Thordal-Christensen, 2003; Mysore and Ryu, 2004). While not well understood, both PTI (Li et al., 2005) and ETI (Nissan et al., 2006; Wei et al., 2007) have been shown to play a role in nonhost resistance to bacterial pathogens. In some cases, P. syringae strains display race cultivar resistance. This is generally due to the resistant cultivar possessing an R protein that can recognize a T3E from the pathogen inducing ETI (Bent and Mackey, 2007). One well-studied P. syringae strain is P. syringae pv tomato DC3000, which causes bacterial speck disease on specific tomato (Solanum lycopersicum) cultivars and disease on all ecotypes of Arabidopsis tested. These interactions have been classically referred to as compatible interactions. However, DC3000 triggers nonhost resistance in tobacco and many other plants.DC3000 contains more than 30 T3Es (Lindeberg et al., 2006; Cui et al., 2009; Cunnac et al., 2009). These are encoded by genes contained within the Hrp pathogenicity island, which also encodes the T3SS apparatus (Alfano et al., 2000), other pathogenicity islands, or as individual genes throughout the genome of DC3000 (Buell et al., 2003; Wei et al., 2007). One molecular tool that has been useful in studying the effect individual T3Es have on plants is the cosmid pHIR11 (Huang et al., 1988). This cosmid encodes a functional T3SS from P. syringae pv syringae 61 and the T3E HopA1. It confers upon nonpathogenic bacteria, such as Pseudomonas fluorescens, the ability to inject HopA1 into plant cells. In tobacco and other plants, injected HopA1 induces ETI, including an HR (Huang et al., 1988; Alfano et al., 1997). The expression of other T3Es in P. fluorescens(pHIR11) enabled them to be screened for the ability to suppress HopA1-induced ETI (Jamir et al., 2004; Guo et al., 2009). Bacterial strains carrying the pHIR11 derivatives pLN18 or pLN1965, both of which lack hopA1 and so no longer induce ETI, were used to determine which T3Es could suppress PTI (Oh and Collmer, 2005; Guo et al., 2009). Collectively, these experiments demonstrated that many P. syringae T3Es possessed the ability to suppress both ETI and PTI.One PTI suppression assay using P. fluorescens(pLN18) employed by Oh and Collmer (2005) took advantage of earlier observations indicating that PTI could inhibit the ability of the plant to mount an HR in response to an ETI-inducing bacterial strain (Newman et al., 2000; Klement et al., 2003). In this assay, the PTI inducers P. fluorescens(pLN18) or a 22-amino-acid peptide from flagellin (flg22) are infiltrated into Nicotiana benthamiana. Six hours later, the ETI inducer DC3000 is infiltrated in a region of the leaf that overlaps with the earlier infiltration. The HR is typically inhibited in the overlapping region that was pretreated with a PTI inducer. Several T3Es suppressed this inhibition when they were separately delivered at time of pretreatment (Oh and Collmer, 2005). It has been speculated that the probable mechanisms for inhibition of the HR caused by PTI include impairment of delivery of T3Es that induce the HR, modification of the events downstream of T3E recognition, or a shutdown of programmed cell death (Newman et al., 2000).Here, we show that PTI inhibits the HR on tobacco because it directly or indirectly restricts the ability of P. fluorescens(pLN1965) or DC3000 to inject T3Es based on injection (translocation) assays using T3E-adenylate cyclase (CyaA) fusions. This was confirmed using an independent injection assay that monitored the amount of the cleaved in planta form of the T3E AvrRpt2. Interestingly, this injection restriction was greatly reduced in the compatible interactions between DC3000 and Arabidopsis or between P. syringae pv tabaci 11258 and tobacco. A DC3000 mutant lacking four clusters of T3E genes, which corresponds to 11 T3Es, was less able to inject a T3E-CyaA fusion into PTI-induced Arabidopsis, suggesting that the PTI suppressing activities of the T3E inventory of DC3000 allow it to overcome the injection restriction. Transgenic Arabidopsis plants separately expressing specific T3Es known to be capable of PTI suppression increased the ability of P. fluorescens(pLN1965) to inject a T3E-CyaA fusion into PTI-induced plant cells. Collectively, these data suggest that PTI can directly or indirectly restrict type III injection and PTI suppression by T3Es can relieve this restriction in susceptible plant cells but not plant cells undergoing ETI.