Báo cáo khoa học: Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis doc

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Natriuretic peptides affect Pseudomonas aeruginosa andspecifically modify lipopolysaccharide biosynthesisWilfried Veron1, Olivier Lesouhaitier1, Xaviera Pennanec2, Karine Rehel2, Philippe Leroux3,Nicole Orange1and Marc G. J. Feuilloley11 Laboratory of Cold Microbiology, UPRES 2123, University of Rouen, Evreux, France2 Laboratoire de Biotechnologie et Chimie Marines, Universite´de Bretagne-Sud B.P. 92116, Lorient, France3 Research Department of Microenvironment and Cell Integrated Renewal, UPRES 3829, University of Rouen, FranceBacteria of the genus Pseudomonas can adapt to a multi-tude of environmental niches, owing to the size of theirgenome and the abundance of regulatory genes [1]. Thenatural resistance to b-lactams and biocides of Pseudo-monas strains and their opportunistic behavior poseconsiderable problems for hospitals [2]. Pseudomonasaeruginosa is one of the principal microorganismsresponsible for nosocomial diseases [3]. P. aeruginosahas considerable infectious potential, as it can infectboth surface tissues, such as skin and wounds, and inter-nal organs, such as the lungs and urinary tract [4]. Cen-tral nervous system infections are observed more rarely,but such infections are steadily increasing in numberand are associated with high morbidity rates [5,6]. Con-sistent with these clinical data, in vitro experiments haveshown that P. aeruginosa has a high specific affinity forneurons and glial cells, and that the binding of the bac-terium or its lipopolysaccharide (LPS) to these targetcells is associated with necrosis [7,8].In the host, bacteria are exposed to various defensemechanisms. Successful adaptation to an ecologicalniche, such as eukaryotic tissues, clearly requiresthe sensing of chemical signals, their integration andthe development of an appropriate response [9,10]. TheKeywordscyclases; cytotoxicity; natriuretic peptides;sensor; vfrCorrespondenceO. Lesouhaitier, Laboratory of ColdMicrobiology, UPRES 2123, University ofRouen, 55 rue Saint Germain,27000 Evreux, FranceFax: +33 232 29 15 55Tel: +33 232 29 15 42E-mail: olivier.lesouhait@univ-rouen.fr(Received 18 July 2007, revised 28 August2007, accepted 18 September 2007)doi:10.1111/j.1742-4658.2007.06109.xNatriuretic peptides of various forms are present in animals and plants,and display structural similarities to cyclic antibacterial peptides. Pretreat-ment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide(BNP) or C-type natriuretic peptide (CNP) increases bacterium-inducedglial cell necrosis. In eukaryotes, natriuretic peptides act through receptorscoupled to cyclases. We observed that stable analogs of cAMP (dibutyrylcAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natri-uretic peptide and CNP on bacteria. Further evidence for the involvementof bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicityby natriuretic peptides is provided by the observed doubling of intrabacte-rial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS)extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and8-bromo-cGMP induces higher levels of necrosis than LPS extracted fromuntreated bacteria. Capillary electrophoresis and MALDI-TOF MS analy-sis have shown that differences in LPS toxicity are due to specific differ-ences in the structure of the macromolecule. Using a strain deleted in thevfr gene, we showed that the Vfr protein is essential for the effect of natri-uretic peptides on P. aeruginosa PAO1 virulence. These data support thehypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriureticpeptide sensor system that may affect virulence by activating the expressionof Vfr and LPS biosynthesis.Abbreviations8BcGMP, 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 FEBSrole of the immune system in the response to Pseudo-monas infection has been investigated in detail [10,11].However, tissue factors or hormones, including natri-uretic peptides, have also been described as havingantimicrobial activity [12,13]. Three types of natriureticpeptide ) atrial natriuretic peptide, brain natriureticpeptide (BNP) and C-type natriuretic peptide (CNP) )were initially characterized. These molecules are majorcardiovascular and osmoregulatory factors in verte-brates, and have been detected both in peripheralorgans, where they are produced as hormones, and innervous tissues, where they act as neuromodulators[14]. Natriuretic peptides include a short loop (Fig. 1)containing an excess of positively charged amino acids.This structure is markedly similar to that of cationiccyclic antimicrobial peptides [15]. The abundance ofBNP and CNP in tissues not involved in regulatingblood pressure or diuresis suggests that these neuro-peptides probably have other activities. Natriureticpeptides have been conserved