Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA1
Biocentrum, Bldg 301, Technical University of Denmark, DK-2800 Lyngby, Denmark2
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA3
Department of Gastrointestinal Infections, Statens Serum Institut, DK 2300 Copenhagen, Denmark4
Department of Microbiology, University of Virginia, Health Sciences Center, Charlottesville, VA 22908, USA5
Author for correspondence: Paul S. Cohen. Tel: +1 401 874 5920. Fax: +1 401 874 2202. e-mail: pco1697u{at}postoffice.uri.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: monopalmitoylphosphatidic acid, cystic fibrosis
Abbreviations: BHL, N-butanoyl homoserine lactone; GFP, green fluorescent protein; LB, Luria broth; LPS, lipopolysaccharide; MPPA, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (also known as monopalmitoylphosphatidic acid); OdDHL, N-(3-oxododecanoyl) homoserine lactone
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The expression of elastase, as well as the expression of a large number of other virulence factors, by P. aeruginosa is regulated by quorum sensing (Van Delden & Iglewski, 1998 ). In P. aeruginosa there are at least two different quorum-sensing systems, las (Gambello & Iglewski, 1991
) and rhl (Ochsner & Reiser, 1995
), which consist of two signal-generating synthetases (LasI/RhlI) and two cognate transcriptional regulators (LasR/RhlR). The major products of the peptides LasI and RhlI are the autoinducers N-(3-oxododecanoyl) homoserine lactone (OdDHL; also referred to as 3OC12-HSL or PAI-1) (Pearson et al., 1994
) and N-butanoyl homoserine lactone (BHL; also referred to as C4-HSL or PAI-2) (Pearson et al., 1995
; Winson et al., 1995
), respectively. These two quorum-sensing systems have been shown to be involved in biofilm differentiation (Davies et al., 1998
). Biofilm differentiation plays an important role in the protection of P. aeruginosa from the host-defence system and from the action of antibiotics (Costerton et al., 1987
). In addition, OdDHL interferes with the host immune system, where it specifically down-regulates the production of the cytokines IL-12 and TNF-
which support the bactericidal Th-1 milieu and protect the host, a finding which has to led the suggestion that the signal molecule is also an important virulence factor (Telford et al., 1998
).
In addition to being regulated by quorum-sensing systems, Pseudomonas spp. virulence factors are produced in response to a variety of environmental factors. For example, several of the extracellular virulence factors accumulate to maximal levels only under conditions of low iron in the growth medium, e.g. the siderophores pyochelin and pyoverdin, exotoxin A, and the exoproteases elastase and alkaline protease (Cox, 1993 ; Vasil & Ochsner, 1999
). In addition, zinc has been shown to enhance pyoverdin production (Höfte et al., 1993
; Rossbach et al., 2000
), and zinc and calcium have been shown to be important for the efficient production and processing of elastase and the LasA protease (Brumlik & Storey, 1992
; Olson & Ohman, 1992
). Evidence also exists suggesting that limiting phosphate availability may also be an environmental signal in inducing phospholipase C synthesis (Shortridge et al., 1992
).
In the present study, evidence is presented which indicates that a specific lysophospholipid, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate [also called monopalmitoylphosphatidic acid (MPPA)], which is generated by the secretory phospholipase A2 and accumulates in inflammatory exudates (Fourcade et al., 1998 ; Paya et al., 1996
), inhibits the extracellular accumulation of alginate, pyoverdin, elastase and LasA protease in cultures of P. aeruginosa PAO1 grown in Luria broth (LB).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Determination of the zinc and iron content of the LB and the LB containing MPPA.
The zinc and iron contents of the LB and the LB containing MPPA (80 µM) were determined by atomic absorption (CEIMIC). The zinc and iron concentrations in LB lot 98H8213 were 10·3 and 7·2 µM, respectively, and in lot 109H8206 they were 10·6 and 7·2 µM, respectively. In the LB (lot 98H8213) containing 80 µM MPPA, the zinc and iron concentrations were 5·7 and 3·6 µM, respectively.
Culture conditions and growth measurements.
P. aeruginosa PAO1 was grown overnight in a rotary-shaking water bath at 37 °C to about 109 c.f.u. ml-1. The next day, P. aeruginosa PAO1 was inoculated at 103 c.f.u. ml-1 into culture tubes (15 mmx100 mm) containing 2 ml aliquots of LB or 2 ml aliquots of LB containing MPPA. Cultures were incubated by standing them in a water bath at 37 °C for periods of up to 48 h, as required. Growth was assayed by optical density measurements at 500 nm and, where indicated, by viable counts. In all experiments in which green fluorescent protein (GFP) synthesis was measured, P. aeruginosa PAO1 containing the plasmid pMRP17R-, which expresses the GFP, was grown in the presence of carbenicillin (60 µg ml-1), which prevents loss of the plasmid.
Quantification of GFP in liquid cultures.
Expression of lasB::gfp was measured by detection of the green fluorescence with a Perkin Elmer LS-5B Luminescence Spectrometer (470 nm excitation wavelength; 515 nm emission wavelength). At the time of measurement, the background fluorescence emitted by un-inoculated LB was used as a control, and this value (routinely about 50) was subtracted from the subsequent sample values. Cultures were diluted fivefold in LB; the fluorescence emitted by these diluted cultures of P. aeruginosa PAO1(pMRP17R-) was routinely about 250. The background fluorescence measured in fivefold-diluted LB-grown cultures of P. aeruginosa PAO1 without pMRP17R- was routinely about 50. In each experiment, fluorescence measurements of a fivefold-diluted 48 h LB-grown culture of P. aeruginosa PAO1(pMRP17R-) diluted additionally over a 20-fold range generated a linear standard curve. This curve was used to calculate the relative amounts of fluorescence in each of the remaining cultures within that experiment.
Assays for pyoverdin and proteolytic activity in culture supernatants.
