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1 Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Searle 6-495, Chicago, IL 60611, USA
2 Department of Medicine, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Searle 6-495, Chicago, IL 60611, USA
Correspondence
Alan R. Hauser
ahauser{at}northwestern.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The net result is that Yersinia bacteria are able to persist in the presence of a robust host immune response. Similar cooperation occurs between Salmonella type III effector proteins to orchestrate bacterial entry into intestinal epithelial cells, an essential step in the development of enteritis. A large number of type III effector proteins working in concert are required to achieve maximal levels of Salmonella bacterial uptake (Raffatellu et al., 2005). These proteins cause a complex series of actin cytoskeletal changes, which result in the transient formation of localized host-cell surface membrane ruffles that engulf the invading bacteria. SopB, SopE and SopE2 activate the Rho family GTPases Cdc42 and Rac1, thereby facilitating actin recruitment and polymerization at the site of bacterial entry (Hardt et al., 1998; Zhou et al., 2001). Two other effector proteins, SipA and SipC, further enhance actin nucleation, polymerization and cross-linking by directly binding to actin (Zhou et al., 1999a,b). Once bacterial entry is complete, another effector protein, SptP, reverses actin restructuring to re-establish normal host-cell morphology (Fu & Galan, 1999). Similar to Salmonella spp., bacteria of the genus Shigella also utilize type III effector proteins to gain access to the intracellular environment of intestinal epithelial cells. IpaC and IpaA coordinately modify the host-cell actin cytoskeleton to modulate entry of this bacterium (Tran Van Nhieu et al., 2000). In each of these cases, type-III-secreted effector proteins work in a coordinated fashion to bring about an important pathogenic consequence.
Experimental evidence for the existence of a cooperative relationship between virulence factors is that each factor alone has a minor effect on measures of virulence, but that together they dramatically enhance disease severity. This is indeed the case for the effector proteins secreted by the Yersinia and Salmonella type III systems (Grosdent et al., 2002; Raffatellu et al., 2005). Regardless of the specific molecular interactions, the true magnitude of the contribution of an individual effector protein to pathogenesis becomes apparent only when the cooperating effector proteins are also present in virulence assays.
It is unclear whether effector proteins of the type III secretion system of Pseudomonas aeruginosa act in a coordinated fashion to facilitate the development and progression of disease. This opportunistic pathogen secretes four effector proteins: ExoS, ExoT, ExoU and ExoY (Frank, 1997). The enzymic and cell biological activities of these proteins are well defined. ExoS is a bifunctional toxin containing a GTPase-activating protein (GAP) domain and an ADP-ribosyltransferase (ADPRT) domain. The amino-terminal GAP activity acts on Rho family GTPases while the carboxyl-terminal ADPRT activity is directed towards Ras and other host-cell proteins (Fraylick et al., 2001; Goehring et al., 1999; Henriksson et al., 2000, 2002; Krall et al., 2002; McGuffie et al., 1999; Olson et al., 1999; Rocha et al., 2003; Vincent et al., 1999). As a result of these enzymic activities, intoxication with ExoS is associated with several observable phenotypes, including cytotoxicity and inhibition of bacterial internalization by both phagocytic and non-phagocytic mammalian cells (Cowell et al., 2000; Fleiszig et al., 1997; Frithz-Lindsten et al., 1997; Henriksson et al., 2000; Kaufman et al., 2000; Olson et al., 1997, 1999; Pederson & Barbieri, 1998; Vincent et al., 1999). (Throughout this discussion, the term ‘cytotoxicity’ will be used to refer to cytolytic cell death.) Like ExoS, ExoT also has GAP activity for Rho GTPases and contains an ADPRT domain. The ADPRT activity of ExoT, however, targets the cellular Crk-I and Crk-II kinases rather than Ras (Sun & Barbieri, 2003). As a result, ExoT intoxication is associated with inhibition of bacterial internalization but not cytotoxicity (Cowell et al., 2000; Garrity-Ryan et al., 2000; Krall et al., 2000). ExoU possesses phospholipase A2 and lysophospholipase activities (Phillips et al., 2003; Rabin & Hauser, 2005; Sato et al., 2003; Tamura et al., 2004) that lead to rapid lysis of mammalian cells (Coburn & Frank, 1999; Finck-Barbançon et al., 1997; Fleiszig et al., 1997; Hauser et al., 1998a; Hauser & Engel, 1999; Vallis et al., 1999). ExoY is an adenylate cyclase that increases intracellular levels of cAMP (Yahr et al., 1998). Several studies have clearly demonstrated that the enzymic activities of ExoS, ExoT and ExoU contribute to pathogenesis by affecting bacterial persistence in infected tissues, bacterial dissemination and host survival (Finck-Barbançon et al., 1997; Garrity-Ryan et al., 2000; Hauser et al., 1998a; Pankhaniya et al., 2004; Shaver & Hauser, 2004). ExoY, in contrast, has not been shown to play an appreciable role in disease progression in animal models (Holder et al., 2001; Lee et al., 2005).
