Functional analysis of the group A streptococcal luxS/AI-2 system in metabolism, adaptation to stress and interaction with host cells
- Maria Siller†1,
- Rajendra P Janapatla†1,
- Zaid A Pirzada1,
- Christine Hassler1,
- Daniela Zinkl1 and
- Emmanuelle Charpentier1, 2Email author
© Siller et al; licensee BioMed Central Ltd. 2008
Received: 14 May 2008
Accepted: 30 October 2008
Published: 30 October 2008
The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19.
Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp).
Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections.
Bacterial cell density-dependent signaling, also termed "quorum sensing", is used by bacteria to collectively modulate gene expression in response to changes in the population density [1–6]. Although signaling can be achieved through a variety of regulatory mechanisms, all systems described to date involve the production, secretion and detection of extracellular low-molecular-weight signaling molecules called "autoinducers" [1, 3, 7, 8]. Due to the specificity of their respective sensors, recognition of acylated homoserine lactones (AHLs) (gram-negative bacteria) and peptide autoinducers (gram-positive bacteria) is restricted for communication within the same species. More recently, a novel quorum sensing system involving a furanone-like signaling molecule termed "autoinducer 2" (AI-2) was described to regulate bioluminescence in Vibrio harveyi [9, 10]. AI-2 is synthesized by the luxS gene product, which has been identified in the genome of over 55 gram-negative and gram-positive bacterial species [1, 11, 12]. Since culture supernatants of several bacterial species had a complementary effect on luxS-deficient V. harveyi, AI-2 has been proposed to function as a "universal" signaling molecule for interspecies communication [1, 9, 10]. In addition to controlling bioluminescence, recent studies show that the luxS/AI-2 signaling is involved in the regulation of pathogenicity in several organisms [5, 12].
Apart from its role in quorum sensing, the enzyme LuxS is tightly coupled to the S-adenosylmethionine (SAM) utilization pathway [11–13]. SAM is an essential donor of methyl groups for DNA, RNA and other methylation reactions. Its utilization by methyl transferases yields S-adenosylhomocysteine (SAH), which is toxic for the cells and is eliminated through hydrolysis by the nucleosidase Pfs to produce S-ribosylhomocysteine (SRH) and adenine. Finally, LuxS cleaves SRH into homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). DPD spontaneously forms the cyclic pro-AI-2 molecule, which in V. harveyi reacts with borate to form a stable cyclic furanosyl borate diester [1, 11, 13].
Knowledge about quorum-sensing systems in the gram-positive human pathogen Streptococcus pyogenes is rather limited. S. pyogenes (Group A Streptococcus, GAS) is responsible for a broad range of diseases including mild illnesses such as pharyngitis, impetigo and scarlet fever and more severe invasive infections such as necrotizing fasciitis, streptococcal toxic shock syndrome and post-infectious rheumatic fever [14, 15]. In GAS, like in other bacteria, pathogenicity is multifactorial and requires the coordinated temporal regulation of virulence factor expression in response to changing environmental conditions and cell population density [14, 16, 17]. Studies have confirmed that expression of several GAS virulence genes is temporal and dependent on growth phase [16–20]. Furthermore, a number of specific regulators (e.g. two-component systems and response regulators) modulate virulence gene expression in a growth phase dependent manner [16, 21–24]. Based on similarities with previously described peptide signaling regulons of S. pneumoniae (com system) and S. aureus (agr system), two putative quorum-sensing systems, sil and fasBCA, have been reported in GAS [21, 25]. Although no signaling peptide could be identified within the fasBCA locus, the putative pheromone peptide SilCR from the sil operon was shown to regulate DNA uptake and the ability of GAS to cause invasive infection [21, 25]. In addition, the observation of a luxS homologue in the GAS genome suggested that quorum sensing via the luxS/AI-2 signaling could have a relevant function in the pathogenesis of this organism [26, 27]. Two recent studies report a role of luxS/AI-2 in the regulation of virulence gene expression in M3 and M6 serotypes [26, 27]. However, the role of the luxS/AI-2 in the AMC-related metabolism and adaptation to stress conditions in GAS remains unknown.
Here, we were interested in investigating further the function of the luxS/AI-2 system in GAS serotypes M1 and M19. Expression of luxS in connection with production of AI-2 like activity was analyzed. The metabolic role of luxS in the activated methyl cycle (AMC) in GAS was examined. Wild-type and isogenic luxS-deficient strains were compared in regard to their adaptation to diverse growth and stress conditions as well as diverse aspects of pathogenicity including interaction with epithelial cells and macrophages. In summary, our data suggest an important function of the luxS/AI-2 system in survival and growth of GAS under conditions that are relevant during infections.