Culture supernatants for assays of proteolytic activity and the presence of pyoverdin were prepared by centrifugation (16000 g for 10 min at room temperature). The relative amounts of pyoverdin present in culture supernatants were measured at A380 (Haas et al., 1991 ). The proteolytic activity of the culture supernatants was measured on nutrient broth casein agar plates [Nutrient agar (Difco) plus 1% skim milk powder] containing 100 µg streptomycin sulfate ml-1 (to prevent growth of residual bacteria). Supernatant samples (50 µl) were added to wells within the plates, which had been made by removing agar plugs (6·5 mm diameter). The plates were incubated for 24 h at 37 °C, after which time the diameter of the zone of casein hydrolysis was measured. To compare the relative amounts of proteolytic activity within the culture supernatants (on the basis of 50 µl aliquots) from each experiment, a standard curve of proteolytic activity was generated. This was done by diluting a fresh LB-grown culture supernatant with LB such that the diameters (mm) of the zones of casein hydrolysis obtained from 50 µl aliquots of the diluted LB-grown culture related to proteolytic activities of 100, 80, 60, 40, 20, 10, 5 and 2·5%. Standard curves for the actual proteolytic activities (mm) versus the log10 of the theoretical activities (%) were linear.
Immunoblot analysis of LasB and LasA.
Washed pellets from 2 ml cultures of P. aeruginosa PAO1 were resuspended in 2 ml of fresh LB and sonicated as described previously (Zhou et al., 1997 ). Sonicates (500 µl) and cell-free filtered (0·22 µm pore size) supernatants (500 µl) were mixed with 100 µl of 6x concentrated sample buffer (0·35 M Tris/HCl, pH 6·8; 10% SDS; 6% 2-mercaptoethanol; 30% (v/v) glycerol; 0·012% bromophenol blue). The mixtures were boiled for 5 min and samples (40 µl) were loaded alongside pre-stained protein standards (BioRad). Samples were separated on 10% acrylamide gels and were transferred to nitrocellulose membranes by standard methods (Gallagher, 1999
; Gallagher et al., 1997
). Blots were treated with 1:8000 dilutions of either rabbit anti-elastase serum (Olson & Ohman, 1992
) or rat anti-LasA serum (Olson & Ohman, 1992
). The chemiluminescent detection of LasB or LasA was performed using the appropriate secondary antibody and the protocol supplied with the ECL chemiluminescence Western-blotting kit (Amersham Pharmacia Biotech). Relative amounts of the 33 kDa elastase protein made in the presence and absence of MPPA were determined by densitometry (Molecular Dynamics Personal Densitometer SI).
Extraction of autoinducers.
Autoinducers were isolated from 2 ml static cultures of P. aeruginosa PAO1 grown at 37 °C. The cultures were homogenized by vortexing, and the cells were pelleted by centrifugation. Cell-free supernatants were extracted twice in glass test tubes with 3 ml acidified ethyl acetate (100 µl glacial acid per 100 ml). The solvent was removed by evaporation under a gentle stream of nitrogen gas. The dried residues were re-dissolved in 100 µl of ethanol and stored at -20 °C.
Assay of autoinducer concentration.
The concentrations of the autoinducers in the extracted cultures were determined by a bioluminescent assay. OdDHL and BHL activities were assayed by use of E. coli JM109-based monitor strains harbouring pMH297 and pSB536, respectively (Table 1). Overnight cultures of the autoinducer monitor strains were subcultured at an OD450 of 0·05 in AB minimal medium (Clark & Maaløe, 1967
) supplemented with 0·1% glucose and Casamino acids. The cultures were grown with shaking (200 r.p.m.) at 37 °C. At OD450 0·3, the cultures were split into aliquots and each aliquot was placed in a 5 ml glass test tube. Autoinducer extracts were added (2 µl per 1·0 ml culture), and the cultures were further incubated for 2 h at 37 °C with shaking (200 r.p.m.). A 100 µl sample of each culture was retrieved for measurement of its bioluminescence (1253 Luminometer, Bio-Orbit Oy, Turku, Finland) and its OD450 value. The autoinducer activity was calculated as the specific bioluminescence. The autoinducer concentration in the extracted cultures was determined from a standard curve generated by use of pure autoinducer standards.
Biofilm formation.
This was assessed using the procedure of OToole et al. (1999) . Twenty-four-well Nunclon polystyrene culture plates (Nunc, Roskilde, Denmark) containing 2 ml LB or 2 ml LB containing 80 µM MPPA were inoculated, as described above. Following 28 or 48 h of incubation at 37 °C, the wells were washed four times with LB and stained with crystal violet. After washing to remove excess stain, the remaining crystal violet dye was solubilized with 2·5 ml of 95% ethyl alcohol. The solubilized dye was then diluted twofold and the level of crystal violet recovered was assessed in a spectrophotometer at 580 nm.
Alginate determination.
Ten static 2 ml LB-grown cultures of P. aeruginosa PAO1 (incubated for 48 h at 37 °C) were pooled and added to 60 ml of 0·9% sterile saline. The mixture was vortexed extensively and centrifuged at 10000 g for 12 min at room temperature. The supernatant was saved and the pellet was resuspended in 5 ml of 0·9% sterile saline, vortexed extensively and then centrifuged as above. The first and second supernatants were combined, ethanol precipitated and then assayed for alginate, as described by May & Chakrabarty (1994) . The pellet was assayed for protein (see below) using the Lowry method. Ten static 2 ml P. aeruginosa PAO1 cultures grown in LB containing MPPA (80 µM) were assayed for the presence of alginate and protein in the same manner as the LB-grown cultures.
Protein determinations.
Statically grown 2 ml cultures of P. aeruginosa PAO1 were centrifuged at room temperature for 10 min at 10000 g. The cell pellets were resuspended in 1 ml of saline and were precipitated with 1 ml of 10% (w/v) trichloroacetic acid (TCA). The precipitates were centrifuged at room temperature for 10 min at 10000 g, and were resuspended in 100 µl of 1 M NaOH. Supernatants from the static cultures were filtered free of P. aeruginosa PAO1 (0·22 µm pore-size filters), TCA-precipitated and then resuspended in 1 M NaOH, as described above. Protein determinations of the cell and supernatant fractions were performed using the Lowry method.
Isolation of LPS and immunological detection.