Recent observations suggest that cooperation between specific P. aeruginosa effector proteins does occur at the cellular level. Expression profiling of an infected pneumocyte cell line indicated that intoxication with ExoS, ExoT and ExoY together altered transcription of a distinct set of genes relative to intoxication with each effector protein alone (Ichikawa et al., 2005). Furthermore, the type III secretion phenotypes of P. aeruginosa clinical isolates are also consistent with cooperation between specific effector proteins. For example, clinical isolates rarely secrete ExoS, ExoT and ExoU individually. Rather, these proteins are usually secreted in specific combinations. The majority of secreting isolates produce ExoS and ExoT, whereas most of the remaining strains secrete ExoU and ExoT (Berthelot et al., 2003; Feltman et al., 2001; Hauser et al., 2002; Jain et al., 2004; Lomholt et al., 2001; Roy-Burman et al., 2001). One explanation for these secretion patterns is that selective pressures have favoured the evolution of strains that secrete specific combinations of cooperative effector proteins. However, the true consequences of interactions between effector proteins to overall virulence are currently unclear.
In an effort to better understand the roles of the P. aeruginosa type III effector proteins in pathogenesis, we have recently determined the relative virulence associated with ExoS, ExoT and ExoU when these proteins are secreted individually (Shaver & Hauser, 2004). Isogenic bacterial mutants that individually secreted ExoS, ExoT or ExoU were constructed in clinical isolate PA99 (Feltman et al., 2001; Shaver & Hauser, 2004). This parent strain was chosen because it naturally secretes all three of these effector proteins. Analysis of ExoY was not performed because this protein does not play a major role in the pathogenesis of P. aeruginosa and because PA99 does not contain the gene encoding ExoY (Feltman et al., 2001; Holder et al., 2001; Lee et al., 2005; Shaver & Hauser, 2004). Comparison of strains secreting ExoS, ExoT or ExoU revealed that ExoU and ExoS each individually increased bacterial persistence in the lung, spread to extrapulmonary sites and mortality, with ExoU having a more pronounced effect than ExoS (Shaver & Hauser, 2004). In this system, ExoT had no detectable effect on mortality or bacterial persistence in the lung and made only a minimal contribution to dissemination. From these results, we concluded that when secreted individually, ExoU had the greatest impact on virulence, ExoS had an intermediate effect and ExoT had a minor effect.
In the work described here, we extend our previous studies to examine whether ExoS, ExoT or ExoU act coordinately during pathogenesis. In other words, we wished to determine whether the contribution to virulence of each individual effector protein was enhanced when secreted in the context of the other effector proteins. If two or more effector proteins act in a coordinated manner to subvert host cells, then secretion of multiple effector proteins would be associated with synergistically enhanced virulence compared to secretion of individual effector proteins. Thus, for these studies, the virulence of isogenic mutants that secreted multiple effector proteins was compared to those secreting individual proteins using a variety of assays, both in vitro and in vivo. PA99 allowed us to systematically compare for the first time the effects of combinations of ExoS, ExoT and ExoU when secreted by a strain that naturally produced all three toxins.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) (Feltman et al., 2001; Shaver & Hauser, 2004). This clinical isolate naturally secretes ExoS, ExoT and ExoU, and therefore is an ideal parent strain for the construction of type III secretion mutants. PA99 does not contain the exoY gene and therefore does not secrete ExoY (Shaver & Hauser, 2004). Mutant strains were named according to the effector proteins secreted. For example, deletion of the exoS gene resulted in a strain that secreted ExoT and ExoU and was designated PA99TU. Detailed descriptions of the construction of these strains have been published previously (Shaver & Hauser, 2004), with the exception of PA99SU. Briefly, chromosomal effector genes were sequentially deleted from the parent strain by allelic replacement using plasmid constructs containing in-frame deletion alleles of each gene. To produce PA99SU, pCM104, an allelic exchange vector containing an in-frame deletion allele of the exoT gene (Shaver & Hauser, 2004), was conjugated into wild-type PA99. Exconjugants in which the chromosomal wild-type exoT allele was replaced with the deleted allele were then selected, as described previously (Shaver & Hauser, 2004). In addition to the effector mutants, two non-secreting strains, PA99null and PA99secr–, were generated previously (Shaver & Hauser, 2004). PA99null lacks secretion of all known effector proteins, but assembles a competent secretion and translocation apparatus, while PA99secr– fails to build a functional secretion apparatus due to a mutation in a required structural gene, pscJ. Appropriate deletion of each gene was confirmed by PCR amplification and the expected secretion phenotype was verified by immunoblot analysis, as described previously (Shaver & Hauser, 2004). Other than the absence of the appropriate deleted proteins, there were no obvious changes in the amounts of the other secreted proteins by immunoblot analysis (data not shown). All mutants had equivalent growth rates in Luria–Bertani (LB) and MINS (Nicas & Iglewski, 1984) media (data not shown). Furthermore, secretion of each deleted gene was restored by complementation with the wild-type allele at a neutral chromosomal locus (data not shown). An advantage of this collection of mutants is that each effector protein is expressed from a single-copy chromosomal gene under control of its endogenous promoter.
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Aminoglycoside exclusion assays of bacterial internalization.
