Targeting mechanisms of Pseudomonas aeruginosa pathogenesis

Médecine et maladies infectieuses 36 (2006) 78–91 http://france.elsevier.com/direct/MEDMAL/ General review Targeting mechanisms of Pseudomonas aeruginosa pathogenesis Thérapeutiques ciblant les mécanismes pathogéniques de Pseudomonas aeruginosa E. Kipnis *, T. Sawa, J. Wiener-Kronish Department of Anesthesia and Perioperative Care, University of California San Francisco, 513 Parnassus Avenue, Room s-261, Medical Science Building, Box 0542, San Francisco, CA 94143, USA Received and accepted 18 October 2005 Available online 19 January 2006 Abstract Pseudomonas aeruginosa is an opportunistic pathogen responsible for ventilator-acquired pneumonia, acute lower respiratory tract infections in immunocompromised patients and chronic respiratory infections in cystic fibrosis patients. High incidence, infection severity and increasing resistance characterize P. aeruginosa infections, highlighting the need for new therapeutic options. One such option is to target the many pathogenic mechanisms conferred to P. aeruginosa by its large genome encoding many different virulence factors. This article reviews the pathogenic mechanisms and potential therapies targeting these mechanisms in P. aeruginosa respiratory infections. © 2006 Elsevier SAS. All rights reserved. Résumé Pseudomonas aeruginosa est un pathogène opportuniste responsable de pneumonies nosocomiales, infections des voies respiratoires basses chez les patients immunodéprimés et infections respiratoires chroniques lors de la mucoviscidose. L’incidence élevée, la sévérité et la résistance croissante sont caractéristiques des infections à P. aeruginosa et soulignent le besoin d’ouvrir de nouvelles voies thérapeutiques. Une voie thérapeutique possible est de cibler un des nombreux mécanismes pathogéniques dont est doté P. aeruginosa grâce à son génome de grande taille codant pour de nombreux facteurs de virulence. Nous exposons les différents mécanismes pathogéniques au cours des infections respiratoires à P. aeruginosa ainsi que les voies thérapeutiques potentielles qui les ciblent. © 2006 Elsevier SAS. All rights reserved. Keywords: Pseudomonas aeruginosa; Pathogenicity; Virulence factors Mots clés : Pseudomonas aeruginosa ; Pathogénicité ; Facteurs de virulence 1. Introduction tance of P. aeruginosa to conventional antimicrobial treatment has increased over the past decade [5,6]. Pseudomonas aeruginosa is the most common pathogen responsible for both acute respiratory infections in ventilated or immunocompromised patients and chronic respiratory infections in cystic fibrosis patients [1,2]. P. aeruginosa is also responsible for excessive mortality in VAP [1,3,4]. Adding to the problems of high incidence and infection severity, the resis- These three problems of high incidence, severity and resistance persist even though they are now broadly recognized and various strategies have been proposed addressing them [1,7–12]. * Corresponding author. Tel.: +1 415 476 1653. E-mail address: ekipnis@gmail.com (E. Kipnis). 0399-077X/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.medmal.2005.10.007 Therefore, it is crucial that new therapeutic options for P. aeruginosa infections be explored. Such new options may come from specifically targeting the pathogenic mechanisms of P. aeruginosa. Indeed, P. aeruginosa is a remarkable pathogen in that it is endowed with a uniquely large genome containing genes for many different virulence factors and regulatory mechanisms allowing it to adapt to hostile environments. E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 79 This article reviews the pathogenic mechanisms and potential therapies targeting these mechanisms in P. aeruginosa respiratory infections. 3. Bacterial cell surface virulence factors (Fig. 1) 2. Schematic overview of P. aeruginosa pathogenesis in respiratory infections Flagella are complex proteic structures forming a filamentous polar appendage at the surface of P. aeruginosa. Flagella are the main motile appendage of gram negative bacteria and allow the swimming movement of P. aeruginosa through a propeller or screw-like motion. Flagella have a critical role in pathogenesis by tethering and adhering to epithelial cells through binding with a common membrane component, asialoGM1 [13]. They also participate in virulence and elicit an NFkB dependent inflammatory response through interactions with Toll-receptors TLR5 and TLR2 and a calcium entry dependent activation of the extracellular regulated kinase pathway leading to IL-8 production [14,15]. However, flagella are also very immunogenic, rendering their presence a liability for P. aeruginosa after successful colonization. This is why P. aeruginosa is capable of adapting