Construction of luxS-deficient mutants
Bacterial strains and plasmids used in this study
Lab strain collection
Isogenic luxS-deficient mutant of RDN29
Isogenic luxS-deficient mutant of RDN02
Host for cloning; AI-2 deficient (contains a frame-shift mutation in luxS)
Lab strain collection
Host for cloning
AI-1 sensor deficient, AI-2 sensor positive
Apr Kmr; pBR322Ω 1.5-kb pJH1 Cla I (aphIII)
ColE1ori, Ampr, lacZ
New England Biolabs
pUC19ΩluxS up-aphIII-luxS down
repDEG-pAMβ1, ermAM/B, ColE1ori
Temporal luxS expression and production of AI-2 like activity
Role of luxS in growth and metabolism
Growth rates and yields, colony and chain morphology of wild-type and luxS-deficient strains in complex THY medium, THY medium supplemented with 10% fetal bovine serum, C-medium and CDM were similar (data not shown). In addition, wild-type and luxS-deficient strains did not significantly differ in their ability to form primary adhesion to uncoated polystyrene surfaces (initial step in biofilm formation) when grown in THY or CDM (data not shown). To investigate whether inactivation of luxS and therefore disruption of the AMC would lead to a metabolic burden that might influence growth or fitness, we analyzed the growth rates of wild-type and luxS- deficient strains in sulfur-limited CDM (CDM-S1 and CDM-S2). Although overall growth rates and yields were significantly reduced in the restricted media compared to those in CDM, they were similar when wild-type strains were compared to the luxS-deficient mutants (data not shown). These results indicate that luxS does not have an essential AMC-related metabolic role in GAS.
Virulence gene expression in luxS mutants
Oligonucleotides used in this study
fasX (effector molecule, FasBCA)
AATTGTGAATTC TATCATAATTGTGG (Eco RI)
Cassette aphIII for luxS- deficient mutants
TAAATCAAGCTT CTAAAACAATTCATCCAG (Hin dIII)
ACTAGTGAATTC TCAAACATAACAATCC (Eco RI)
Fragment luxS down for luxS- deficient mutants
TTCTGAGGATCC CCACCATCCAGCC (Bam HI)
AATCAAGCATGC TTACTTGGAAAAGAACCCAACC (Sph I)
Fragment luxS up for luxS- deficient mutants
AAGTGGAAGCTT GTGGAGGAGAAAAGGC (Hin dIII)
Confirmation of the luxS- deficient mutants
GTCAATGGATCC CCAGCTCTATTGCACC (Bam HI)
PluxS-luxS-TT insert for luxS complementation plasmid pEC83
TATCTAGAGCTC TAGATTACTGAGAAAATC (Sac I)
Among the quorum sensing systems described to date, the luxS/AI-2 pathway has been shown to be involved in the regulation of virulence in a number of gram-negative and gram-positive bacteria [1, 5, 11, 12]. In this study, we analyzed the expression of the streptococcal luxS/AI-2 system, its possible role in the AMC metabolic pathway and its function in adaptation to diverse host-induced stress conditions in two GAS clinical isolates of serotypes M1 and M19.
Expression analysis of luxS indicated that the luxS transcript is monocistronic (644 bases) in GAS serotypes M1 and M19. In a previous report, which was based on sequence analysis, the luxS transcript in GAS (M6 strain) was predicted to be polycistronic as a distal member of the fatty acid metabolism operon . In our study, Northern blot analysis of luxS expression did not reveal any additional transcript of higher size. Furthermore, we showed that the monocistronic transcript is expressed in a growth phase-dependent manner, a finding already reported for bacterial species like Streptococcus bovis, whereas transcription of luxS in species like Salmonella typhimurium and Vibrio fischeri has been shown to be constitutive [37–39]. Accordingly, production of AI-2 like activity occurred in a temporal fashion in the serotypes analyzed, however, shifted out of phase compared to luxS expression. AI-2 like activity peaked at late-logarithmic phase when the luxS transcript had already declined being just above the detectable expression level. We also investigated the possibility of an auto-regulation mechanism of luxS expression in GAS. Induction experiments by Northern blot analysis failed to show a regulation of luxS expression by AI-2 dependent CM. These findings are in accordance with previous work in S. typhimurium, where AI-2 production and luxS transcription were not in phase either, also suggesting that luxS expression was not regulated by AI-2 .
Despite intensive research on luxS and AI-2 during the last years, a clear separation of the possible metabolic function of AI-2 from its possible signaling activity could not be achieved. A recent comparative genomic and phylogenetic analysis of synthesis and signal transduction pathways showed that S. pyogenes contains the two-step enzymatic pathway catalyzed by the Pfs and LuxS enzymes to detoxify SAH leading to AI-2 production [13, 40]. Interestingly, in S. pyogenes the AMC is incomplete as it lacks the homocysteine methyltransferases (MetE or MetH) that convert the SAH-derived metabolic product homocysteine to methionine. Nevertheless, S. pyogenes encodes the SAM synthetase ortholog (MetK) that permits conversion of methionine to SAM . Therefore, either GAS cannot recycle homocysteine to methionine (as it has been described in Borrelia burgdorferi ) or recycling occurs through other types of methyltransferases. In the former case of a de novo methionine synthesis defect, bacterial growth would exclusively depend on sulfur provided from external sources. In our study, decreasing media concentrations of cysteine and methionine reduced to a similar extent bacterial growth and yields in both wild-type and luxS-deficient strains. These data indicate that GAS, as a poly-auxotrophic organism, is unable to recycle homocysteine to methionine. Moreover, we showed that inactivation of luxS does not lead to a metabolic burden that would influence growth or fitness. Although AI-2 can certainly be considered as a metabolite, the viability and lack of growth defects of the investigated luxS-deficient mutants in various media including minimal media depleted of sulfur sources argues against an essential role of luxS in AMC-related metabolism.