Bacterial internalization was quantified using antibiotic protection assays. All ExoU-secreting strains were excluded from these assays to avoid confounding effects of rapid ExoU-mediated cytotoxicity, which allows the antibiotic access to intracellular bacteria. In addition, all assays were conducted prior to the onset of ExoS-dependent cell lysis, as indicated by the results of LDH release assays. A549 cells were seeded onto transwell filters in 12-well plates (Corning) and grown to 70–80 % confluence in Waymouth's medium supplemented with 10 % FBS. Bacteria were grown overnight in LB medium at 37 °C with shaking (250 r.p.m.). Bacterial concentrations were estimated using optical density measurement and diluted to an OD600 of 0·1, which corresponds to 1·5x108 c.f.u. ml–1. A549 cells on the filter were washed and placed in 0·5 ml of the appropriate medium. The bottom well was filled with 1·5 ml medium. Bacteria were added to the top well in 150 µl medium (m.o.i. approximately 45) and were incubated at 37 °C in 5 % CO2. Following 3 h of infection, cells were washed with Waymouth's medium to remove non-adherent bacteria and then incubated in fresh Waymouth's medium supplemented with 10 % FBS and amikacin (400 µg ml–1; Sigma) for 2 h to kill extracellular bacteria. Filters were removed from transwells using a sterile scalpel, soaked in 0·25 % Triton X-100 in medium and vortexed with sterile 3 mm glass beads for 2 min to ensure complete cell disruption. The resulting solution was serially diluted and plated onto LB agar to determine the number of viable internalized bacteria per well. All strains were tested in triplicate on each of several different days. Results were pooled from all assays.
Mouse model of acute pneumonia.
Studies of acute pneumonia were conducted using the aspiration mouse model (Comolli et al., 1999). Briefly, bacterial cultures were grown overnight in MINS medium at 37 °C with shaking (250 r.p.m.) and were then diluted and regrown to exponential phase. Bacteria were collected by centrifugation and resuspended to the appropriate concentration in PBS. Six- to eight-week-old female BALB/c mice were anaesthetized by intraperitoneal injection of a mixture of ketamine (100 mg ml–1) and xylazine (20 mg ml–1). Mice were inoculated with 1·2x106 c.f.u. bacteria in 50 µl PBS, as determined by optical density. Inocula were confirmed by plating of serial dilutions onto Vogel Bonner minimal (VBM) agar (Vogel & Bonner, 1956). Preliminary experiments using this dose indicated that severe illness in response to the wild-type PA99STU occurred between 22 and 28 h post-infection. To ensure day-to-day reproducibility, animals infected with PA99STU and PA99secr– were included in each daily experiment. All experiments were performed in accordance with the guidelines of the Northwestern University Animal Care and Use Committee.
For determination of bacterial numbers in individual organs, mice were sacrificed at 18 h post-infection. Lungs, spleens and livers were aseptically removed and individually homogenized in PBS. Bacteria in each organ were enumerated following plating of serial dilutions on VBM agar. For each experiment, at least 5 mice per strain were infected for each time point.
For determination of 50 % lethal dose (LD50) values, mice were anaesthetized with inhaled methoxyfluorane and were intranasally infected with a range of bacterial inocula. Mice were monitored for a total of 7 days and animal mortality was recorded. In all experiments, mice were sacrificed when severe illness developed and were scored as dead. A minimum of 10 mice were used for each strain. The LD50 value was calculated according to the method of Reed & Muench (1938).
Statistical methods.
The significance of differences in bacterial load, cytotoxicity and bacterial internalization was determined using ANOVA followed by multiple unplanned comparisons among all strains tested using the Tukey–Kramer HSD test with an 
value of 0·05. Prior to analysis, bacterial load and bacterial internalization data were natural log-transformed to fit a normal distribution. Cytotoxicity data were arcsin-transformed prior to statistical analysis. 
2 tests were performed on frequencies of dissemination. For all statistics, P<0·05 was considered significant.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Schulert et al., 2003). Since nearly all P. aeruginosa isolates that secrete ExoS or ExoU also secrete ExoT, we wished to examine whether secretion of ExoT could augment the cytotoxic capacity of ExoU or ExoS. Cytotoxicity towards A549 bronchial epithelial-like cells, which are derived from a cell type encountered by P. aeruginosa during respiratory infections, was measured using LDH release assays.
Markedly different levels of cytotoxicity resulted from infection with the mutant strains (Fig. 1). Non-secreting strains PA99null and PA99secr– were only minimally cytotoxic after 24 h. As expected, PA99T was no more cytotoxic than the non-secreting mutants. In contrast, PA99U was associated with rapid death of the majority of cells. Fifty percent of cells were lysed by 3 h and maximal killing of more than 80 % of cells had occurred by 9 h post-infection. PA99S also resulted in significant cytotoxicity, lysing 25 and 80 % of cells at 9 and 24 h, respectively. In general, additional secretion of ExoS, ExoT or both proteins along with ExoU did not appreciably increase ExoU-mediated cytotoxicity. A small trend towards increased cytotoxicity at the 3 h time-point was noted when ExoS and ExoT were both secreted together with ExoU relative to ExoU secretion alone. This difference, however, was not statistically significant. It was somewhat unexpected that secretion of ExoS along with ExoU did not result in a measurable increase in cytotoxicity, since both proteins can induce cell lysis. One possible explanation for this result is that the cytotoxic properties of ExoU are so robust that they mask any contribution by ExoS. Alternately, ExoU may simply act before the cytotoxic consequences of intoxication with ExoS become apparent. Similarly, ExoS-dependent cytotoxicity was unaffected by secretion of ExoT in that PA99ST and PA99S had similar cytotoxicity at all time-points tested.