by selecting aflagellar mutants to evade host response in chronic infections [16]. It is therefore not surprising that flagella have been considered interesting targets for immunotherapy. P. aeruginosa pneumonia was attenuated in rats receiving human antiflagellar monoclonal antibodies [17]. Similar findings led to the development of a P. aeruginosa flagella vaccine for cystic fibrosis patients that successfully completed phase I and II trials and is undergoing phase III evaluation [18,19]. P. aeruginosa is an opportunistic pathogen that, after being acquired from the environment, colonizes the respiratory epithelium in patients with predisposing conditions such as cystic fibrosis, mechanical ventilation, immunodeficiency or preexisting respiratory disease. Flagella and pili, the motile surface appendages of P. aeruginosa are responsible for bacterial motility and progression towards epithelial contact. These appendages also act as initial tethers in facilitating bacteria to epithelial cell contact by binding to the epithelial surface glycolipid asialo-GM1. Additionally, lipopolysaccharide (LPS) also plays a similar role in bacterial adhesion through asialoGM1 binding. These appendages then play a major role in the irreversible adhesion to epithelial cells, which is the initial critical step in colonization of the respiratory epithelium. Upon cell contact, the type III secretion system, a major virulence determinant, is activated. The type III secretion system allows P. aeruginosa to inject secreted toxins through a syringe-like apparatus directly into the eukaryotic cytoplasm. Four effector proteins are known: ExoY, ExoS, ExoT, and ExoU and all participate, at varying levels, in the cytotoxicity of P. aeruginosa leading to invasion and dissemination of P. aeruginosa. Other virulence factors secreted via type II secretion system into the extracellular space such as elastase, alkaline phosphatase, exotoxin A, and phospholipase C also participate in invasion by destroying the protective glycocalix of the respiratory epithelium and exposing epithelial ligands to P. aeruginosa. These secretins also participate in cytotoxicity. A similar role also exists for pyoverdine and pyocyanin. In acute infections, invasion, dissemination and extensive tissue damage predominate. However, in chronic infections, particularly in cystic fibrosis patients, P. aeruginosa may also adapt, by losing its most immunogenic features such as pili and flagella to avoid clearance, and by isolating itself from host defenses and adhering to the respiratory epithelium by forming biofilms. In chronic infections, a persistent inflammatory state is maintained by extracellular secreted virulence factors. Whether in acute or chronic infections, P. aeruginosa possesses a multiplicity of regulating systems allowing it to adapt to its environment and notably to host defenses. Among these systems, quorum-sensing (QS) showcases P. aeruginosa adaptability. Quorum sensing systems are complex bacterial cell-tocell signaling systems that allow the bacteria to sense their own cell density and to communicate with each other resulting in coordinated production of virulence factors depending on bacterial density. QS has been shown to be critical to maintaining airway inflammation through virulence factor production and to the formation of biofilm in chronic infections. This schematic overview shows that there are many levels in the pathogenesis of P. aeruginosa infections that may represent therapeutic targets, which we will now describe in detail. 3.1. Flagella 3.2. Pili Pili or fimbriae are smaller filamentous surface appendages of P. aeruginosa. Multiple pili are usually present on the surface. P. aeruginosa pili are among the rare prokaryotic pili involved in bacterial motility. This motility, called twitching is due to the retractile properties of P. aeruginosa pili and allows P. aeruginosa to “spread” along hydrated surfaces rather than “swim” [20]. This feature facilitates the rapid colonization of the airway [20]. Like flagella, pili are crucial to the adhesion phase of colonization through binding to asialoGM1 of the epithelial cell membrane [21,22]. Furthermore, studies have shown that both pili-mediated adherence and twitching motility are critical to P. aeruginosa virulence [23,24]. In an infant mouse model of lung infection, piliated strains of P. aeruginosa caused more severe and diffuse pneumonia than corresponding non-piliated mutants [25]. Therefore, pili, like flagella seem to be legitimate targets in developing antipseudomonal immunotherapy [26–28]. Among such strategies, a chimeric vaccine incorporating both pilin and non-toxic modified exotoxin A successfully reduced bacterial adherence [29]. However, there are issues linked to the lack of cross reactivity of antigens targeting pili across different P. aeruginosa strains that need to be resolved [26,28]. 