Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis Wilfried Veron1, Olivier Lesouhaitier1, Xaviera Pennanec2, Karine Rehel2, Philippe Leroux3, Nicole Orange1 and Marc G J Feuilloley1 Laboratory of Cold Microbiology, UPRES 2123, University of Rouen, Evreux, France ´ Laboratoire de Biotechnologie et Chimie Marines, Universite de Bretagne-Sud B.P 92116, Lorient, France Research Department of Microenvironment and Cell Integrated Renewal, UPRES 3829, University of Rouen, France Keywords cyclases; cytotoxicity; natriuretic peptides; sensor; vfr Correspondence O Lesouhaitier, Laboratory of Cold Microbiology, UPRES 2123, University of Rouen, 55 rue Saint Germain, 27000 Evreux, France Fax: +33 232 29 15 55 Tel: +33 232 29 15 42 E-mail: olivier.lesouhait@univ-rouen.fr (Received 18 July 2007, revised 28 August 2007, accepted 18 September 2007) doi:10.1111/j.1742-4658.2007.06109.x Natriuretic peptides of various forms are present in animals and plants, and display structural similarities to cyclic antibacterial peptides Pretreatment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide (BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced glial cell necrosis In eukaryotes, natriuretic peptides act through receptors coupled to cyclases We observed that stable analogs of cAMP (dibutyryl cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natriuretic peptide and CNP on bacteria Further evidence for the involvement of bacterial cyclases in the regulation of P aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacterial cAMP concentration after exposure to CNP Lipopolysaccharide (LPS) extracted from P aeruginosa PAO1 treated with both dibutyryl cAMP and 8-bromo-cGMP induces higher levels of necrosis than LPS extracted from untreated bacteria Capillary electrophoresis and MALDI-TOF MS analysis have shown that differences in LPS toxicity are due to specific differences in the structure of the macromolecule Using a strain deleted in the vfr gene, we showed that the Vfr protein is essential for the effect of natriuretic peptides on P aeruginosa PAO1 virulence These data support the hypothesis that P aeruginosa has a cyclic nucleotide-dependent natriuretic peptide sensor system that may affect virulence by activating the expression of Vfr and LPS biosynthesis Bacteria of the genus Pseudomonas can adapt to a multitude of environmental niches, owing to the size of their genome and the abundance of regulatory genes [1] The natural resistance to b-lactams and biocides of Pseudomonas strains and their opportunistic behavior pose considerable problems for hospitals [2] Pseudomonas aeruginosa is one of the principal microorganisms responsible for nosocomial diseases [3] P aeruginosa has considerable infectious potential, as it can infect both surface tissues, such as skin and wounds, and internal organs, such as the lungs and urinary tract [4] Central nervous system infections are observed more rarely, but such infections are steadily increasing in number and are associated with high morbidity rates [5,6] Consistent with these clinical data, in vitro experiments have shown that P aeruginosa has a high specific affinity for neurons and glial cells, and that the binding of the bacterium or its lipopolysaccharide (LPS) to these target cells is associated with necrosis [7,8] In the host, bacteria are exposed to various defense mechanisms Successful adaptation to an ecological niche, such as eukaryotic tissues, clearly requires the sensing of chemical signals, their integration and the development of an appropriate response [9,10] The Abbreviations 8BcGMP, 8-bromo-cGMP; BNO, ordinary nutrient broth medium; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; dbcAMP, dibutyryl cAMP; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MECK, micellar electrokinetic chromatography 5852 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS W Veron et al Effect of natriuretic peptides on Pseudomonas role of the immune system in the response to Pseudomonas infection has been investigated in detail [10,11] However, tissue factors or hormones, including natriuretic peptides, have also been described as having antimicrobial activity [12,13] Three types of natriuretic peptide ) atrial natriuretic peptide, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) ) were initially characterized These molecules are major cardiovascular and osmoregulatory factors in vertebrates, and have been detected both in peripheral organs, where they are produced as hormones, and in nervous tissues, where they act as neuromodulators [14] Natriuretic peptides include a short loop (Fig 1) containing an excess of positively charged amino acids NH2- S P K M V Q G S G R K M G D F R I S C hBNP HOOC- H R R L V K CNP C G S S L G S NH2- G L G L K L S K D G CF R I G HOOC- C S G L G S M O NH N This structure is markedly similar to that of cationic cyclic antimicrobial peptides [15] The abundance of BNP and CNP in tissues not involved in regulating blood pressure or diuresis suggests that these neuropeptides probably have other activities Natriuretic peptides have been conserved throughout the evolution of animals, with different forms being present in vertebrates [16] and invertebrates [17] Structural analogs of animal natriuretic peptides exist in plants [18], and a gene encoding a plant natriuretic peptide-like molecule has recently been identified in a Gram-negative bacterium [19] Similarly, a class III cyclase with a similar function to the GMP cyclase receptors of eukaryotic natriuretic peptides was recently described in prokaryotes [20] These observations suggest that bacteria could also employ natriuretic peptide-like molecules as communication tools In the present study, we investigated the effects of BNP and CNP (Fig 1) on the virulence of P aeruginosa PAO1 We evaluated the virulence of unexposed bacteria and of bacteria exposed to these peptides in vitro, using primary cultures of glial cells as the study model We were able to reproduce the effects of BNP and CNP by treating the bacteria with stable analogs of cyclic nucleotides (Fig 1) The effects of BNP and CNP on the intrabacterial concentrations of cAMP and cGMP were determined, and a vfr null mutant of P aeruginosa PAO1 was used to investigate the mechanism of action of these factors Finally, the impact of stable analogs of cyclic nucleotides on the structure and cytotoxicity of LPS was determined by complementary approaches N dbcAMP N N Results O O H H O O P O OH O O N N 8BcGMP H2N Br N N H O HO H H Preliminary studies showed that the treatment of P aeruginosa PAO1 with BNP or CNP (10)6 m) had no effect on bacterial growth (data not shown) The peptides were administered at various time points, at the start of culture or during the early part of the stationary phase In some experiments, we added an aliquot of peptide to the culture hourly, to take into account possible degradation In all cases, even on solid medium, BNP and CNP had no effect on bacterial growth or cultivability O P O O OH Fig Structure of human BNP and CNP and cyclic analogs of nucleotides used in the present study Human BNP (hBNP) is composed of 32 amino acids whereas CNP contains only 22 amino acids dbcAMP and 8BcGMP are cell-permeable cAMP and cGMP analogs, respectively Effect of natriuretic peptides on the cytotoxic activity of P aeruginosa PAO1 The effect of BNP and CNP on the virulence of P aeruginosa PAO1 was studied by treating bacteria with these peptides before mixing them with cells and comparing the resulting cytotoxicity with that of FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5853 Effect of natriuretic peptides on Pseudomonas W Veron et al nontreated bacteria The model of eukaryotic cells used here ) primary cultures of glial cells ) has been validated in previous studies [7,8] In all experiments, bacteria were rinsed before exposure to glial cells, to remove all traces of peptide The virulence of the bacteria was evaluated by determining lactate dehydrogenase (LDH) levels in the incubation medium LDH is a stable enzyme present in the cytosol of eukaryotic cells, and is released into the medium when the membrane is destabilized This destabilization is considered to be a marker of necrosis However, as some bacterial strains can also generate LDH, we also checked for the spontaneous production of this enzyme by P aeruginosa PAO1 When P aeruginosa PAO1 was incubated alone in the culture medium used for glial cells, LDH remained undetectable (Fig 2) In the same conditions, glial cells cultured alone also released a limited amount of LDH The amount of LDH released in these conditions indicates that only a small proportion of the glial cell population (7.6 ± 1.5%; n ¼ 18) spontaneously underwent cell death during the course of the experiment (6 h) This result (fewer than 10% of the cells dying over a h period) is an indicator of the quality of the culture, and all experiments in which the controls displayed spontaneous necrosis levels exceeding this value were excluded from the analysis When glial cells were exposed to P aeruginosa PAO1 (106 CFmL)1, h), the percentage of the cell population displaying signs of necrosis increased to 39.0 ± 2.2% The prior treatment of P aeruginosa with natriuretic peptides (10)6 m, 14 h, 37 °C) increased the cytotoxic activity of the bacteria significantly In the presence of P aeruginosa PAO1 treated with BNP, the percentage of the cell population displaying signs of necrosis reached 52.4 ± 3.1% (P < 0.001) An even higher percentage was reached (73.4 ± 4.5%, P < 0.001) when bacteria were exposed to CNP (Fig 2) A shorter period of prior treatment of P aeruginosa PAO1 with natriuretic peptides (4.5 h) had similar effects (data not shown) CNP had a significantly stronger effect than BNP (P < 0.001; Fig 2) We carried out control experiments in which glial cells were exposed only to BNP or CNP (10)6 m) The percentage of glial cells displaying signs of necrosis was identical to that of a control culture (data not shown) Thus, the effects observed were due entirely to the direct action of BNP or CNP on the physiology of P aeruginosa PAO1 LDH (% of release) 90 Effect of stable analogs of cAMP and cGMP on the cytotoxic activity of P aeruginosa PAO1 70 50 30 10 Glial Cells - + + + + - + - - - - - + - - - - - + PAO1 control + PAO1 BNP treated PAO1 CNP treated Fig Cytotoxic activity of P aeruginosa PAO1 treated with natriuretic peptides Effect of BNP and CNP (10)6 M) on the cytotoxicity of P aeruginosa PAO1 The cytotoxic effect of the bacterium was determined by measuring LDH accumulation in the medium due to the rupture of the cytoplasmic membrane of glial cells and the release of this stable cytosolic enzyme Values were calculated as the mean concentration of LDH in the culture medium after h of incubation with nontreated (control) (n ¼ 47) or treated (n ¼ 24) bacteria Data are the means of four independent experiments P < 0.001, significantly different 5854 The effect of natriuretic peptides in eukaryotic cells is mediated by different receptor subtypes with intrinsic guanylate cyclase activity [21] or through receptors coupled to an adenylate cyclase [22] We therefore investigated the effects of cell-permeable stable analogs of cGMP [8-bromo-cGMP (8BcGMP)] and cAMP [dibutyryl cAMP (dbcAMP)] on the virulence of P aeruginosa PAO1 As observed with natriuretic peptides, exposure of the bacteria to dbcAMP or 8BcGMP 14 h; 37 °C) significantly increased (10)5 m; (P < 0.001) the potential of P aeruginosa PAO1 to induce necrosis in glial cells Necrosis levels reached 57.9 ± 4.6% of the cell population when the bacteria were exposed to dbcAMP, and 59.6 ± 5.1% when the bacteria were first treated with 8BcGMP, versus only 39.0 ± 2.2% for nontreated P aeruginosa PAO1 (Fig 3) Control studies were carried out with glial cells directly exposed to dbcAMP or 8BcGMP (10)5 m) The percentage of glial cells displaying signs of necrosis was identical to that of a control culture, indicating that the stable analogs of cyclic nucleotides had no intrinsic effect on the viability of glial cells (data not shown) FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS W Veron et al Effect of natriuretic peptides on Pseudomonas treatment was determined by ELISA The value obtained in early stationary phase (10.0 ± 1.4 pmolỈ mL)1 of bacterial extract) was used as the 100% basal level (Fig 4A) As we hypothesized a role for cyclic nucleotides in the transduction of a signal mediated by natriuretic peptides, and a rapid and early effect of these peptides, we exposed the bacteria for a short time (30 min) to BNP and CNP and then determined their intracellular cAMP content The treatment of P aeruginosa PAO1 with BNP (10)6 m) did not modify cAMP levels in the bacteria (Fig 4A) In contrast, CNP (10)6 m) treatment significantly increased intrabacterial cAMP concentrations, which reached 174.8 ± 6.3% of control values (P < 0.001) The basal concentration of cGMP in P aeruginosa PAO1 (0.55 ± 0.16 pmolỈmL)1) was lower than that of cAMP, and was at the limit of the sensitivity of the assay This value was used as the 100% basal level (Fig 4B) The exposure of P aeruginosa PAO1 to BNP or CNP (10)6 m, 30 min) had no significant effect on cGMP levels in the bacteria (Fig 4B) 70 50 30 10 Glial Cells + + + + PAO1 control - + - - - + - - - + PAO1 dbcAMP treated PAO1 8BcGMP treated Fig Cytotoxic activity of P aeruginosa PAO1 treated with cyclic nucleotide analogs Effect of dbcAMP and 8BcGMP (10)5 M) on the cytotoxicity of P aeruginosa PAO1 Values were calculated as the mean concentration of LDH in the culture medium after h of incubation with nontreated (control) (n ¼ 47) or treated (n ¼ 14) bacteria Data are the means of four independent experiments P < 0.001, significantly different Effect of natriuretic peptides on intrabacterial concentrations of cAMP and cGMP As exogenous cyclic nucleotides were found to have an effect on P aeruginosa PAO1, we investigated the possible action of natriuretic peptides on the intrabacterial concentrations of cAMP and cGMP The concentration of cAMP in P aeruginosa PAO1 grown in ordinary nutrient broth medium (BNO) in the absence of Effects of stable analogs of cAMP and cGMP on the cytotoxicity of the LPS of P aeruginosa PAO1 LPS is largely responsible for the cytotoxicity of Pseudomonas in glial cells [8] We therefore investigated the impact of stable analogs of cyclic nucleotides on the endotoxic activity of this macromolecule The cytotoxicity of the LPS extracted from P aeruginosa PAO1 exposed to dbcAMP or 8BcGMP (10)5 m; h; 37 °C) was compared with that of the LPS extracted from control bacteria in the same growth phase (early stationary phase) LPS was extracted from control and treated bacteria, and its concentration was determined by a 2-keto-3-deoxyoctonate (KDO) assay The mean concentrations of LPS measured by this technique in bacterial cultures grown in the absence (0.65 mgỈmL)1) or Fig Intrabacterial concentration of monophosphate cyclic nucleotides in P aeruginosa PAO1 after exposure to natriuretic peptides Effect of BNP and CNP (10)6 M) on the intrabacterial concentration of cAMP (A) and cGMP (B) in P aeruginosa PAO1 Data are the means of four independent experiments P < 0.001, significantly different; NS, not significantly different cAMP (% of control level) A B NS 200 150 100 50 PAO1 PAO1 PAO1 control BNP CNP treated treated FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS cGMP (% of control level) LDH (% of release) 90 NS NS 200 150 100 50 PAO1 PAO1 PAO1 control BNP CNP treated treated 5855 Effect of natriuretic peptides on Pseudomonas W Veron et al presence of cAMP or cGMP (0.53 mgỈmL)1 and 0.64 mgỈmL)1, respectively) were in the same range An equivalent amount of the molecule (500 ngỈmL)1) was added to the growth medium for glial cells, and its cytotoxic activity was determined by measuring LDH release, as previously described The spontaneous basal level of necrosis in glial cells was very low (4.9 ± 0.3% of the cell population in 24 h; n ¼ 10) (Fig 5) When glial cells were exposed to LPS extracted from control P aeruginosa PAO1, 10.5 ± 0.3% of the glial cells displayed signs of necrosis The necrotic potential of the endotoxin extracted from P aeruginosa PAO1 exposed to dbcAMP and 8BcGMP was significantly higher, with 13.4 ± 0.8% (P < 0.001) and 15.7 ± 0.7% (P < 0.001) of the cells, respectively, displaying signs of necrosis Control studies were carried out with extracts of bacterial culture medium obtained with the same protocol used for LPS extraction and purification These experiments demonstrated that the effect of LPS on glial cell viability was not due to contaminants from the extraction buffers used to purify the endotoxin (data not shown) LDH (% of release) 16 12 Glial Cells + + + + LPS PAO1 control - + - - LPS PAO1 dbcAMP treated - + - LPS PAO1 8BcGMP treated - - + Fig Cytotoxic activity of LPS from P aeruginosa PAO1 treated with cyclic nucleotide analogs Effect of dbcAMP and 8BcGMP (10)5 M) on the cytotoxicity of the LPS from P aeruginosa PAO1 Values are expressed as the mean concentration of LDH in the culture medium after 24 h of incubation with LPS (500 ngặmL)1) from nontreated (control) (n ẳ 12) or treated (n ¼ 12) bacteria Data are the means of three independent experiments P < 0.001, significantly different 5856 Effect of stable analogs of cAMP and cGMP on the structure of the LPS of P aeruginosa PAO1 The effect of stable analogs of cAMP and cGMP on the structure of the LPS was studied using two complementary techniques, micellar electrokinetic chromatography (MEKC) and MALDI-TOF MS In both cases, LPS was extracted from P aeruginosa PAO1, with or without exposure to dbcAMP or 8BcGMP (10)5 m; h; 37 °C), and purified for analysis as described by Darveau & Hancock [23] LPS was first analyzed by MEKC, as this technique separates hydrophobic molecules differing little in size and polarity more efficiently than HPLC We have previously shown that it is possible to differentiate between LPS molecules from the same strain of Pseudomonas grown at different temperatures by MEKC [24] The LPS of P aeruginosa PAO1 grown in control conditions gave three major peaks (Fig 6A) The first and major peak (peak 1) had a retention time of 8.2 The second peak was associated with a series of compounds of very similar structure and had a retention time of 11.8 The third major peak was well separated, with a retention time of 22.5 min, and seemed to correspond to a single molecular form that was strongly retained and highly hydrophobic The electropherograms of LPS extracted from P aeruginosa PAO1 treated with dbcAMP and 8BcGMP were considerably modified (Fig 6B,C) Peak 1, eluted close to the electro-osmotic flux, corresponded to poorly charged and nonhydrophobic molecules ) presumably a form of LPS with a large osidic chain and a small number of ionized phosphate groups The main differences between LPS extracted from bacteria with and without cyclic nucleotide treatment concerned the levels of peaks and In dbcAMP-treated bacteria, peak was much smaller and a fourth peak appeared at 17.1 (Fig 6B) Peak was also broadened and accompanied by secondary traces between 22.2 and 23.0 The difference between LPS extracted from control and 8BcGMP-treated bacteria was even greater LPS from bacteria exposed to 8BcGMP displayed a signal corresponding to peak 1, no peak 2, and only a small, broadened peak (Fig 6C) The changes observed in the LPS from dbcAMP-treated and 8BcGMP-treated bacteria suggest a decrease in the strongly charged or very hydrophobic forms of LPS MALDI-TOF MS of the LPS of P aeruginosa PAO1 identified a large number of compounds (Fig 7A) The lack of a mass spectrum library for the LPS of P aeruginosa PAO1 made it difficult to interpret the results A study of MS data [25–30] for FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS W Veron et al Effect of natriuretic peptides on Pseudomonas PAO1 control 0.2 EOF A 0.1 PAO1 dbcAMP treated Absorbance Units (203 nm) 0.2 B Effect of natriuretic peptides on the cytotoxic activity of P aeruginosa PAO9002 0.1 0.2 C PAO1 8BcGMP treated 0.1 0 10 20 Retention time (min) IV correspond to compounds associated with oligosaccharide cores with different O-antigen repeating units The LPS extracted from P aeruginosa PAO1 treated with dbcAMP presented many structural variations (Fig 7B) In particular, the multiple peaks in the region between 2000 and 3200 Da, corresponding to the region of the complete oligosaccharide core with repeated O-units [26,30], were numerous for control LPS (Fig 7A) and much more limited for LPS from bacteria treated with dbcAMP (Fig 7B) Treatment with 8BcGMP had no obvious effect on this part of the LPS molecule (Fig 7C) In contrast, the profiles of the LPS molecules from P aeruginosa PAO1 treated with dbcAMP and 8BcGMP generated a large number of new compounds (36 and 18, respectively) with molecular masses between 1000 and 1900 Da, a zone normally attributed to the components of lipid A [25] 30 Fig MECK analysis of LPS treated with cyclic nucleotide analogs MECK analysis of the LPS extracted from control (A), dbcAMP (10)5 M)-treated (B) or 8BcGMP (10)5 M)-treated (C) P aeruginosa PAO1 EOF, electro-osmotic flux Arrows and numbers refer to the different molecular forms identified in the LPS from P aeruginosa PAO1 P aeruginosa PAO1 LPS fragments led to the identification of four principal clusters corresponding to different components of the molecule Cluster I seems to be generated by the fragments of lipid A, whereas the molecular masses associated with clusters II, III and In P aeruginosa, the protein Vfr has been shown to be activated by both cAMP and cGMP [31] We investigated the possible involvement of Vfr in the action of natriuretic peptides on P aeruginosa, using strain PAO9002, a vfr null mutant of P aeruginosa PAO1 obtained by insertion of the accC1 gene into the vfr site [32] As for P aeruginosa PAO1, we checked that the strain did not spontaneously produce any LDHrelated molecule that might interfere with our assay (data not shown) The basal cytotoxicity of P aeruginosa PAO9002 for glial cells was intrinsically much higher than that of P aeruginosa PAO1 We therefore reduced the time for which glial cells were incubated with P aeruginosa PAO9002 to 2.5 h In these conditions, the percentage of cells displaying signs of necrosis following the exposure of glial cells to P aeruginosa PAO9002 (106 CFmL)1) was in a similar range to that following the exposure of cells to P aeruginosa PAO1, i.e 39.0 ± 2.2% In contrast to the results obtained with P aeruginosa PAO1, the prior treatment of P aeruginosa PAO9002 with BNP or CNP (10)6 m; 14 h; 37 °C) had no significant effect on the cytotoxic potential of this strain (Fig 8) Cytotoxicity and chemical properties of the LPS extracted from P aeruginosa PAO9002 The LPS of P aeruginosa PAO9002 was extracted and purified, and its cytotoxicity was determined in primary cultures of glial cells as described above The LPS extracted from P aeruginosa PAO9002 was less cytotoxic than that extracted from P aeruginosa PAO1 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5857 Effect of natriuretic peptides on Pseudomonas W Veron et al Intens [a.u.] A X104 2777.91 LPS PAO1 control 1.25 III 1.00 0.75 IV II 0.50 I 6456.87 0.25 7212.73 8367.22 0.00 2000 4000 6000 8000 10000 12000 14000 16000 18000 Intens [a.u.] B X104 2778.75 m/z LPS PAO1 dbcAMP treated 6458.12 5337.87 12165.87 2000 4000 6000 8000 10000 12000 14000 16000 18000 Intens [a.u.] C X104 m/z 3062.49 LPS PAO1 8BcGMP treated 3.0 2.5 2.0 1.5 6458.10 1.0 7317.12 0.5 0.0 2000 4000 6000 8000 10000 12000 14000 16000 18000 m/z Fig MALDI-TOF analysis of LPS treated with cyclic nucleotide analogs MALDI-TOF mass spectra obtained from the LPS extracted from control (A), dbcAMP (10)5 M)-treated (B) or 8BcGMP (10)5 M)-treated (C) P aeruginosa PAO1 Roman numerals (A) indicate clusters of peaks corresponding to compounds with similar molecular masses (Fig 9A) The percentage of the cell population undergoing necrosis in the presence of LPS from P aeruginosa PAO9002 (14.56 ± 0.98%) was higher than that normally measured in a control culture (9.02 ± 0.24%), but significantly lower (P < 0.01) than that in cultures exposed to LPS from P aeruginosa PAO1 (18.05 ± 0.42%) MEKC analysis of this LPS revealed marked differences from the LPS extracted from P aeruginosa PAO1 (Fig 9B) Indeed, the diversity of molecular forms identified in the LPS from P aeruginosa PAO9002 was very low Most of 5858 the signal was concentrated in peak Another form, denoted peak 5, appeared at 20.3 A small signal was also detected around peak The electropherogram profile of the LPS from P aeruginosa PAO9002 closely resembled that of the LPS from P aeruginosa PAO1 after treatment with 8BcGMP Discussion The structure of BNP and CNP and the results published by Krause et al [13] suggested that human FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS W Veron et al Effect of natriuretic peptides on Pseudomonas 50 NS + - - PAO9002 BNP treated + - PAO9002 CNP treated - + LDH (% of release) NS 40 30 20 10 PAO9002 control Fig Cytotoxic activity of P aeruginosa PAO9002 treated with natriuretic peptides Effect of BNP and CNP (10)6 M) on the cytotoxicity of P aeruginosa PAO9002 Values were calculated as the mean concentration of LDH in the culture medium after 2.5 h of incubation with nontreated (control) (n ¼ 16) or treated (n ¼ 16) bacteria Data are the means of four independent experiments NS, not significantly different natriuretic peptides, and not rat peptides, might have antibacterial activity Our preliminary studies showed that, in contrast, at micromolar concentrations similar to those encountered in animal tissues, BNP and CNP have no antibacterial effect As antibacterial agents are also known to have indirect effects on bacterial virulence [33], we used a well-characterized in vitro assay developed in our laboratory [7,8] to investigate the effects of BNP and CNP on the cytotoxicity of bacteria We cultured P aeruginosa in the presence of BNP or CNP Bacteria were exposed to the peptides during the exponential growth phase and were then rinsed to remove any trace of BNP or CNP before mixing with primary cultures of glial cells As confirmed by controls, in which natriuretic peptides were directly mixed with glial cells, our results can only be explained by changes in bacterial virulence The observation that treating P aeruginosa with BNP and CNP increases the cytotoxic effect of the bacteria suggests that natriuretic factors can affect the physiology of microorganisms sufficiently to modify virulence factor production, but not enough to affect the growth and survival of P aeruginosa These results are entirely consistent with published data suggesting that antimicrobial peptides can exert multiple effects, not simply inhibiting bacterial growth, but also interfering with microbial physiology, including the capacity to produce virulence factors or even toxin activity [33] Peptides cannot penetrate cells freely Thus, in eukaryotic cells, natriuretic peptides exert their effects through three different cytoplasmic membrane receptor subtypes with intrinsic guanylate cyclase activity [21] or indirectly coupled to an adenylate cyclase [22] As in investigations of the possible involvement of cyclic nucleotide phosphates in the mechanism of action of neurohormones in eukaryotes [34], we tested the effect of stable analogs of cGMP and cAMP on P aeruginosa The exposure of bacteria to 8BcGMP and dbcAMP fully reproduced the effects of BNP and CNP, increasing necrosis As exogenous cyclic nucleotides affected P aeruginosa PAO1 virulence, we investigated the possible effects of natriuretic peptides on intrabacterial cAMP and cGMP concentrations The level of cAMP in P aeruginosa was unaffected by exposure to BNP In contrast, CNP induced a marked 20 LDH (% of release) Fig Characteristics of the LPS extracted from the vfr null mutant P aeruginosa PAO9002 Cytotoxic activity (A) and MEKC electropherogram (B) of the LPS extracted from the vfr null mutant of P aeruginosa PAO9002 Cytotoxicity values were calculated as the means of three independent experiments P < 0.001, significantly different; P < 0.01, significantly different; EOF, electro-osmotic flux Arrows and numbers refer to the different molecular forms identified in the LPS from P aeruginosa PAO9002 B Absorbance Units (203 nm) A 15 10 Glial cells LPS LPS PAO1 PAO9002 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS EOF 0.2 0.1 0 10 20 Retention time (min) 30 5859 Effect of natriuretic peptides on Pseudomonas W Veron et al increase in cAMP concentration The basal concentration of cGMP in P aeruginosa was very low, at the limit of the sensitivity of the assay We observed only a slight, nonsignificant increase in cGMP concentration in bacteria treated with CNP We cannot exclude the possibility of an increase in cyclic nucleotide concentrations due to mechanisms involving a membrane phosphotransferase system and catabolic repression [35], but the similarity of the effects of natriuretic peptides in eukaryotes and prokaryotes and the growing number of adenylyl and guanylyl cyclases identified in bacteria [36] are consistent with the involvement of bacterial cyclases in the response of P aeruginosa to natriuretic peptides Consistent with these results, it appears that in addition to the well-characterized bacterial secondary messenger, cyclic di-GMP, levels of cyclic monophosphate nucleotides probably also play a crucial role in integrating the environmental signals transmitted to the bacterial cell surface [37] In Pseudomonas, as in all other Gram-negative bacteria, the endotoxin is a major virulence factor released on the death of the bacterium, but also produced in vesicular forms as an offensive weapon, particularly in response to antimicrobial molecules [38,39] We investigated the possible role of LPS in the response of P aeruginosa to natriuretic peptides, by studying the impact of stable analogs of cAMP and cGMP on the cytotoxic activity and chemical properties of the LPS from P aeruginosa PAO1 We used cyclic nucleotides rather than the natriuretic peptides themselves, because of technical limitations, as large volumes of bacterial culture were required, and the correspondingly large quantities of peptide required would have been unreasonably expensive P aeruginosa is known to respond rapidly to stress and to antimicrobial drugs by modulating the structure of its LPS [40] P aeruginosa should react similarly to natriuretic peptides or stable analogs of cyclic nucleotides This hypothesis is supported by the results of MEKC analysis Indeed, in bacteria treated with cyclic nucleotides, the greater cytotoxicity of the LPS appeared to be associated with a decrease in expression of the strongly charged or very hydrophobic forms of LPS by P aeruginosa This change was particularly marked in 8BcGMP-treated bacteria, in which an overall decrease in the diversity of LPS isoforms was observed In the absence of a mass spectrum database for P aeruginosa PAO1 LPS, the complexity of this molecule made it very difficult to interpret the spectra obtained by MALDI-TOF MS However, many essential subfragments of the LPS were found to have been modified in bacteria treated with dbcAMP and 8BcGMP The more obvious 5860 changes concerned the 1200–2050 Da zone, in which new peaks appeared, with molecules of masses between 2200 and 3100 Da tending to disappear Compounds with a molecular mass of around 1300– 1500 Da are generally considered to be signature components of lipid A [25], and molecules of 2500– 2900 Da are considered to be fragments of the oligosaccharide core with repeat O-units [26] We can therefore speculate that treatment with dbcAMP and 8BcGMP induces the reorganization of both the hydrophobic and polar regions of LPS, leading to an increase in the heterogeneity or number of acylated components of lipid A and a simplification or decrease in the length of the oligosaccharide core As lipid A plays a large part in the toxicity of LPS [41,42], these modifications may account for the greater cytotoxicity of the LPS extracted from dbcAMP-treated and 8BcGMP-treated bacteria We investigated the possible involvement of cyclic nucleotides in the action of natriuretic peptides in P aeruginosa in more detail by carrying out the same series of experiments with P aeruginosa PAO9002, a vfr null mutant of P aeruginosa PAO1 [32] The protein Vfr is a cAMP-binding protein that controls the production of many virulence factors [43,44] The basal cytotoxicity of P aeruginosa PAO9002 was particularly high, and this strain appeared to be totally insensitive to BNP and CNP The very high virulence of P aeruginosa PAO9002 suggests that the knock-down of vfr fixes the bacterium in a maximum virulence configuration, in which the bacterium cannot respond to natriuretic peptides This confirms the importance of cAMP in the mechanism of action of natriuretic peptides in P aeruginosa In this species, Vfr may be activated by both cAMP and cGMP, and is unlikely to discriminate between the two types of cyclic nucleotide [31] This may account for the identical effects of stable analogs of cAMP and cGMP in P aeruginosa PAO1 This hypothesis is also entirely consistent with the results of MEKC analysis, showing that the LPS of P aeruginosa PAO9002 is very similar to that of P aeruginosa PAO1 treated with 8BcGMP This study demonstrates, for the first time, that natriuretic peptides can modulate the virulence of P aeruginosa Our data strongly suggest that the action of BNP and CNP in the bacteria is linked to cyclic nucleotide production and the induction of Vfr protein activation The signal transduction cascade generated by this mechanism should regulate bacterial virulence, at least partly by modulating LPS structure Our results suggest that P aeruginosa has a membrane-associated natriuretic peptide sensor, and open up new areas of research into the evolution of the FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS W Veron et al physiological role of natriuretic peptides This discovery also provides an opportunity for the development of new therapeutic agents Experimental procedures Reagents and test substances DMEM and Ham’s F12 culture medium, Hepes buffer, poly(l-lysine), insulin, dbcAMP, 8BcGMP and human BNP were purchased from Sigma-Aldrich (St Quentin Fallavier, France) CNP peptide was obtained from Neosystem (Strasbourg, France) a-Cyano-4-hydroxycinnamic acid, trifluoroacetic acid and molecules for MS calibration were obtained from Sigma-Aldrich Corp (St Louis, MO, USA) Acetonitrile was purchased from Fisher Scientific (Loughborough, UK) Fetal bovine serum, l-glutamine and antibiotic ⁄ antimycotic solutions were obtained from Cambrex (Emerainville, France) The Cytotox 96 kit was purchased ` from Promega (Charbonnieres, France) Effect of natriuretic peptides on Pseudomonas Glial cell culture Newborn rats were decapitated 48–72 h after birth, in sterile conditions The brain was quickly extracted and rinsed in glial culture medium consisting of DMEM ⁄ Ham’s medium (2 : 1) supplemented with 10% fetal bovine serum, mm glutamine, 0.001% insulin, mm Hepes, 0.3% glucose and 1% antibiotic ⁄ antimycotic solution The meninges were removed, and the telencephalon was carefully dissected, immersed in glial culture medium, and mechanically dispersed for by gentle aspiration through a sterile needle The suspension was filtered through a sterile nylon filter with 82 lm pores to remove the remaining tissue fragments Cells were counted and layered, at a concentration of 105 cells per slide, on glass slides coated with 50 lgỈmL)1 poly(l-lysine) Glial cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and were allowed to grow for 9–14 days, to obtain a confluent culture in all culture wells The culture medium was changed the day after plating and every days thereafter Bacterial cultures Measurement of the release of cytosolic LDH by glial cells P aeruginosa PAO1 was obtained from an international collection The vfr null mutant of P aeruginosa PAO1, P aeruginosa 9002, was generously provided by S J Suh (University of Wisconsin-Madison, WI, USA) Both strains were grown in BNO (Merck, Darmstadt, Germany) at 37 °C For treatment with natriuretic peptides or cyclic nucleotides, bacteria were transferred to 10 mL of BNO containing (or not containing in the case of the control) the test substances, and were cultured overnight until the start of stationary phase Just before the infection assays, bacteria in early stationary phase were harvested by centrifugation in an Eppendorf centrifuge tube (2500 g, min, 20 °C) and resuspended at a cell density of 106 CFmL)1 in glial cell culture medium without antibiotics and antimycotics The density of the bacterial suspension was determined by absorption at 580 nm, using a spectrophotometer (ThermoSpectronics, Cambridge, UK) Bacterial density and the absence of contamination were checked by plating LDH is a stable cytosolic enzyme released into the culture medium upon cell lysis Its use as an indicator of necrosis in glial cells has been validated [8] The amount of LDH released by eukaryotic cells incubated with bacteria was determined with the Cytotox 96 enzymatic assay (Promega) Glial cells were incubated for h with control or treated P aeruginosa PAO1 at a concentration of 106 CFmL)1 For studies with P aeruginosa PAO9002, the incubation time was reduced to 2.5 h, because this strain is highly cytotoxic A lysis buffer, consisting of 9% Triton X-100 in water, was used to determine the maximum amount of LDH released by glial cells in our experimental conditions (100% LDH release) A background level, corresponding to 0% LDH release, was established using culture medium alone, making it possible to subtract the contribution to LDH activity of the medium used for glial cell culture The percentage of LDH release in the cell population was then determined by the equation: % LDH ¼ ðD sample À D 0%Þ Â 100= ðD 100% À D 0%Þ Animals Adult Wistar rats (180–200 g) were purchased from a com´ ´ mercial source (Depre, St Doulchard, France) Newborn Wistar rats were obtained by mating in the laboratory Animals were housed in an animal house (Agreement no AGEXP27.01) in which the temperature (20 °C) and photoperiod (12 h day ⁄ 12 h night) were artificially controlled Animal manipulations were performed under the supervision of authorized investigators and according to the European Communities Council Directive of 24 November 1986 (86 ⁄ 609 ⁄ EEC) where D is attenuance at 490 nm The assay was sensitive enough for determination of a concentration of LDH equivalent to the lysis of 1% of the cell population Measurement of intrabacterial cAMP and cGMP concentrations The levels of cAMP and cGMP in bacteria exposed to natriuretic peptides were determined using cAMP and a FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5861 Effect of natriuretic peptides on Pseudomonas W Veron et al cGMP direct enzyme immunoassay kits from SigmaAldrich P aeruginosa (106 CFmL)1) in early stationary phase was incubated for 30 at 37 °C in 10 mL of BNO supplemented with BNP or CNP (10)6 m) Bacteria were then centrifuged (6 min; 2500 g), and the pellets were resuspended in saline The samples were boiled for 10 min, ultracentrifuged (1 h; 62 000 g) to remove cell debris, and freeze-dried The resulting residues were resuspended in mL of water and assayed for cAMP or cGMP, according to the kit supplier’s protocol was purchased from Beckman Coulter (Villepinte, France) The capillary was installed in a refrigerated cartridge and conditioned with m NaOH for h before rinsing and equilibration with running buffer The separation buffer for MEKC consisted of 25 mm boric acid (pH 9) supplemented with SDS (80 mm) The potential used for separation (25 kV) was determined as the limit of linearity of the curve for the current–voltage relationships Samples were injected by N2 hydrostatic pressure (0.5 lbỈin)2, 20 s) and analyzed over a 30 period MALDI-TOF MS analysis of LPS LPS extraction and purification LPS was purified from P aeruginosa PAO1 according to a procedure based on that of Darveau & Hancock [23] Briefly, bacteria in early stationary phase were harvested by centrifugation (6000 g, 10 min, 20 °C) P aeruginosa PAO1 pellets were suspended in 10 mm Tris buffer containing mm MgCl2, 200 lgỈmL)1 pancreatic DNase and 50 lgỈmL)1 pancreatic RNase, and were submitted to four bursts of sonication of 30 s at a probe density of 70 The suspension was then incubated for h at 37 °C After this period, tetrasodium-EDTA, Tween-20 and Tris ⁄ HCl were added The sample was centrifuged (10 000 g, 30 min, 20 °C) to remove peptidoglycan The supernatant was incubated overnight with protease, at 37 °C, with constant shaking Two volumes of 0.375 m MgCl2 in 95% ethanol were added, and the mixture was cooled to °C The sample was then centrifuged (12 000 g, 15 min, °C), and the pellet was sonicated in a solution of Tween-20 and tetrasodium-EDTA The pH of the solution was lowered to 7, to prevent lipid saponification The solution was incubated for 30 at 85 °C, to ensure that outer membrane proteins were denatured, and the pH of the solution was increased to 9.5 Protease was then added, and the sample was incubated overnight at 37 °C Two volumes of 0.375 m MgCl2 in 95% ethanol were added, and the sample was centrifuged (12 000 g, 15min, °C) The pellet was resuspended in 10 mm Tris ⁄ HCl, sonicated, and centrifuged twice to remove insoluble Mg2+-EDTA crystals The supernatant was then ultracentrifuged (62 000 g, h, 15 °C), and the pellet, which contained LPS, was resuspended in distilled water and used for the KDO assay The control extraction buffer was prepared following the same procedure, but starting with sterile BNO Capillary electrophoresis of LPS Purified bacterial LPS molecules were analyzed by MEKC, using a Beckman (Gagny, France) P ⁄ ACE 5510 system equipped with a diode array detector (detection range, 190– 600 nm; wavelength of the curves presented, k ¼ 203 nm) and refrigerated injection system A fused-silica capillary tube (50 lm internal diameter · 57 cm; 50 cm to detector) 5862 For each test, lL of purified LPS was carefully spotted onto each target well and allowed to dry in air A solution of a-cyano-4-hydroxycinnamic acid matrix (14 mgỈmL)1) was prepared in acetronitrile ⁄ 2.5% trifluoroacetic acid (1 : 1, v ⁄ v) The matrix was sonicated for 30 min, and lL of matrix solution was added to each well and allowed to dry at room temperature MS was performed in linear mode on a Microflex Time-of-Flight machine (Bruker Daltonik GmbH, Leipzig, Germany) with a pulsed ultraviolet laser at 337 nm The machine was run in positive mode and calibrated with lL of a 10 pm protein standard mixture of angiotensin II, adrenocorticotrophic hormone Clip (18– 39), human insulin, oxidized B-chain, bovine insulin and cytochrome c spotted onto the target plate Each final spectrum consisted of 200 laser shots Statistics Assay results were determined for a minimum of 10 sample measurements, from three or four independent preparations Student’s t-test was used to compare the means of values within sets of experiments Acknowledgements We wish to 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E & Lory S (2003) Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway Dev Cell 4, 253–263 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS ... transduction of a signal mediated by natriuretic peptides, and a rapid and early effect of these peptides, we exposed the bacteria for a short time (30 min) to BNP and CNP and then determined their intracellular... to natriuretic peptides This confirms the importance of cAMP in the mechanism of action of natriuretic peptides in P aeruginosa In this species, Vfr may be activated by both cAMP and cGMP, and. .. Lam JS & Beveridge TJ (1993) Interaction of gentamicin with the A band and B band lipopolysaccharides of Pseudomonas aeruginosa and its possible lethal effect Antimicrob Agents Chemother 37, 715–721