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Quorum sensing in the Burkholderia cepacia complex

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Quorum sensing in the Burkholderia cepacia complex
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  Research in Microbiology 155 (2004) 238–244www.elsevier.com/locate/resmic Mini-review Quorum sensing in the  Burkholderia cepacia  complex Vittorio Venturi ∗ , Arianna Friscina, Iris Bertani, Giulia Devescovi, Claudio Aguilar  Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy Received 29 December 2003; accepted 22 January 2004Available online 30 January 2004 Abstract Quorum sensing is a cell-density-dependent regulatory mechanism which, in Gram-negative bacteria, usually involves the production anddetection of   N  -acyl homoserine lactones (HSLs). In the last four years HSL-dependent quorum sensing has been identified in members of the  Burkholderia cepacia  complex, and this mini-review summarizes initial findings and discusses future perspectives. © 2004 Elsevier SAS. All rights reserved. Keywords:  Quorum sensing;  Burkholderia cepacia  complex; Homoserine lactone 1. Cell–cell communication in bacteria The regulation of gene expression allows a bacterium toeffectively adapt to rapid changes in the surrounding en-vironment; consequently several regulatory systems haveevolved which permit adaptation to environmental fluctua-tions. Another major level of regulation has recently beendiscovered which involves cell–cell communication via theproduction of small signaling molecules called autoinduc-ers. Many signaling molecules have to date been identifiedand most are involved in a form of regulation known as quo-rum sensing, allowing bacteria to monitor their populationdensity by responding to the extracellular concentration of the autoinducer they produce [28]. Many quorum-sensing-regulated phenotypes are often most beneficial only when acommunityof bacteria expresses them, as is the case, for ex-ample, in biofilm formation, conjugation, bioluminescencesecretion of enzymes,virulencefactors and pigmentproduc-tion [31].In Gram-negative bacteria,  N  -acyl homoserine lactone(HSL) autoinducers appear to be the most commonly usedsignaling molecules produced by an autoinducer synthasebelonging to the LuxI protein family. A transcriptional reg-ulator, belonging to the LuxR family, then forms a complexwith the cognate autoinducer at high threshold levels affect-ing the transcriptional activity of target genes. The HSLs * Corresponding author.  E-mail address:  venturi@icgeb.org (V. Venturi). produced by different bacterial species are believed to befreely diffusible across the cell envelope and to differ inthe length and structure of the acyl chain. The paradigmfor HSL-dependent quorum sensing is bioluminescence  lux activation in the marine symbiotic bacterium  Vibrio fis-cheri ; the LuxI synthase synthesizes  N  -(3-oxo-hexanoyl)- L -homoserine lactone (3oxoC6HSL) which then interactswith its receptor LuxR, to form an active complex resultingin activation of the  lux  regulon. Many LuxIR-type quo-rum sensing systems have now been found, and amongthe most studied are the  Agrobacterium tumefaciens  systemcontrolling conjugation and Ti plasmid replication,  Erwiniacarotovora  controlling exoenzymes and antibiotic produc-tion, and the  Pseudomonas aeruginosa  systems regulatingbiofilm and virulence gene expression [31]. The purposeof this mini-review is to summarize and discuss currentknowledge of quorum sensing in the  Burkholderia cepa-cia  complex (BCC), as these bacterial species have recentlyreceived increasing attention due to their emergence as im-portant opportunistic pathogens of humans in the cysticfibrosis (CF) lung [5]. 2. The BCC The  Burkholderia  genus is a very heterogenous groupof species (Gram-negative,  β -proteobacteria and consist-ing of over 30 species) and was first designated in 1992as accommodating almost all the former rRNA group II 0923-2508/$ – see front matter  © 2004 Elsevier SAS. All rights reserved.doi:10.1016/j.resmic.2004.01.006  V. Venturi et al. / Research in Microbiology 155 (2004) 238–244  239 pseudomonads [5,32]. Species belonging to  Burkholderia colonize different ecological niches including, soil, wa-ter, and root rhizosphere; they may have both pathogenicand symbiotic interactions with plants, and are also patho-genic to humans [5]. The species  B. cepacia  was orig-inally described by Burkholder in 1950 as the causativeagent of bacterial rot of onions, causing a disease calledsour skin [3]. In the last decade, however, several tax-onomic studies have revealed that strains of   B. cepacia in reality represent a complex of closely related speciesor genomovars. In fact, to date, the term ‘  Burkholderiacepacia  complex (BCC)’ refers to a group of nine closelyrelated species all of which have been isolated from envi-ronmental and clinical sources [5]. These nine species are  B. cepacia  (or genomovarI),  B. multivorans (genomovarII),  B. cenocepacia (genomovarIII),  B. stabilis  (genomovarIV),  B. vietnamiensis  (genomovarV),  B. dolosa  (genomovarVI),  B. amifaria  (genomovar VII),  B. anthina  (genomovar VIII)and  B. pyrrocina  (genomovar IX). Research interest in thisbacterial complex has grown considerably in the last fewyearswithinthescientificcommunity,sincepatientswithCFare particularly susceptible to pulmonary infections causedby these bacteria, increasing the risk of mortality. Infec-tion by BCC bacteria usually occurs in patients who arealready colonized with  Pseudomonas aeruginosa,  which isthe main cause of chronic lung infection in CF patients [4].Some strains belonging to the BCC also display potentialas biocontrol agents, as they can efficiently colonize theroot rhizosphere of several important crops and antagonizegrowthofmicrobialplantpathogens.Finally,theratherlargegenomes of BCC bacteria provide them with extraordinarynutritional versatility and adaptability, making them poten-tially useful for bioremediation purposes ([5] and referencestherein). 3. Quorum sensing genes and signals in the BCC The first indication that cell–cell communication waspresent in the BCC was the observation that  P. aeruginosa concentrated stationary phase spent culture fluids markedlyaffected the production of siderophore, protease and lipasein a BCC strain [19]. These studies were initiated be-cause  P. aeruginosa  and BCC strains co-aggregate in theCF lung, and thus it was of interest to determine whetherone species synergistically enhances the virulence determi-nants of the other (see below). The first quorum sensingsystem of a BCC strain was then isolated following thecharacterization of a transposon mutant which, in stationaryphase, hyperproduced the siderophore ornibactin in clinicalisolate  B. cenocepacia  strain K56-2 [15]. The quorum sens-ing system consisted of LuxIR homologs CepI (22.2 kDa)and CepR (26.5 kDa) displaying highest identity (64 and67%, respectively) to the SolIR quorum sensing proteins of closely related  Ralstonia solanacearum . CepI in  B. ceno-cepacia  K56-2 synthesizes two HSL molecules, a C6HSL( N  -hexanoyl- L -homoserine lactone, also called HHL) anda C8HSL ( N  -octanoyl- L -homoserine lactone or OHL). The cepI   and  cepR  genes are divergently transcribed, separatedby an intergenic region of 727 base pairs; the  cepI   promotercontains a putative 20-bp  lux -box like sequence that par-tially overlaps the putative − 35 region of the promoter. Thisbox could be recognized by CepR–HSL, since  cepI   is pos-itively regulated by CepR at high cell densities providing amechanism of signal amplification via a positive feed-back control [16].Another quorum sensing system from a  B. cenocepa-cia  clinical isolate was identified on the basis of its re-quirement for biofilm formation on abiotic surfaces [13].The system was close to that identified in  B. cenocepa-cia  K56-2 (Table 1) consisting of CepIR proteins and alsoproducing C8- and C6HSL [12,13]. In this  B. cenocepacia strain, H111, it was determined that C8HSL and C6HSLwere produced at a molar ratio of approximately 10:1 withCepR responding most efficiently to C8HSL ([12,13,23],see below). The quorum sensing system of   B. cepacia type strain ATCC 25416 T srcinally isolated from onionrot [3] was also recently identified and characterized anddisplayed almost identical characteristics to the other twoCepIR systems of   B. cenocepacia  clinical isolates K56-2and H111 ([1], Table 1). The identification and charac-terization of these three CepIR quorum sensing systemsdemonstrated that the proteins, gene organization and struc-ture of HSLs are highly identical (Table 1), indicating thatwithin the BCC the quorum sensing loci are basically iden-tical.The CepIR system has thus been demonstrated to bewidely distributed throughout the BCC, since autoinducerprofiles of tested strains showed that they all make C6-and C8HSLs, and PCR has allowed the cloning of several cepIR  homologs (Table 1, [12,18]). Interestingly,  B. viet-namiensis  strains, as well as producing C6- and C8HSLs,the autoinducer profiles, displayed additional HSL mole-cules. In fact,  B. vietnamiensis  strain G4, an environmentalisolate from a waste treatment facility, produced C6-, C8-,C10-, C12HSLs, and 3oxoC10HSLs [6]. The concentrationsof these HSLs detected in the culture fluid varied con-siderably, with C10HSL being the most abundant, C8HSLless abundant, and C6-, C12- and 3OC10HSLs presentonly in small amounts. Similarly, another environmentalisolate of   B. vietnamiensis  displayed similar HSL autoin-ducer profiles, producing C10- and C12HSLs as well asC6- and C8HSLs [18]. In these strains an additional quo-rum sensing system was shown to be present, designatedBviIR (BviI and BviR are only 36% identical to CepIand CepR, respectively), which is responsible for the pro-duction of the additional HSLs. It therefore appears that,unlike other species of BCC,  B. vietnamiensis  containstwo quorum sensing systems, and it remains to be deter-mined whether these two systems are hierarchically orga-nized.  V. Venturi et al. / Research in Microbiology 155 (2004) 238–244  241 4. CepIR-dependent phenotypes, gene expressionand pathogenicity 4.1. CepI-regulated phenotypes The CepIR quorum sensing system was first identi-fied in two  B. cenocepacia  clinical isolates due to its in-volvement in siderophore production and biofilm formation[13,15,16]. A knock-outmutantin  cepI   and  cepR  in  B. ceno-cepacia  K56-2 produced 100% more ornibactin, the majorsiderophore of the four types produced by  B. cenocepacia .Siderophore production appears to be maximal at station-ary phase and the Cep quorum sensing system negativelyregulatesornibactinproductioninstationaryphase.Theneg-ative effect of CepIR on ornibactin biosynthesis was alsoobserved via the increased expression of two ornibactinbiosynthetic genes in  cepIR  mutants. It has been suggestedthat this negative regulation of ornibactin biosynthesis mayserve to reduce energy-expended ornibactin biosynthesis athigh cell densities, as iron is not required in high amountsin nongrowing cells [16]. The molecular mechanism of thisnegative regulation is currently unknown; the CepR–HSLcomplex could act directly by binding to siderophore genepromoters and repressing transcription or possibly via acurrentlyunknownintermediateregulator.Thequorumsens-ing system of another  B. cenocepacia  clinical isolate wasidentified following the identification and characterizationof a transposon mutant impaired in biofilm formation [13].Biofilms are surface-associated sessile bacterial communi-ties whichare oftenlinkedto virulenceas theycan withstandthe immune response as well as being significantly more re-sistant to antibiotics [21]. In fact, it is believed that biofilmgrowth occurs in CF lung infections caused by  P. aerugi-nosa  and  B. cepacia  [9,23]. Interestingly, the CepIR systemin  B. cenocepacia  H111 is not involved in the initial at-tachment of cells to solid surfaces; however, it is essentialfor the differentiation of microcolonies, a process requiredfor the development of a mature biofilm [13]. The geneswhich are CepIR-regulated, necessary for these later stagesof biofilm development in  B. cepacia,  are currently un-known. Similarly, in  P. aeruginosa  the LasIR–C123oxoHSLquorum sensing system ( P. aeruginosa  possesses two quo-rum sensing systems, the LasIR and RhlIR–C4HSL system)is also necessary for the formation of mature differentiatedbiofilms [9], highlighting the fact that in both  B. cepacia and  P. aeruginosa  cell–cell communication is important forbiofilm growth.In  B. cepacia  strain ATCC 25416 T the CepIR systemis associated with onion pathogenicity since the  cepI   and cepR  mutants were less virulent in an onion-rot model,as demonstrated by attenuated tissue maceration comparedto the parent strain [1]. The attenuated maceration inthe onion is attributed mainly to the production of theextracellular enzyme polygalacturonase [11]. Extracellularpolygalacturonase activity in the  cep  knock-out mutantsdecreased by 40% and this activity could be complementedby introducing the  cep  locus in  trans . The attenuation of onion maceration in  cep  mutants can therefore be at leastin part due to polygalacturonaseproduction. This enzyme isencoded by the  pehA  gene and again it is unknown whetherCepIRregulatesitsexpressiondirectlyorviaanintermediateregulator. The fact that the  pehA  promoter is not active in  E.coli  in the presence of CepR and C8HSL points to indirectregulation by quorum sensing (C. Aguilar and V. Venturi,unpublished data).In summary, several CepIR-dependent phenotypes of three BCC strains were studied (summarized in Table 1); in  B. cenocepacia  K56-2 the ornibactin siderophore is nega-tively regulated, in  B. cenocepacia  H111 biofilm maturationneeds a functional CepIR and in  B. cepacia  ATCC 25416 T onion maceration via extracellular polygalacturonase activ-ity requires CepIR. In addition, in all three strains, an ex-tracellularproteaseis CepIR-regulated,whereas siderophoreproduction is not regulated in strain H111 or in ATCC25416 T . In strain H111 swarming motility is also quorum-sensing-regulated; this type of motility is not associatedwith biofilm development. From these studies it is evidentthat certain CepIR-regulated phenotypesappear to be strain-specific. 4.2. CepIR-dependent gene expression The phenotypic studies of   cepIR  knockout mutants re-vealed that quorum sensing represses siderophore biosyn-thesis and positively regulates biofilm formation, swarmingmotility and the production of several extracellular enzymes([2,13,15,16], see Table 1). The regulation of such diversephenotypes could be an indication that CepIR–HSL consti-tutes a global regulatory system modulating the transcrip-tional expression of a large set of genes. Studies designed todecipher the CepIR regulon are difficult to perform to datesince there is no annotated BCC strain genome sequencecurrently available. The bacterial strains which have beenstudied for their quorum sensing so far are not sequenced;the DNA sequence of a  B. cenocepacia  strain is currentlyavailable (http://www.sanger.ac.uk/projects); however, thisstrain has proved difficult to manipulate genetically and atpresent, to our knowledge, no studies have been reported onquorumsensingusingthis strain.Riedelandco-workers[22]recently performed a proteomic study using  B. cenocepa-cia  H111 and  cep  mutant derivatives in order to identifyproteins which are quorum-sensing-regulated.Extracellular,cell-surface and intracellular protein profiles of strain H111andthe cepI   mutantderivativeweredeterminedby2-DEandshowed that quorum sensing controls the expression, eitherpositively or negatively, of 55 out of 585 proteins. Some N  -terminal amino acid sequences for eleven of the regu-lated proteins were determined and most displayed identityto known proteins of other bacteria. This study will benefitfrom future complete annotated sequences of BCC strains;however, it is clear that, just as in  P. aeruginosa , in  B. ceno-cepacia , quorum sensing is a global regulatory system. This  242  V. Venturi et al. / Research in Microbiology 155 (2004) 238–244 proteomic analysis may also result in the identification of proteins which are indirectly regulated by quorum sensingas they can be regulated via an intermediate regulator theexpression of which is CepIR-controlled.A molecularapproachwas carried out by Aguilar and co-workers[2] whichresulted in the isolationof severaldirectlypositively regulated CepIR–C8HSL-dependent promotersof   B. cepacia  ATCC 25416 T . Genomic DNA fragmentsdigested with different restriction enzymes were cloned andscreened in  E. coli  in a plasmid vector containing the  cepR gene, a promoterless  lacZ   gene downstream from a multiplecloning site. The cloned fragments were screened for theirability to activate transcription of the  lacZ   gene in thepresence of C8HSL [2]. This led to the identification of 28 putative gene promoters; however, again, the absenceof an annotated  B. cepacia  genome rendered this analysisdifficult. This number of quorum-sensing-regulatedgenes isby no means complete, as known regulated promoters werenot identified and negatively regulated promoters cannot beisolated using this approach. This study, however, has alsoconfirmed that quorum sensing in  B. cepacia  is a globalregulatory system.The genes and proteins identified as being CepIR-regula-ted using the two above-mentioned approaches are listed inTable 1 and we have indicated that the quorum sensing reg-ulon contains a representation of several functional classesincluding adaptation, protection, secreted factors and also, itappears, general metabolic functions. There was little over-lap using the two approaches,indicatingthat neitherresultedin complete identification of the regulon. Other genomicstudies need to be performed in order to precisely definethe regulon. Indeed, transcriptome analysis in  P. aeruginosa revealed that quorum sensing controls the expression of sev-eral hundred genes, indicating that this response may bemore complex than first anticipated [25]. 4.3. CepIR-dependent pathogenicity Respiratory infections with  P. aeruginosa  and BCCbacteria play a major role in the pathogenesis of CF,and BCC strains are usually acquired late in the courseof the disease when patients are already colonized with P. aeruginosa  [4]. The important role of quorum sensingin  P. aeruginosa  pathogenicity has been reported for severalanimal models such as the burned[24] and acute-pneumoniamouse models [26], as well as a  Caenorhabditis elegans nematode model [8,29].  P. aeruginosa  pathogenicity of  C. elegans occursviaslowkilling(2–3days)bycolonizationof the intestine and rapid paralytic killing through cyanidepoisoning. Interestingly,  B. cenocepacia  also causes slowand fast killing of   C. elegans ; however,the fast killing modeof action does not occur via hydrogen cyanide, as it is notproduced by  B. cenocepacia  and is thought to occur viathe production of a yet unidentified exotoxin [14]. Quorumsensing mutants of   B. cenocepacia  H111 are attenuated intheir virulence in both modes of killing, probably throughthe decreased CepIR-dependent expression of virulencefactors which are currently unknown and are not secretedby the general type II secretion pathway [14].The contribution of CepIR to virulence has also beenrecently demonstrated in two murine animal models using  B. cenocepacia  clinical isolate K56-2 and quorum sensingmutant derivatives K56-I2 ( cepI   knock-out) and K56-R2( cepR  knock-out) [27]. A respiratory infection model inwhichratswereinfectedwithagarbeadscontainingthethree  B. cenocepacia strains demonstratedthat, as bacterial countsdid not vary considerably, the extent of lung histopathologi-calchangesonday10was significantlylowerinratsinfectedwith either K56-I2 or K56-R2 compared to the parent strain.This is probably due to CepIR regulating production of vir-ulence factors, which results in increased lung injury. Theeffects of   cepIR  mutations were also tested in a mouseintranasal infection model using  Cftr  ( − / − ) mice and their Cftr  ( + / + ) littermate controls [27]. Parent strain K56-2 wasmorevirulentthan cepIR mutantderivativesin boththewild-type and  Cftr  ( − / − ) mice. In addition it was confirmedthat inthe lungs of these mice infected with K56-2, C8HSL wasdetected, whereas it was not when infected with  cepIR  mu-tantderivatives.All theseinitial results usingtwopathogenicmodel systems ( C. elegans  and mice) show the importantroleofquorumsensingin  Burkholderia pathogenicity.Inad-dition, it was demonstrated that human sputum samples of CF patients infected with BCC bacteria contained C8HSL,the principal HSL produced by BCC strains [20]. 5. Quorum-sensing-dependent interspeciescommunication between  Burkholderia and  Pseudomonas Bacterial human infection in the CF lung not only at-tests to the complex interaction between the pathogen andthe host, but is also an outcome of the communication of pathogenic microorganisms and resident microflora [10].This interspecies communication is not well understood andthe discovery of bacterial communication via quorum sens-ing has important implications for the relationship betweendifferent species. In order to understand the pathogenesis of the CF infection, it is therefore important to study cell–cellcommunication between  Burkholderia  and  Pseudomonas species, since co-colonizationcould possibly result in a syn-ergistic infection throughquorumsensing. In fact, cross-talk via HSLs produced by different species can occur due tothe conserved LuxR family of proteins, and similarity in thestructure of the signaling molecules. Several studies haverecently begun to address this interspecies communicationand indications are that  Burkholderia  and  Pseudomonas  un-dergo interspecies communication though HSL-dependentquorum sensing [17,19,23]. McKenney et al. [19] observedthat production of siderophores, lipase and protease in a  Burkholderia  strain was stimulated when growth mediumof concentrated spent culture supernatant of   P. aeruginosa
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