3.3. LPS Although the inner face of the outer membrane resembles a typical phospholipid bilayer, the outer face of the outer mem- 80 E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 Fig. 1. Overview of P. aeruginosa cell-surface virulence factors. Fig. 1. Schéma des facteurs de virulence de surface cellulaire de P. aeruginosa. After reaching the respiratory epithelium through flagellum-dependent “swimming” and spreading along the epithelium through pili-dependent “twitching” P. aeruginosa is tethered to respiratory epithelial cells through binding of flagellum and pili to asialoGM1. Further adherence is conferred by interactions between LPS and asialoGM1, CD14 and/or CFTR. Flagellar interaction with TLR5 activates a Calcium entry-dependent MAP-kinase pathway leading to increased IL-8 transcription. Both flagellar interaction with TLR4 and LPS interaction with TLR4/MD2 activate MyD88-dependent NFκB pathways leading to increased IL-8 transcription. In cystic fibrosis patients, alginate increases adhesion and through an unknown mechanism depresses host response. LPS: lipopolysaccharide, asialoGM1: asialoganglioside gangliotetraosylceramide, CD14: cluster of differentiation receptor 14, CFTR: cystic fibrosis transmembrane conductance regulator, TLR: toll-like receptor, MAP-kinase: mitogen-activated protein kinase, IL-8: interleukin 8, PKC: protein kinase C, c-raf: src/ras-dependent kinase, MEK1/2: dual specificity MAP kinase/extracellular signal-regulated kinase kinase, ERK 1/2: dual specificity extracellular signal-regulated kinase, PKR: protein kinase regulated by RNA, IKK: I-kappa-B kinase, NFκB: nuclear factor kappa B, MyD88: myeloid differentiation primary response 88, TRAF6: tumor necrosis factor receptor-associated factor 6, IRAK: IL-1 receptor-associated kinase. brane is mainly composed of LPS. LPS associates a hydrophobic domain, Lipid A inserted into the phospholipid bilayer to a hydrophilic tail composed of the core polysaccharide and the O-specific polysaccharide. The variable O-specific polysaccharide chains are the basis of antigenic identification of P. aeruginosa serotypes. This immunogenicity makes them obvious targets for immunotherapy. However, the active immunization elicited by O-antigen based vaccines is lacking in protectiveness even when multiple Oantigens from different serotypes are conjugated [30–32]. Thus, various O-antigen based vaccines have been tested over decades with limited success [32]. To circumvent this problem several strategies have been developed. Multiple serotype conjugates can be further conjugated with another target such as exotoxin A [30]. Such a vaccine has successfully completed phase I and II trials and is currently undergoing a phase III trial [30,33]. Equally promising, live attenuated vaccines from deletional mutants of P. aeruginosa or Salmonella enteritica expressing P. aeruginosa O-antigens have been developed and confer protection in animal models [34–36]. E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 Passive immunization therapies against O-specific polysaccharides have been explored for many years [37]. The main obstacle of administering monoclonal antibodies mainly obtained from mice to humans has been recently overcome with the advent of transgenic mice producing human monoclonal antibodies protective against P. aeruginosa [38,39]. Similarly, human hybridoma derived monoclonal antibodies are also under development against P. aeruginosa [30]. Other strategies have consisted in the use of synthetic antimicrobial peptides known to inhibit LPS such as CAP-18. Although CAP-18 was a potent in vitro antipseudomonal, in vivo administration did not improve survival [40]. LPS is a substance biologically active to the extent that it is used in many models of sepsis or acute lung injury even though it has recently been shown that its potency in mimicking acute lung injury depends mostly on preexisting damage [41] and on structure. Indeed, the Lipid A component of P. aeruginosa LPS activates multiple pro-inflammatory pathways [42–44]. LPS is also critical to virulence [45], mainly by its role in adhesion through asialoGM1 binding [22], TLR4/CD14 or TLR4/MD-2 recognition [43,44] or binding to CFTR [46]. In cystic fibrosis patients, P. aeruginosa adapts by selecting mutants with specific Lipid A modifications that allow resistance to host antimicrobial peptides and increase TLR4 activation [44,47,48]. Some immunotherapeutic strategies target Lipid-A, although their activity is also based on O-antigen reactivity [49]. However, new strategies specifically target Lipid-A because of its role in pathogenesis and not for recognition by immune-based methods. These strategies aim to inhibit the first or second step in synthesis of Lipid-A [50,51]. However, these inhibitors have not been tested in P. aeruginosa infections. 