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are consistent with our findings; they observed that after 4 h, little difference existed in membrane permeability following infection with strains secreting ExoU alone or in combination with ExoT. Similarly, Vance et al. (2005) noted little change in ExoS-mediated cytotoxicity towards macrophages when ExoT was secreted along with ExoS. Interestingly, Lee et al. (2005) reported that ExoT had a slight protective effect against ExoS- or ExoU-mediated killing of CHO cells. Our results show a small degree of ExoT-mediated protection against killing, but this did not achieve statistical significance. These quantitative differences may be due to the use of different cell lines or bacterial strains.
Bacterial internalization
ExoS and ExoT act as anti-internalization factors, inhibiting bacterial uptake by both epithelial and phagocytic cells in culture (Cowell et al., 2000; Garrity-Ryan et al., 2000). Thus, strains with defective type III secretion systems are internalized at relatively high frequencies compared to strains that secrete either ExoS or ExoT (Cowell et al., 2000; Evans et al., 1998; Frithz-Lindsten et al., 1997; Garrity-Ryan et al., 2000; Ha & Jin, 2001; Hauser et al., 1998b; Rocha et al., 2003). These observations have led to the hypothesis that type III secretion may function as a virulence determinant by allowing bacteria to escape clearance by phagocytic cells during infection. To determine whether combinations of effector proteins dramatically enhance inhibition of bacterial uptake relative to individual effector proteins, we conducted aminoglycoside protection invasion assays using several bacterial mutants and A549 cells. For these assays, all ExoU-secreting strains were excluded, since aminoglycoside exclusion assays are not interpretable in the presence of toxins that disrupt host-cell plasma membrane integrity. For the same reason, all assays were performed following only 3 h of infection, prior to the appearance of ExoS-mediated cytotoxicity (Fig. 1
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As expected, PA99null and PA99secr– had the highest levels of bacterial uptake, confirming that significant internalization occurs in the absence of secretion of ExoS or ExoT (Fig. 2). PA99S and PA99T each were internalized to a lesser degree, with PA99T exhibiting the smallest amount of bacterial uptake. ExoT secretion reduced bacterial uptake 51-fold compared to fourfold by ExoS secretion. Secretion of ExoS and ExoT together resulted in an intermediate 15-fold reduction in internalization. Thus, rather than enhancing ExoT activity, the secretion of ExoS with ExoT actually attenuated the potent anti-internalization activity of ExoT in vitro. Garrity-Ryan et al. (2000) also observed that ExoT displayed greater anti-internalization activity than ExoS and noted intermediate levels of bacterial internalization when ExoS was secreted with ExoT. Other studies, however, did not detect differences in invasion with secretion of ExoS, ExoT or both (Cowell et al., 2000; Ha & Jin, 2001). These variable results may be due to differences in the cell types or bacterial strains tested.
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). Briefly, mice were infected with 1·2x106 c.f.u. bacteria in 50 µl PBS. Bacterial numbers in the lung at 18 h post-infection were determined by plating homogenized lungs on VBM agar.
Secretion of multiple toxins had little additional effect on bacterial numbers in the lung as compared to secretion of ExoS, ExoT or ExoU individually (Fig. 3). As we previously reported, PA99U and PA99S, but not PA99T, were able to persist at relatively high numbers in the lung through 18 h (Shaver & Hauser, 2004). Secretion of ExoS, ExoT or both along with ExoU did not increase bacterial numbers in the lung relative to secretion of ExoU alone. In fact, a trend towards decreased bacterial persistence with PA99SU, PA99TU and PA99STU compared to PA99U was observed, although this did not achieve statistical significance. Likewise, secretion of ExoS with ExoT did not result in a statistically significant increase in bacterial numbers in the lung relative to secretion of ExoS alone, although there was a small trend in this direction. This last result has been confirmed by other reports showing that secretion of ExoS with additional effector proteins did not affect the bacterial loads in the lungs compared to secretion of ExoS alone (Lee et al., 2005; Vance et al., 2005). We conclude that effector proteins do not have a marked synergistic effect on bacterial persistence in the lung during acute pneumonia.
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Individually, ExoS, ExoT and ExoU each contributed to bacterial dissemination (Table 2). As we reported previously (Shaver & Hauser, 2004), PA99S, PA99T and PA99U each had an increased frequency of dissemination compared to PA99null and PA99secr–, which had minimal extrapulmonary spread. In general, secretion of multiple effector proteins resulted in an increase in dissemination, but the net effect was additive rather than synergistic. For example, PA99STU, PA99SU and PA99TU were detected in the spleen and liver more frequently than PA99U, although these differences did not reach statistical significance. Likewise, there was a trend towards increased dissemination of PA99ST to the spleen compared to PA99S or PA99T. Similar trends were evident when bacterial load in each organ was measured (Table 2). Vance et al. (2005) have reported results in agreement with ours. Using a strain that naturally secreted ExoS, ExoT and ExoY, they demonstrated that secretion of two or three effector proteins was associated with the greatest dissemination to the spleen, secretion of one protein with intermediate amounts of dissemination and secretion of no effector proteins with the least dissemination. In contrast, Lee et al. (2005) found that dissemination was not increased by secretion of ExoT and ExoY along with ExoS. This may be due to differences in inoculum sizes or in the bacterial strains used in these two studies. Some P. aeruginosa strains may produce additional factors not encoded by others that enhance dissemination, thus obviating the need for multiple type III effector proteins. In any case, the additive rather than synergistic increases in dissemination observed when multiple effector proteins are secreted in combination suggest that ExoS, ExoT and ExoU enhance spreading by acting independently instead of functioning in concert.