The previously described regulatory role of luxS in the production of virulence factors in GAS [26, 27] prompted us to investigate the role of the luxS/AI-2 system in the adaptation to host-induced stress conditions. During an on-going infection, bacteria often have to face challenging conditions in particular niches of the host, including changes in pH, and therefore are forced to develop quickly an adaptive response, which requires fine-tuning of pathogenicity gene expression. Although we could not identify a possible involvement of the GAS luxS system in adaptation to oxidative and salt stress, we showed an increased survival of S. pyogenes under acidic conditions when the luxS/AI-2 system was down-regulated. This is in accordance with luxS expression and AI-2 production being significantly lowered when GAS cells were grown in low pH conditions. Interestingly, previous studies in S. mutans showed that acid sensitivity was enhanced in luxS deficient mutants [42, 43] and that luxS expression was increased at low pH [44, 45]. These reverse effects of luxS on tolerance to acidic conditions in S. pyogenes and S. mutans need to be considered in regard to their different living habitats and pathogenesis. Thus, our data reveal a link between the S. pyogenes luxS/AI-2 system and pathways involved in adaptation of the organism to stress [46, 47]. In addition, we also observed a reduction of luxS expression and AI-2 like activity in GAS cells grown in serum enriched RPMI, a medium with a composition similar to that of human plasma. In a previous study, Marouni et al. reported that in an M6 serotype, the survival of a luxS-deficient mutant in epithelial cells at 4 h after infection was higher compared to the wild-type parent . Here we analyzed further the role of luxS in interaction of S. pyogenes with host cells. No differences in adhesion rates to human pharyngeal epithelial cells were observed when comparing wild-type and luxS-deficient mutants in both M1 and M19 serotypes. However, the luxS-deficient mutants had a significant advantage to survive intracellularly in both epithelial cells (over a period of 7 h after infection) and macrophages (over a period of 3 h after infection). Taken together, we show that low level of luxS and AI-2 expression seems to provide a competitive advantage for GAS survival under specific conditions encountered during infection.
With luxS being linked to stress and adaptation to the host, we were interested in investigating additional effects of luxS on virulence factor expression in GAS. In an M6 serotype, luxS was shown to regulate streptolysin S (SLS) expression at the transcriptional level and SpeB cysteine protease activity . In an M3 serotype, luxS was reported to regulate expression of SpeB and M protein at the transcriptional level and hyaluronic acid capsule at the post-transcriptional level . In our report, we show that luxS had a positive effect on fasX expression and a negative effect on sibA (psp) expression at the transcriptional level. fasX is a small RNA molecule, effector of the fasBCA operon, which has regulatory functions on virulence factor expression. fasX was also suggested to be involved in local tissue destruction and general bacterial aggressiveness towards host cells [21, 48]. sibA (psp) is a virulence gene encoding a secreted immunoglobulin binding protein . Remarkably, the above described regulatory effects were only observed in the M19 strain, thus suggesting a strain or serotype dependent effect of luxS on the expression of these two targets. The strain- or serotype-dependent effect of luxS observed in this study emphasizes differences in regulatory pathways among different GAS isolates. Along these lines, previous reports demonstrated that mutations in GAS regulators might alter disease progression [49, 50]. This is well illustrated with the two-component regulatory system CovRS where mutations in the sensor encoding gene covS have been shown to correlate with human disease severity [51, 52]. Mutations in this gene can occur under selective pressure encountered in the host and can generate hypervirulent GAS variants with increased risk of systemic dissemination [51, 52]. In the case of luxS, strain specificity manifestation of virulence-associated gene regulation by luxS has been reported previously in Neisseria meningitidis and Serratia marcescens [53, 54]. Additionally, Marouni et al. attributed the dissimilar results of the effect of luxS on bacterial growth and the level of SpeB regulation in GAS to a possible strain-specific effect . The finding that luxS can affect the expression of fasX RNA also provides an additional evidence for the notion that small RNAs have the ability to integrate cell density signals together with other environmental stimuli to affect gene expression .
Our data together with previous reports suggest a complex role of the luxS/AI-2 system in S. pyogenes. Here, we showed that expression of both luxS and AI-2 occurs in a temporal fashion but AI-2 like activity does not seem to have a regulatory effect on luxS expression. Analysis of the role of luxS in metabolism demonstrated a limited role of luxS in the AMC-related metabolism. However, studying the possible function of the luxS/AI-2 system in adaptation to stress revealed that a down-regulation of luxS expression provides an advantage for S. pyogenes to tolerate acidic conditions and grow in a host environment-mimicking medium. Accordingly, there was an increased ability of the luxS-deficient mutants to survive intracellularly in both epithelial cells and macrophages. Altogether, our data suggest an important function for the luxS/AI-2 system in survival and growth of GAS under conditions that are relevant during infections. Based on the data outlined in this article, it is tempting to speculate that in GAS, luxS is not exclusively a key part of a detoxification pathway but rather modulation of luxS expression levels would allow adjusting bacterial fitness in response to changing host conditions. Finally, our study revealed two novel virulence-associated targets of luxS in S. pyogenes: the regulator fasX RNA and the virulence gene sibA.