O-serotyping was performed at the Statens Seruminstitut, as described by Liu et al. (1983) . LPS was isolated from 48 h static cultures of P. aeruginosa PAO1, as described previously (Coyne et al., 1994
). LPS samples were analysed for O5 antigen by immunoblotting SDS polyacrylamide gels, as described previously (Coyne et al., 1994
).
Statistics.
Students t-test was used to evaluate the effect of MPPA on P. aeruginosa PAO1 growth, pyoverdin accumulation and exoprotease synthesis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of MPPA on the growth of P. aeruginosa PAO1 and on the extracellular accumulation of pyoverdin and exoprotease
Specific phospholipids enhance the activity of ß-lactam antibiotics against LB-grown P. aeruginosa PAO1 and against LB-grown P. aeruginosa strains isolated from the sputum of cystic fibrosis patients (Krogfelt et al., 2000 ). The most active phospholipid is MPPA (Krogfelt et al., 2000
). Experiments were performed to determine whether MPPA has any effect on P. aeruginosa PAO1 growth and on pyoverdin and exoprotease production. LB-grown cultures of P. aeruginosa PAO1 (2 ml), inoculated at about 103 c.f.u. ml-1, were grown statically at 37 °C for 48 h in the presence of 40 and 80 µM MPPA. The presence of 40 µM MPPA in the growth medium had no effect on P. aeruginosa PAO1 growth nor on the accumulation of extracellular pyoverdin; however, extracellular exoprotease activity was reduced by about 10-fold (P<0·05) (Table 2
). In the presence of 80 µM MPPA, the P. aeruginosa PAO1 doubling time was increased by about 1·5-fold (3·62 h when grown in LB alone compared to 5·33 h when grown in LB+MPPA) (Fig. 1
), the final growth yield (measured at OD500) was reduced by 40% (P<0·001), the extracellular accumulation of pyoverdin was reduced by 67% (P<0·001), and extracellular protease activity was undetectable (Table 2
). In control experiments, MPPA (80 µM) was added to the cell-free supernatants containing exoprotease activity and the mixture was incubated at 37 °C for 24 h. Under these conditions, MPPA had no effect on exoprotease activity, ruling out the possibility that the exoproteases were inactivated by MPPA.
|
|
lasB::gfp expression in the presence of MPPA
To investigate the effect of MPPA on the transcription of lasB, a lasB::gfp transcriptional fusion, containing the regulatory region upstream of lasB (Rust et al., 1996 ; Dr Matthew R. Parsek, personal communication) and supplied to P. aeruginosa PAO1 in trans on pMRP17R- (Table 1
), was used to evaluate the activity of the lasB promoter. The data from one of two experiments performed with essentially identical results are illustrated in Fig. 1
. The LB-grown P. aeruginosa PAO1(pMRP17R-) cultures entered stationary phase about 24 h post-inoculation (Fig. 1a
). The lasB::gfp fusion was expressed in two cycles. The first cycle of lasB::gfp-directed GFP synthesis began between 12 and 16 h post-inoculation, and continued for about 12 h (Fig. 1b
). The second cycle of GFP synthesis began between 36 and 40 h post-inoculation, and stopped between 44 and 48 h post-inoculation (Fig. 1b
). The accumulation of active extracellular elastase followed the pattern of lasB::gfp expression, except that extracellular elastase accumulation lagged several hours behind the start of the first cycle of transcription (compare Fig. 1b
, c
), suggesting either that the lasB::gfp transcriptional fusion does not accurately reflect first cycle lasB transcription or that first cycle lasB transcripts are post-transcriptionally regulated. Post-transcriptional iron regulation of lasB expression has, in fact, been demonstrated (Brumlik & Story, 1998
). In any case, the first cycle of lasB::gfp expression accounted for 40% of the total GFP synthesis and 20% of the total elastase activity (compare Fig. 1b
, c). The second cycle of lasB::gfp expression and the extracellular accumulation of elastase were essentially simultaneous (compare Fig. 1b
, c
).
In the presence of 80 µM MPPA, P. aeruginosa PAO1 also entered stationary phase at about 24 h post-inoculation, and reached a level of growth of about 60% of the stationary-phase level obtained when grown in LB only, as measured at OD500 (Fig. 1a) and by viable counts [48 h incubation in LB alone, c.f.u. ml-1=5·23x108 (±1·70x108, n=4); 48 h incubation in LB+MPPA, c.f.u. ml-1=3·35x108 (±0·64x108, n=4)]. The expression of lasB::gfp, as measured by GFP synthesis, began between 32 and 36 h post-inoculation, and stopped within 4 h at a level 10-fold lower than that observed in cultures grown in LB only (Fig. 1b
), despite a less than twofold decrease in the growth yield (Fig. 1a
). As in previous experiments, in the presence of 80 µM MPPA, active extracellular elastase did not accumulate (Fig. 1c
). Therefore, these data suggest that MPPA in the culture medium resulted in the inhibition of lasB promoter activity, when compared to cultures grown in LB only.
P. aeruginosa PAO1 grown in the presence of 80 µM MPPA synthesizes a low level of inactive elastase
Elastase is synthesized as a 53 kDa pre-proenzyme which is processed to a 51 kDa inactive proenzyme as it translocates across the inner membrane (Iglewski et al., 1990 ; Kessler & Safrin, 1988
). Proelastase is then further processed to its mature 33 kDa form in the periplasm, but remains non-covalently bound to its 18 kDa propeptide and remains inactive (Kessler & Safrin, 1988
; Kessler et al., 1998
). The propeptideelastase complex is then secreted from the periplasm across the outer membrane by the P. aeruginosa type II general Xcp secretion pathway (Martinez et al., 1998
; Tommassen et al., 1992
). Elastase is activated extracellularly by degradation of the propeptide (Kessler et al., 1998
).