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). As reported previously (Shaver & Hauser, 2004), PA99U was associated with the highest virulence (i.e. the lowest LD50 value), PA99S was of intermediate virulence and PA99T was the least pathogenic. PA99SU, PA99TU or PA99STU did not increased in virulence compared to PA99U (LD50=8·2x105, 9·2x105, 8·6x105 and 5·5x105 c.f.u., respectively). Comparison of PA99ST with PA99S indicated that secretion of ExoS and ExoT together did not result in significantly higher levels of virulence than secretion of ExoS alone (LD50=4·4x106 vs 3·1x106, respectively), although there was a small trend in this direction. Together, these results indicate that P. aeruginosa effector proteins act individually rather than synergistically to cause mortality in the mouse model of acute pneumonia.
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; Guiney & Lesnick, 2005; Tran Van Nhieu et al., 2000). A hallmark of such cooperation is that individual effector proteins alone are associated with little virulence, but multiple effector proteins together have a marked pathogenic consequence. In this study, we did not find evidence suggesting that cooperation between ExoS, ExoT and ExoU of the P. aeruginosa type III system led to enhanced disease severity. In vitro cytotoxicity and internalization assays and an in vivo acute pneumonia model did not reveal dramatically augmented effects when ExoS, ExoT and ExoU were secreted together rather than individually. Instead, each of these proteins acted independently, causing at best additive but not synergistic effects. The absence of significant synergistic interactions between ExoS, ExoT and ExoU indicates that the pathogenic effects of these proteins can be adequately characterized by using a reductionist approach of studying each when expressed individually. Thus, these findings validate the results of earlier reports looking at effector proteins expressed alone (Finck-Barbançon & Frank, 2001; Henriksson et al., 2002; Pederson & Barbieri, 1998; Pederson et al., 1999; Phillips et al., 2003; Rabin & Hauser, 2003, 2005; Sato & Frank, 2004).
Interestingly, the results of some of our assays suggested that ExoS, ExoT and ExoU may in fact antagonize each other to a small degree. For example, ExoS and ExoT together were less efficient at preventing bacterial internalization than ExoT alone (Fig. 2). Secretion of ExoS or ExoT with ExoU resulted in slightly lower bacterial loads in the lungs of infected mice (Fig. 3). Likewise, secretion of ExoU alone was associated with slightly lower LD50 values (and therefore enhanced virulence) relative to secretion of ExoU with ExoS and/or ExoT (Fig. 4). Although many of these differences were not statistically significant, the observation of similar trends in multiple assays by us and others (Lee et al., 2005) suggests that there may be subtle antagonism when multiple P. aeruginosa effector proteins are secreted together. Whether this occurs during the secretion process or after the effector proteins are in the host cell is unclear.
Importantly, our results do not imply that ExoS, ExoT and ExoU lack interactions at the molecular level, but merely that such interactions, if they occur, do not increase virulence to a degree measurable in our assays. Additional work is necessary to determine whether these effector proteins do indeed modify or associate with each other, but if they do, it is unlikely that such interactions are of major pathogenic significance in acute pneumonia.
Given the general lack of increased pathogenicity with multiple effectors, it is unclear why secretion of more than one effector protein has been maintained in P. aeruginosa populations. It is conceivable that the assays we used did not adequately represent the range of activities required for P. aeruginosa to cause disease. Alternatively, because P. aeruginosa is an environmental organism for which humans are an accidental host (Rhame, 1979), it is possible that selective pressures driving the maintenance of multiple-effector phenotypes exist in the environment rather than in humans or mice. In this regard, it would be interesting to test whether ExoS, ExoT and ExoU cooperate in other models of P. aeruginosa disease, such as insects or amoebae (Miyata et al., 2003; Pukatzki et al., 2002). Each distinct set of effector proteins may have been maintained to provide defence against a different environmental threat. In this case, each strain of P. aeruginosa would maintain the set of effector proteins necessary for combating the spectrum of environmental threats present in its particular niche.
There are several limitations to our analysis. First, the cytotoxicity and internalization assays were performed using a single cell type, A549. It is conceivable that P. aeruginosa bacteria behave differently in assays with other cell types. This was, however, the same cell line used by Ichikawa et al. (2005) to demonstrate synergistic effects of ExoS, ExoT and ExoY on transcriptional profiles (Ichikawa et al., 2005). Second, only a single bacterial strain, PA99, was used in these experiments and this strain has the phenotype of secreting ExoS, ExoT and ExoU, which is rare among P. aeruginosa isolates (Feltman et al., 2001). Thus it is possible that synergistic interactions between effector proteins are strain-dependent and would be apparent with other P. aeruginosa strains. However, the results of our virulence and cytotoxicity assays suggest that PA99 is representative of P. aeruginosa strains. For example, the LD50 of PA99 was 9x105, which is similar to that of the frequently used ExoU-secreting strain PA103 (7x105) (Schulert et al., 2003). Likewise, the cytotoxicity of PA99 (55 %) was similar to that of PA103 (78 %) at 3 h (Schulert et al., 2003). Furthermore, many of the trends observed in our experiments were also noted by other investigators using strains PAO1 or PAK, as described in the preceding paragraphs. Together, these observations suggest that our findings are reflective of P. aeruginosa strains in general, but additional studies with other P. aeruginosa strains are necessary to confirm this. Third, the assays that we used may not measure all aspects of virulence associated with effector proteins or may not be sensitive enough to detect modest synergistic contributions to virulence. In this regard, it is interesting that in measurements of both bacterial burdens in the lungs and LD50 values, PA99ST was slightly more virulent than PA99S (Figs 3 and 4). Although these differences did not approach statistical significance, they may reflect small functional interactions between these two proteins, which are nearly always secreted together.
