1 School of Optometry, University of California, Berkeley 94720-2020, CA, USA
2 Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA
3 Department of Microbiology, Wayne State University, Detroit, MI, USA
Correspondence
Suzanne M. J. Fleiszig
fleiszig{at}socrates.berkeley.edu
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ABSTRACT |
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INTRODUCTION |
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The transcriptional activator ExsA regulates the production of ExoS, ExoT, ExoU and ExoY by P. aeruginosa. These proteins, each capable of altering mammalian cell function, are normally type III secreted by the bacteria (Cheng & Schneewind, 2000; Yahr et al., 1996
). Mutations in ExsA reduce the ability of the pathogen to damage the cornea and the lung (Cowell et al., 1999
; Kudoh et al., 1994
). Two of the proteins secreted by this system (ExoS and ExoT) are able to inhibit uptake of P. aeruginosa by epithelial cells or macrophages (Cowell et al., 2000
; Garrity-Ryan et al., 2000
; Ha & Jin, 2001
). Cytotoxic strains, which poorly invade host cells, encode ExoT, but not ExoS. Interestingly, invasive strains encode both ExoT and ExoS, yet they invade more efficiently than cytotoxic strains (Fleiszig et al., 1997a
). The aim of this study was to understand this apparent paradox.
The major role of proteases in P. aeruginosa virulence in various infection models is thought to involve tissue penetration (Twining et al., 1993; Tang et al., 1996
). Proteases synthesized by this organism include LasB encoded by the lasB gene, LasA encoded by the lasA gene, alkaline protease encoded by the aprA gene, and protease IV (GenBank accession number AY062882). LasA possesses a low level of elastolytic activity, but is important as an enzyme that enhances the elastolytic activity of LasB (Kessler et al., 1997
). When investigating the differences between invasive and cytotoxic strains of P. aeruginosa, we noted degradation products of both ExoS and ExoT produced by invasive strains of P. aeruginosa on Western blots of bacterial culture supernatants (Fleiszig et al., 1997a
). The results of this study show that LasA/LasB play a role in the regulation of steady-state levels of ExoS and ExoT released under conditions for toxin production, and modulate invasion of the bacteria into mammalian cells.
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METHODS |
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Protease assays.
LasB activity was determined using Congo Red elastin (Sigma) (Kessler et al., 1997) and the activity was expressed as µg elastin degraded (108 organisms)-1 h-1. General proteolytic activity was determined using FITC-casein (Twining, 1984
) and the results were expressed as relative fluorescence increase (108 organisms)-1 h-1. Experiments were performed in triplicate and confirmed using two different sets of P. aeruginosa conditioned broths. All experiments were performed within the linear range of the assays.
Preparation of epithelial cells.
Immortalized rabbit corneal epithelial cells were maintained and passaged as previously described (Fleiszig et al., 1996). Cells were passaged onto 24-well plates or glass coverslips in modified SHEM medium (Fleiszig et al., 2001
), and grown at 37 °C in 5 % CO2 for 37 days before each assay.
Bacterial mutants.
Two different stocks of PAO1 were used for these studies. Although both are virulent in vivo, vast differences in the ID50 between these PAO1 stocks from different laboratories have been observed by us and by other researchers (personal communications). Thus, the two PAO1 wild-type isolates were distinguished as PAO1 and PAO1V, and were used as controls for their respective mutants. PAO1 wild-type and PAO1lasB were obtained from Dr Barbara Iglewski (University of Rochester, Rochester, NY, USA). Protease-deficient mutants of PAO1V were constructed by allelic replacement. Suicide vectors for allelic exchange contained P. aeruginosa chromosomal DNA where the ORF of the targeted protease gene was either replaced or disrupted with an antibiotic resistance gene. Bacillus subtilis sacB provided a counter-selectable marker for excision of vector sequence after a recombination event occurred between the mutant allele on the vector and homologous wild-type chromosomal DNA. A LasA protease-deficient mutant of PAO1V was constructed by replacing the entire lasA allele with a gentamicin resistance gene as described by Gustin (1998). To construct a mutant deficient in both LasA and LasB, exchange vector pSKM40-ST was introduced by a triparental mating essentially as described by McIver et al. (1995)
, resulting in replacement of the wild-type lasB allele with a streptomycin resistance gene. The allelic exchange of aprA was achieved as described by Pillar et al. (2000)
. Allelic exchange was confirmed in all mutants by a PCR analysis or Southern blotting. Loss of LasA protease activity was confirmed with a staphylolytic assay as described by Kessler et al. (1993)
. Loss of LasB was confirmed with gelatin zymography (Twining et al., 1993
).