throughout the evolutionof animals, with different forms being present in verte-brates [16] and invertebrates [17]. Structural analogs ofanimal natriuretic peptides exist in plants [18], and agene encoding a plant natriuretic peptide-like moleculehas recently been identified in a Gram-negative bacte-rium [19]. Similarly, a class III cyclase with a similarfunction to the GMP cyclase receptors of eukaryoticnatriuretic peptides was recently described in prokary-otes [20]. These observations suggest that bacteriacould also employ natriuretic peptide-like molecules ascommunication tools.In the present study, we investigated the effects ofBNP and CNP (Fig. 1) on the virulence of P. aerugin-osa PAO1. We evaluated the virulence of unexposedbacteria and of bacteria exposed to these peptidesin vitro, using primary cultures of glial cells as thestudy model. We were able to reproduce the effects ofBNP and CNP by treating the bacteria with stableanalogs of cyclic nucleotides (Fig. 1). The effects ofBNP and CNP on the intrabacterial concentrations ofcAMP and cGMP were determined, and a vfr nullmutant of P. aeruginosa PAO1 was used to investigatethe mechanism of action of these factors. Finally, theimpact of stable analogs of cyclic nucleotides on thestructure and cytotoxicity of LPS was determined bycomplementary approaches.ResultsPreliminary studies showed that the treatment ofP. aeruginosa PAO1 with BNP or CNP (10)6m) hadno effect on bacterial growth (data not shown). Thepeptides were administered at various time points, atthe start of culture or during the early part of the sta-tionary phase. In some experiments, we added analiquot of peptide to the culture hourly, to take intoaccount possible degradation. In all cases, even onsolid medium, BNP and CNP had no effect on bacte-rial growth or cultivability.Effect of natriuretic peptides on the cytotoxicactivity of P. aeruginosa PAO1The effect of BNP and CNP on the virulence ofP. aeruginosa PAO1 was studied by treating bacteriawith these peptides before mixing them with cells andcomparing the resulting cytotoxicity with that ofGLSKGCCFGLKLDRIGSMSGGLNH2-HOOC-hBNPCNPdbcAMP8BcGMPNHONNNNOOOHHOOPOOHONNNNOHOHHOOPOOHH2NBrHMVQGSGCCFGRKMDRISSSSGGLKVLRRHNH2-HOOC-SPKFig. 1. Structure of human BNP and CNP and cyclic analogs of nu-cleotides used in the present study. Human BNP (hBNP) is com-posed of 32 amino acids whereas CNP contains only 22 aminoacids. dbcAMP and 8BcGMP are cell-permeable cAMP and cGMPanalogs, respectively.W. Veron et al. Effect of natriuretic peptides on PseudomonasFEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5853nontreated bacteria. The model of eukaryotic cellsused here ) primary cultures of glial cells ) has beenvalidated in previous studies [7,8]. In all experiments,bacteria were rinsed before exposure to glial cells, toremove all traces of peptide. The virulence of the bac-teria was evaluated by determining lactate dehydroge-nase (LDH) levels in the incubation medium. LDH isa stable enzyme present in the cytosol of eukaryoticcells, and is released into the medium when the mem-brane is destabilized. This destabilization is consideredto be a marker of necrosis. However, as some bacterialstrains can also generate LDH, we also checked forthe spontaneous production of this enzyme by P. aeru-ginosa PAO1. When P. aeruginosa PAO1 was incu-bated alone in the culture medium used for glial cells,LDH remained undetectable (Fig. 2). In the same con-ditions, glial cells cultured alone also released a limitedamount of LDH. The amount of LDH released inthese conditions indicates that only a small proportionof the glial cell population (7.6 ± 1.5%; n ¼ 18) spon-taneously underwent cell death during the course ofthe experiment (6 h). This result (fewer than 10% ofthe cells dying over a 6 h period) is an indicator of thequality of the culture, and all experiments in which thecontrols displayed spontaneous necrosis levels exceed-ing this value were excluded from the analysis. Whenglial cells were exposed to P. aeruginosa PAO1(106CFUÆmL)1, 6 h), the percentage of the cell popu-lation displaying signs of necrosis increased to39.0 ± 2.2%. The prior treatment of P. aeruginosawith natriuretic peptides (10)6m,14h,37°C)increased the cytotoxic activity of the bacteria signifi-cantly. In the presence of P. aeruginosa PAO1 treatedwith BNP, the percentage of the cell populationdisplaying 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 exposedto CNP (Fig. 2). A shorter period of prior treatmentof P. aeruginosa PAO1 with natriuretic peptides (4.5 h)had similar effects (data not shown). CNP had asignificantly stronger effect than BNP (P<0.001;Fig. 2). We carried out control experiments in whichglial cells were exposed only to BNP or CNP (10)6m).The percentage of glial cells displaying signs of necro-sis was identical to that of a control culture (data