3.4. Alginate Alginate is a mucoid exopolysaccharide produced by P. aeruginosa that is made up of repeating polymers of mannuronic and glucuronic acid. Alginate, like LPS, functions as an adhesin, anchoring P. aeruginosa to the colonized respiratory epithelium. Surrounding conditions in cystic fibrosis patient’s lungs and host inflammatory response increase alginate synthesis leading to conversion to an alginate overproducing mucoid phenotype [52,53]. This mucoid alginate-producing phenotype is commonly found in cystic fibrosis airways over the course of P. aeruginosa infections. Overexpressed alginate protects P. aeruginosa from phagocytosis, antibiotics and even attenuates the host response [54,55]. Although alginate has been widely considered to participate in the architecture of P. aeruginosa biofilm in cystic fibrosis [56] and probably does have a role in some of the properties of biofilm [54], it has been recently shown that alginate is not crucial to biofilm development [57]. However, because of this excessive production in cystic fibrosis, alginate has been a recent target in immunotherapy [58, 59]. Human monoclonal antibodies directed against alginate were effective in a murine model of lethal pneumonia and sur- 81 prisingly even against non-mucoid strains producing undetectable levels of alginate [59]. However, active immunization against alginate is in the process of being optimized, particularly through conjugation with exoA [58], in order to compensate for the variable efficacy of antibodies produced in response to alginate [60]. 4. Secreted virulence factors (Fig. 2) 4.1. Pyocyanin Pyocyanin is a blue pigment metabolite of P. aeruginosa that has been shown to have numerous pathogenic effects such as increasing IL-8 [61,62], depressing host-response [61,63], and inducing apoptosis in neutrophils [63]. In animal models of acute and chronic lung infection, pyocyanin was shown to be essential to P. aeruginosa virulence [64]. The same study also showed that pyocyanin instillation caused an influx of neutrophils into the lung. Additionally, due to it’s known oxidoreductive properties, pyocyanin oxidizes glutathione and inactivates catalase in respiratory epithelial cells thus participating in oxidative-stress related damage [65,66]. Furthermore, in yeast cells, pyocyanin disrupts vacuolar ATPase and mitochondrial electron transport that may impair CFTR chloride channels in cystic fibrosis [67]. Although no therapeutic strategies directly target pyocyanin, several antioxidant therapies have proved useful in cystic fibrosis [68–70]. Glutathione delivery through aerosolization improved FEV in cystic fibrosis patients [68,70]. Lung function was improved in cystic fibrosis patients after an 8-week antioxidant supplemented regimen [68]. Recently, authors have suggested inhibiting the synthesis of pyocyanin as a therapeutic strategy, although such studies have yet to be conducted [67,71]. 4.2. Pyoverdine Pyoverdine is a siderophore, a small molecule chelating iron from the environment for use in P. aeruginosa metabolism. Pyoverdine has also been shown to play a role in P. aeruginosa virulence [72,73]. One explanation for this role has recently emerged when it was found that pyoverdine regulates the secretion of other P. aeruginosa virulence factors, exotoxin A and an endoprotease and its own secretion [74]. Although there are yet no therapeutic strategies aimed at pyoverdine, this virulence regulating function that doubles as a cell-to-cell signaling mechanism makes it an attractive target. 4.3. Alkaline protease Alkaline protease is a fibrin lysing protease secreted by P. aeruginosa through a type I secretion system [75]. Although its pathogenic role is only clear in corneal infections as is the case for most P. aeruginosa proteases [75], it may participate in the pathogenesis of acute lung injury. Indeed, it has been shown that there is an early massive intra-alveolar formation of fibrin in 82 E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 Fig. 2. Overview of secreted P. aeruginosa virulence factors. Fig. 2. Schéma des facteurs sécrétoires de virulence de P. aeruginosa. Pyocyanin, an oxidative reactive pigment increases reactive oxygen species (ROS) within host cells and impairs host cell antioxydant capacities by decreasing glutathione stock catalase activity. Pyocyanin also increases production of IL-8. Type I secretion system secreted alkaline phosphatase lyses the fibrin matrix limiting acute lung injury. Elastase disrupts epithelial