Invasion of P. aeruginosa in a rabbit corneal epithelial (RCE) cell line.
RCE cells were inoculated with 2x105 c.f.u. bacteria (m.o.i. 1 : 1), and incubated for 3 h at 37 °C in 5 % CO2. This suspension was then carefully aspirated and the wells washed with two sequential 0·5 ml aliquots of phosphate-buffered saline (PBS, Sigma) to remove non-associated bacteria. Associated extracellular bacteria were killed by incubation for 1 h with 1 ml amikacin (200 µg ml-1 in MEM). The antibiotic was aspirated, and cells were washed with 1 ml PBS, before lysis with Triton X-100 (0·25 %, v/v, in PBS). Intracellular bacteria were enumerated by culturing serial dilutions of the lysate on MacConkey agar (PML Microbiologicals) overnight at 37 °C. Invasion was expressed either as c.f.u. per well or as a percentage relative to that for the invasive strain 6294, taken as 100 %.
To study bacterial invasion after disruption of tight junctions, RCE cells were incubated with 30 mM EGTA in MEM for 30 min at 37 °C in 5 % CO2 and then quickly washed twice with PBS prior to adding the bacterial inoculum (Fleiszig et al., 1997b). Invasion of cells was determined as described above.
To determine the effect of exogenous LasB on invasion of epithelial cells, RCE cells that had been passaged onto 10 cm diameter culture dishes were infected with PAO1, PAO1lasB or MEM alone for 3 h at 37 °C in 5 % CO2. Culture supernatant was then collected, centrifuged and filtered through a 0·22 µm filter to remove bacteria. A MEM control was included to control for factors produced by epithelial cells over this time period. The culture supernatants (containing secreted LasB or not, depending on supernatant origin) were then added to fresh cells along with either PAO1 or PAO1lasB for 3 h at 37 °C in 5 % CO2. Invasion of these cells was then determined as described above. In additional experiments, the amount of LasB was determined by comparing band intensities of the supernatant of PAO1-infected cells with bands of various amounts of purified LasB (Nagase Chemicals). LasB produced was estimated to be 15 ng ml-1. Invasion of PAO1, PAO1lasA and PAO1lasAlasB was compared to that when 15 ng ml-1 LasB was added.
Association of P. aeruginosa with RCE cells.
RCE cells were cultured on 3 µm pore polycarbonate filters (Costar) in 12-well culture dishes. Bacterial suspensions were added to the upper chamber and MEM to the lower chamber. Postinfection (3 h at 37 °C in 5 % CO2) filters were vigorously rinsed twice with PBS. Filters were then carefully cut from their plastic holder and placed in glass grinders containing 0·25 % (v/v) Triton X-100 in PBS. To quantify the number of bacteria associated with cells, filters were macerated and bacteria enumerated by culturing serial dilutions as described above.
Measurement of transepithelial resistance (TER) of infected RCE cells.
RCE cells were cultured on 3 µm pore filters as above. One filter was left without cells as a control. TER was measured daily, using an EVOM (Epithelial Voltohmmeter; World Precision Instruments). Before TER measurement, the upper and lower chambers were aspirated, and fresh MEM was carefully added. TER measurement was made after 10 min of equilibration. This procedure was repeated on the day of assay. Bacteria were carefully added to the upper chamber of appropriate wells, while MEM was added to control uninfected wells. TER readings were taken at the time of inoculation, and then hourly for 4 h.
Degradation of recombinant ExoS by purified LasB and AprA.
Partially purified active recombinant ExoS produced in Escherichia coli with a deletion of the hydrophobic region at 5172 was obtained from Dr Joseph Barbieri (Medical College of Wisconsin, Milwaukee, WI, USA). P. aeruginosa LasB and AprA were purchased from Nagase Chemicals. Three micrograms of recombinant ExoS was incubated with 6 ng LasB or 6 ng AprA at 23 °C in 50 mM Tris buffer (pH 8·0). Samples were collected immediately, and again at 1 min and 4 min. The degradation products were separated by PAGE and stained with Coomassie brilliant blue.