notshown). Thus, the effects observed were due entirely tothe direct action of BNP or CNP on the physiology ofP. aeruginosa PAO1.Effect of stable analogs of cAMP and cGMP onthe cytotoxic activity of P. aeruginosa PAO1The effect of natriuretic peptides in eukaryotic cells ismediated by different receptor subtypes with intrinsicguanylate cyclase activity [21] or through receptorscoupled to an adenylate cyclase [22]. We thereforeinvestigated the effects of cell-permeable stable analogsof cGMP [8-bromo-cGMP (8BcGMP)] and cAMP [di-butyryl cAMP (dbcAMP)] on the virulence of P. aeru-ginosa PAO1. As observed with natriuretic peptides,exposure of the bacteria to dbcAMP or 8BcGMP(10)5m;14h;37°C) significantly increased(P<0.001) the potential of P. aeruginosa PAO1 toinduce necrosis in glial cells. Necrosis levels reached57.9 ± 4.6% of the cell population when the bacteriawere exposed to dbcAMP, and 59.6 ± 5.1% when thebacteria were first treated with 8BcGMP, versus only39.0 ± 2.2% for nontreated P. aeruginosa PAO1(Fig. 3). Control studies were carried out with glialcells directly exposed to dbcAMP or 8BcGMP(10)5m). The percentage of glial cells displaying signsof necrosis was identical to that of a control culture,indicating that the stable analogs of cyclic nucleotideshad no intrinsic effect on the viability of glial cells(data not shown).+PAO1 control- Glial Cells-+++++PAO1 BNPtreated-+-PAO1 CNPtreated +LDH (% of release)03050709010Fig. 2. Cytotoxic activity of P. aeruginosa PAO1 treated with natri-uretic peptides. Effect of BNP and CNP (10)6M) on the cytotoxicityof P. aeruginosa PAO1. The cytotoxic effect of the bacterium wasdetermined by measuring LDH accumulation in the medium due tothe rupture of the cytoplasmic membrane of glial cells and therelease of this stable cytosolic enzyme. Values were calculated asthe mean concentration of LDH in the culture medium after 6 h ofincubation 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.Effect of natriuretic peptides on Pseudomonas W. Veron et al.5854 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBSEffect of natriuretic peptides on intrabacterialconcentrations of cAMP and cGMPAs exogenous cyclic nucleotides were found to have aneffect on P. aeruginosa PAO1, we investigated the pos-sible action of natriuretic peptides on the intrabacterialconcentrations of cAMP and cGMP. The concentra-tion of cAMP in P. aeruginosa PAO1 grown in ordin-ary nutrient broth medium (BNO) in the absence oftreatment was determined by ELISA. The valueobtained in early stationary phase (10.0 ± 1.4 pmolÆmL)1of bacterial extract) was used as the 100% basallevel (Fig. 4A). As we hypothesized a role for cyclicnucleotides in the transduction of a signal mediated bynatriuretic peptides, and a rapid and early effect ofthese peptides, we exposed the bacteria for a shorttime (30 min) to BNP and CNP and then determinedtheir intracellular cAMP content. The treatment ofP. aeruginosa PAO1 with BNP (10)6m) did not mod-ify cAMP levels in the bacteria (Fig. 4A). In contrast,CNP (10)6m) treatment significantly increased intra-bacterial cAMP concentrations, which reached174.8 ± 6.3% of control values (P<0.001). Thebasal concentration of cGMP in P. aeruginosa PAO1(0.55 ± 0.16 pmolÆmL)1) was lower than that ofcAMP, and was at the limit of the sensitivity of theassay. This value was used as the 100% basal level(Fig. 4B). The exposure of P. aeruginosa PAO1 toBNP or CNP (10)6m, 30 min) had no significant effecton cGMP levels in the bacteria (Fig. 4B).Effects of stable analogs of cAMP and cGMP onthe cytotoxicity of the LPS of P. aeruginosa PAO1LPS is largely responsible for the cytotoxicity of Pseu-domonas in glial cells [8]. We therefore investigated theimpact of stable analogs of cyclic nucleotides on theendotoxic activity of this macromolecule. The cytotox-icity of the LPS extracted from P. aeruginosa PAO1exposed to dbcAMP or 8BcGMP (10)5m; 4 h; 37 °C)was compared with that of the LPS extracted from con-trol bacteria in the same growth phase (early stationaryphase). LPS was extracted from control and treatedbacteria, and its concentration was determined by a2-keto-3-deoxyoctonate (KDO) assay. The mean con-centrations of LPS measured by this technique in bacte-rial cultures grown in the absence (0.65 mgÆmL)1)orLDH (% of release)01030709050PAO1 control-- Glial Cells+++++PAO1 dbcAMPtreated-+-PAO1 8BcGMPtreated +Fig. 3. Cytotoxic activity of P. aeruginosa PAO1 treated with cyclicnucleotide analogs. Effect of dbcAMP and 8BcGMP (10)5M) on thecytotoxicity of P. aeruginosa PAO1. Values were calculated as themean concentration of LDH in the culture medium after 6 h ofincubation 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.cAMP (% of control level)100200PAO1controlPAO1BNPtreated treatedPAO1CNPPAO1controlPAO1BNPtreated treatedPAO1CNPNSAB015050cGMP (% of control level)100200015050NS NSFig. 4. Intrabacterial concentration of mono-phosphate cyclic nucleotides in P. aerugin-osa PAO1 after exposure to natriureticpeptides. Effect of BNP and CNP (10)6M)on the intrabacterial concentration of cAMP(A) and cGMP (B) in P. aeruginosa PAO1.Data are the means of four independentexperiments. P<0.001, significantly differ-ent; NS, not significantly different.W. Veron et al. Effect of natriuretic peptides on PseudomonasFEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5855presence of cAMP or cGMP (0.53 mgÆmL)1and0.64 mgÆmL)1, respectively) were in the same range. Anequivalent amount of the molecule (500 ngÆmL)1) wasadded to the growth medium for glial cells, and itscytotoxic activity was determined by measuring LDHrelease, as previously described. The spontaneous basallevel of necrosis in glial cells was very low (4.9 ± 0.3%of the cell population in 24 h; n ¼ 10) (Fig. 5). Whenglial cells were exposed to LPS extracted from controlP. aeruginosa PAO1, 10.5 ± 0.3% of the glial cells dis-played signs of necrosis. The necrotic potential of theendotoxin extracted from P. aeruginosa PAO1 exposedto 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 signsof necrosis. Control studies were carried out withextracts of bacterial culture medium obtained with thesame protocol used for LPS extraction and purification.These experiments demonstrated that the effect of LPSon glial cell viability was not due to contaminants fromthe extraction buffers used to purify the endotoxin(data not shown).Effect of stable analogs of cAMP and cGMP onthe structure of the LPS of P. aeruginosa PAO1The effect of stable analogs of cAMP and cGMP onthe structure of the LPS was studied using two com-plementary techniques, micellar electrokinetic chroma-tography (MEKC) and MALDI-TOF MS. In bothcases, LPS was extracted from P. aeruginosa PAO1,with or without exposure to dbcAMP or 8BcGMP(10)5m; 4 h; 37 °C), and purified for analysis asdescribed by Darveau & Hancock [23]. LPS was firstanalyzed by MEKC, as this technique separates hydro-phobic molecules differing little in size and polaritymore efficiently than HPLC. We have previouslyshown that it is possible to differentiate between LPSmolecules from the same strain of Pseudomonas grownat different temperatures by MEKC [24]. The LPS ofP. aeruginosa PAO1 grown in control conditions gavethree major peaks (Fig. 6A). The first and major peak(peak 1) had a retention time of 8.2 min. The secondpeak was associated with a series of compounds ofvery similar structure and had a retention time of11.8 min. The third major peak was well separated,with a retention time of 22.5 min, and seemed to cor-respond to a single molecular form that was stronglyretained and highly hydrophobic. The electrophero-grams of LPS extracted from P. aeruginosa PAO1 trea-ted with dbcAMP and 8BcGMP were considerablymodified (Fig. 6B,C). Peak 1, eluted close to the elec-tro-osmotic flux, corresponded to poorly charged andnonhydrophobic molecules ) presumably a form ofLPS with a large osidic chain and a small number ofionized phosphate groups. The main differencesbetween LPS extracted from bacteria with and withoutcyclic nucleotide treatment concerned the levels ofpeaks 2 and 3. In dbcAMP-treated bacteria, peak 2was much smaller and a fourth peak appeared at17.1 min (Fig. 6B). Peak 3 was also broadened andaccompanied by secondary traces between 22.2 and23.0 min. The difference between LPS extracted fromcontrol and 8BcGMP-treated bacteria was evengreater. LPS from bacteria exposed to 8BcGMP dis-played a signal corresponding to peak 1, no peak 2,and only a small, broadened peak 3 (Fig. 6C). Thechanges observed in the LPS from dbcAMP-treatedand 8BcGMP-treated bacteria suggest a decrease inthe strongly charged or very hydrophobic forms ofLPS.MALDI-TOF MS of the LPS of P. aeruginosaPAO1 identified a large number of compounds(Fig. 7A). The lack of a mass spectrum library for theLPS of P. aeruginosa PAO1 made it difficult to inter-pret the results. A study of MS data [25–30] forGlial CellsLPS PAO1LPS PAO1controldbcAMP treatedLPS PAO18BcGMP treatedLDH (% of release)0481612-- +++++-+- +Fig. 5. Cytotoxic activity of LPS from P. aeruginosa PAO1 treatedwith cyclic nucleotide analogs. Effect of dbcAMP and 8BcGMP(10)5M) on the cytotoxicity of the LPS from P. aeruginosa PAO1.Values are expressed as the mean concentration of LDH in the cul-ture medium after 24 h of incubation with LPS (500 ngÆmL)1) fromnontreated (control) (n ¼ 12) or treated (n ¼ 12) bacteria. Data arethe means of three independent experiments. P<0.001, signifi-cantly different.Effect of natriuretic peptides on Pseudomonas W. Veron et al.5856 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBSP. aeruginosa PAO1 LPS fragments led to the identifi-cation of four principal clusters corresponding to dif-ferent components of the molecule. Cluster I seems tobe generated by the fragments of lipid A, whereas themolecular masses associated with clusters II, III andIV correspond to compounds associated with oligosac-charide cores with different O-antigen repeating units.The LPS extracted from P. aeruginosa PAO1 treatedwith dbcAMP presented many structural variations(Fig. 7B). In particular, the multiple peaks in theregion between 2000 and 3200 Da, corresponding tothe region of the complete oligosaccharide core withrepeated O-units [26,30], were numerous for controlLPS (Fig. 7A) and much more limited for LPS frombacteria treated with dbcAMP (Fig. 7B). Treatmentwith 8BcGMP had no obvious effect on this part ofthe LPS molecule (Fig. 7C). In contrast, the profiles ofthe LPS molecules from P. aeruginosa PAO1 treatedwith dbcAMP and 8BcGMP generated a large numberof new compounds (36 and 18, respectively) withmolecular masses between 1000 and 1900 Da, a zonenormally attributed to the components of lipid A [25].Effect of natriuretic peptides on the cytotoxicactivity of P. aeruginosa PAO9002In P. aeruginosa, the protein Vfr has been shown to beactivated by both cAMP and cGMP [31]. We investi-gated the possible involvement of Vfr in the actionof natriuretic peptides on P. aeruginosa, using strainPAO9002, a vfr null mutant of P. aeruginosa PAO1obtained by insertion of the accC1 gene into the vfrsite [32]. As for P. aeruginosa PAO1, we checked thatthe strain did not spontaneously produce any LDH-related molecule that might interfere with our assay(data not shown). The basal cytotoxicity of P. aerugin-osa PAO9002 for glial cells was intrinsically muchhigher than that of P. aeruginosa PAO1. We thereforereduced the time for which glial cells were incubatedwith P. aeruginosa PAO9002 to 2.5 h. In these condi-tions, the percentage of cells displaying signs of necro-sis following the exposure of glial cells to P. aeruginosaPAO9002 (106CFUÆmL)1) was in a similar range tothat following the exposure of cells to P. aeruginosaPAO1, i.e. 39.0 ± 2.2%. In contrast to the resultsobtained with P. aeruginosa PAO1, the prior treatmentof P. aeruginosa PAO9002 with BNP or CNP (10)6m;14 h; 37 °C) had no significant effect on the cytotoxicpotential of this strain (Fig. 8).Cytotoxicity and chemical properties of the LPSextracted from P. aeruginosa PAO9002The LPS of P. aeruginosa PAO9002 was extracted andpurified, and its cytotoxicity was determined in pri-mary cultures of glial cells as described above. TheLPS extracted from P. aeruginosa PAO9002 was lesscytotoxic than that extracted from P. aeruginosa PAO1PAO1controlPAO1dbcAMPtreatedPAO18BcGMPtreatedRetention time (min)Absorbance Units (203 nm)01020300.20.100.20.100.20.1023ABC4EOF1Fig. 6. MECK analysis of LPS treated with cyclic nucleotide ana-logs. MECK analysis of the LPS extracted from control (A),dbcAMP (10)5M)-treated (B) or 8BcGMP (10)5M)-treated (C)P. aeruginosa PAO1. EOF, electro-osmotic flux. Arrows and num-bers refer to the different molecular forms identified in the LPSfrom P. aeruginosa PAO1.W. Veron et al. Effect of natriuretic peptides on PseudomonasFEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5857(Fig. 9A). The percentage of the cell population under-going necrosis in the presence of LPS from P. aerug-inosa PAO9002 (14.56 ± 0.98%) was higher thanthat 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. aerugin-osa PAO1 (18.05 ± 0.42%). MEKC analysis of thisLPS revealed marked differences from the LPSextracted from P. aeruginosa PAO1 (Fig. 9B). Indeed,the diversity of molecular forms identified in the LPSfrom P. aeruginosa PAO9002 was very low. Most ofthe signal was concentrated in peak 1. Another form,denoted peak 5, appeared at 20.3 min. A small signalwas also detected around peak 2. The electrophero-gram profile of the LPS from P. aeruginosa PAO9002closely resembled that of the LPS from P. aeruginosaPAO1 after treatment with 8BcGMP.DiscussionThe structure of BNP and CNP and the results pub-lished by Krause et al. [13] suggested that human2777.91ABC6456.877212.738367.220.000.250.500.751.001.25X104X104X104Intens. [a.u.]Intens. [a.u.]Intens. [a.u.]2000 4000 6000 8000 10000 12000 14000 16000 18000m/z2000 4000 6000 8000 10000 12000 14000 16000 18000m/z2000 4000 6000 8000 10000 12000 14000 16000 18000m/z3062.496458.107317.120.00.51.01.52.02.53.02778.756458.125337.8712165.8701234LPS PAO1 controlLPS PAO1 dbcAMP treatedLPS PAO1 8BcGMP treatedIIIIIIIVFig. 7. MALDI-TOF analysis of LPS treated with cyclic nucleotide analogs. MALDI-TOF mass spectra obtained from the LPS extracted fromcontrol (A), dbcAMP (10)5M)-treated (B) or 8BcGMP (10)5M)-treated (C) P. aeruginosa PAO1. Roman numerals (A) indicate clusters of peakscorresponding to compounds with similar molecular masses.Effect of natriuretic peptides on Pseudomonas W. Veron et al.5858 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBSnatriuretic peptides, and not rat peptides, might haveantibacterial activity. Our preliminary studies showedthat, in contrast, at micromolar concentrations similarto those encountered in animal tissues, BNP and CNPhave no antibacterial effect. As antibacterial agentsare also known to have indirect effects on bacterialvirulence [33], we used a well-characterized in vitroassay developed in our laboratory [7,8] to investigatethe effects of BNP and CNP on the cytotoxicity ofbacteria.We cultured P. aeruginosa in the presence of BNPor CNP. Bacteria were exposed to the peptides duringthe exponential growth phase and were then rinsed toremove any trace of BNP or CNP before mixing withprimary cultures of glial cells. As confirmed by con-trols, in which natriuretic peptides were directly mixedwith glial cells, our results can only be explained bychanges in bacterial virulence. The observation thattreating P. aeruginosa with BNP and CNP increasesthe cytotoxic effect of the bacteria suggests that natri-uretic factors can affect the physiology of microorgan-isms sufficiently to modify virulence factor production,but not enough to affect the growth and survival ofP. aeruginosa. These results are entirely consistent withpublished data suggesting that antimicrobial peptidescan exert multiple effects, not simply inhibiting bacte-rial growth, but also interfering with microbial physiol-ogy, including the capacity to produce virulencefactors or even toxin activity [33].Peptides cannot penetrate cells freely. Thus, ineukaryotic cells, natriuretic peptides exert their effectsthrough three different cytoplasmic membrane receptorsubtypes with intrinsic guanylate cyclase activity [21]or indirectly coupled to an adenylate cyclase [22]. Asin investigations of the possible involvement of cyclicnucleotide phosphates in the mechanism of action ofneurohormones in eukaryotes [34], we tested the effectof stable analogs of cGMP and cAMP on P. aerugin-osa. The exposure of bacteria to 8BcGMP anddbcAMP fully reproduced the effects of BNP andCNP, increasing necrosis. As exogenous cyclic nucleo-tides affected P. aeruginosa PAO1 virulence, we inves-tigated the possible effects of natriuretic peptides onintrabacterial cAMP and cGMP concentrations. Thelevel of cAMP in P. aeruginosa was unaffected byexposure to BNP. In contrast, CNP induced a markedLDH (% of release)1020ABGlialcellsLPSPAO1LPSPAO900201551250.20.10Absorbance Units (203 nm)EOFRetention time (min)0 102030Fig. 9. Characteristics of the LPS extractedfrom the vfr null mutant P. aeruginosaPAO9002. Cytotoxic activity (A) and MEKCelectropherogram (B) of the LPS extractedfrom the vfr null mutant of P. aeruginosaPAO9002. Cytotoxicity values were calcu-lated as the means of three independentexperiments. P<0.001, significantly differ-ent; P<0.01, significantly different; EOF,electro-osmotic flux. Arrows and numbersrefer to the different molecular forms identi-fied in the LPS from P. aeruginosaPAO9002.PAO9002control-PAO9002 BNPtreatedPAO9002 CNPtreated-+-+-+--LDH (% of release)030405020NS NS10Fig. 8. Cytotoxic activity of P. aeruginosa PAO9002 treated withnatriuretic peptides. Effect of BNP and CNP (10)6M) on the cyto-toxicity of P. aeruginosa PAO9002. Values were calculated as themean concentration of LDH in the culture medium after 2.5 h ofincubation with nontreated (control) (n ¼ 16) or treated (n ¼ 16)bacteria. Data are the means of four independent experiments. NS,not significantly different.W. Veron et al. Effect of natriuretic peptides on PseudomonasFEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5859increase in cAMP concentration. The basal concentra-tion of cGMP in P. aeruginosa was very low, at thelimit of the sensitivity of the assay. We observed onlya slight, nonsignificant increase in cGMP concentra-tion in bacteria treated with CNP. We cannot excludethe possibility of an increase in cyclic nucleotide con-centrations due to mechanisms involving a membranephosphotransferase system and catabolic repression[35], but the similarity of the effects of natriuretic pep-tides in eukaryotes and prokaryotes and the growingnumber of adenylyl and guanylyl cyclases identified inbacteria [36] are consistent with the involvement ofbacterial cyclases in the response of P. aeruginosa tonatriuretic peptides. Consistent with these results, itappears that in addition to the well-characterized bac-terial secondary messenger, cyclic di-GMP, levels ofcyclic monophosphate nucleotides probably also play acrucial role in integrating the environmental signalstransmitted to the bacterial cell surface [37].In Pseudomonas, as in all other Gram-negative bac-teria, the endotoxin is a major virulence factorreleased on the death of the bacterium, but also pro-duced in vesicular forms as an offensive weapon,particularly in response to antimicrobial molecules[38,39]. We investigated the possible role of LPS inthe response of P. aeruginosa to natriuretic peptides,by studying the impact of stable analogs of cAMPand cGMP on the cytotoxic activity and chemicalproperties of the LPS from P. aeruginosa PAO1. Weused cyclic nucleotides rather than the natriuretic pep-tides themselves, because of technical limitations, aslarge volumes of bacterial culture were required, andthe correspondingly large quantities of peptiderequired would have been unreasonably expensive.P. aeruginosa is known to respond rapidly to stressand to antimicrobial drugs by modulating the struc-ture of its LPS [40]. P. aeruginosa should react simi-larly to natriuretic peptides or stable analogs of cyclicnucleotides. This hypothesis is supported by theresults of MEKC analysis. Indeed, in bacteria treatedwith cyclic nucleotides, the greater cytotoxicity of theLPS appeared to be associated with a decrease inexpression of the strongly charged or very hydropho-bic forms of LPS by P. aeruginosa. This change wasparticularly marked in 8BcGMP-treated bacteria, inwhich an overall decrease in the diversity of LPS iso-forms was observed. In the absence of a mass spec-trum database for P. aeruginosa PAO1 LPS, thecomplexity of this molecule made it very difficult tointerpret the spectra obtained by MALDI-TOF MS.However, many essential subfragments of the LPSwere found to have been modified in bacteria treatedwith dbcAMP and 8BcGMP. The more obviouschanges concerned the 1200–2050 Da zone, in whichnew peaks appeared, with molecules of massesbetween 2200 and 3100 Da tending to disappear.Compounds with a molecular mass of around 1300–1500 Da are generally considered to be signaturecomponents of lipid A [25], and molecules of 2500–2900 Da are considered to be fragments of the oligo-saccharide core with repeat O-units [26]. We cantherefore speculate that treatment with dbcAMP and8BcGMP induces the reorganization of both thehydrophobic and polar regions of LPS, leading to anincrease in the heterogeneity or number of acylatedcomponents of lipid A and a simplification ordecrease in the length of the oligosaccharide core. Aslipid A plays a large part in the toxicity of LPS[41,42], these modifications may account for thegreater cytotoxicity of the LPS extracted fromdbcAMP-treated and 8BcGMP-treated bacteria.We investigated the possible involvement of cyclicnucleotides in the action of natriuretic peptides inP. aeruginosa in more detail by carrying out the sameseries of experiments with P. aeruginosa PAO9002, avfr null mutant of P. aeruginosa PAO1 [32]. The pro-tein Vfr is a cAMP-binding protein that controls theproduction of many virulence factors [43,44]. The basalcytotoxicity of P. aeruginosa PAO9002 was particularlyhigh, and this strain appeared to be totally insensitiveto BNP and CNP. The very high virulence of P. aeru-ginosa PAO9002 suggests that the knock-down of vfrfixes the bacterium in a maximum virulence configura-tion, in which the bacterium cannot respond to natri-uretic peptides. This confirms the importance of cAMPin the mechanism of action of natriuretic peptides inP. aeruginosa. In this species, Vfr may be activated byboth cAMP and cGMP, and is unlikely to discriminatebetween the two types of cyclic nucleotide [31]. Thismay account for the identical effects of stable analogsof cAMP and cGMP in P. aeruginosa PAO1. Thishypothesis is also entirely consistent with the results ofMEKC analysis, showing that the LPS of P. aerugin-osa PAO9002 is very similar to that of P. aeruginosaPAO1 treated with 8BcGMP.This study demonstrates, for the first time, thatnatriuretic peptides can modulate the virulence ofP. aeruginosa. Our data strongly suggest that theaction of BNP and CNP in the bacteria is linked tocyclic nucleotide production and the induction of Vfrprotein activation. The signal transduction cascadegenerated by this mechanism should regulate bacterialvirulence, at least partly by modulating LPS structure.Our results suggest that P. aeruginosa has a mem-brane-associated natriuretic peptide sensor, and openup new areas of research into the evolution of theEffect of natriuretic peptides on Pseudomonas W. Veron et al.5860 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBSphysiological role of natriuretic peptides. This discov-ery also provides an opportunity for the developmentof new therapeutic agents.Experimental proceduresReagents and test substancesDMEM and Ham’s F12 culture medium, Hepes buffer,poly(l-lysine), insulin, dbcAMP, 8BcGMP and humanBNP were purchased from Sigma-Aldrich (St Quentin Fal-lavier, France). CNP peptide was obtained from Neosystem(Strasbourg, France). a-Cyano-4-hydroxycinnamic acid,trifluoroacetic acid and molecules for MS calibration wereobtained from Sigma-Aldrich Corp. (St Louis, MO, USA).Acetonitrile was purchased from Fisher Scientific (Lough-borough, UK). Fetal bovine serum, l-glutamine and anti-biotic ⁄ antimycotic solutions were obtained from Cambrex(Emerainville, France). The Cytotox 96 kit was purchasedfrom Promega (Charbonnie`res, France).Bacterial culturesP. aeruginosa PAO1 was obtained from an internationalcollection. 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 strainswere grown in BNO (Merck, Darmstadt, Germany) at37 °C. For treatment with natriuretic peptides or cyclicnucleotides, bacteria were transferred to 10 mL of BNOcontaining (or not containing in the case of the control) thetest substances, and were cultured overnight until the startof stationary phase. Just before the infection assays, bacteriain early stationary phase were harvested by centrifugation inan Eppendorf centrifuge tube (2500 g, 4 min, 20 °C) andresuspended at a cell density of 106CFUÆmL)1in glial cellculture medium without antibiotics and antimycotics. Thedensity of the bacterial suspension was determined byabsorption at 580 nm, using a spectrophotometer (Thermo-Spectronics, Cambridge, UK). Bacterial density and theabsence of contamination were checked by plating.AnimalsAdult Wistar rats (180–200 g) were purchased from a com-mercial source (De´pre´, St Doulchard, France). NewbornWistar rats were obtained by mating in the laboratory.Animals were housed in an animal house (Agreementno. AGEXP27.01) in which the temperature (20 °C) andphotoperiod (12 h day ⁄ 12 h night) were artificially con-trolled. Animal manipulations were performed under thesupervision of authorized investigators and according to theEuropean Communities Council Directive of 24 November1986 (86 ⁄ 609 ⁄ EEC).Glial cell cultureNewborn rats were decapitated 48–72 h after birth, in ster-ile conditions. The brain was quickly extracted and rinsedin glial culture medium consisting of DMEM ⁄ Ham’s med-ium (2 : 1) supplemented with 10% fetal bovine serum,2mm glutamine, 0.001% insulin, 5 mm Hepes, 0.3% glu-cose and 1% antibiotic ⁄ antimycotic solution. The meningeswere removed, and the telencephalon was carefully dis-sected, immersed in glial culture medium, and mechanicallydispersed for 5 min by gentle aspiration through a sterileneedle. The suspension was filtered through a sterile nylonfilter with 82 lm pores to remove the remaining tissue frag-ments. Cells were counted and layered, at a concentrationof 105cells per slide, on glass slides coated with 50 lgÆmL)1poly(l-lysine). Glial cells were incubated at 37 °Cinahumidified atmosphere containing 5% CO2and wereallowed to grow for 9–14 days, to obtain a confluent cul-ture in all culture wells. The culture medium was changedthe day after plating and every 2 days thereafter.Measurement of the release of cytosolic LDH byglial cellsLDH is a stable cytosolic enzyme released into the culturemedium upon cell lysis. Its use as an indicator of necrosis inglial cells has been validated [8]. The amount of LDH releasedby eukaryotic cells incubated with bacteria was determinedwith the Cytotox 96 enzymatic assay (Promega). Glial cellswere incubated for 6 h with control or treated P. aeruginosaPAO1 at a concentration of 106CFUÆmL)1. For studies withP. aeruginosa PAO9002, the incubation time was reduced to2.5 h, because this strain is highly cytotoxic. A lysis buffer,consisting of 9% Triton X-100 in water, was used to deter-mine the maximum amount of LDH released by glial cells inour experimental conditions (100% LDH release). A back-ground level, corresponding to 0% LDH release, was estab-lished using culture medium alone, making it possible tosubtract the contribution to LDH activity of the medium usedfor glial cell culture. The percentage of LDH release in the cellpopulation was then determined by the equation:% LDH ¼ðD sample À D 0%ÞÂ100=ðD 100% À D 0%Þwhere D is attenuance at 490 nm.The assay was sensitive enough for determination of aconcentration of LDH equivalent to the lysis of 1% of thecell population.Measurement of intrabacterial cAMP and cGMPconcentrationsThe levels of cAMP and cGMP in bacteria exposed tonatriuretic peptides were determined using cAMP and aW. Veron et al. Effect of natriuretic peptides on PseudomonasFEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5861[...]... 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Krause A, Liepke C, Meyer M, Adermann K, Forssmann WG & Maronde E (2001) Human natriuretic peptides exhibit antimicrobial activity Eur J Med Res 6, 215–218 Jamison RL, Canaan-Kuhl S & Pratt R (1992) The natriuretic peptides and their receptors Am J Kidney Dis 20, 519–530 Bulet P, Stocklin R & Menin L (2004) Anti-microbial ¨ peptides: from invertebrates to vertebrates Immunol Rev 198, 169–184 Inoue K, . Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis Wilfried Veron1,. mediated by natriuretic peptides, and a rapid and early effect ofthese peptides, we exposed the bacteria for a shorttime (30 min) to BNP and CNP and then
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