tight junctions, cleaves surfactant proteins A and D, inactivates proteinase-activated receptor 2 and increases production of IL-8. Phospholipase C lyses mebrane phospholipids and inactivates surfactant. Exotoxin A (ExoA) inhibits elongation factor 2 (EF2) leading to transcriptional arrest. Pyoverdin increases the secretion of ExoA and itself. P. aeruginosa acute lung injury and that inhibition of this initial fibrinoformation is deleterious in an animal model [76]. Furthermore, the efficacy of passive and active immunization therapies targeting alkaline protease in a murine model of sepsis shows that alkaline protease may have a larger role in P. aeruginosa pathogenesis and is thus a legitimate target [77–79]. P. aeruginosa keratitis [80], it has only recently been established that protease IV is also involved in the pathogenesis of lung infection through degradation of surfactant proteins A, D and B [81]. This may indicate that protease IV could be a new therapeutic target. 4.5. Elastase 4.4. Protease IV Other proteases secreted by P. aeruginosa such as protease IV also have a role in pathogenesis. Although, protease IV is particularly known to participate in the pathogenesis of Elastase, or lasB, is a metalloproteinase secreted by P. aeruginosa into the extracellular space through a type II secretion system. Elastase has been shown to have a role in the pathogenesis of P. aeruginosa respiratory infections by ruptur- E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 ing the respiratory epithelium through tight-junction destruction, thus increasing epithelial permeability and facilitating neutrophil recruitment [82–84]. Elastase is also pro-inflammatory, increasing IL-8 levels in a rat air pouch inflammation model [85]. P. aeruginosa elastase can also decrease host immune response through cleavage of respiratory tract surfactant proteins A and D and Proteinase-activated receptor 2 into inactive forms [86–88]. Elastase is a candidate for immune based targeting of P. aeruginosa as attested by the efficacy of immunization with synthetic epitopes of elastase in decreasing the severity of infection in rats [89]. Likewise, monoclonal antibodies directed against P. aeruginosa elastase decreased epithelial permeability in a monolayer of polarized respiratory epithelial cells [90]. The inhibition of elastase secreted by P. aeruginosa with protease inhibitors could be a potential therapeutic option. However, current protease inhibitor therapeutic strategies are aimed at the more overwhelming secretion of elastase by inflammatory neutrophils and have been shown to be effective in rat models of chronic respiratory infection [91,92]. 4.6. Phospholipase C Phospholipase C, more specifically hemolytic phospholipase C, is a phospholipase secreted by P. aeruginosa into the extracellular space through a type II secretion system. Hemolytic phospholipase C targets eukaryotic membrane phospholipids and has been shown to participate in the pathogenesis of P. aeruginosa acute lung injury [93] and in inflammation [94, 95]. Like elastase, part of the pathogenic effect of hemolytic phospholipase C may be due to surfactant inactivation [96]. Furthermore, hemolytic phospholipase can suppress the host neutrophil oxidative burst response [97]. However, no therapeutic strategy to date specifically targets phospholipase C mediated pathogenicity although it is interesting to note that various antipseudomonal antibiotics can decrease the secretion of phospholipase C in a clinical isolate of P. aeruginosa [98]. 4.7. Exotoxin A Exotoxin A (ExoA), secreted into the extracellular space through a type II secretion system is an ADP-ribosyl transferase [99] inhibiting elongation factor-2 (EF-2) thereby inhibiting protein synthesis and leading to cell death [100]. ExoA has also been shown to depress host response to infection [101, 102]. Among extracellular toxins, ExoA has a major role in P. aeruginosa virulence [103] although to a lesser extent than type III secretion system effectors such as ExoS [104]. Indeed, an ExoA deficient mutant was 20 times less virulent than the wild strain in mice [103]. Passive immunization targeting ExoA increased survival in a murine model of P. aeruginosa infection [105] and in a murine model of gut-derived sepsis [77]. Because of these immunogenetic properties, non-toxic altered ExoA has been used particularly successfully as a target for multivalent immu- 83 notherapy in chimeric vaccine with outer membrane proteins I and F [106] or pilin protein [29]. An alternative to using altered ExoA to avoid toxicity has been immunization with ExoA-coding DNA, successfully tested in animals [107,108]. 5. Type III secretion system (Fig. 3) Type III secretion systems are shared among Yersinia, Salmonella, Shigella and Pseudomonas species as a mechanism to directly inject toxins into the host cells. The type III secretion system (TTSS) of P. aeruginosa is a complex pilus-like structure allowing the translocation of effector proteins from the bacteria, across the bacterial membranes and into the eukaryotic cytoplasm through a needle-like appendage forming a pore in the eukaryotic membrane [109]. There are four known toxins, variably expressed in different strains and isolates, injected into host cells by P. aeruginosa through the TTSS: ExoY, ExoS, ExoT and ExoU. 5.1. ExoS ExoS is a bifunctional cytotoxin with two active domains, a C-terminal ADP-ribosyltranferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain. The ADP-ribosyltransferase activity necessitates a eukaryotic cell cofactor: 14-3-3 protein [110]. The pathogenic role of ExoS is mainly attributable to the ADP-ribosyltranferase activity leading to disruption of normal cytoskeletal organization [111,112], although GAP activity also plays a similar role [111,113]. Additionally, it has recently been shown that the C-terminal domain binds to TLR2 and the N-terminal domain binds to TLR4, showing that ExoS may also modulate the host immune and inflammatory response [114]. Although it is generally recognized that ExoS is less cytotoxic than ExoU [111,115], their secretion is mutually exclusive in wild strains of P. aeruginosa, implying that ExoS is still a major cytotoxin in certain strains [115,116]. Furthermore, the relative risk of mortality increased to 8.7 in patients who were infected with a strain expressing a functional TTSS and secreting either ExoS or ExoU [117] underlining the importance of this toxin in the pathogenesis of P. aeruginosa infection. However, there are no therapeutic strategies that target the toxin in itself. 5.2. ExoT ExoT is similar to ExoS, with dual ADP-ribosyltranferase and GAP activities, although the ExoT ADP-ribosyltranferase, initially thought to be deficient compared to that of ExoS, targets different pathways [118–122]. Although, ExoT has similar effects on the eukaryotic cytoskeleton as ExoS, and has been shown to inhibit both Pseudomonas internalization and wound repair [123,124], it is considered only a minor cytotoxin [111]. Surprisingly, ExoT was even found to diminish the cytotoxicity due to ExoU in a specific strain [115]. Therefore, unless this protective effect yields a specific therapeutic pathway, 84 E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 Fig. 3. Overview of P. aeruginosa type three secretion system. Fig. 3. Schéma du système sécrétoire de type III de P. aeruginosa. The type 3 secretion system is constituted of a secretion apparatus(PscC proteins) and a translocator or “needle” (PopD, PopB, PcrV proteins) allowing the “injection” of Exotoxins (ExoY, ExoS, ExoT and ExoU) into the host cell cytoplasm. ExoY is an adenylate cyclase that causes an increase in cytoplasmic camp leading to increased epithelial barrier permeability. Exo S and Exo T both have N-terminal GTPase activating protein (GAP) domains that inactivate actin polymerization disrupting the cytoskeleton. They also have C-terminal ADP-ribosyltransferase (ADPRT) activities on different substrates. ExoS ADPRT, in the presence of 14-3-3 protein, inhibits the interaction of Ezrin-Radixin-Moesin (E, R and M) and cytoskeletal proteins. ExoT ADPRT inhibits the Rac/integrin cytoskeletal wound healing pathway. ExoU, a phospholipase/ lysophospholipase destroys epithelial membranes in the presence of an unknown host cell cofactor. ExoT in itself does not seem to be an interesting candidate in targeting P. aeruginosa pathogenesis. [115]. Therefore, ExoY could be a potential, albeit minor, therapeutic target. 5.3. ExoY 5.4. ExoU ExoY is an adenylate cyclase [125] injected directly into the host cytosol by the TTSS and increases cytosolic cAMP, enhanced by a eukaryotic cofactor. This increased cytosolic cAMP leads to increased pulmonary microvascular intercellular gap formation and increased lung permeability [126]. However, this role of ExoY in overall P. aeruginosa pathogenesis may need to be explored further as most in vitro and in vivo models of cytotoxicity only show a minor effect of ExoY ExoU was recently found to have a phospholipase/lysophospholipase activity disrupting eukaryotic cell membranes after translocation into the cell by the TTSS and activation by a yet unknown eukaryotic cofactor [127–129]. ExoU is widely recognized as being the major cytotoxin secreted via the TTSS, even though a recent study has shown that the cumulative ExoU-independent/TTSS-mediated pathogenicity is far from minor [130]. ExoU is 100 times more cytotoxic than ExoS E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 [115] and ExoU secretion alone by the TTSS confers cytotoxicity in animal models of lung injury leading to sepsis [111,131, 132]. Furthermore, ExoU is responsible for decompartmentalization of the inflammatory response in a model of acute lung injury leading to sepsis [133]. In patients infected with a strain expressing ExoU the relative risk of mortality increased to 2.3 [117]. In P. aeruginosa-induced ventilator associated pneumonia, ExoU was secreted by isolates from more severely ill patients [134]. Because ExoU plays such a major role in the pathogenesis of P. aeruginosa infection, it represents a very attractive therapeutic target. Indeed, anti-ExoU immunotherapy has been explored but deemed disappointing. Immunization of mice with recombinant ExoU only conferred 80% survival against PA103 intratracheal challenge compared to 100% survival with recombinant PcrV [135]. Likewise, passive immunization with Anti-ExoU IgG only allowed 50% survival. Although we are just beginning to understand the mechanism of ExoU cytotoxicity, phospholipase A2 inhibitors have already successfully been used in vitro [136]. Additionally, elucidating the mechanism of activation of ExoU by unknown eukaryotic cofactors could present new therapeutic options. As we have seen, the TTSS is involved in pathogenesis through the translocation of multiple toxins into host cells, however, it also appears that it may participate in P. aeruginosa virulence in itself. Indeed, mutants expressing the TTSS but not the toxins are cytotoxic [115]. This cytotoxicity imputable to the TTSS apparatus itself may explain, in part, why ExoU-independent pathogenicity as a whole is quite major [130] although ExoS is considered moderately cytotoxic and ExoT and ExoY are considered minor cytotoxins. Therefore, the TTSS is a major cancdidate for pathogenesis-targeting therapeutics both because it translocates cytotoxins into the eukaryotic cell and because the apparatus itself may play a part in virulence. Several proteins composing this system have been studied, among which PcrV is a probable structural protein of the translocation structure, or “needle”, of the TTSS. PcrV plays a role in the assembly of the translocation pore in the eukaryotic cell membrane [137]. Early studies showed 100% survival in mice receiving either active or passive anti-PcrV immunotherapy before an intratracheal instillation of PA103 [135]. Subsequent studies have continued to prove the efficacy of anti-PcrV immunotherapy in animal models of P. aeruginosa infection and monoclonal antibodies have been successfully generated [138– 141]. 85 sion in an entire bacterial population. This coordinated gene expression concerns survival genes and more importantly, genes coding for virulence factors and biofilm formation. The discovery of these systems has created an entire field of research and several in-depth reviews are available [142,143]. For our purposes, it is sufficient to know that there are two such QS systems in P. aeruginosa, las and rhl that interact in a hierarchical manner, the first activating the second and both following similar overall pathways (Fig. 4). Briefly, a gene encoding an autoinducer synthase (“I” genes, lasI or rhlI) is activated, the synthesized autoinducer (Oxododecanyl or oxohexanoyl-homoserine lactone) diffuses into the environment, reaches a threshold concentration allowing it to bind to transcriptional activators forming a complex that activates, among others, genes coding virulence factors such as elastase, ExoA, type II secretion system apparatus proteins, alkaline protease, alginate, pyocyanin and pyoverdine. This ability of P. aeruginosa to coordinate the upregulation of virulence genes in a whole population translates into increased pathogenicity of QS capable strains compared to QS deficient mutants throughout a variety of animal models [45, 144–146]. Furthermore, several studies have shown that the autoinducer N-(3-oxododecanoyl)-L-homoserine-lactone is immunomodulatory and can depress host responses [147–150]. These roles of QS in virulence and immunomodulation make it a highly interesting candidate for pathogenesis targeting therapeutics to the extent that potential strategies involving each 6. Quorum-sensing (Fig. 4) QS is a mechanism shared by Gram-negative bacteria that allows bacteria-to-bacteria cell signaling through small molecules, acyl homoserine lactones (AHL), also known as autoinducers that diffuse freely across bacterial membranes. When a certain bacterial density or “quorum” is obtained, these molecules reach a threshold concentration at which, as cofactors of transcriptional regulators, they allow coordinated gene expres- Fig. 4. Overview of P. aeruginosa QS. Fig. 4. Schéma du QS de P. aeruginosa. “I” genes (lasI or rhlI), encoding an autoinducer synthase are activated, the synthesized autoinducer (acy-homoserine lactone, AHL) diffuses into the environment. An increase in bacterial density during infection leads to an increase in autoinducer concentration past a threshold allowing it to bind to transcriptional activators (LasR, RhlR) forming a complex that activates genes coding virulence factors. 86 E. Kipnis et al. / Médecine et maladies infectieuses 36 (2006) 78–91 step of QS have been mapped out in an in-depth review [151]. Among such strategies, several have been explored experimentally. QS can be successfully inhibited by competitive antagonist analogs of acyl-homoserine-lactones (AHLs) leading to decreased elastase production and biofilm formation [152]. The signaling AHLs are degraded by AHL-lactonases. Plants naturally expressing these lactonases have increased resistance to QS capable pathogens [153]. Insertion of an AHL-lactonase gene into PAO1 led to decreased production of virulence factors [154]. In this search for QS inhibitors, following the discovery that antibacterial furanones produced by marine macroalga were capable of inhibiting QS, synthetic furanones were developed [155]. Synthetic furanones successfully inhibit QS and reduce virulence factor expression, increase bacterial clearance and survival in murine models of P. aeruginosa lung infection [155–157]. Pursuing the quest for QS inhibitors has led to the development of screening strategies that have successfully shown QS inhibiting properties of novel compounds from garlic extracts [158]. Closer to clinical practice, it has been found that azithromycin inhibits QS, decreasing virulence factor expression and biofilm production [159,160]. This effect may account, in part, for the antipseudomonal effects of subinhibitory concentrations of macrolides [161,162]. 7. Genetics The source of the pathogenic versatility of P. aeruginosa is undoubtedly it’s unique genome. Sequencing of this genome was achieved in 2000 and revealed a uniquely large genome of 6.3 million base pairs in 5570 predicted genes(PAO1 strain). Among these, 8.4% are involved in gene regulation, making P. aeruginosa the pathogen with the greatest number of genes devoted to regulation [163]. This in itself could explain the adaptability of P. aeruginosa to so many hostile environments and its impressive array of pathogenic mechanisms. Additionally, although the genome of P. aeruginosa is remarkably conserved in strains among different isolates [164] and even among different strains [163–165], 10% of the genome is variable and organized in blocks, or islands in the conserved core [164]. These islands have been successively shown to comprise material coding for known virulence factors such as ExoU [165], and up to 19 genes involved in virulence in a model of mouse thermal injury [166]. These islands, thus dubbed “pathogenicity islands” provide P. aeruginosa with even greater pathogenic versatility. Is pathogenesis accessible to therapeutic targeting on a genetic level in P. aeruginosa? Given the above information it would be highly desirable. In fact, antisense oligonucleotides or antisense peptide nucleic acids inhibit specific gene expression in Escherichia coli [167] and in Staphylococcus aureus [168]. In an in vivo animal study in which anti-NF-kappaB antisense therapy increased survival in endotoxic shock and peritonitis [169]. However, this approach has yet to be developed in P. aeruginosa infections. 8. Conclusions Although we have limited the scope of this review to the therapeutic targeting of direct bacterial mechanisms of pathogenesis, P. aeruginosa also interacts with host cells in triggering or modulating a multitude of pathways leading to further impairment of host defenses or injury [170]. These interactions may also be accessible to specific therapies as well, as the success of activated protein C in modulating the inflammation/ coagulation disorders of sepsis has shown [171]. Among the therapeutic strategies we have reviewed, many, including the most promising, are either active or passive immunotherapy-based. The advantages of immune-based therapeutics are notably the high specificity that allows targeting of these very specific pathogenic mechanisms and thus, few adverse effects. Additionally, active immunization strategies against P. aeruginosa are being pursued with the development of an anti-PcrV vaccine that could potentially transform the evolution of patients highly susceptible to P. aeruginosa such as cystic fibrosis patients. A current review addressing the entire range of vaccine development strategies against P. aeruginosa has recently been published [172]. 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