The results of these studies show that the secretion of ExoU or ExoS primarily dictates the virulence associated with the type III secretion system of P. aeruginosa during acute pneumonia. Secretion of ExoT has only a modest effect on pathogenesis and does not significantly augment the toxicities associated with ExoS or ExoU. Based on these results, we propose that strains of P. aeruginosa causing acute pneumonia can be divided into three major groups according to their type III secretion phenotypes. The first group consists of ExoU-secreting bacteria. These strains have the potential to cause particularly severe infections in acutely ill patients (Hauser et al., 2002; Roy-Burman et al., 2001) due to the potent activity of ExoU. The second group includes ExoS-secreting bacteria, which may be associated with intermediate levels of virulence. Finally, the third group of strains consists of those that do not secrete type III proteins and therefore lack the toxicities of ExoS and ExoU. These strains would thus be expected to cause the least serious illnesses unless they also encode other virulence determinants that compensate for the lack of ExoU and ExoS. Stratification of patients based upon the type III secretion properties of the strains they harbour may have important clinical utility. For example, more aggressive therapies may be warranted in patients infected with ExoU-secreting strains. Additional studies are necessary to better define the prognostic significance of type III secretion phenotypes in human patients.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Coburn, J. & Frank, D. (1999). Macrophages and epithelial cells respond differently to the Pseudomonas aeruginosa type III secretion system. Infect Immun 67, 3151–3154.
Comolli, J. C., Hauser, A. R., Waite, L., Whitchurch, C. B., Mattick, J. S. & Engel, J. N. (1999). Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect Immun 67, 3625–3630.
Cornelis, G. R. (2002). The Yersinia Ysc-Yop ‘type III’ weaponry. Nat Rev Mol Cell Biol 3, 742–752.[CrossRef][Medline]
Cowell, B. A., Chen, D. Y., Frank, D. W., Vallis, A. J. & Fleiszig, S. M. J. (2000). ExoT of cytotoxic Pseudomonas aeruginosa prevents uptake by corneal epithelial cells. Infect Immun 68, 403–406.
Evans, D. J., Frank, D. W., Finck-Barbancon, V., Wu, C. & Fleiszig, S. M. (1998). Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity. Infect Immun 66, 1453–1459.
Feltman, H., Schulert, G., Khan, S., Jain, M., Peterson, L. & Hauser, A. R. (2001). Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology 147, 2659–2669.
Finck-Barbançon, V. & Frank, D. W. (2001). Multiple domains are required for the toxic activity of Pseudomonas aeruginosa ExoU. J Bacteriol 183, 4330–4344.
Finck-Barbançon, V., Goranson, J., Zhu, L., Sawa, T., Wiener-Kronish, J. P., Fleiszig, S. M. J., Wu, C., Mende-Mueller, L. & Frank, D. (1997). ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol Microbiol 25, 547–557.[CrossRef][Medline]
Fleiszig, S. M. J., Wiener-Kronish, J. P., Miyazaki, H., Vallas, V., Mostov, K., Kanada, D., Sawa, T., Yen, T. S. B. & Frank, D. (1997). Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun 65, 579–586.[Abstract]
Frank, D. W. (1997). The exoenzyme S regulon of Pseudomonas aeruginosa. Mol Microbiol 26, 621–629.[CrossRef][Medline]
Fraylick, J. E., La Rocque, J. R., Vincent, T. S. & Olson, J. C. (2001). Independent and coordinate effects of ADP-ribosyltransferase and GTPase-activating activities of exoenzyme S on HT-29 epithelial cell function. Infect Immun 69, 5318–5328.
Frithz-Lindsten, E., Du, Y., Rosqvist, R. & Forsberg, A. (1997). Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III-dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments. Mol Microbiol 25, 1125–1139.[CrossRef][Medline]
Fu, Y. & Galan, J. E. (1999). A salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion. Nature 401, 293–297.[CrossRef][Medline]
Garrity-Ryan, L., Kazmierczak, B., Kowal, R., Comolli, J., Hauser, A. & Engel, J. N. (2000). The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages. Infect Immun 68, 7100–7113.
Goehring, U.-M., Schmidt, G., Pederson, K. J., Aktories, K. & Barbieri, J. T. (1999). The N-terminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein for Rho GTPases. J Biol Chem 274, 36369–36372.
Grosdent, N., Maridonneau-Parini, I., Sory, M. P. & Cornelis, G. R. (2002). Role of Yops and adhesins in resistance of Yersinia enterocolitica to phagocytosis. Infect Immun 70, 4165–4176.
Guiney, D. G. & Lesnick, M. (2005). Targeting of the actin cytoskeleton during infection by Salmonella strains. Clin Immunol 114, 248–255.[CrossRef][Medline]
Ha, U. & Jin, S. (2001). Growth phase-dependent invasion of Pseudomonas aeruginosa and its survival with HeLa cells. Infect Immun 69, 4398–4406.
