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ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca2+-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.

ABSTRACTFrancisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensissubspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pilgenes. We show here that deletion of the pilFputative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilTputative disassembly ATPase also caused a complete loss of pili, indicating that pilTfunctions differently in F. tularensisthan in model Tfp systems such as those found in Pseudomonas aeruginosaand Neisseriaspp. The LVS pilFand pilTmutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilFand pilTmutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pilgenes and provides evidence that the Francisellapili contribute to host cell adhesion and virulence.

Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence.

A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A2(PLA2) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA2activity of ExoU in vitro, whereas eukaryotic secreted PLA2and cytosolic PLA2were not inhibited. This novel inhibitor of ExoU-specific PLA2activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.

ABSTRACTA number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosaassociated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A2(PLA2) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA2activity of ExoU in vitro, whereas eukaryotic secreted PLA2and cytosolic PLA2were not inhibited. This novel inhibitor of ExoU-specific PLA2activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.

ABSTRACTFrancisella tularensisis a facultative intracellular pathogen and is the etiological agent of tularemia. It is capable of escaping from the phagosome, replicating to high numbers in the cytosol, and inducing apoptosis in macrophages of a variety of hosts. F. tularensishas received significant attention recently due to its potential use as a bioweapon. Currently, there is no licensed vaccine against F. tularensis, although a partially protective live vaccine strain (LVS) that is attenuated in humans but remains fully virulent for mice was previously developed. An F. tularensisLVS mutant deleted in the purMCDpurine biosynthetic locus was constructed and partially characterized by using an allelic exchange strategy. The F. tularensisLVS ΔpurMCDmutant was auxotrophic for purines when grown in defined medium and exhibited significant attenuation in virulence when assayed in murine macrophages in vitro or in BALB/c mice. Growth and virulence defects were complemented by the addition of the purine precursor hypoxanthine or by introduction of purMCDNin trans. The F. tularensisLVS ΔpurMCDmutant escaped from the phagosome but failed to replicate in the cytosol or induce apoptotic and cytopathic responses in infected cells. Importantly, mice vaccinated with a low dose of the F. tularensisLVSΔ purMCDmutant were fully protected against subsequent lethal challenge with the LVS parental strain. Collectively, these results suggest that F. tularensismutants deleted in the purMCDbiosynthetic locus exhibit characteristics that may warrant further investigation of their use as potential live vaccine candidates.

ABSTRACTNonvertebrate 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 mellonellais a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosapathogenesis. Based on the observation that a mutation in the TTSS pscDgene of P. aeruginosastrain 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. aeruginosastrain PA14, only ExoT and ExoU play a significant role in G. mellonellakilling. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoUΔexoY, ΔexoTΔexoY, and ΔexoTΔexoUdouble mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonellakilling. On the other hand, data obtained with a ΔexoTΔexoUΔexoYtriple mutant and a ΔpscDmutant suggested that additional, as-yet-unidentified P. aeruginosacomponents of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonellamodel and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonellafor the study of the P. aeruginosaTTSS.

ABSTRACTExpression of type III proteins of Pseudomonas aeruginosain patients with cystic fibrosis (CF) was investigated by measuring the immune response against components of the type III pathway. Twenty-three of the 33 sera contained antibodies against PcrV, a protein involved in translocation of type III cytotoxins into eukaryotic cells, and 11 of 33 had antibodies against ExoS, while most CF sera contained antibodies against PopB and PopD, components of the type III apparatus. These data indicate that P. aeruginosacommonly expresses components of the type III translocation apparatus in adult CF patients.

ABSTRACTThe presence of invasion-inhibitory activity that is regulated by the transcriptional activator ExsA of cytotoxic Pseudomonas aeruginosahas previously been proposed. The results of this study show that both ExoT and ExoS, known type III secreted effector proteins of P. aeruginosathat are regulated by ExsA, possess this activity. Invasion was reduced 94.4% by ExoT and 96.0% by ExoS. Invasion-inhibitory activity is not linked to ADP-ribosylation activity, at least for ExoS, since a noncatalytic mutant also inhibits uptake by an epithelial cell line (invasion was reduced 96.0% by ExoSE381A).

ABSTRACTThe multiple effects of Pseudomonas aeruginosatype III secretion have largely been attributed to variations in cytotoxin expression between strains. Here we show that the target cell type is also important. While lung epithelial cells showed significant changes in morphology but not viability when infected with P. aeruginosa, macrophages were efficiently killed by P. aeruginosa. Both responses were dependent on the type III secretion system.

ABSTRACTExoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P. aeruginosa388 and the non-ExoS-producing strain 388ΔS. Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388ΔS and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388. Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras. ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras. While microvillus effacement was detected in both 388- and 388ΔS-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388. We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras. The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the P. aeruginosainfectious process by rendering cells nonfunctional.

ABSTRACTA strain of Pseudomonas aeruginosathat fails to express known type III-secreted effector proteins was constructed as an expression host. Individual effectors were expressed intrans, and their biological effects on CHO cells were assessed in an acute cellular infection model. Intoxication with ExoS, ExoT, or ExoY resulted in alterations in cell morphology. As shown in previous genetic studies, ExoU expression was linked to acute cytotoxicity.

ABSTRACTThis study was initiated to characterize the regulation and secretion of ExoS by Pseudomonas aeruginosaduring contact with eukaryotic cells. The production of ExoS was monitored by a sensitive ADP-ribosyltransferase activity assay, and specific activities were calculated for supernatant and cell-associated fractions. Time course analysis indicated that ExoS was produced after a lag period, suggesting that induction of the regulon is necessary for the expression of detectable amounts of enzyme activity. Under tissue culture growth conditions, ExoS was induced when P. aeruginosawas in contact with Chinese hamster ovary (CHO) cells or after growth in tissue culture medium with serum. The serum induction of ExoS appeared to result in generalized type III secretion, while induction by contact with CHO cells appeared to result in polarized type III secretion. Mutants in the type III secretory system that express a null phenotype for ExoS production in bacteriological medium produced but did not secrete the enzyme when P. aeruginosawas grown under inducing conditions in tissue culture medium. These results suggest that both induction and secretion of ExoS may differ when the bacteria are exposed to different growth environments. The putative type III translocation proteins and secretion apparatus of P. aeruginosawere required for translocation of bacterial factors that mediate changes in CHO cell morphology during infection.

ABSTRACTThe role of quorum sensing by Pseudomonas aeruginosain producing cytotoxicity has not been fully investigated. Strains ofP. aeruginosahave been characterized as having an invasive or a cytotoxic phenotype (S. M. J. Fleiszig et al., Infect. Immun. 65:579–586, 1997). We noted that the application of a large inoculum of the invasive strain 6294 caused cytotoxicity of cultured epithelial cells. To investigate this dose-related cytotoxicity, we compared the behavior of 6294 to that of another invasive strain, PAO1, and determined whether the cytotoxicity could be related to quorum sensing. Both invasive strains, 6294 and PAO1, appear to have quorum-sensing systems that were operative when large doses of bacteria were applied to cultured lung epithelial cells or instilled into the lungs of animals. Nonetheless, only 6294 was cytotoxic. Cytotoxicity induced by 6294 correlated with increased elastase production. These experiments suggest that there are multiple mechanisms for the induction of cytotoxicity, pathology, and mortality in vivo. However, in vivo cytotoxicity and mortality, but not pathology, could be predicted by quantitative in vitro cellular damage experiments utilizing a range of bacteria-to-cell ratios. It appears that quorum sensing may inversely correlate with virulence in that strains that produced PAI [N-(3-oxododecanoyl) homoserine lactone] also appeared to attract more polymorphonuclear leukocytes in vivo and were possibly eliminated more quickly. In addition, exoproduct production in bacteriological medium in vitro may differ significantly from exoproduct expression from infections in vivo or during cocultivation of bacteria with tissue culture cells.

ABSTRACTGenetic and functional data suggest that Pseudomonas aeruginosaexoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa388 and an isogenic mutant strain, 388ΔexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388ΔexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388ΔexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.

ABSTRACTPseudomonas aeruginosaclinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene,exsA, that controls the expression of several extracellular proteins, is inactivated. exsAmutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specificexsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factorexoUwas neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by anexsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.

Achromobacter xylosoxidansis increasingly recognized as a colonizer of cystic fibrosis (CF) patients, but the role that A. xylosoxidansplays in pathology remains unknown. This knowledge gap is largely due to the lack of model systems available to study the toxic potential of this bacterium. Recently, a phospholipase A2(PLA2) encoded by a majority of A. xylosoxidansgenomes, termed AxoU, was identified.