To examine the effect of MPPA on translational and processing events, the relative amounts of intracellular and extracellular elastase in 48 h static P. aeruginosa PAO1 cultures incubated in the presence and absence of MPPA (80 µM) were determined by immunoblotting. Neither intracellular nor extracellular unprocessed pre-proelastase (53 kDa) and proelastase (51 kDa) were detected in control P. aeruginosa PAO1 LB-grown cultures or in cultures grown in the presence of 80 µM MPPA (data not shown). In 48 h LB-grown cultures, the intracellular level of the 33 kDa elastase protein was about 40% that of the extracellular level (determined by densitometry, also compare Fig. 2a, c
). For cultures grown in the presence of MPPA (80 µM), intracellular levels of the 33 kDa elastase protein were found to be about 7% those of the intracellular levels detected in cultures grown in LB (determined by densitometry, also compare Fig. 2ac
). When corrected for differences in growth, the intracellular levels of the 33 kDa elastase protein in the presence of MPPA were 11·7% of those detected in the LB-grown cultures. Extracellular 33 kDa elastase protein was also detected in cultures grown in the presence of 80 µM MPPA, but at levels of about 6% those detected in supernatants of cultures grown in LB (determined by densitometry, also compare Fig. 2b
, c
), 10% when corrected for differences in growth. Thus, it appears that 80 µM MPPA inhibited both the intracellular and extracellular accumulation of the 33 kDa elastase protein by about 10-fold. In addition, it appears that the 33 kDa elastase present in MPPA supernatants was present in an inactive form.
|
To this point, these data suggest that cells grown in the presence of MPPA (80 µM) show reduced lasB transcription and correspondingly reduced levels of elastase accumulation. Overall this appears to result in the production of low levels of inactive elastase, which can be activated under the appropriate conditions.
LasA synthesis in the presence of MPPA
LasA, a second P. aeruginosa PAO1 zinc metalloprotease, is thought to be selective for Gly-Ala peptide bonds within Gly-Gly-Ala sequences in elastin (Kessler et al., 1997 ), and it is required for maximal elastase activity on elastin (Kessler et al., 1997
). LasA synthesis is positively regulated by quorum sensing (Brint & Ohman, 1996
; Passador et al., 1993
). LasA is synthesized as a 42 kDa proenzyme that is processed extracellularly via a transient 28 kDa intermediate and a 14 kDa propeptide fragment (Kessler et al., 1998
). The 28 kDa intermediate is then further processed to the mature 20 kDa LasA protein (Kessler et al., 1998
). The relative amounts of intracellular and extracellular LasA protein present in 48 h LB-grown cultures of P. aeruginosa PAO1 and in cultures incubated in the presence of MPPA (80 µM) were determined by immunoblotting, using antibodies to LasA. Although 48 h LB-grown culture supernatants contained amounts of the 20 kDa mature LasA protein that could be diluted fourfold and still be within the detection limit for the immunoblotting procedure, the 20 kDa mature LasA protein was not found in P. aeruginosa PAO1 MPPA cell extracts nor in culture supernatants (Fig. 3
). Neither the 42 kDa proenzyme, the 28 kDa intermediate nor the 14 kDa propeptide fragment were found in 48 h LB-grown P. aeruginosa PAO1 culture cell extracts or in their culture supernatants, neither were they found in the MPPA P. aeruginosa PAO1 culture cell extracts or in their culture supernatants (data not shown). It would appear, therefore, that in addition to inhibiting the intracellular and extracellular accumulation of elastase, MPPA also inhibited the intracellular and extracellular accumulation of the LasA protease.
|
|
O-antigen synthesis in the presence of MPPA
P. aeruginosa strains isolated from cystic fibrosis patients produce reduced levels of exoproteases (Luzar & Montie, 1985 ) and O-antigen (Hancock et al., 1983
). Since MPPA inhibited the accumulation of pyoverdin, elastase, LasA and alginate, it was of interest to determine whether growth of P. aeruginosa PAO1 in the presence of 80 µM MPPA had an effect on the O-serotype or on O-antigen synthesis. P. aeruginosa PAO1 grown in static 48 h LB cultures and in LB cultures containing 80 µM MPPA were tested with 20 polyclonal sera specific to each of the 20 international antigenic typing system (IATS) O-serotypes of P. aeruginosa. When grown in either the presence or the absence of MPPA, P. aeruginosa PAO1 serotyped as O2/O5. LPS was also purified from P. aeruginosa PAO1 cells grown in the presence and the absence of 80 µM MPPA. This was examined for O5 antigen by immunoblotting. No major difference was observed in the amount of O5 antigen produced by P. aeruginosa PAO1 when grown in the presence or in the absence of MPPA (data not shown).
Autoinducer synthesis in the presence of MPPA
The data presented above indicate that MPPA has a broad range of inhibitory effects relating to the production of virulence factors by P. aeruginosa PAO1. Since the synthesis of elastase and a number of other factors are believed to be regulated by quorum sensing, we speculated that differences in the concentration of signal molecules in the presence and the absence of MPPA could, at least in part, account for the observed differences in the extracellular accumulation of virulence factors. To examine this possibility, signal molecules were extracted at 4 h intervals between 12 and 48 h post-inoculation from 2 ml cultures of P. aeruginosa PAO1 grown statically in LB and in LB containing 80 µM MPPA. The concentration of the signal molecules was estimated by means of OdDHL- and BHL-specific indicator bacteria which expressed bioluminescence in a concentration-dependent manner. The data from one of two experiments performed with essentially identical results are illustrated in Fig. 4. Extracellular BHL began to accumulate between 12 and 16 h post-inoculation in LB-grown cultures, and accumulation peaked at about 20 h post-inoculation. The BHL level dropped thereafter (Fig. 4
). Extracellular OdDHL began to accumulate between 16 and 20 h post-inoculation in LB-grown cultures, and its accumulation levelled off at about 28 h post-inoculation (Fig. 4
). In the presence of 80 µM MPPA, the extracellular levels of both OdDHL and BHL reached LB-grown culture levels; however, the extracellular appearance of each lactone was delayed by about 8 h, relative to the LB control (Fig. 4
). Therefore, enough OdDHL and BHL accumulated in P. aeruginosa PAO1 cultures grown in the presence of MPPA to stimulate lasB::gfp transcription. The failure of OdDHL and BHL to do so suggests either that these signal molecules are unable to re-enter P. aeruginosa PAO1 cells grown in the presence of 80 µM MPPA or that lasB::gfp transcription is inhibited because other regulators required for lasB expression are affected by MPPA.