Hardt, W. D., Chen, L. M., Schuebel, K. E., Bustelo, X. R. & Galan, J. E. (1998). Salmonella typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 93, 815–826.[CrossRef][Medline]
Hauser, A. R. & Engel, J. N. (1999). Pseudomonas aeruginosa induces type III secretion-mediated apoptosis of macrophages and epithelial cells. Infect Immun 67, 5530–5537.
Hauser, A. R., Kang, P. J. & Engel, J. (1998a). PepA, a novel secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence. Mol Microbiol 27, 807–818.[CrossRef][Medline]
Hauser, A. R., Kang, P. J., Fleiszig, S. J. M., Mostov, K. & Engel, J. (1998b). Defects in type III secretion correlate with internalization of Pseudomonas aeruginosa by epithelial cells. Infect Immun 66, 1413–1420.
Hauser, A. R., Cobb, E., Bodí, M., Mariscal, D., Vallés, J., Engel, J. N. & Rello, J. (2002). Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit Care Med 30, 521–528.[CrossRef][Medline]
Henriksson, M. L., Rosqvist, R., Telepnev, M., Wolf-Watz, H. & Hallberg, B. (2000). Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras. Biochem J 347, 217–222.[Medline]
Henriksson, M. L., Sundin, C., Jansson, A. L., Forsberg, A., Palmer, R. H. & Hallberg, B. (2002). Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo. Biochem J 367, 617–628.[CrossRef][Medline]
Holder, I. A., Neely, A. N. & Frank, D. W. (2001). Type III secretion/intoxication system important in virulence of Pseudomonas aeruginosa infections in burns. Burns 27, 129–130.[CrossRef][Medline]
Ichikawa, J. K., English, S. B., Wolfgang, M. C., Jackson, R., Butte, A. J. & Lory, S. (2005). Genome-wide analysis of host responses to the Pseudomonas aeruginosa type III secretion system yields synergistic effects. Cell Microbiol 7, 1635–1646.[CrossRef][Medline]
Jain, M., Ramirez, D., Seshadri, R. & 7 other authors (2004). Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis. J Clin Microbiol 42, 5229–5237.
Kaufman, M. R., Jia, J., Zeng, L., Ha, U., Chow, M. & Jin, S. (2000). Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of ExoS. Microbiology 146, 2531–2541.
Krall, R., Schmidt, G., Aktories, K. & Barbieri, J. T. (2000). Pseudomonas aeruginosa ExoT is a Rho GTPase-activating protein. Infect Immun 68, 6066–6068.
Krall, R., Sun, J., Pederson, K. J. & Barbieri, J. T. (2002). In vivo rho GTPase-activating protein activity of Pseudomonas aeruginosa cytotoxin ExoS. Infect Immun 70, 360–367.
Lee, V. T., Smith, R. S., Tummler, B. & Lory, S. (2005). Activities of Pseudomonas aeruginosa effectors secreted by the Type III secretion system in vitro and during infection. Infect Immun 73, 1695–1705.
Lomholt, J. A., Poulsen, K. & Kilian, M. (2001). Epidemic population structure of Pseudomonas aeruginosa: evidence for a clone that is pathogenic to the eye and that has a distinct combination of virulence factors. Infect Immun 69, 6284–6295.
McGuffie, E. M., Fraylick, J. E., Hazen-Martin, D. J., Vincent, T. S. & Olson, J. C. (1999). Differential sensitivity of human epithelial cells to Pseudomonas aeruginosa exoenzyme S. Infect Immun 67, 3494–3503.
Miyata, S., Casey, M., Frank, D. W., Ausubel, F. M. & Drenkard, E. (2003). Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis. Infect Immun 71, 2404–2413.
Nicas, T. I. & Iglewski, B. H. (1984). Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun 45, 470–474.
Olson, J. C., McGuffie, E. M. & Frank, D. W. (1997). Effects of differential expression of the 49-kilodalton exoenzyme S by Pseudomonas aeruginosa on cultured eukaryotic cells. Infect Immun 65, 248–256.[Abstract]
Olson, J. C., Fraylick, J. E., McGuffie, E. M., Dolan, K. M., Yahr, T. L., Frank, D. W. & Vincent, T. S. (1999). Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S. Infect Immun 67, 2847–2854.
Pankhaniya, R. R., Tamura, M., Allmond, L. R., Moriyama, K., Ajayi, T., Wiener-Kronish, J. P. & Sawa, T. (2004). Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU. Crit Care Med 32, 2293–2299.[Medline]
Pederson, K. J. & Barbieri, J. T. (1998). Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas aeruginosa exoenzyme S is cytotoxic to eukaryotic cells. Mol Microbiol 30, 751–759.[CrossRef][Medline]
Pederson, K. J., Vallis, A. J., Aktories, K., Frank, D. W. & Barbieri, J. T. (1999). The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins. Mol Microbiol 32, 393–401.[CrossRef][Medline]
Phillips, R. M., Six, D. A., Dennis, E. A. & Ghosh, P. (2003). In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors. J Biol Chem 278, 41326–41332.
Pukatzki, S., Kessin, R. H. & Mekalanos, J. J. (2002). The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. Proc Natl Acad Sci U S A 99, 3159–3164.
Rabin, S. D. P. & Hauser, A. R. (2003). Pseudomonas aeruginosa ExoU, a toxin transported by the type III secretion system, kills Saccharomyces cerevisiae. Infect Immun 71, 4144–4150.
Rabin, S. D. P. & Hauser, A. R. (2005). Functional regions of the Pseudomonas aeruginosa cytotoxin ExoU. Infect Immun 73, 573–582.
Raffatellu, M., Wilson, R. P., Chessa, D., Andrews-Polymenis, H., Tran, Q. T., Lawhon, S., Khare, S., Adams, L. G. & Baumler, A. J. (2005). SipA, SopA, SopB, SopD, and SopE2 contribute to Salmonella enterica serotype typhimurium invasion of epithelial cells. Infect Immun 73, 146–154.
Reed, L. & Muench, J. (1938). A simple method for estimating fifty percent endpoints. Am J Hyg 27, 493–497.
Rhame, F. S. (1979). The ecology and epidemiology of Pseudomonas aeruginosa. In Pseudomonas aeruginosa: the Organism, Diseases it Causes, and their Treatment, pp. 31–51. Edited by L. D. Sabath. Bern: Hans Huber Publishers.
Rocha, C. L., Coburn, J., Rucks, E. A. & Olson, J. C. (2003). Characterization of Pseudomonas aeruginosa exoenzyme S as a bifunctional enzyme in J774A.1 macrophages. Infect Immun 71, 5296–5305.
Roy-Burman, A., Savel, R. H., Racine, S., Swanson, B. L., Revadigar, N. S., Fujimoto, J., Sawa, T., Frank, D. W. & Wiener-Kronish, J. P. (2001). Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J Infect Dis 183, 1767–1774.[CrossRef][Medline]
Sato, H. & Frank, D. W. (2004). ExoU is a potent intracellular phospholipase. Mol Microbiol 53, 1279–1290.[CrossRef][Medline]
Sato, H., Frank, D. W., Hillard, C. J. & 9 other authors (2003). The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU. EMBO J 22, 2959–2969.[CrossRef][Medline]
Sawa, T., Ohara, M., Kurahashi, K., Twining, S. S., Frank, D., Doroques, D. B., Long, T., Gropper, M. A. & Wiener-Kronish, J. P. (1998). In vitro cellular toxicity predicts Pseudomonas aeruginosa virulence in lung infections. Infect Immun 66, 3242–3249.
Schulert, G. S., Feltman, H., Rabin, S. D. P., Martin, C. G., Battle, S. E., Rello, J. & Hauser, A. R. (2003). Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J Infect Dis 188, 1695–1706.[CrossRef][Medline]
Shaver, C. M. & Hauser, A. R. (2004). Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect Immun 72, 6969–6977.
Sun, J. & Barbieri, J. T. (2003). Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins. J Biol Chem 278, 32794–32800.
Tamura, M., Ajayi, T., Allmond, L. R., Moriyama, K., Wiener-Kronish, J. P. & Sawa, T. (2004). Lysophospholipase A activity of Pseudomonas aeruginosa type III secretory toxin ExoU. Biochem Biophys Res Commun 316, 323–331.[CrossRef][Medline]
Tran Van Nhieu, G., Bourdet-Sicard, R., Dumenil, G., Blocker, A. & Sansonetti, P. J. (2000). Bacterial signals and cell responses during Shigella entry into epithelial cells. Cell Microbiol 2, 187–193.[CrossRef][Medline]
Vallis, A. J., Finck-Barbancon, V., Yahr, T. L. & Frank, D. W. (1999). Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells. Infect Immun 67, 2040–2044.
Vance, R. E., Rietsch, A. & Mekalanos, J. J. (2005). Role of the type III secreted exoenzymes S, T, and Y in systemic spread of Pseudomonas aeruginosa PAO1 in vivo. Infect Immun 73, 1706–1713.
Vincent, T. S., Fraylick, J. E., McGuffie, E. M. & Olson, J. C. (1999). ADP-ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo. Mol Microbiol 32, 1054–1064.[CrossRef][Medline]
Vogel, H. J. & Bonner, D. M. (1956). Acetylornithinase of Escherichia coli partial purification and some properties. J Biol Chem 218, 97–106.
Yahr, T. L., Vallis, A. J., Hancock, M. K., Barbieri, J. T. & Frank, D. W. (1998). ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc Natl Acad Sci U S A 95, 13899–13904.
Zhou, D., Mooseker, M. S. & Galan, J. E. (1999a). An invasion-associated Salmonella protein modulates the actin-bundling activity of plastin. Proc Natl Acad Sci U S A 96, 10176–10181.
Zhou, D., Mooseker, M. S. & Galan, J. E. (1999b). Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science 283, 2092–2095.
Zhou, D., Chen, L. M., Hernandez, L., Shears, S. B. & Galan, J. E. (2001). A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization. Mol Microbiol 39, 248–259.[CrossRef][Medline]
Received 23 July 2005;
revised 17 October 2005;
accepted 18 October 2005.
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indicates a significant difference as compared to PA99S and PA99ST. For statistical analysis, data were arcsin-transformed prior to testing with Tukey–Kramer HSD, 
, STU; 
, TU; 
, SU; 
, ST; 
, S; 
, T; 
, U; x, null; |, secr–.
denotes statistical difference from PA99ST; 
