| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1INSERM CPTP-U563, CHU Purpan, 1 Place du Dr Baylac, TSA 40031, 31059 Toulouse Cedex 9, France 2,4Laboratoire de Bactériologie-Hygiène2 and INSERM U5834, CHU Rangueil, 1 Avenue Jean Poulhes, TSA 50032, 31059 Toulouse Cedex 9, France 3Laboratoire de Microbiologie, Faculté de Pharmacie, 27 Chemin des Maraîchers, 31400 Toulouse, France
Correspondence Anne Dubouix dubouix.a{at}chu-toulouse.fr
Received April 25, 2003
Accepted August 11, 2003
It has been shown that human group IIa secreted phospholipase A2 (sPLA2), found at high levels in inflammatory fluids, displays direct bactericidal properties against Gram-positive bacteria, while activity against Gram-negative bacteria requires the complement system or additional co-factors produced by neutrophils. Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen, is the most common Gram-negative rod found in cystic fibrosis lung infections, where it is associated with an inflammatory environment. Because murine intestinal group II sPLA2 produced by Paneth cells has been shown to be directly bactericidal against Gram-negative bacteria, IIa sPLA2 activity against P. aeruginosa clinical isolates was evaluated and provides the first evidence that the enzyme can be fully bactericidal in a concentration- and time-dependent manner against Gram-negative rods. Furthermore, it was demonstrated that these bactericidal properties were unaffected by high protein and salt concentrations, as observed in cystic fibrosis secretions, and that bacterial killing paralleled phospholipid hydrolysis. Finally, no cytotoxicity was observed when IIa sPLA2 was incubated with human pulmonary cells, highlighting its potential use to synergize bactericidal antibiotics by promoting sublethal alterations of the bacterial cell wall.
 |
INTRODUCTION |
|---|
 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Biochemical studies have shown that the bactericidal properties of sPLA2 rely on cell-wall phospholipid hydrolysis. While destruction of Gram-positive rods has been mostly attributed to direct group IIa sPLA2 activity, Gram-negative bacteria are considered to require both sPLA2 and co-factors. These co-factors [e.g. complement system or antimicrobial peptides secreted by polymorphonuclear neutrophils such as bactericidal/permeability-increasing protein (BPI)] are thought to be responsible for an acute disruption of the bacterial envelope (Elsbach et al., 1994). Nevertheless, the structurally related murine group II sPLA2 secreted by Paneth cells of the intestine is able to display, on its own, effective bactericidal properties against Gram-negative pathogens such as Salmonella typhimurium and E. coli (Harwig et al., 1995). Therefore, group II sPLA2 is one of the first intestinal host-defence mechanisms.
Among Gram-negative rods, Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen and is the most prominent rod isolated from cystic fibrosis (CF) lungs. In airway secretions, it is associated with increased inflammation and a highly salt-enriched microenvironment known to impair innate immunity components such as defensins (Smith et al., 1996). Almost 80 % of CF patients beyond their teens are infected by P. aeruginosa and are more likely to die than patients with other kinds of pneumonia. Furthermore, P. aeruginosa is one of the few bacterial species that is naturally resistant to several ß-lactam antibiotics and frequently evolves to multiresistant antibiotypes. Therefore, in the past decade, a great deal of effort has been put into attempts to make the P. aeruginosa outer membrane permeable to antibiotics in order to promote bactericidal activity.
The aim of the present work was to assess whether IIa sPLA2 could display direct bactericidal properties against P. aeruginosa clinical isolates and to study the impact of the enzyme on the P. aeruginosa cell wall.
|
METHODS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
Group IIa sPLA2.
Recombinant human group IIa sPLA2 was produced in E. coli as described by Fourcade et al. (1995).
Assay for bactericidal activity.
Bacteria were grown overnight at 37 °C in trypticase soy broth (TSB) and then diluted 1 : 10 in fresh medium and subcultured to mid-exponential phase. After harvesting, the bacteria were sedimented by centrifugation at 14 000 g for 5 min, washed twice with 10 mM HEPES/NaOH (pH 7.6), 0.15 M NaCl, 10 mg BSA ml-1 and 2.5 mM CaCl2 and resuspended in the same solution at a concentration of 108 c.f.u. ml-1.
Bactericidal activity was assayed as follows. Bacterial suspensions (108 c.f.u.) were incubated with appropriate dilutions (8, 16 and 32 µg ml-1) of group IIa sPLA2 in the saline buffer described previously containing either BSA or serum. Mixtures were incubated at 37 °C with shaking for 1 or 3 h and then filtered on 0.20 µm Millipore filters. Membranes were then plated onto trypticase soy agar (TSA) and grown at 37 °C for 18 h in an aerobic atmosphere.
The bactericidal effect of group IIa sPLA2 was expressed as the residual number of c.f.u. with respect to the initial inoculum and the EC50 (concentration eliciting 50 % effect) corresponding to the enzyme concentration able to kill 50 % of the initial inoculum. The results presented are means of at least three independent experiments.
Effects of salt concentration and proteins on bactericidal effects of sPLA2.
The bactericidal properties of sPLA2 were tested in the presence of various incubation media. These media were BSA in the range 0 to 10 mg ml-1 and NaCl from 0 to 0.25 M. Results were expressed as means ± SD of triplicate determinations. Statistical analysis was then performed using a paired t-test.
Radiolabelling of bacterial lipids.
Bacteria were subcultured to mid-exponential phase in TSB supplemented with 2 µCi [1-32P]oleic acid ml-1 (Amersham Lifescience France) (1 Ci = 3.7 x 107 Bq) and 0.1 % BSA (w/v). Bacteria were then harvested and cultured in the same medium without [1-32P]oleic acid at 37 °C for 30 min in order to remove unesterified fatty acid precursor incorporated into ester positions. Finally, the bacteria were washed with saline buffer.
Lipid analysis.
Bacteria (108 c.f.u.) were exposed to 5 or 10 µg sPLA2 ml-1 for up to 3 h. The composition of the lipids was then determined by extraction in CHCl3/CH3OH as described by Bligh & Dyer (1959) and analysis of the radiolabelled material recovered in the CHCl3 phase (> 95 % of total) by TLC in the presence of standards. Results were expressed as the percentage hydrolysis of phospholipids compared with a control performed in the absence of the enzyme. Data are means ± SD of three independent experiments.
Assay for cytotoxicity to human pulmonary cells.
In order to test the possible cytotoxic effect of group IIa sPLA2 on human cells, the viability of tracheal epithelial cells incubated with an appropriate dilution of the enzyme was evaluated for 24 h.
Immortalized and characterized tracheal epithelial cells from fetuses were a gift from URA CNRS 1283 (Paris, France) (Lemnaouar et al., 1993). Cells were grown to confluence at 37 °C under 5 % CO2 in 1 : 1 DMEM/Ham's F12 medium supplemented with 2 % (w/v) Ultroser G, 100 IU penicillin ml-1 and 100 µg streptomycin ml-1.
Solutions containing 8, 16 or 32 µg sPLA2 ml-1 were mixed with DMEM while the same volume of saline buffer was used as a negative control. A 10 % (v/v) solution of Triton X-100 in DMEM was used as a positive control. At 0, 6, 12 and 24 h, the supernatant was carefully removed and centrifuged at 14 000 g for 5 min in order to remove cells. Lactate dehydrogenase (LDH) in the supernatant was evaluated and a ratio LDHassay/LDHpositive control > 5 % was considered to indicate cytotoxicity. The cytotoxicity test was performed three times for each treatment.
|
RESULTS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
|
Group IIa sPLA2 bactericidal activity is time-dependent
In order to investigate whether bactericidal activity could be enhanced by longer incubation, all strains were subjected to sPLA2 for up to 3 h. Table 2 lists the relative EC50 values for bactericidal activity against all the strains tested. Recombinant sPLA2 was thus more effective against both Staphylococcus aureus and P. aeruginosa strains when the incubation time was increased. The minimal concentrations required for EC50 were mostly close to 2–8 µg ml-1 when incubated for 3 h, compared with 5–12 µg ml-1 after only 1 h. These results suggest that bactericidal activity is time-dependent and that increased contact between enzyme and bacterial strains, as would occur under physiological conditions, dramatically enhances the antibacterial properties of sPLA2.
Group IIa sPLA2 bactericidal properties are not affected by high concentrations of protein or NaCl
In order to test whether the abnormal composition of CF airway secretions could affect sPLA2, we tested whether the enzyme bactericidal properties could be modified by protein and salt variations. As shown in Table 3, similar bactericidal effects were observed for concentrations as high as 32 µg sPLA2 ml-1, whatever the test conditions; no significant difference was observed either when the protein content was increased or when high salt concentrations were added to the incubation medium (P > 0.05). When human serum was used instead of BSA, no inhibitory effect could be observed (data not shown). These results demonstrate that, even if an unfavourable microenvironment is present, as observed in CF lungs, sPLA2 is able to display potent bactericidal activity against both Staphylococcus aureus and P. aeruginosa strains.
|
Group IIa sPLA2 bactericidal activity parallels phospholipid hydrolysis
In order to evaluate whether the antimicrobial properties of group IIa sPLA2 against P. aeruginosa paralleled significant hydrolysis of the bacterial phospholipids, as demonstrated for Staphylococcus aureus strains, the ability of the enzyme to liberate oleic acid from the bacterial membrane was also studied.
As shown in Fig. 2, incubation of radiolabelled bacteria with 5 µg sPLA2 ml-1 led to the degradation of 60 % of the major phospholipids within 3 h. Furthermore, increased degradation of phospholipids was observed for higher concentrations (10 µg ml-1). These results indicate that the ability of IIa sPLA2 to hydrolyse phospholipids of the bacterial envelope correlates with the concentration- and time-dependent bactericidal activity.
|
Group IIa sPLA2 is not cytotoxic to human cells
Human tracheal epithelial cells were incubated with saline buffer or sPLA2 concentrations identical to those used to display bactericidal activity in order to investigate possible cytotoxic effects. As shown in Table 4, for all samples, the ratio LDHassay/LDHpositive control was <5 %, even after 24 h incubation and was considered not to indicate cytotoxicity. These results show that sPLA2 concentrations able to display bactericidal activity are totally innocuous to human pulmonary cells.
|
|
DISCUSSION |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
In this report, we have examined the effects of recombinant IIa sPLA2 on the viability of P. aeruginosa clinical isolates. Several investigators had shown previously that IIa sPLA2, initially described as part of the inflammatory response, also displays bactericidal activity against Gram-positive bacteria such as Staphylococcus aureus, while activity against E. coli strains requires co-factors. The latter include the complement system or BPI and p15s, produced by neutrophils (Weinrauch et al., 1995), which lead to synergistic disruption of the bacterial envelope. Nevertheless, the closely related murine type II sPLA2 produced by Paneth cells is able to exert a direct antimicrobial effect against Gram-negative rods (Harwig et al., 1995).
The first step in our study was to determine recombinant IIa sPLA2 activity against P. aeruginosa in parallel with Staphylococcus aureus strains in order to validate the efficacy of the recombinant enzyme. We found that IIa sPLA2 could be fully bactericidal against the P. aeruginosa and Staphylococcus aureus clinical isolates in a concentration-dependent fashion and at a similar range of concentrations for both species without the addition of any co-factors. To our knowledge, these results provide the first demonstration that IIa sPLA2 can be intrinsically microbicidal to a Gram-negative species. It is interesting to note that sPLA2 displayed comparable properties against both smooth and mucoid strains, indicating that bacterial alginate does not necessarily interfere with the bactericidal properties of the enzyme. Furthermore, most of the P. aeruginosa strains tested expressed antibiotic-multiresistant phenotypes, which did not interfere with their susceptibility to sPLA2.
The abnormal composition of CF airway secretions is known to affect the action of antimicrobial peptides, since antimicrobial function is often dependent on ion strength and salt sensitivity is crucial for cationic peptides (Smith et al., 1996). We therefore tested the effects of protein and salt variations as well as acidic pH (data not shown) in order to mimic the CF lung microenvironment. No difference in activity was found among the various groups. This is of particular interest because it shows that, unlike many host-defence antimicrobial peptides. which are totally ineffective in CF because of airway secretion hyperosmolarity, sPLA2 remains active.
We finally decided to evaluate the time-course of bactericidal properties and demonstrated that incubation for up to 3 h significantly enhanced the reduction of the initial inoculum. Taken together, these results show that IIa sPLA2 can be an effective microbicide, the direct antibacterial potency of which against P. aeruginosa approximates to that against Staphylococcus aureus even under unfavourable conditions close to those observed in CF lungs.
We also observed that the enzyme was responsible for phospholipid hydrolysis and concomitant liberation of free fatty acids, as already demonstrated for Gram-positive bacteria. Indeed, incubation of radiolabelled bacteria with 5 µg sPLA2 ml-1 led to degradation of 60 % of the major phospholipids within 3 h. This observation is consistent with the amount expected (e.g. 50 %) when only the phospholipids of the extracellular leaflet of the bilayered membrane (which is accessible to sPLA2) are hydrolysed (Koduri et al., 2002), and is in agreement with observations on Micrococcus luteus strains (Buckland et al., 2000). Moreover, the degree and time-course of phospholipid hydrolysis correlated well with bacterial viability, even for mucoid and antibiotic-multiresistant strains.
These findings suggest that IIa sPLA2 is intrinsically able to cause disruption of bacterial envelope integrity, thus leading to impairment of bacterial viability. One could then imagine that the enzyme could potentially synergize the effects of antibiotics such as ß-lactam in combination with natural antibacterial peptides such as BPI. Because no cytotoxicity could be observed for concentrations comparable to those necessary to display bactericidal activity against P. aeruginosa strains, IIa sPLA2 may be considered as a potential therapeutic agent synergizing antibiotics. However, one of the main problems when administering antibiotics to CF patients is how to achieve targeted drug delivery to the diseased tissue in order to obtain microbicidal concentrations as high as possible while limiting side effects. Therefore, new administration routes such as tobramycin nebulization have been proposed in recent years (Doring et al., 2000). In this context, the dual potency of IIa sPLA2 to hydrolyse not only bacterial phospholipids but also micelles of phospholipid bilayers should be of particular interest. Hence, new targeting systems such as liposomal drug-carrier systems are being developed. The action of IIa sPLA2 on liposomes can be used to release their molecular content (e.g. drugs) directly where needed. This method has in fact been demonstrated to be fully efficient for anti-cancer drug delivery, while tumorous tissues are known to hyperexpress IIa sPLA2 (Davidsen et al., 2003). One could easily imagine that this method could be used in CF therapy.
Additional work is obviously needed to clarify the various possible strategies. Nevertheless, these findings may prove interesting with regards to new therapeutic strategies, particularly since more drug-resistant bacterial strains are emerging.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
REFERENCES |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  B. P. Hurley and B. A. McCormick Multiple Roles of Phospholipase A2 during Lung Infection and Inflammation Infect. Immun., June 1, 2008; 76(6): 2259 - 2272. [Full Text] [PDF]
|
|
|
|
|
|
S. Kirschnek and E. Gulbins Phospholipase A2 Functions in Pseudomonas aeruginosa- Induced Apoptosis Infect. Immun., February 1, 2006; 74(2): 850 - 860. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
) ml-1 or saline buffer (
) and then grown on TSA. Bactericidal effect was evaluated by counting c.f.u. after 24 h and is expressed as the percentage of inoculum remaining (c.f.u. %). Strong activity was observed for both Staphylococcus aureus strains ATCC 25923 (a) and 0412 (b) and P. aeruginosa strains ATCC 27583 (c) and 7844 (d) over the same range of sPLA2 concentrations, while B. cepacia strains ATCC 25416T (e) and 235 (f) were more resistant. Data shown are means ± SD (n = 3).