|
RpoS plays no role in the MPPA-mediated inhibition of the accumulation of alginate, pyoverdin and elastase
RpoS, the stationary phase factor, regulates the expression of a number of P. aeruginosa PAO1 genes, the products of which confer increased tolerance to various forms of stress (Suh et al., 1999
). Expression of the P. aeruginosa PAO1 rpoS gene increases as cultures reach stationary phase (Whiteley et al., 2000
). Moreover, P. aeruginosa PAO1 rpoS mutants have been reported to accumulate increased levels of pyoverdin, pyocyanin and BHL (Suh et al., 1999
; Whiteley et al., 2000
). To determine whether the expression of rpoS was involved in the MPPA-induced inhibition of virulence factor production, P. aeruginosa PAO-MW20, a P. aeruginosa PAO1 rpoS mutant, was grown in 2 ml static cultures for 48 h and the effects of 80 µM MPPA on growth and on alginate, pyoverdin and elastase production were determined. Although PAO-MW20 appeared to be less susceptible to the growth-inhibiting effects of MPPA (LB, OD500=1·50±0·08, n=5; MPPA, OD500=1·33±0·05, n=5), pyoverdin production was inhibited by about 65% (LB, A380=3·12±0·22, n=5; MPPA, A380=1·07±0·08, n=5), alginate production was reduced by more than 10-fold [LB, 44·9 (experiment 1) and 68·3 (experiment 2); MPPA, 0·59 (experiment 1) and 6·1 (experiment 2); results expressed as µg alginate (mg cell protein)-1] and elastase was essentially completely inhibited (LB diameter of zone of casein hydrolysis, 22·5±0·39 mm, n=5; MPPA, no detectable activity, n=5; MPPA upon activation, about 2% of LB activity, i.e. 9·06±0·05 mm, n=5). While the data suggest a role for rpoS expression in limiting the growth of P. aeruginosa PAO1 in the presence of MPPA, it would appear that rpoS expression plays no role in the MPPA-mediated inhibition of pyoverdin, elastase and alginate accumulation.
Effect of the addition of calcium, iron, magnesium and zinc to LB containing MPPA
MPPA binds divalent cations in LB (Krogfelt et al., 2000 ). LB contains 10·4 µM zinc (see Methods), 7·2 µM iron (see Methods), 263 µM calcium (Krogfelt et al., 2000
) and 225 µM magnesium (Krogfelt et al., 2000
). In the presence of 80 µM MPPA, the zinc concentration in LB is reduced to 5·7 µM (see Methods), the calcium concentration is reduced to 167 µM (Krogfelt et al., 2000
), the magnesium concentration is reduced to 128 µM (Krogfelt et al., 2000
) and the iron concentration is reduced to 3·6 µM (see Methods). In the presence of 80 µM MPPA, the transcription of lasB::gfp, as measured by GFP synthesis, was reduced to about 38% of the LB culture control level when corrected for growth, pyoverdin accumulation was reduced to 55% of the LB level and elastase was undetectable (Table 4
). When LB containing 80 µM MPPA was supplemented with ferrous sulfate (10 µM), calcium chloride (100 µM), magnesium chloride (100 µM) and zinc chloride (100 µM), P. aeruginosa PAO1 growth levels returned to LB levels, pyoverdin accumulated at about 75% of the LB levels, lasB::gfp expression was about 90% of the LB levels and the extracellular accumulation of active elastase returned to the LB levels (Table 4
). It therefore appears that the effects of MPPA on P. aeruginosa PAO1 can be reversed to a large extent by supplementing the medium with divalent cations.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies have shown that a variety of ions are involved in regulating virulence factor gene expression and in the efficient production and processing of virulence factors in P. aeruginosa (Brumlick & Storey, 1992 ; Cox, 1993
; Hofte et al., 1993
; Olson & Ohman, 1992
; Rossbach et al., 2000
; Vasil & Ochsner, 1999
). In addition, MPPA has been shown to bind divalent cations (Krogfelt et al., 2000
). In LB containing 80 µM MPPA, the calcium, iron, magnesium and zinc concentrations are reduced by 5060% relative to the concentrations of the same divalent cations in LB (see Methods and Krogfelt et al., 2000
). Replenishing MPPA cultures with magnesium, calcium, iron and zinc restored their growth, their lasB::gfp expression and their extracellular accumulation of elastase and pyoverdin to levels close to those of the LB-grown cultures (Table 4
), suggesting that the effects of MPPA are due, at least in part, to its ability to bind divalent cations.
EDTA (125 µM) also inhibited lasB::gfp expression and the accumulation of extracellular elastase (Table 5); however, unlike MPPA, EDTA stimulated rather than inhibited the extracellular accumulation of pyoverdin. EDTA chelates zinc about 102-fold better than it does iron (Fe2+), about 106-fold better than it does calcium and about 108-fold better than it does magnesium (Hodgins, 1961
). Therefore, at an EDTA concentration of 125 µM it would be expected that all of the zinc (10·4 µM) and iron (7·2 µM), about 45% of the calcium (117 µM) and none of the magnesium would be chelated. It might therefore be that the inhibition of pyoverdin accumulation in the presence of MPPA, but not in the presence of EDTA, is due to the differences in the cation concentrations in the two culture media. A detailed study of the effect of individual divalent cation additions to LB containing either MPPA or EDTA on the accumulation of P. aeruginosa PAO1 extracellular virulence factors will be the subject of a future communication.
While it appears that divalent cations can reverse or compensate for the inhibitory effects of MPPA, since lysophosphatidic acid is known to insert into membranes (Christiansen & Carlsen, 1983 ), the possibility that MPPA may inhibit the accumulation of extracellular virulence factors by physically disrupting the P. aeruginosa PAO1 membrane structure cannot be excluded. Such membrane disruption could affect cellular secretory processes in general or alter the response of the bacterium to environmental signals.