Western blotting of ExsA-regulated proteins of P. aeruginosa.
PA103, PAO1, PAO1lasB, PAO1V and PAO1VlasA, PAO1VlasAlasB and PAO1VlasAlasBaprA were grown under conditions suitable for production of ExoS and ExoT. Briefly, the strains were grown on VogelBonner minimal agar medium (Vogel & Bonner, 1956). The organisms were then inoculated into deferrated trypticase soy broth containing 100 mM monosodium glutamate, 1 % (v/v) glycerol and 10 mM nitrilotriacetic acid (NTA) a chelator (Sigma), and grown for 14 h at 32 °C. These conditions allow maximum toxin and protease production.
The number of organisms per ml varied less than 10 % between cultures based on the OD600. The cultures were normalized and the organisms pelleted by centrifugation. The proteins in an aliquot of the supernatant fraction were precipitated with saturated ammonium sulfate at 4 °C for 2 h then centrifuged. The pellet was resuspended in PAGE sample buffer. The samples were used for Western blotting using rabbit anti-exoenzyme S (a gift from Dr Dara Frank, Medical College of Wisconsin, Milwaukee, WI, USA) using standard techniques. The samples were loaded at levels within the linear range of the ECL reagent used to visualize ExoS and ExoT. ExoT cross-reacts with the antibody. The ExoS bands were quantified using SigmaGel (SPSS). The experiment was confirmed using cultures from two additional sets of single colonies.
Statistics.
At least three wells of cells were used for each strain in each invasion and association assay, and all experiments were repeated at least twice. Results are presented as means and standard deviations of the data collected from one representative series of experiments. Whole assay groups were analysed with an ANOVA test; individual groups were directly compared using Student's t-test.
Elastolytic and caseinolytic activity was determined in triplicate on two different sets of conditioned broths. Results were presented as means and standard deviations. Correlations between invasion and caseinolytic activity, elastolytic activity or ExoS levels of the conditioned broths were determined by linear regression analysis using SigmaPlot (SPSS).
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RESULTS |
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Elastolytic activity present in the conditioned medium of P. aeruginosa organisms grown to the stationary phase was better correlated with the level of invasion of the 11 different strains into RCE cells over a 3 h period (r=0·74) than with the caseinolytic activity of these strains (r=0·06) (compare Fig. 1a and 1b). This result suggested that LasA and LasB may be involved in the regulation of invasion of P. aeruginosa.
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The role of LasB in modulating invasion is not explained by disruption of tight junctions
LasB has previously been shown to disrupt tight junctions of epithelial cells, and has been found to cause reduced TER across cellular monolayers (Azghani, 1996). Basolateral cell membranes of epithelial cells are more susceptible to P. aeruginosa invasion than are apical cell surfaces (Fleiszig et al., 1997b
). Thus, we explored whether reduced invasion of the protease mutants might be related to a reduced capacity to disrupt epithelial cell tight junctions, which would decrease bacterial access to the basolateral membranes as compared to wild-type PAO1. The results showed that epithelial cell TER was slightly reduced after 4 h of infection with wild-type PAO1, but that LasB was not involved, since there was no significant difference between the TER drop caused by the lasB mutant and wild-type PAO1 (TER of control, 170·5±0·7; PAO1, 154±3·2; PAO1lasB, 155·5±5·8; P>0·05).
In other experiments, corneal epithelial cells were pretreated with EGTA to deliberately disrupt tight junctions before bacterial infection. Under those conditions, PAO1VlasA and PAO1lasB mutants were taken up to a similar degree as wild-type PAO1 (invasion values 5·81x104±5·45x103, 8·28x104±9·93x103 and 7·40x104±8·65x103 c.f.u. per well, respectively; P>0·05), while the PAO1VlasAlasB double and PAO1VlasAlasBaprA triple mutants remained significantly (P<0·05) less invasive than the wild-type PAO1V (invasion values 3·27x104±4·91x103, 3·76x104±3·07x103 and 7·40x104±8·65x103 c.f.u. per well, respectively). Taken together, these data showed that the role of LasA and LasB in invasion involved factors additional to disruption of tight junctions.
Exogenous LasB does not restore normal invasiveness to a lasB mutant
To determine whether LasB might be affecting invasion via an extracellular mechanism, supernatants of RCE cells infected with PAO1 or PAO1lasB were filtered to remove bacteria, and were then inoculated to a fresh batch of PAO1 or PAO1lasB bacteria before being added to unexposed RCE cells. The presence of LasB in the PAO1 conditioned medium and the absence in PAO1lasB-conditioned medium was confirmed by zymography on gelatin PAGE gels. Neither type of conditioned medium affected invasion by the lasB mutant of PAO1 (tissue culture control medium 3·21±0·15x104 c.f.u. ml-1, LasB+ conditioned medium 3·34±0·31x104 c.f.u. ml-1, LasB- conditioned medium 2·97±0·57x104 c.f.u. ml-1; P>0·05). Similarly, neither conditioned medium affected invasion by wild-type PAO1 (P>0·05 in all cases). These results showed that exogenous elastase produced by wild-type PAO1 did not modulate invasion.
Similar results were obtained when purified LasB was added rather than LasB-containing culture supernatant, i.e. exogenous LasB failed to increase invasion of the PAO1VlasAlasB double mutant to levels noted with either PAO1VlasA mutant or wild-type PAO1V (2·09±0·32x103 c.f.u. ml-1 vs 6·66±0·49x103 c.f.u. ml-1, and 7·90±1·17x103 c.f.u. ml-1, respectively; P<0·0001 and P=0·0004). Invasion of PAO1VlasAlasB was the same with or without added purified LasB (2·09±0·32x103 c.f.u. ml-1 vs 1·81±0·19x103 c.f.u. ml-1; P=0·21). These results suggest a cell-associated mechanism in LasB effects on invasion.
lasA/lasB mutations correlate with higher steady-state levels of intact ExoS and ExoT
Because ExoS and ExoT inhibit invasion of P. aeruginosa into epithelial cells, a mechanism by which LasA and LasB could alter invasion would be by decreasing levels of intact ExoS and ExoT, either by direct degradation or by an indirect mechanism. When grown under divalent-cation-depleted conditions, P. aeruginosa releases type III secreted products into the culture supernatant (Yahr et al., 1997). ExoS and ExoT can be detected on a Western blot using appropriate antibodies. The invasive strain PAO1 expresses ExoS and ExoT (Fleiszig et al., 1997a
), while the cytotoxic strain PA103 expresses ExoU and ExoT, but not ExoS (Yahr et al., 1997
). Using an antibody raised against ExoS that also recognizes ExoT, intact ExoT was detected in the concentrated supernatant fractions of PA103 (which is defective in lasR), the double mutant PAO1VlasAlasB and the triple mutant PAO1VlasAlasBaprA (Fig. 2
a). The PAO1VlasA and PAO1lasB samples contained significantly more intact ExoS than wild-type PAO1. Mutation of both genes together resulted in a greater increase in the level of intact ExoS. Further mutation of the aprA gene did not increase the level of intact ExoS. The levels of intact ExoS secreted by these mutants significantly correlated with invasion (Fig. 2b, r
=0·98). Multiple degradation bands were observed for all samples. Levels of other proteins were not affected by mutation, as determined by Coomassie blue staining of gels (data not shown). These results showed that mutation of protease genes was associated with increased steady-state levels of intact ExoS and ExoT.
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DISCUSSION |
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To explore the relationship between proteolytic activity and invasion, the well-studied strain PAO1 was chosen. This was a suitable strain for this study because it produces multiple active proteases including LasA, LasB and alkaline protease, and it readily invades RCE cells. Mutation of the proteases individually or in combination allowed for the exploration of the relationship between proteolytic activity and invasion. The results showed that loss of either LasA or LasB decreased invasion about 70 % while loss of both proteases decreased invasion further. Mutation of the alkaline protease gene, aprA, in addition to the genes for LasA and LasB did not further decrease invasion. These results suggested that LasA and LasB might be involved in the regulation of invasion.