Bacterial strains and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1. S. pyogenes was routinely cultured in Todd Hewitt Broth (THB, Bacto, Becton Dickinson) supplemented with 0.2% yeast extract (Oxoid) (THY) without agitation or on trypticase soy agar supplemented with 3% sheep blood (TSA, BBL, Becton Dickinson). C-medium (peptide rich and carbohydrate poor), THY or RPMI medium supplemented with 10% foetal bovine serum (Gibco), metal ion-restricted medium, chemically defined medium (CDM) and sulfur-restricted CDM (CDM-S) were used in specific experiments [56, 57]. For the eukaryotic-like environment, RPMI 1640 (PAA) was supplemented with 10% foetal bovine serum (Gibco) or 10% foetal bovine serum and 50 μM FeCl3. For metal-ion restricted medium, THY was treated overnight with 30 g of chelating resin Chelex 100 (Sigma)/liter, supplemented with 1 mM MgCl2 or 1 mM MgCl2and 1 mM FeCl3. Sulfur-restricted CDM (CDM-S) consisted of CDM with sulfur containing salts replaced with their chloride equivalents . CDM contained L-methionine (670 μM), L-cysteine (4,125 μM) and L-cystine (208 μM). In sulfur-limiting conditions, L-cystine was eliminated and only L-cysteine (10 μM or 350 μM) and L-methionine (1 μM or 100 μM) were provided as sulfur source. All S. pyogenes cultures were incubated at 37°C in an atmosphere supplemented with 5% CO2. Escherichia coli was grown aerobically at 37°C in Luria-Bertani (LB) medium either in liquid with shaking or on agar plates. V. harveyi was cultured aerobically at 28°C in an Autoinducer Bioassay (AB) medium . Transformation of E. coli and S. pyogenes was performed as previously described [59, 60]. Whenever required, suitable antibiotics were added to the medium to the following final concentrations: erythromycin 300 μg/ml for E. coli and 3 μg/ml for S. pyogenes; kanamycin 25 μg/ml for E. coli and 300 μg/ml for S. pyogenes. Bacterial cell growth was monitored periodically by measuring the optical density of culture aliquots at 620 nm using a microplate reader (SLT Spectra Reader).
DNA manipulations including DNA preparation, amplification, digestion, ligation, purification, agarose gel electrophoresis and Southern blot analysis were performed according to standard techniques . Synthetic oligonucleotides used as primers in this study (Table 2) were supplied by VBC-Biotech Services GmbH. Sequencing reactions were performed at VBC-Biotech Services GmbH.
Construction of the luxS-deficient mutants
Replacement of the luxS coding sequence with a kanamycin resistance cassette, aphIII , was performed selecting for a double cross-over event. For this purpose, a 1209 bp fragment upstream of luxS (luxS-up) and a 1107 bp fragment downstream of luxS (luxS-down) were amplified using wild-type genomic DNA as template and primers (containing flanking restriction sites) oliRN22/oliRN21 and oliRN20/oliRN19, respectively. The aphIII cassette was amplified using plasmid pAT21 as template and primers oliRN23/oliRN24. After digestion with the respective restriction enzymes, the three fragments were ligated and cloned into pUC19 (suicide vector for S. pyogenes). The resulting plasmid pEC131 was purified, linearized with the restriction enzyme Sca I (which cuts within the ampicillin resistance cassette) and used to transform electro-competent wild-type S. pyogenes. Kanamycin resistant clones were selected and the correct replacement event was checked by PCR analysis using combinations of primers oliRN267 and oliRN268 derived from flanking regions upstream and downstream of the luxS-up and luxS-down fragments and primers oliRN69 and oliRN70 specific to the aphIII cassette. Southern blot analysis was done to further confirm that the recombination events had not affected the DNA regions located upstream and downstream of luxS. The S. pyogenes mutants were grown in liquid medium without antibiotic unless otherwise specified.
Construction of plasmids for complementation studies
Plasmid pEC82 contains repDEG-pAMβ1 (the origin of replication of pAMβ1), ermAM (an erythromycin resistance gene with its own promoter and transcriptional terminator), ColE1 (a pUC19-based ColE1 origin of replication for E. coli) and an expanded MCS (multiple cloning site). To create the pEC83 luxS-complementation vector, a 1152 bp large DNA fragment (PluxS-luxS-TT) containing the luxS coding sequence, its putative promoter region and putative transcriptional terminator, was amplified from wild-type genomic DNA using primers (containing flanking restriction sites) oliRN205/oliRN280 and cloned into pEC82. Plasmids pEC82 and pEC83 were introduced in competent E. coli and S. pyogenes strains selecting for erythromycin resistant clones.
Total RNA was prepared from culture samples harvested at different time points during growth and further processed for normalization and Northern blot analysis as previously described  with minor modifications. Specific α-32P-dATP labeled DNA probes corresponding to internal fragments of genes were created by amplification of wild-type genomic DNA using primers described in Table 2. Primer extension analysis of the luxS transcript and DNA sequencing reactions were carried out according to standard protocols .
Assays for bioluminescence, acid killing, hydrogen peroxide killing, salt stress and biofilm formation were performed as described previously [9, 28, 29, 43, 63] with some minor modifications. For the acid tolerance assay, mid-logarithmic phase bacterial cultures were harvested by centrifugation and washed once with 0.1 M glycine buffer (pH 7.0). Culture aliquots were removed for CFU determination and the remaining cultures were subjected to killing by incubating the cells in 0.1 M glycine buffer (pH 4) for 6 h. The number of viable bacteria was determined by plating appropriate dilutions in triplicate on TSA media. For each individual experiment, the ability of strains to survive the acid challenge was reported as survival in percent, defined by the ratio of average CFU/ml (triplicate measurements) recovered after challenge versus the average number of CFU/ml (triplicate measurements) present immediately before challenge × 100.
Preparation of conditioned medium for induction assays
Conditioned medium (CM) was prepared from bacterial cultures grown in THY buffered with 0.1 M HEPES (Sigma) (pH 7.5) to early-, mid- and late-logarithmic phase. Culture supernatants were separated from bacterial pellets by centrifugation at 9,500 × g for 30 min at 4°C, filter sterilized (0.45 μm) and stored at -20°C. For induction assays, early-logarithmic wild-type cultures were induced for 60 and 90 min with THY adjusted to pH 7.5, 6.0 and 5.0 or with CM of wild-type and luxS-deficient cultures from early-, mid- and late-logarithmic growth phase and then subjected to Northern blot analysis.
Bacterial adhesion, internalization and survival assays in host cells. HEp-2 cells and RAW 264.7 mouse macrophages were maintained in RPMI medium (HEp-2 cells) or Dulbecco's modified Eagle's medium (D-MEM, Gibco) (RAW 264.7 cells) supplemented with 10% foetal bovine serum (Gibco), penicillin (100 μg/ml) and streptomycin (100 μg/ml), and grown at 37°C in an atmosphere containing 5% CO2. Prior the infection assay, the cells were cultured overnight to semi-confluency (1.5 × 105 cells per well for HEp-2 cells and 2 × 105 cells per well for RAW264.7 cells) in 24-well tissue culture plates containing medium without antibiotic. GAS strains were grown to the same OD620 nm corresponding to mid-logarithmic (RAW 264.7) or late-logarithmic (HEp-2) phase, washed with PBS, suspended in fresh cell culture medium and added to the semi-confluent host cell monolayers at a multiplicity of infection (MOI) of 5:1 (HEp-2) or 100:1 (RAW 264.7) in triplicate. Times of incubation were 30 min, 1 h and 2 h (adhesion, HEp-2 cells), 2 h (internalization, HEp-2 cells) and 30 min (RAW 264.7 cells). For adhesion assays (HEp-2 cells), infected monolayers were washed extensively with PBS before lysing. For internalization assays (RAW 264.7 cells), extracellularly adhered bacteria were killed by incubation with fresh medium containing either 100 μg/ml gentamicin and 5 μg/ml penicillin (HEp-2 cells) or 60 μg/ml penicillin (RAW 264.7 cells). At indicated time points, the infected monolayers were washed extensively with PBS to remove the antibiotics. Cells were then lysed with chilled sterile distilled water. The number of viable intracellular bacteria released from the lysed cells was determined by plating appropriate dilutions of the lysates on TSA-blood plates in triplicates followed by 24 h incubation at 37°C. The number of bacteria that survived intracellularly was calculated using the following equation: average number of bacteria recovered (cfu/ml) per well (triplicate) at designated time point/inoculated number of bacteria (cfu/ml)) × 100. Average values were plotted.
We thank Dr. Bonnie Bassler (Princeton University, USA) for her generous gift of V. harveyi strains. We thank Nina Gratz and Pavel Kovarik for their help with the macrophage work. We are grateful to Dr. Eszter Nagy (Intercell AG, Vienna, Austria) for helpful discussions and critical review of the manuscript. We thank Dr. Jason Rosch (St. Jude Children's Research Hospital, Memphis, USA) for helpful discussions. This work was supported by grant No. P17238-B09 from the Austrian Science Fund (Fonds zur Förderung der wissenschaftlichen Forschung, FWF; to EC), grant No. H-1020-2004 from the University Jubilee Foundation of the City of Vienna (Hochschuljubilaümsstiftung der Stadt Wien, HJST; to EC) and grant No. 10802 from the Austrian Nationalbank (die Österreichische Nationalbank, ÖNB; to EC). RPJ and ZAP were recipients of scholarships from the Austrian Exchange Service (Der Österreichische Austauschdienst, ÖAD) and the Higher Education Commission of Pakistan (HEC), respectively.
- Henke JM, Bassler BL: Bacterial social engagements. Trends Cell Biol. 2004, 14 (11): 648-656.View ArticlePubMedGoogle Scholar
- Lazazzera BA: Quorum sensing and starvation: signals for entry into stationary phase. Curr Opin Microbiol. 2000, 3 (2): 177-182.View ArticlePubMedGoogle Scholar
- Lyon GJ, Novick RP: Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria. Peptides. 2004, 25 (9): 1389-1403.View ArticlePubMedGoogle Scholar
- Parsek MR, Greenberg EP: Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol. 2005, 13 (1): 27-33.View ArticlePubMedGoogle Scholar
- Walters M, Sperandio V: Quorum sensing in Escherichia coli and Salmonella. Int J Med Microbiol. 2006, 296 (2–3): 125-131.View ArticlePubMedGoogle Scholar
- Williams P, Winzer K, Chan WC, Camara M: Look who's talking: communication and quorum sensing in the bacterial world. Philos Trans R Soc Lond B Biol Sci. 2007, 362 (1483): 1119-1134.PubMed CentralView ArticlePubMedGoogle Scholar
- Podbielski A, Kreikemeyer B: Cell density-dependent regulation: basic principles and effects on the virulence of Gram-positive cocci. Int J Infect Dis. 2004, 8 (2): 81-95.View ArticlePubMedGoogle Scholar
- Suntharalingam P, Cvitkovitch DG: Quorum sensing in streptococcal biofilm formation. Trends Microbiol. 2005, 13 (1): 3-6.View ArticlePubMedGoogle Scholar
- Bassler BL, Greenberg EP, Stevens AM: Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol. 1997, 179 (12): 4043-4045.PubMed CentralPubMedGoogle Scholar
- Surette MG, Miller MB, Bassler BL: Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production. Proc Natl Acad Sci USA. 1999, 96 (4): 1639-1644.PubMed CentralView ArticlePubMedGoogle Scholar
- De Keersmaecker SC, Sonck K, Vanderleyden J: Let LuxS speak up in AI-2 signaling. Trends Microbiol. 2006, 14 (3): 114-119.View ArticlePubMedGoogle Scholar
- Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR: Making 'sense' of metabolism: autoinducer-2, LuxS and pathogenic bacteria. Nat Rev Microbiol. 2005, 3 (5): 383-396.View ArticlePubMedGoogle Scholar
- Winzer K, Hardie KR, Williams P: LuxS and autoinducer-2: their contribution to quorum sensing and metabolism in bacteria. Adv Appl Microbiol. 2003, 53: 291-396.View ArticlePubMedGoogle Scholar
- Bisno AL, Brito MO, Collins CM: Molecular basis of group A streptococcal virulence. Lancet Infect Dis. 2003, 3 (4): 191-200.View ArticlePubMedGoogle Scholar
- Cunningham MW: Pathogenesis of group A streptococcal infections. Clin Microbiol Rev. 2000, 13 (3): 470-511.PubMed CentralView ArticlePubMedGoogle Scholar
- Kreikemeyer B, McIver KS, Podbielski A: Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions. Trends Microbiol. 2003, 11 (5): 224-232.View ArticlePubMedGoogle Scholar
- Salim KY, de Azavedo JC, Bast DJ, Cvitkovitch DG: Role for sagA and siaA in quorum sensing and iron regulation in Streptococcus pyogenes. Infect Immun. 2007, 75 (10): 5011-5017.PubMed CentralView ArticlePubMedGoogle Scholar
- Beyer-Sehlmeyer G, Kreikemeyer B, Horster A, Podbielski A: Analysis of the growth phase-associated transcriptome of Streptococcus pyogenes. Int J Med Microbiol. 2005, 295 (3): 161-177.View ArticlePubMedGoogle Scholar
- Chaussee MS, Phillips ER, Ferretti JJ: Temporal production of streptococcal erythrogenic toxin B (streptococcal cysteine proteinase) in response to nutrient depletion. Infect Immun. 1997, 65 (5): 1956-1959.PubMed CentralPubMedGoogle Scholar
- Smoot LM, Smoot JC, Graham MR, Somerville GA, Sturdevant DE, Migliaccio CA, Sylva GL, Musser JM: Global differential gene expression in response to growth temperature alteration in group A Streptococcus. Proc Natl Acad Sci USA. 2001, 98 (18): 10416-10421.PubMed CentralView ArticlePubMedGoogle Scholar
- Kreikemeyer B, Boyle MD, Buttaro BA, Heinemann M, Podbielski A: Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule. Mol Microbiol. 2001, 39 (2): 392-406.View ArticlePubMedGoogle Scholar
- Mangold M, Siller M, Roppenser B, Vlaminckx BJ, Penfound TA, Klein R, Novak R, Novick RP, Charpentier E: Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule. Mol Microbiol. 2004, 53 (5): 1515-1527.View ArticlePubMedGoogle Scholar
- Neely MN, Lyon WR, Runft DL, Caparon M: Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes. J Bacteriol. 2003, 185 (17): 5166-5174.PubMed CentralView ArticlePubMedGoogle Scholar
- McIver KS, Scott JR: Role of mga in growth phase regulation of virulence genes of the group A streptococcus. J Bacteriol. 1997, 179 (16): 5178-5187.PubMed CentralPubMedGoogle Scholar