The transcription of lasB is positively regulated by RpoS, and by the lasR lasI and rhlR rhlI quorum-sensing systems at high cell densities (Brint & Ohman, 1996 ; Gambello & Iglewski, 1991
; Passador et al., 1993
; Pearson et al., 1994
; Suh et al., 1999
; Whiteley et al., 1999
, 2000
). However, the inhibition of lasB expression by MPPA can not be explained by the small reduction in P. aeruginosa PAO1 growth in the presence of MPPA, since increasing the cell density did not reverse the effects of MPPA. Neither can it be explained by the amounts of OdDHL and BHL synthesized, since, in the presence of MPPA, OdDHL and BHL accumulation in culture supernatants was normal (Fig. 4
). It is possible, however, that in the presence of MPPA, the signal molecules were unable to re-enter P. aeruginosa PAO1. Although there is, as yet, no evidence to support this hypothesis, if true, OdDHL and BHL would not be able to interact with their cognate R-proteins. As a consequence, the signals could fail to fully activate the transcription of their target genes. Experiments designed to test this hypothesis are presently under way. Similarly, it appears that the effect of MPPA on the production of P. aeruginosa PAO1 virulence factors does not involve rpoS expression, since in PAO-MW20 cultures incubated in the presence of MPPA, the levels of pyoverdin, elastase and alginate production were all inhibited to the same extent as in P. aeruginosa PAO1 cultures.
At the present time, it is unclear as to why the elastase protein accumulates to a level of only about 10% of that seen in LB in the presence of MPPA (Fig. 2) when the lasB::gfp fusion is expressed at a level of 38% of that observed in LB (Table 4
). However, it should be noted that the lasB::gfp fusion used here is a transcriptional fusion that may not accurately reflect the degree to which the rate of transcription of the wild-type lasB gene in the presence of MPPA is reduced relative to its absence or the rate at which it is translated. Nevertheless, the data show that the lasB promoter is about threefold less active when P. aeruginosa PAO1 is grown in the presence of MPPA than when it is grown in LB only.
Our interest in MPPA originated from the finding that it enhances the activity of ß-lactam antibiotics against P. aeruginosa PAO1 and against P. aeruginosa strains isolated from cystic fibrosis patients (Krogfelt et al., 2000 ). MPPA is a member of a class of phospholipids called lysophosphatidic acids, which contain one fatty acid esterified to the C1 atom of the glycerol moiety, but which are lacking the fatty acid normally esterified to the C2 atom of the glycerol moiety. Lysophosphatidic acids are believed to be generated from the phosphatidic acid present in the inner leaflet of damaged cell membranes (Fourcade et al., 1998
) by secretory phospholipase A2, which accumulates in inflammatory exudates (Paya et al., 1996
). Lysophosphatidic acids have recently been implicated in blocking neutrophil recruitment to damaged lung tissue (Abraham et al., 1995
) and in inhibiting the metabolic burst of human neutrophils (Chettibi et al., 1994
). MPPA may therefore be viewed as a natural anti-inflammatory agent.
It has been suggested that during the initial stages of infection with P. aeruginosa, antibodies to O-antigen, extracellular proteases and other virulence factors are synthesized, and these then interact with their cognate antigens to form immune complexes in the lungs of cystic fibrosis patients (Kronborg, 1995 ). The immune complexes, in turn, attract polymorphonuclear leukocytes (PMNLs) (Kronborg, 1995
). The PMNLs then release elastase, cathepsin, oxygen radicals, etc., which leads to the initial inflammation and tissue damage (Kronborg, 1995
; Kronborg et al., 1992
). Later on, when chronic infection sets in and P. aeruginosa is protected against phagocytosis by biofilm formation, PMNLs continue to infiltrate the lungs and, in attempting to eradicate the infection, cause further inflammation and tissue damage (Kronborg, 1995
; Kronborg et al., 1992
). Therefore, in infected cystic fibrosis patients, when the lung is relatively healthy, it appears likely that P. aeruginosa would synthesize high levels of pyoverdin, elastase and LasA protease, thereby initiating inflammation and tissue damage. Extensive tissue damage would lead to increased levels of lysophosphatidic acids in inflammatory exudates which might lower the local divalent cation concentrations. Low divalent cation concentrations could then result in inhibition of the extracellular accumulation of P. aeruginosa pyoverdin, elastase and the LasA protease, in the inhibition of the further recruitment of PMNLs to the site of infection and in the prevention of the metabolic burst of those PMNLs that are already present. When the tissue is repaired and the lysophosphatidic acid concentration is reduced, the synthesis of pyoverdin, elastase and the LasA protease would resume, PMNLs would be recruited and the cycle would repeat. If this scenario is correct, it may be that treating cystic fibrosis patients with MPPA will limit the deleterious effects of chronic infection by reducing inflammation and by limiting the extracellular accumulation of P. aeruginosa pyoverdin, elastase and the LasA protease. In addition, since MPPA inhibits the accumulation of alginate, it might reduce P. aeruginosa biofilm formation in the lung, making the infection more amenable to antibiotic treatment. Furthermore, if high enough concentrations of MPPA can be achieved in the lungs, its ability to enhance the activities of ß-lactam antibiotics (Krogfelt et al., 2000
) might help to limit infection. However, it should be noted that one limiting factor in the use of MPPA in the lungs may be that the concentration of calcium in the sputum of cystic fibrosis patients is 0·7 mM, about 2·5-fold higher than in LB, and the sputum concentration of magnesium is 1·2 mM, about 5·3-fold higher than in LB (Halmerbauer et al., 2000
). Animal experiments, presently in progress, should tell us whether MPPA shows clinical promise.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baltimore, R. S. (1993). Mucoid colony variants: the exopolysaccharide of Pseudomonas aeruginosa and microcolony formation. In Pseudomonas aeruginosa the Opportunist: Pathogenesis and Disease, pp. 2640. Edited by R. B. Fick, Jr. Boca Raton, FL: CRC Press.
Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. (1983). Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5, 279-313.[Medline]
Brint, J. M. & Ohman, D. E. (1996). Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlRRhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxRLuxI family. J Bacteriol 177, 7155-7163.[Abstract]
Brumlik, M. J. & Storey, D. G. (1992). Zinc and iron regulate translation of the gene encoding Pseudomonas aeruginosa elastase. Mol Microbiol 6, 337-344.[Medline]
Brumlik, M. J. & Storey, D. G. (1998). Post-transcriptional control of Pseudomonas aeruginosa lasB expression involves the 5' untranslated region of the mRNA. FEMS Microbiol Lett 159, 233-239.[Medline]
Charlton, T. S., de Nys, R., Netting, A., Kumar, N., Hentzer, M., Givskov, M. & Kjelleberg, S. (2000). A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. Environ Microbiol 2, 530-541.[Medline]
Chettibi, S., Sawrence, A. J., Stevenson, R. D. & Young, J. D. (1994). Effect of lysophosphatidic acid on motility, polarisation, and metabolic burst of human neutrophils. FEMS Immunol Med Microbiol 8, 271-281.[Medline]
Christiansen, K. & Carlsen, J. (1983). Reconstitution of a protein into lipid vesicles using natural detergents. Biochim Biophys Acta 735, 225-233.[Medline]
Clark, D. J. & Maaløe, O. (1967). DNA replication and the division cycle in Escherichia coli. J Mol Biol 23, 99-112.
Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M. & Marrie, T. J. (1987). Bacterial biofilms in nature and disease. Annu Rev Microbiol 41, 435-464.[Medline]
Cox, C. D. (1993). Iron and the virulence of Pseudomonas aeruginosa. In Pseudomonas aeruginosa the Opportunist: Pathogenesis and Disease, pp. 4158. Edited by R. B. Fick, Jr. Boca Raton, FL: CRC Press.
Coyne, M. J.Jr, Russell, K. S., Coyle, C. L. & Goldberg, J. B. (1994). The Pseudomonas aeruginosa algC gene encodes phosphoglucomutase, required for the synthesis of a complete lipopolysaccharide core. J Bacteriol 176, 3500-3507.[Abstract]
Cryz, S. J.Jr, Pitt, T. L., Furer, E. & Germanier, R. (1984). Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa. Infect Immun 44, 508-513.[Medline]
Davies, D., Parsek, M., Pearson, J., Iglewski, B., Costerton, J. & Greenberg, E. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295-298.
Ediger, T. L. & Towes, M. L. (2001). Dual effects of lysophosphatidic acid on human airway smooth muscle cell proliferation and survival. Biochim Biophys Acta 1531, 59-67.[Medline]
Fourcade, O., Le Balle, F., Fauvel, J., Simon, M. F. & Chap, H. (1998). Regulation of secretory type-II phospholipase A2 and of lysophosphatidic acid synthesis. Adv Enzyme Regul 38, 99-107.[Medline]
Gallagher, S. R. (1999). One-dimensional SDS gel electrophoresis of proteins. In Current Protocols in Molecular Biology, pp. 10.2A.110.2A.34. New York: Wiley.
Gallagher, S., Winston, S. E., Fuller, S. A. & Hurrell, J. G. R. (1997). Immunoblotting and immunodetection. In Current Protocols in Molecular Biology, pp. 10.8.110.8.21. New York: Wiley.
Gambello, M. J. & Iglewski, B. H. (1991). Cloning and characterization of the Pseudomonas aeruginosa lasR gene: a transcriptional activator of elastase expression. J Bacteriol 173, 3000-3009.[Medline]
Haas, B., Kraut, J., Marks, J., Zanker, S. C. & Castignetti, D. (1991). Siderophore presence in sputa of cystic fibrosis patents. Infect Immun 59, 3997-4000.[Medline]
Halmerbauer, G., Arri, S., Schierl, M., Strauch, E. & Koller, D. Y. (2000). The relationship of eosinophil granule proteins to ions in the sputum of patients with cystic fibrosis. Clin Exp Allergy 30, 1771-1776.[Medline]
Hancock, R. E. W., Mutharia, L. M., Chan, L., Darveau, R. P., Speert, D. P. & Pier, G. B. (1983). Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains. Infect Immun 42, 170-177.[Medline]
Hodgins, G. R. (1961). Chelating agents. In Handbook of Chemistry and Physics , pp. 1476-1491. Edited by C. D. Hodgman, R. C. Weast & S. M. Selby. Cleveland, OH:The Chemical Rubber Publishing Company.
Höfte, M., Buysens, A., Koedam, N. & Cornelis, P. (1993). Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2. Biometals 6, 85-91.[Medline]
Howe, T. R. & Iglewski, B. H. (1984). Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model. Infect Immun 43, 1058-1063.[Medline]
Iglewski, B. H., Rust, L. & Bever, R. (1990). Molecular analysis of elastase. In Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology , pp. 36-43. Edited by S. Silver, A. M. Chakrabarty, B. Iglewski & S. Kaplan. Washington, DC:American Society for Microbiology.
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, 615-621.[Abstract]
Kessler, E. & Safrin, M. (1988). Synthesis, processing, and transport of Pseudomonas aeruginosa elastase. J Bacteriol 170, 5241-5247.[Medline]
Kessler, E., Safrin, M., Abrams, W. R., Rosenbloom, J. & Ohman, D. E. (1997). Inhibitors and specificity of Pseudomonas aeruginosa LasA. J Biol Chem 272, 9884-9889.
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, 30225-30231.
Krogfelt, K. A., Utley, M., Krivan, H. C., Laux, D. C. & Cohen, P. S. (2000). Specific phospholipids enhance the activity of ß-lactam antibiotics against Pseudomonas aeruginosa. J Antimicrob Chemother 46, 377-384.
Kronborg, G. (1995). Lipopolysaccharide (LPS), LPS immune complexes and cytokines as inducers of inflammation in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection. APMIS 103, 1-30.[Medline]
Kronborg, G., Fomsgaard, A., Galanos, C., Freudenberg, M. A. & Hoiby, N. (1992). Antibody response to lipid A, core, and O sugars of the Pseudomonas aeruginosa lipopolysaccharide in chronically infected cystic fibrosis patients. J Clin Microbiol 30, 1848-1855.[Abstract]
Liu, P. V., Matsumoto, H., Kusama, H. & Bergan, T. (1983). Survey of heat-stable, major somatic antigens of Pseudomonas aeruginosa. Int J Syst Bacteriol 33, 256-264.