Elastase activity of P. aeruginosa has been shown to have effects on tight junctions of epithelial cells, which might expose their more susceptible basolateral membranes (Fleiszig et al., 1997b). After EGTA was used to disrupt epithelial cell tight junctions, mutation of both lasA and lasB still reduced epithelial cell invasion, indicating that factors additional to exposure of basolateral membranes were involved in the effects of LasA/LasB on invasion.
Exogenously added conditioned medium containing LasA and LasB did not restore normal invasion levels to the lasB mutant. Moreover, addition of purified LasB did not affect invasion levels of wild-type or protease mutants. These results suggest that modulation of invasion by LasA/B probably does not involve secreted protease(s). Alternative mechanisms of delivery of these proteases to cells could involve vesicles that bud off from P. aeruginosa and fuse with cell membranes (Kadurugamuwa & Beveridge, 1997), or the attachment of the proteases to the outer membrane of the organism. Vesicle delivery of proteases is a viable possibility since the vesicles contain periplasmic proteins including LasA and LasB. In Yersinia pestis, Pla, a membrane serine protease, is required for invasion but not for association with HeLa cells (Cowan et al., 2000
). Although not yet reported, LasA and LasB may associate with membranes. The annotated list of Pseudomonas proteins places LasA on the outer membrane (http://www.pseudomonas.com). Alternatively, these proteases may activate a membrane protease during its passage through the periplasm. LasB is autocatalytically cleaved in the periplasm but has little activity until the activation peptide, which serves as a chaperone and inhibitor, dissociates or is degraded (Kessler & Safrin, 1994
; Braun et al., 1996
). This latter step usually occurs following secretion; however, some LasB activity is present in the periplasm as shown for the activation of diphosphate kinase by LasB (Kamath et al., 1998
). LasA may also have partial activity in the periplasm because LasB, as well as proteinase IV and alkaline protease, can activate this enzyme (Kessler et al., 1998
).
Possible mechanisms by which LasA and LasB may induce invasion include degradation of invasion inhibitors or of proteins involved in the synthesis, processing or delivery of the inhibitors. Two type III secreted proteins of P. aeruginosa, ExoS and ExoT, can inhibit bacterial invasion via disruption of the host cell cytoskeleton (Cowell et al., 2000; Garrity-Ryan et al., 2000
). Mutation of lasA and/or lasB increased the steady-state levels of these two toxins secreted by the organisms grown under conditions for toxin production. Further mutation of aprA did not increase the levels of these toxins. The level of ExoS was inversely proportional to the invasion of the mutant strains. This relationship was highly significant, suggesting that regulation of invasion by LasA and LasB might include regulating the level of this protein, which can inhibit invasion. This could involve direct degradation of the toxin, since LasB degraded ExoS in vitro. However, in vivo LasA and LasB may not have access to this substrate. ExoS is directly secreted from the cytoplasm through a pore that passes through the inner membrane, periplasm and outer membrane by a type III secretion mechanism (Cheng & Schneewind, 2000
; Cornelis & Van Gijsegem, 2000
). Because LasB is partially active in the periplasm (Kamath et al., 1998
) and it is conceivable that LasA is activated by LasB in the periplasm, these enzymes may cleave components of the type III secretion machinery that pass through the periplasm and thus inhibit delivery of toxins to the target cells. Another intriguing possibility is the association of the zymogen forms of LasA and LasB with the secretion apparatus during assembly, followed by activation of LasA and LasB and direct cleavage of ExoS and ExoT as they pass through the secretion apparatus. Alternatively, LasA and LasB may control levels of ExoS and ExoT indirectly through degradation of a protein involved in the regulation/functioning of type III secretion, or by activation of other proteases that directly degrade the toxins. Studies to elucidate the mechanisms by which LasA and LasB might alter levels of ExoS and ExoT are under way.
The results of this study suggest a mechanism by which an invasive strain of P. aeruginosa might express ExoS and ExoT while remaining invasive. We presented three hypotheses in a previous paper (Cowell et al., 2000). The first was that invasive P. aeruginosa might differ from cytotoxic strains in their ability to activate ExsA-regulated type III secretion upon contact with corneal epithelial cells. Since then, Allewelt et al. (2000)
found that an invasive strain of P. aeruginosa became cytotoxic to corneal cells when ExoU was supplied via a plasmid, showing that invasive strains were capable of secreting type III ExsA-regulated proteins directly into corneal cells. Another mechanism we proposed to explain invasion by P. aeruginosa strains possessing both exoS and exoT was that ExoS and ExoT may interfere with one another in their ability to inhibit invasion due to their significant homology and reported secretion as a heterologous aggregate (Kulich et al., 1993
; Yahr et al., 1996
). Research published by Ha & Jin (2001)
demonstrated a lack of interference between these proteins using exoS and exoT mutants in the invasive strain PAK. Our third hypothesis was that invasive strains of P. aeruginosa might possess other factor(s) that interfere with ExoS and ExoT invasion-inhibitory activity (Cowell et al., 2000
). These factors may be LasA and LasB because mutation of lasA and/or lasB increases the steady-state levels of ExoS and ExoT produced by the bacteria and inhibits invasion of the organisms into epithelial cells.
This study also provides insight into the results of previously published studies on invasion of P. aeruginosa into epithelial cells. Ha & Jin (2001) observed decreased levels of ExoS in PAK-infected HeLa cells and increased invasion when bacteria grown to stationary phase were used for infection as compared to bacteria grown to exponential phase. Based on our studies, these results could be explained by the difference in the levels of LasA and LasB produced under these two growth conditions. In bacteria grown to stationary phase, the Las and Rhl quorum-sensing systems are stimulated to a much greater degree than in exponential-phase bacteria, resulting in increased synthesis of autoinducers, which upregulate the synthesis of LasA and LasB (Toder et al., 1991
; Pesci et al., 1997
; Calfee et al., 2001
). Elevated levels of LasA and LasB under stationary growth conditions would decrease the levels of ExoS and ExoT, and thus lead to increased invasion relative to organisms grown under exponential conditions.
Zhu et al. (2001) showed that deletion of both lasR and rhlR resulted in markedly reduced corneal epithelial cell invasion, and an almost total loss of virulence in a corneal infection model. Because the quorum-sensing regulators, lasR and rhlR, regulate LasA and LasB synthesis, deletion of these genes would decrease LasA and LasB to nearly undetectable levels. Our studies would predict that the decreased invasion observed may involve the downregulation of LasA and LasB leading to increased levels of ExoS and ExoT, which would increase inhibition of invasion.
Invasion and invasion inhibition are both potentially useful traits for a pathogen. Since shedding of epithelial cells with internalized P. aeruginosa is thought to be a host defence against infection (Fleiszig et al., 1995; Pier et al., 1996
), reduced phagocytosis by either professional or non-professional phagocytes could potentially contribute to pathogenicity. In other settings, internalization might be more important. Thus, the ability to regulate invasion in response to prevailing environmental conditions would seem to be valuable. The P. aeruginosa genome contains approximately 5500 ORFs, of which almost 10 % appear to be regulatory genes (McCarthy, 2000
). This is one of the highest proportions of regulatory genes for any sequenced bacterial genome. It also has a large number of distinct gene families, indicating considerable environmental diversity (Stover et al., 2000
). Thus, it would not be surprising if invasion levels were regulated by more than one system. In P. aeruginosa, both the ExsA-regulated type III secretion system and the LasR and RhlR systems are regulated by environmental conditions, and both systems modulate invasion. Although P. aeruginosa clinical isolates have been broadly classified as invasive or cytotoxic, the interplay of different effectors and regulators under prevailing environmental conditions encountered in a mammalian host may blur the lines drawn by this classification system.
The studies presented here suggest a mechanism by which invasive strains that synthesize the invasion inhibitors ExoS and ExoT can invade epithelial cells. By this mechanism, LasA and LasB directly or indirectly decrease the levels of these toxins. Indirect mechanisms could include increased turnover of proteins involved in regulation of their synthesis or their transport into target cells. The proposed model might also explain the marked variability in the cytotoxic capacity of cytotoxic P. aeruginosa strains (those strains that encode ExoU and ExoT, but not ExoS), and the significant inverse correlation between invasion and cytotoxic capacity (Fleiszig et al., 1996). For these strains, ExoU is required for acute cytotoxicity and ExoT for invasion inhibition. Less cytotoxic and more invasive ExoU/ExoT-encoding strains might be those with greater elastase activity, leading to lower steady-state levels of ExoU and ExoT, and therefore reduced cytotoxic/invasion inhibitory activity. Indeed, published protein profiles confirm lower steady-state levels of these toxins with these strains (Fleiszig et al., 1997a
; Finck-Barbançon et al., 1997
). How these findings might relate to effects of P. aeruginosa on mammalian cells in vivo is yet to be explored.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Azghani, A. O. (1996). Pseudomonas aeruginosa and epithelial permeability: role of virulence factors elastase and exotoxin A. Am J Resp Cell Mol Biol 15, 132140.[Abstract]
Braun, P., Tommassen, J. & Filloux, A. (1996). Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa. Mol Microbiol 19, 297306.[Medline]
Calfee, M. W., Coleman, J. P. & Pesci, E. C. (2001). Interference with Pseudomonas quinolone signal synthesis inhibits virulence factor expression by Pseudomonas aeruginosa. Proc Natl Acad Sci 98, 1163311637.
Carmeli, Y., Troillet, N., Karchmer, A. W. & Samore, M. J. (1999). Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa. Arch Intern Med 159, 127132.
Cheng, L. W. & Schneewind, O. (2000). Type III machines of Gram-negative bacteria: delivering the goods. Trends Microbiol 8, 214220.[CrossRef][Medline]
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, 36253630.
Cornelis, G. R. & Van Gijsegem, F. (2000). Assembly and function of type III secretory systems. Annu Rev Microbiol 54, 735774.[CrossRef][Medline]
Cowan, C., Jones, H. A., Kaya, Y. H., Perry, R. D. & Straley, S. C. (2000). Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin. Infect Immun 68, 45234530.
Cowell, B. A., Wu, C. & Fleiszig, S. M. J. (1999). Use of an animal model in studies of bacterial corneal infection. Inst Lab Animal Res J 40, 4350.
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, 403406.
Cowell, B. A., Weissman, B. A., Yeung, K. K., Johnson, L., Ho, S., Van, R., Bruckner, D., Mondino, B. & Fleiszig, S. M. J. (2003). Phenotype of Pseudomonas aeruginosa isolates causing corneal infection between 1997 and 2000. Cornea 22, 131134.[CrossRef][Medline]
Finck-Barbançon, V., Goranson, J., Zhu, L., Wiener-Kronish, J. P., Fleiszig, S. M. J., Wu, C., Mende-Mueller, L. & Frank, D. W. (1997). ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol Microbiol 25, 547557.[Medline]
Fleiszig, S. M. J., Zaidi, T. S. & Pier, G. B. (1995). Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro. Infect Immun 63, 40724077.[Abstract]
Fleiszig, S. M. J., Zaidi, T. S., Preston, M. J., Grout, M., Evans, D. J. & Pier, G. B. (1996). Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa. Infect Immun 64, 22882294.[Abstract]
Fleiszig, S. M. J., Wiener-Kronish, J. P., Miyazaki, H., Vallas, V., Mostov, K. E., Kanada, D., Sawa, T., Yen, T. S. B. & Frank, D. W. (1997a). Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun 65, 579586.[Abstract]
Fleiszig, S. M. J., Evans, D. J., Do, N., Vallas, V., Shin, S. & Mostov, K. E. (1997b). Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity. Infect Immun 65, 28612867.[Abstract]
Fleiszig, S. M. J., Arora, S. K., Van, R. & Ramphal, R. (2001). FlhA, a component of the flagellum assembly apparatus of Pseudomonas aeruginosa, plays a role in internalization by corneal epithelial cells. Infect Immun 69, 49314937.
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, 71007113.
Gustin, J. K. (1998). Analysis of the Pseudomonas aeruginosa LasA protease. PhD Dissertation, University of Tennessee, Memphis, TN.
Ha, U. & Jin, S. (2001). Growth phase-dependent invasion of Pseudomonas aeruginosa and its survival within HeLa cells. Infect Immun 69, 43984406.