- Eran Y, Getter Y, Baruch M, Belotserkovsky I, Padalon G, Mishalian I, Podbielski A, Kreikemeyer B, Hanski E: Transcriptional regulation of the sil locus by the SilCR signalling peptide and its implications on group A streptococcus virulence. Mol Microbiol. 2007, 63 (4): 1209-1222.View ArticlePubMedGoogle Scholar
- Lyon WR, Madden JC, Levin JC, Stein JL, Caparon MG: Mutation of luxS affects growth and virulence factor expression in Streptococcus pyogenes. Mol Microbiol. 2001, 42 (1): 145-157.View ArticlePubMedGoogle Scholar
- Marouni MJ, Sela S: The luxS gene of Streptococcus pyogenes regulates expression of genes that affect internalization by epithelial cells. Infect Immun. 2003, 71 (10): 5633-5639.PubMed CentralView ArticlePubMedGoogle Scholar
- Brenot A, King KY, Caparon MG: The PerR regulon in peroxide resistance and virulence of Streptococcus pyogenes. Mol Microbiol. 2005, 55 (1): 221-234.View ArticlePubMedGoogle Scholar
- Dalton TL, Scott JR: CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes. J Bacteriol. 2004, 186 (12): 3928-3937.PubMed CentralView ArticlePubMedGoogle Scholar
- Loughman JA, Caparon M: Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a model for in vivo gene expression. J Bacteriol. 2006, 188 (2): 399-408.PubMed CentralView ArticlePubMedGoogle Scholar
- Bates CS, Toukoki C, Neely MN, Eichenbaum Z: Characterization of MtsR, a new metal regulator in group A streptococcus, involved in iron acquisition and virulence. Infect Immun. 2005, 73 (9): 5743-5753.PubMed CentralView ArticlePubMedGoogle Scholar
- Fagan PK, Reinscheid D, Gottschalk B, Chhatwal GS: Identification and characterization of a novel secreted immunoglobulin binding protein from group A streptococcus. Infect Immun. 2001, 69 (8): 4851-4857.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu M, Hanks TS, Zhang J, McClure MJ, Siemsen DW, Elser JL, Quinn MT, Lei B: Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR. Microbiology. 2006, 152 (Pt 4): 967-978.PubMed CentralView ArticlePubMedGoogle Scholar
- Malke H, Steiner K, McShan WM, Ferretti JJ: Linking the nutritional status of Streptococcus pyogenes to alteration of transcriptional gene expression: the action of CodY and RelA. Int J Med Microbiol. 2006, 296 (4–5): 259-275.View ArticlePubMedGoogle Scholar
- Reid SD, Montgomery AG, Musser JM: Identification of srv, a PrfA-like regulator of group A streptococcus that influences virulence. Infect Immun. 2004, 72 (3): 1799-1803.PubMed CentralView ArticlePubMedGoogle Scholar
- Shelburne SA, Sumby P, Sitkiewicz I, Granville C, DeLeo FR, Musser JM: Central role of a bacterial two-component gene regulatory system of previously unknown function in pathogen persistence in human saliva. Proc Natl Acad Sci USA. 2005, 102 (44): 16037-16042.PubMed CentralView ArticlePubMedGoogle Scholar
- Asanuma N, Yoshii T, Hino T: Molecular characterization and transcription of the luxS gene that encodes LuxS autoinducer 2 synthase in Streptococcus bovis. Curr Microbiol. 2004, 49 (5): 366-371.View ArticlePubMedGoogle Scholar
- Beeston AL, Surette MG: pfs-dependent regulation of autoinducer 2 production in Salmonella enterica serovar Typhimurium. J Bacteriol. 2002, 184 (13): 3450-3456.PubMed CentralView ArticlePubMedGoogle Scholar
- Lupp C, Ruby EG: Vibrio fischeri LuxS and AinS: comparative study of two signal synthases. J Bacteriol. 2004, 186 (12): 3873-3881.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun J, Daniel R, Wagner-Dobler I, Zeng AP: Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. BMC Evol Biol. 2004, 4: 36-PubMed CentralView ArticlePubMedGoogle Scholar
- von Lackum K, Babb K, Riley SP, Wattier RL, Bykowski T, Stevenson B: Functionality of Borrelia burgdorferi LuxS: the Lyme disease spirochete produces and responds to the pheromone autoinducer-2 and lacks a complete activated-methyl cycle. Int J Med Microbiol. 2006, 40: 92-102.View ArticleGoogle Scholar
- Sztajer H, Lemme A, Vilchez R, Schulz S, Geffers R, Yip CY, Levesque CM, Cvitkovitch DG, Wagner-Dobler I: Autoinducer-2-regulated genes in Streptococcus mutans UA159 and global metabolic effect of the luxS mutation. J Bacteriol. 2008, 190 (1): 401-415.PubMed CentralView ArticlePubMedGoogle Scholar
- Wen ZT, Burne RA: LuxS-mediated signaling in Streptococcus mutans is involved in regulation of acid and oxidative stress tolerance and biofilm formation. J Bacteriol. 2004, 186 (9): 2682-2691.PubMed CentralView ArticlePubMedGoogle Scholar
- Hasona A, Zuobi-Hasona K, Crowley PJ, Abranches J, Ruelf MA, Bleiweis AS, Brady LJ: Membrane composition changes and physiological adaptation by Streptococcus mutans signal recognition particle pathway mutants. J Bacteriol. 2007, 189 (4): 1219-1230.PubMed CentralView ArticlePubMedGoogle Scholar
- Lemos JA, Brown TA, Burne RA: Effects of RelA on key virulence properties of planktonic and biofilm populations of Streptococcus mutans. Infect Immun. 2004, 72 (3): 1431-1440.PubMed CentralView ArticlePubMedGoogle Scholar
- Azcarate-Peril MA, McAuliffe O, Altermann E, Lick S, Russell WM, Klaenhammer TR: Microarray analysis of a two-component regulatory system involved in acid resistance and proteolytic activity in Lactobacillus acidophilus. Appl Environ Microbiol. 2005, 71 (10): 5794-5804.PubMed CentralView ArticlePubMedGoogle Scholar
- DeLisa MP, Bentley WE: Bacterial autoinduction: looking outside the cell for new metabolic engineering targets. Microb Cell Fact. 2002, 1 (5): 1-9.Google Scholar
- Klenk M, Koczan D, Guthke R, Nakata M, Thiesen HJ, Podbielski A, Kreikemeyer B: Global epithelial cell transcriptional responses reveal Streptococcus pyogenes Fas regulator activity association with bacterial aggressiveness. Cell Microbiol. 2005, 7 (9): 1237-1250.View ArticlePubMedGoogle Scholar
- Kreikemeyer B, Nakata M, Koller T, Hildisch H, Kourakos V, Standar K, Kawabata S, Glocker MO, Podbielski A: The Streptococcus pyogenes serotype M49 Nra-Ralp3 transcriptional regulatory network and its control of virulence factor expression from the novel eno ralp3 epf sagA pathogenicity region. Infect Immun. 2007, 75 (12): 5698-5710.PubMed CentralView ArticlePubMedGoogle Scholar
- Luo F, Lizano S, Bessen DE: Heterogeneity in the Polarity of Nra Regulatory Effects on Streptococcal Pilus Gene Transcription and Virulence. Infect Immun. 2008, 76 (6): 2490-2497.PubMed CentralView ArticlePubMedGoogle Scholar
- Sumby P, Whitney AR, Graviss EA, DeLeo FR, Musser JM: Genome-wide analysis of group A streptococci reveals a mutation that modulates global phenotype and disease specificity. PLoS Pathog. 2006, 2 (1): e5-PubMed CentralView ArticlePubMedGoogle Scholar
- Walker MJ, Hollands A, Sanderson-Smith ML, Cole JN, Kirk JK, Henningham A, McArthur JD, Dinkla K, Aziz RK, Kansal RG: DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat Med. 2007, 13 (8): 981-985.View ArticlePubMedGoogle Scholar
- Coulthurst SJ, Kurz CL, Salmond GP: luxS mutants of Serratia defective in autoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulence and production of carbapenem and prodigiosin. Microbiology. 2004, 150 (Pt 6): 1901-1910.View ArticlePubMedGoogle Scholar
- Dove JE, Yasukawa K, Tinsley CR, Nassif X: Production of the signalling molecule, autoinducer-2, by Neisseria meningitidis : lack of evidence for a concerted transcriptional response. Microbiology. 2003, 149 (Pt 7): 1859-1869.View ArticlePubMedGoogle Scholar
- Bejerano-Sagie M, Xavier KB: The role of small RNAs in quorum sensing. Curr Opin Microbiol. 2007, 10 (2): 189-198.View ArticlePubMedGoogle Scholar
- Lyon WR, Gibson CM, Caparon MG: A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes. EMBO J. 1998, 17 (21): 6263-6275.PubMed CentralView ArticlePubMedGoogle Scholar
- Rijn van de I, Kessler RE: Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun. 1980, 27 (2): 444-448.PubMed CentralPubMedGoogle Scholar
- Doherty N, Holden MT, Qazi SN, Williams P, Winzer K: Functional analysis of luxS in Staphylococcus aureus reveals a role in metabolism but not quorum sensing. J Bacteriol. 2006, 188 (8): 2885-2897.PubMed CentralView ArticlePubMedGoogle Scholar
- Caparon MG, Scott JR: Genetic manipulation of pathogenic streptococci. Methods Enzymol. 1991, 204: 556-586.View ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: . Molecular Cloning: a Laboratory Manual. 1989, Cold Spring Harbor, NY edn: Cold Spring Harbor Laboratory Press, 2Google Scholar
- Trieu-Cuot P, Courvalin P: Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III. Gene. 1983, 23 (3): 331-341.View ArticlePubMedGoogle Scholar
- Herbert S, Barry P, Novick RP: Subinhibitory clindamycin differentially inhibits transcription of exoprotein genes in Staphylococcus aureus. Infect Immun. 2001, 69 (5): 2996-3003.PubMed CentralView ArticlePubMedGoogle Scholar
- Lembke C, Podbielski A, Hidalgo-Grass C, Jonas L, Hanski E, Kreikemeyer B: Characterization of biofilm formation by clinically relevant serotypes of group A streptococci. Appl Environ Microbiol. 2006, 72 (4): 2864-2875.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.