Dara W. Frank

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.

The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non‐covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non‐cytotoxic derivative PA103 exsA::Ω and several cytotoxic and non‐cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70‐kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70‐kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non‐cytotoxic strains or PA103 exsA::Ω. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70‐kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non‐cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co‐ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

Type III Protein Secretion Is Associated with Death in Lower Respiratory and Systemic Pseudomonas aeruginosa Infections

The ability of Pseudomonas aeruginosa to secrete specific toxins using the type III-mediated pathway has been reported. To determine the association of this phenotype with human illness, immunoblot analysis was used to detect expression of type III secretory proteins in P. aeruginosa isolates from respiratory tract or blood cultures of 108 consecutive patients. Relative risk of mortality was 6-fold greater with expression of the type III secretory proteins ExoS, ExoT, ExoU, or PcrV. Phenotype was independently correlated with toxicity in cellular and murine models. Prevalence of this phenotype was significantly higher in acutely infected patients than in chronically infected patients with cystic fibrosis. These results suggest that the type III protein secretion system is integral to increased P. aeruginosa virulence. A positive phenotype is a predictor of poor clinical outcome. In the future, such analyses may help distinguish potentially lethal infection from colonization and help determine appropriate therapy for critically ill patients.

The exoenzyme S regulon of Pseudomonas aeruginosa

Pseudomonas aeruginosa can cause severe life‐threatening infections in which the bacterium disseminates rapidly from epithelial colonization sites to the bloodstream. In experimental models, the ability of P. aeruginosa to disseminate is linked to epithelial injury, in vitro cytotoxicity and expression of the exoenzyme S regulon. Using the expression of ExoS as a model, a series of genes that are important for regulation, secretion and, perhaps, intoxication of eukaryotic cells have been identified. Proteins encoded by the exoenzyme S regulon and the Yersinia Yop virulon show a high level of amino acid homology, suggesting that P. aeruginosa may use a contact‐mediated translocation mechanism to transfer anti‐host factors directly into eukaryotic cells. Potential anti‐host factors that may disrupt eukaryotic signal transduction through ADP‐ribosylation include ExoS and ExoT. Expression of ExoU, another candidate anti‐host factor, has been correlated with acute cytotoxicity and lung epithelial injury. Members of the exoenzyme S regulon represent only a portion of the virulence factor arsenal possessed by P. aeruginosa. It will be important to understand how the exoenzyme S regulon contributes to pathogenesis and whether these factors could serve as potential therapeutic targets.

Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia.

The pathogenesis of septic shock occurring after Pseudomonas aeruginosa pneumonia was studied in a rabbit model. The airspace instillation of the cytotoxic P. aeruginosa strain PA103 into the rabbit caused a consistent alveolar epithelial injury, progressive bacteremia, and septic shock. The lung instillation of a noncytotoxic, isogenic mutant strain (PA103DeltaUT), which is defective for production of type III secreted toxins, did not cause either systemic inflammatory response or septic shock, despite a potent inflammatory response in the lung. The intravenous injection of PA103 did not cause shock or an increase in TNF-alpha, despite the fact that the animals were bacteremic. The systemic administration of either anti-TNF-alpha serum or recombinant human IL-10 improved both septic shock and bacteremia in the animals that were instilled with PA103. Radiolabeled TNF-alpha instilled in the lung significantly leaked into the circulation only in the presence of alveolar epithelial injury. We conclude that injury to the alveolar epithelium allows the release of proinflammatory mediators into the circulation that are primarily responsible for septic shock. Our results demonstrate the importance of compartmentalization of inflammatory mediators in the lung, and the crucial role of bacterial cytotoxins in causing alveolar epithelial damage in the pathogenesis of acute septic shock in P. aeruginosa pneumonia.

ACTIVE AND PASSIVE IMMUNIZATION WITH THE PSEUDOMONAS V ANTIGEN PROTECTS AGAINST TYPE III INTOXICATION AND LUNG INJURY

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Damage to the lung epithelium is associated with the expression of toxins that are directly injected into eukaryotic cells through a type III-mediated secretion and translocation mechanism. Here we show that the P. aeruginosa homolog of the Yersinia V antigen, PcrV, is involved in the translocation of type III toxins. Vaccination against PcrV ensured the survival of challenged mice and decreased lung inflammation and injury. Antibodies to PcrV inhibited the translocation of type III toxins.

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium‐independent (iPLA2) and cytosolic phospholipase A2 (cPLA2) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA2 and cPLA2 align or are similar to ExoU sequences. Site‐specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.

Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway

Exoenzyme S is an extracellular ADP‐ribosyltransferase of Pseudomonas aeruginosa. Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn5 Tc insertion mutants were isolated which exhibited an exoenzyme S‐deficient phenotype (388::Tn5 Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn5 Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb EcoRI fragment that is not contiguous with the exoenzyme S trans‐regulatory operon. 388::Tn5 Tc 469 and 550 mapped to a region downstream of the trans‐regulatory operon which has been previously shown to contain a promoter region that is co‐ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn5 Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB–L proteins of the yersiniaeYop type III export apparatus. RNase‐protection analysis of wild‐type and mutant strains indicated that exsD and pscB–L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino‐ or carboxy‐terminal residues were expressed in P. aeruginosa. Deletion analyses indicated that the amino‐terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co‐ordinately regulated proteins.

ExoU is a potent intracellular phospholipase.

The combination of a large genome encoding metabolic versatility and conserved secreted virulence determinants makes Pseudomonas aeruginosa a model pathogen that can be used to study host–parasite interactions in many eukaryotic hosts. One of the virulence regulons that likely plays a role in the ability of P. aeruginosa to avoid innate immune clearance in mammals is a type III secretion system (TTSS). Upon cellular contact, the P. aeruginosa TTSS is capable of delivering a combination of at least four different effector proteins, exoenzyme S (ExoS), ExoT, ExoU, and ExoY. Two of the four translocated proteins, ExoS and ExoU, are cytotoxic to cells during infection and transfection. The mechanism of cytotoxicity of ExoS is unclear. ExoU, however, has recently been characterized as a member of the phospholipase A family of enzymes, possessing at least phospholipase A2 activity. Similar to ExoS, ExoT and ExoY, ExoU requires either a eukaryotic‐specific modification or cofactor for its activity in vitro. The biologic effects of minimal expression of ExoU in yeast can be visualized by membrane damage to different organelles and fragmentation of the vacuole. In mammalian cells, the direct injection of ExoU causes irreversible damage to cellular membranes and rapid necrotic death. ExoU likely represents a unique enzyme and is the first identified phopholipase virulence factor that is translocated into the cytosol by TTSS.

Use of the galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

ABSTRACT Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 × 107 CFU μg of DNA−1 in F. tularensis LVS, Francisella novicida U112, and E. coli DH5α. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.