Kadurugamuwa, J. L. & Beveridge, T. J. (1997). Natural release of virulence factors in membrane vesicles by Pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release. J Antimicrob Chemother 40, 615621.[Abstract]
Kamath, S., Kapatral, V. & Chakrabarty, A. M. (1998). Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis. Mol Microbiol 30, 933941.[CrossRef][Medline]
Kessler, E. & Safrin, M. (1994). The propeptide of Pseudomonas aeruginosa elastase acts as an elastase inhibitor. J Biol Chem 269, 2272622731.
Kessler, E., Safrin, M., Olson, J. C. & Ohman, D. E. (1993). Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease. J Biol Chem 268, 75037508.
Kessler, E., Safrin, M., Abrams, W. R., Rosenbloom, J. & Ohman, D. E. (1997). Inhibitors and specificity of Pseudomonas aeruginosa LasA. J Biol Chem 272, 98849889.
Kessler, E., Safrin, M., Gustin, J. K. & Ohman, D. E. (1998). Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 273, 3022530231.
Kudoh, I., Wiener-Kronish, J. P., Hashimoto, S., Pittet, J.-F. & Frank, D. (1994). Exoproduct secretions of Pseudomonas aeruginosa strains influence severity of alveolar epithelial injury. Am J Physiol 267, L551556.[Medline]
Kulich, S. M., Frank, D. W. & Barbieri, J. T. (1993). Purification and characterization of recombinant exoenzyme S from Pseudomonas aeruginosa 388. Infect Immun 61, 307313.[Abstract]
McCarthy, M. (2000). Pseudomonas genome reveals a formidable foe. Lancet 356, 918.[Medline]
McIver, K. S., Kessler, E., Olson, J. C. & Ohman, D. E. (1995). The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol 18, 877889.[CrossRef][Medline]
O'Brien, T. & Hazlett, L. (1996). Pathogenesis of ocular microbial infection. In Ocular Infection and Immunity, pp. 200214. Edited by J. Pepose, G. Holland & K. Wilhelmus. Baltimore, MD: Mosby Year Book.
Pesci, E. C., Pearson, J. P., Seed, P. C. & Iglewski, B. H. (1997). Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179, 31273132.[Abstract]
Pier, G. B., Grout, M., Zaidi, T. S., Olsen, J. C., Johnson, L. G., Yankaskas, J. R. & Goldberg, J. B. (1996). Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 271, 6467.[Abstract]
Pillar, C. M., Hazlett, L. D. & Hobden, J. A. (2000). Alkaline protease-deficient mutants of Pseudomonas aeruginosa are virulent in the eye. Curr Eye Res 21, 730739.[CrossRef][Medline]
Stover, C. K., Pham, X. Q., Erwin, A. L. & 28 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959964.[CrossRef][Medline]
Tang, H. B., Dimango, E., Brian, M. J., Gambello, M. J., Iglewski, B. J., Goldberg, J. B. & Prince, A. (1996). Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infect Immun 64, 3743.[Abstract]
Toder, D. S., Gambello, M. J. & Iglewski, B. H. (1991). Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR. Mol Microbiol 5, 20032010.[Medline]
Twining, S. S. (1984). Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal Biochem 143, 3034.[Medline]
Twining, S. S., Kirschner, S. E., Mahnke, L. A. & Frank, D. W. (1993). Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. Invest Ophthalmol Vis Sci 34, 26992712.[Abstract]
Vogel, H. J. & Bonner, D. M. (1956). Acetylornithase of Escherichia coli: partial purification and some properties. J Biol Chem 218, 97106.
Yahr, T. L., Barbieri, J. T. & Frank, D. W. (1996). Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. J Bact 178, 14121419.[Abstract]
Yahr, T. L., Mende-Mueller, L. M., Friese, M. B. & Frank, D. W. (1997). Identification of type III secreted products of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol 179, 71657168.[Abstract]
Zaidi, T. S., Fleiszig, S. M. J., Preston, M. J., Goldberg, J. B. & Pier, G. B. (1996). Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa. Invest Ophthalmol Vis Sci 37, 976986.[Abstract]
Zhu, H., Thuruthyil, S. J., Kjelleberg, S., Rice, S., Givskov, M. & Willcox, M. D. P. (2001). Contribution of quorum-sensing systems to the virulence of Pseudomonas aeruginosa during corneal infections. Invest Ophthalmol Vis Sci 42, S514.
Received 4 February 2003;
revised 14 April 2003;
accepted 29 April 2003.