Luzar, M. A. & Montie, T. C. (1985). Avirulence and altered physiological properties of cystic fibrosis strains of Pseudomonas aeruginosa. Infect Immun 50, 572-576.[Medline]
Martinez, A., Ostrovsky, P. & Nunn, D. N. (1998). Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion. Mol Microbiol 28, 1235-1246.[Medline]
May, T. B. & Chakrabarty, A. M. (1994). Isolation and assay of Pseudomonas aeruginosa alginate. Methods Enzymol 235, 295-298.[Medline]
Nicas, T. I., Frank, D. W., Stenzel, P., Bradley, J. & Iglewski, B. H. (1985). Role of exoenzyme S in chronic Pseudomonas aeruginosa lung infections. Eur J Clin Microbiol Infect Dis 4, 175-179.
Ochsner, U. A. & Reiser, J. (1995). Autoinducer mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 92, 6424-6428.[Abstract]
Ojeniji, B. (1994). Polyagglutinable Pseudomonas aeruginosa from cystic fibrosis patients: a survey. APMIS 102 (Suppl. 46), 144.
Olson, J. C. & Ohman, D. E. (1992). Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions. J Bacteriol 174, 4140-4147.[Abstract]
OToole, G. A., Pratt, L. A., Watnick, P. I., Newman, D. K., Weaver, V. B. & Kolter, R. (1999). Genetic approaches to study of biofilms. Methods Enzymol 130, 91-109.
Passador, L., Cook, J. M., Gambello, M. J., Rust, L. & Iglewski, B. H. (1993). Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260, 1127-1130.[Medline]
Paya, M., Terencio, M. C., Ferrandiz, M. L. & Alcaraz, M. J. (1996). Involvement of secretory phospholipase A2 activity in the zymosan rat air pouch model of inflammation. Br J Pharmacol 117, 1773-1779.[Abstract]
Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard, A., Iglewski, B. H. & Greenberg, E. P. (1994). Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci USA 91, 197-201.[Abstract]
Pearson, J. P., Passador, L., Iglewski, B. H. & Greenberg, E. P. (1995). A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc Natl Acad Sci USA 92, 1490-1494.[Abstract]
Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in the control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 5756-5767.[Abstract]
Preston, M. J., Seed, P. C., Toder, D. S., Iglewski, B. H., Ohman, D. E., Gustin, J. K., Goldberg, J. B. & Pier, G. B. (1997). Contribution of proteases and LasR to the virulence of Pseudomonas aeruginosa during corneal infections. Infect Immun 65, 3086-3090.[Abstract]
Rossbach, S., Wilson, T. L., Kukuk, M. L. & Carty, H. A. (2000). Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens. FEMS Microbiol Lett 191, 61-70.[Medline]
Rust, L., Pesci, E. C. & Iglewski, B. H. (1996). Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region. J Bacteriol 178, 1134-1140.[Abstract]
Shortridge, V. D., Lazdunski, A. & Vasil, M. L. (1992). Osmoprotectants and phosphate regulate expression of phospholipase C in Pseudomonas aeruginosa. Mol Microbiol 6, 863-871.[Medline]
Speert, D. P., Dimmick, J. E., Pier, G. B., Saunders, J. M., Hancock, R. E. W. & Kelly, N. (1987). An immunohistological evaluation of Pseudomonas aeruginosa pulmonary infection in two patients with cystic fibrosis. J Clin Microbiol 22, 743-747.
Suh, S., Silo-Suh, L., Woods, D., Hassett, D. J., West, S. & Ohman, D. E. (1999). Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J Bacteriol 181, 3890-3897.
Swift, S., Karlyshev, A. V., Fish, L., Durant, E. L., Winson, M. K., Chhabra, S. R., Williams, P., MacIntyre, S. & Stewart, G. S. (1997). Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol 179, 5271-5281.[Abstract]
Tang, H., Kays, M. & Prince, A. (1995). Role of Pseudomonas aeruginosa pili in acute pulmonary infection. Infect Immun 63, 1278-1285.[Abstract]
Tang, H. B., DiMango, D., Bryan, R., Gambello, M., Iglewski, B. H., 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, 37-43.[Abstract]
Telford, G., Wheeler, D., Williams, P., Tomkins, P. T., Appleby, P., Sewell, H., Stewart, G. S. A. B., Bycroft, B. W. & Pritchard, D. I. (1998). The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity. Infect Immun 66, 36-42.
Toder, D. S., Ferrell, S. J., Nezezon, J. L., Rust, L. & Iglewski, B. H. (1994). lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity. Infect Immun 62, 1320-1327.[Abstract]
Tommassen, J., Fillous, A., Bally, M., Murgier, M. & Lazdunski, A. (1992). Protein secretion in Pseudomonas aeruginosa. FEMS Microbiol Rev 9, 73-90.[Medline]
Van Delden, C. & Iglewski, B. (1998). Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg Infect Dis 4, 551-560.[Medline]
Vasil, M. L. & Ochsner, U. A. (1999). The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol 34, 399-413.[Medline]
Vasil, M. L., Prince, R. W. & Shortridge, V. D. (1993). Exoproducts: Pseudomonas exotoxin A and phospholipase C. In Pseudomonas aeruginosa the Opportunist: Pathogenesis and Disease, pp. 5977. Edited by R. B. Fick, Jr. Boca Raton, FL: CRC Press.
Whiteley, M., Lee, K. M. & Greenberg, E. P. (1999). Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 96, 13904-13909.
Whiteley, M., Parsek, M. R. & Greenberg, E. P. (2000). Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa. J Bacteriol 182, 4356-4360.
Winson, M., Camara, M., Latifi, A. & 10 other authors (1995). Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 92, 94279431.[Abstract]
Woods, D. E., Cryz, S. J., Friedman, R. L. & Iglewski, B. H. (1982). Contribution of extoxin A and elastase to virulence of Pseudomonas aeruginosa in chronic lung infections of rats. Infect Immun 36, 1223-1228.[Medline]
Zhou, X., George, S. E., Frank, D. W., Utley, M., Gilmour, I., Krogfelt, K. A., Claxton, L. D., Laux, D. C. & Cohen, P. S. (1997). Isolation and characterization of an attenuated strain of Pseudomonas aeruginosa, a 3,5-dichlorobenzoate degrader. Appl Environ Microbiol 63, 1389-1395.[Abstract]
Received 10 December 2001;
revised 14 February 2002;
accepted 15 February 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |