LuxS Involvement in the Regulation of Genes Coding for Hemin and Iron Acquisition Systems in Porphyromonas gingivalis
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《感染与免疫杂志》
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida 32610
Department of Periodontics, Endodontics and Dental Hygiene, School of Dentistry, University of Louisville, Louisville, Kentucky 40292
ABSTRACT
The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe2+ ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.
INTRODUCTION
A number of bacterial intercommunication systems have been identified that are based on the production and detection of external signals and that enable coordinated responses throughout a population. Quorum sensing is a system by which a bacterial population can monitor its cell density through release of specific signaling molecules called autoinducers. As the population grows, autoinducers accumulate in the local environment until a critical detection threshold level is reached, at which point changes in gene expression are triggered. In this manner bacterial populations can synchronize expression of specific genes required for community survival under the prevailing environmental conditions (2, 27, 81).
Gram-negative bacteria frequently use acylated homoserine lactones (AHLs) as autoinducers, and this signal is classified as autoinducer 1 (AI-1). In the archetypal quorum-sensing system of the marine symbiotic bacterium Vibrio fischeri, AHL is synthesized by the LuxI protein, and transcriptional activation of the target gene is mediated by LuxR (26). In gram-positive bacteria, quorum sensing is achieved through peptide autoinducers that modulate two-component phosphorelay cascades (19). Both the AI-1 and peptide signaling systems are considered species specific and control a wide range of biological activities including bioluminescence, sporulation, biofilm formation, conjugation, motility, competence, and antibiotic production (13, 17, 25, 51, 61, 73). By contrast, the AI-2 system, originally identified in Vibrio harveyi, is considered non-species specific and may function as a universal bacterial language (81). AI-2 is produced through the action of the LuxS enzyme, which cleaves S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). It is now suspected that AI-2 may comprise a family of structures generated from rearrangements of DPD (49, 69). In V. harveyi, AI-2 is detected by LuxPQ, which channel information to the phosphotransferase protein LuxU. LuxU, in turn, transfers the signal to LuxO, a 54-dependent transcriptional activator (4, 23, 24). Other bacterial species may process AI-2 by different mechanisms; for example, in Salmonella enterica serovar Typhimurium, AI-2 is internalized via the Lsr ABC transporter (76).
The luxS gene is widespread among both gram-positive and gram-negative bacteria, many of which are pathogens (79). Culture supernatants from organisms possessing luxS are generally able to stimulate bioluminescence in a sensor 1– sensor 2+ reporter strain of V. harveyi (3). Despite the cross-species-reactive nature of AI-2, the cellular processes controlled by AI-2 differ among organisms. Some examples include type III secretion in enterohemorrhagic and enteropathogenic Escherichia coli (71), motility in E. coli and Helicobacter pylori (44, 70), biofilm formation in Vibrio cholerae, H. pylori, Streptococcus gordonii, and Streptococcus mutans (7, 14, 32, 47, 48, 84), protease and hemolysin production in Vibrio vulnificus and Streptococcus pyogenes (39, 45), and leukotoxin production and iron uptake in Actinobacillus actinomycetemcomitans (22). Thus, individual organisms appear capable of adapting LuxS signaling for their own particular needs.
Porphyromonas gingivalis is an important human pathogen involved in the initiation and progression of periodontal disease. P. gingivalis can colonize numerous surfaces and form biofilms in the oral cavity but especially thrives in the deep periodontal pockets that develop as periodontal disease progresses (50). P. gingivalis is able to invade gingival epithelial cells and persist intracellularly for extended periods (43, 82). Virulence factors of the organism include fimbriae and other surface adhesins, as well as an abundance of proteinases that can degrade host proteins including immune components and iron-sequestering compounds (42, 57). P. gingivalis possesses the luxS gene and produces functional AI-2 (9, 12). Furthermore, in an early demonstration of the cross-species nature of AI-2, P. gingivalis was shown to respond to heterologous AI-2 produced by A. actinomycetemcomitans (22). LuxS-deficient mutants of P. gingivalis have been found to produce lower levels of proteases (9) and to exhibit altered expression of genes involved in hemin uptake (12). P. gingivalis shows a strong preference for iron in the form of hemin and expresses a number of heme/hemin uptake systems (55). These hemin uptake mechanisms include the TonB-linked outer membrane hemin binding proteins HmuR and Tlr and the hemin binding lipoprotein FetB (IhtB). P. gingivalis also possesses numerous operons encoding ABC transport systems and permeases, in order to transport iron sources across the cytoplasmic membrane (65). Strategies employed by P. gingivalis for the uptake of Fe2+ include the use of the ferrous iron transport protein FeoB (16).
In the present study, we undertook a systematic investigation of the role of LuxS in the transcriptional regulation of P. gingivalis hemin and Fe2+ acquisition mechanisms. Quantitative real-time reverse transcription-PCR (RT-PCR) was used to determine differential expression of these genes in wild-type and luxS mutant isotypes. Further confirmation of the involvement of LuxS and AI-2 signal was demonstrated by complementing the luxS-null mutant in trans with the intact luxS gene and by treatment of the mutant strain with purified AI-2. Expression of luxS was found to be controlled by the GppX two-component signal transduction pathway. Additionally, growth of the LuxS mutant was impaired under conditions of hemin limitation.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. P. gingivalis, E. coli, and V. harveyi strains, along with plasmids utilized in this study, are listed in Table 1. P. gingivalis 33277 and its derivatives LS, CLS, GPPX-M, and GPPX-E were grown anaerobically (85% N2, 10% H2, 5% CO2) at 37°C in sTSBY medium (Trypticase soy broth [BBL] supplemented with yeast extract [1 mg ml–1], hemin [5 μg ml–1], and menadione [1 μg ml–1]). For bioluminescence assays, P. gingivalis was cultured in AI bioassay (AB) medium (0.3 M NaCl, 0.05 M MgSO4, 0.2% Casamino Acids, 10 mM KH2PO4, 1 mM L-arginine, 1% glycerol [pH 7.5]) (31) modified by the addition of 50% sTSBY. E. coli DH5 and SURE (Stratagene) strains were grown aerobically with shaking in Luria-Bertani (LB) broth (Difco) or on LB agar at 37°C. The V. harveyi reporter strain BB170 (sensor 1– sensor 2+) was kindly provided by B. Bassler (Princeton University) and was grown aerobically in AB medium at 30°C. Antibiotics, when necessary, were used at the following concentrations: for P. gingivalis, erythromycin at 15 μg ml–1 and tetracycline at 1 μg ml–1; for E. coli, erythromycin at 30 μg ml–1 and ampicillin at 100 μg ml–1.
DNA manipulation. General recombinant DNA techniques were performed as described by Sambrook et al. (60) unless otherwise noted and in accordance with the manufacturers' recommendations for DNA isolation, restriction enzyme digestion (Promega), ligation (New England Biolabs), and plasmid purification (QIAGEN). For Southern blotting, DNA probes were labeled and hybridization detected using the Gene Images AlkPhos direct labeling and detection system (Amersham Biosciences). Standard PCR experiments were performed using the Taq polymerase system (Eppendorf). For cloning and sequencing experiments, a high-fidelity enzyme, PfuTurbo (Stratagene), was used. All primers and their sequences are listed in Table 2. PCR products that required sequencing were cloned into pUC19, and nucleotide sequencing was performed using M13 primers by the University of Florida Sequencing Core with an ABI Prism 377XL automated DNA sequencer.
Construction of P. gingivalis luxS mutant and complemented mutant strains. A luxS gene mutant in P. gingivalis was constructed by allelic replacement. A DNA sequence containing 96 bp of the 5' end of luxS and 784 bp upstream of the ATG initiation codon was amplified from chromosomal DNA by PCR using primers 5'UP784-LS and 3'LS-96, which introduced EcoRI and BamHI sites, respectively. A DNA sequence containing 162 bp of the 3' end of luxS and 678 bp downstream of the luxS stop codon was amplified using primers 5'LS-324 and 3'DOWN678LS, which introduced BamHI and PstI sites, respectively. Both fragments were ligated into the multiple cloning site of pUC19 in E. coli SURE cells (Stratagene) to create plasmid pLS (Table 1), with a 226-bp section (nucleotides 97 to 323) missing from the luxS gene. A BamHI fragment containing ermF was cloned into the BamHI site between the two luxS fragments in pLS to create pLS::ermF (Table 1). Plasmid pLS::ermF was linearized with ScaI and introduced into P. gingivalis 33277 by electroporation. A double-crossover recombination event was selected for by plating on sTSBY agar containing erythromycin. Mutation was confirmed by PCR using primers 5'luxS-1 and 3'luxS-466 and by Southern blotting with luxS and ermF gene probes. The resulting mutant was designated LS.
For genetic complementation of the mutation in LS, the intact luxS gene was expressed in trans under the control of the P. gingivalis mfa1 promoter (56). The mfa1 regulatory region and the luxS open reading frame were fused by a series of PCR amplifications using primers MFA-HIND, MFA-SPH, LUX-SAL, and LUX-SPH (Table 2), which introduced HindIII, SphI, and SalI sites into directed locations. These PCR products were digested with SphI and ligated for gene fusion. The ligation mixture was subjected to another PCR, to generate a fused DNA fragment, using primers MFA-HIND and LUX-SAL. The fused PCR product was cloned into HindIII and SalI sites of shuttle vector plasmid pT-COW (kindly provided by N. Shoemaker, University of Illinois), to create pT-MLUX, which was introduced into LS by conjugation. Erythromycin- and tetracycline-resistant transconjugants were selected, and the presence of plasmid was confirmed by restriction analysis. The complemented strain was designated CLS.
RNA isolation and cDNA synthesis. Total RNA was isolated from triplicate, independent bacterial cultures. Bacterial cells were lysed using Trizol LS reagent (Invitrogen), and RNA was extracted with phenol-chloroform and precipitated with isopropanol. RNA preparations were washed with 70% ethanol, dissolved in RNase-free H2O, and treated with RNase-free DNase I (Ambion). Treated RNA samples were further purified with a second on-column DNase I treatment using the RNeasy minikit (QIAGEN). The iScript cDNA synthesis kit (Bio-Rad) was used to generate cDNA from RNA (1-μg) templates.
Quantitative real-time PCR. (i) Real-time primers. Primers for real-time PCR were designed using Beacon Designer software, version 2.0, and are listed in Table 2. Predicted product sizes were in the 100- to 200-bp range.
(ii) Preparation of standards. Specific DNA products for each gene under investigation were synthesized from chromosomal DNA using standard PCR methods and visualized by gel electrophoresis to verify that a single specific product had been generated. Each product was purified using the QIAquick PCR purification kit (QIAGEN) and quantified using an Eppendorf BioPhotometer. The DNA product copy number (per microliter) (also called the starting quantity [SQ]) was calculated using the formula of Yin et al. (83) as [(6.023 x 1023 x g of DNA/ml)/(molecular weight of product)]/1,000. The molecular weight of the product is calculated as base pairs x 6.58 x 102 g. A 10-fold dilution series of each DNA standard was prepared for SQs of 108 to 104 copies μl–1. These were used in duplicate in each real-time PCR assay to allow the real-time PCR software to estimate the SQ of that gene in cDNA samples.
(iii) Real-time PCR. The standard DNA dilution series (SQ, 108 to 104 copies μl–1) or cDNA templates (2 μl) were added in duplicate to an iCycler iQ 96-well PCR plate (Bio-Rad). To each well, the following were added: 1 μl each 5' and 3' specific primer (50 pmol each); 12.5 μl iQ SYBRGreen Supermix (Bio-Rad) containing 100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), iTaq DNA polymerase (50 U ml–1), 6 mM MgCl2, 20 nM fluorescein, and stabilizers; and 8.5 μl distilled H2O. The 96-well plate was sealed with optical tape (Bio-Rad), and samples were quantified in the iCycler machine (Bio-Rad) using a standard thermal cycling program. Real-time results were analyzed using iCycler iQ Optical System software, version 3.0a (Bio-Rad). The melt curve profile was analyzed to verify a single peak for each sample, indicating primer specificity.
Kgp protease assay. Lysine protease activity was measured as previously described (9, 59, 68, 72). Briefly, P. gingivalis cultures were resuspended in substrate buffer (140 mM NaCl, 100 mM Tris-HCl [pH 7.5]) containing 0.4 mM -N-acetyl-L-lysine-p-nitroanilide (Ac-Lys-pNA.HCl) (Bachem). Samples were incubated in triplicate at 37°C for 1 h, and the increase in A405 due to release of p-nitroaniline was measured.
Autoinducer bioluminescence assay. P. gingivalis strains were grown overnight in AB-sTSBY (50:50), because the organism does not grow in AB medium. This initial culture was diluted to an optical density at 600 nm (OD600) of 0.1 in fresh AB-sTSBY and grown to appropriate optical densities. Cell-free culture supernatants were prepared by centrifugation at 10,000 x g for 10 min and filtration using 0.22-μm-pore-size MillexGP filter units (Millipore). The ability of these cell-free preparations to induce the AI-2 signaling system in the V. harveyi reporter strain BB170 was determined using the bioluminescence assay previously described (31, 74). Briefly, an overnight culture of BB170 (OD600, 1.0) was diluted 1:1,000 in AB medium, and 900 μl was added to 24-well plates (Costar). Cell-free P. gingivalis culture supernatants were added (100 μl per well) and incubated at 30°C with agitation. The cell-free culture supernatant from BB170 grown in 50:50 AB-sTSBY medium was used as a source of AI-2 in positive-control wells, and sterile medium was used in negative-control wells. Luminescence was measured using a VictorDB plate reader.
Purification of P. gingivalis AI-2. AI-2 was partially purified from P. gingivalis 33277 cultures according to the method of Sperandio et al. (69). Briefly, early-exponential-phase cultures (OD600, 0.5) were centrifuged (5,000 x g, 30 min) and filtered (pore size, 0.22 μm; Millipore). Cell-free supernatants were size fractionated for compounds of <3 kDa using YM-3 Centriprep centrifugal filter devices (Millipore). The crude extracts were freeze-dried, and 200 mg was dissolved in 1.5 ml cold 5 mM NaPO4 buffer (pH 6.2) and passed through a C18 Sep-PakPlus cartridge (Waters). The flowthrough that eluted with 5 mM NaPO4 buffer (pH 6.2) contained AI-2, and activity was confirmed in a bioluminescence assay with the V. harveyi reporter strain BB170.
AI-2 complementation of LS. P. gingivalis LS cultures were grown in sTSBY to late-lag phase (OD600, 0.3). The culture medium was then supplemented with 2.5% (vol/vol) AI-2 extract purified from wild-type 33277. Negative controls were supplemented with 2.5% (vol/vol) 5 mM NaPO4 buffer (pH 6.2) or 2.5% (vol/vol) of the AI-2 fraction from LS cultures. At appropriate time intervals after addition of AI-2 or buffer, bacterial cells were collected by centrifugation and RNA was isolated.
RESULTS
Construction of isogenic luxS-null mutant and complemented mutant strains. To analyze the effects of luxS on the phenotype of P. gingivalis, a deletion mutant strain (LS) was constructed by allelic replacement. To ensure that the observed phenotype of the mutant strain results from disruption of luxS, LS was complemented in trans with the wild-type allele on plasmid pTCOW (strain CLS). Because the luxS gene in P. gingivalis comprises part of an operon with pfs, and no promoter sequences were identified in the upstream region of luxS, the regulatory region of the P. gingivalis mfa1 gene was used to promote transcription of luxS in the CLS complemented strain. LS and CLS were compared to the wild-type parental strain 33277 using the AI-2 bioluminescence assay with the V. harveyi reporter strain BB170 (3). Light production by cell-free culture supernatants from LS was significantly decreased compared to light production by the parental strain (Fig. 1). Bioluminescence activity was restored in CLS, but not to wild-type levels. This may be a consequence of aberrant regulation of luxS transcription from the mfa1 promoter or deficient processing of the monocistronic luxS mRNA resulting from plasmid expression.
FIG. 1. Effects of the luxS mutation (strain LS) and it complementation by the wild-type gene (strain CLS) on AI-2 production, tested using the V. harveyi reporter strain BB170. AI-2 production was evaluated as fold activation (activity induced by test supernatant/activity induced by medium alone). Bioluminescence induced by a homologous V. harveyi culture supernatant (not shown) was more than 1 log unit higher than that for wild-type (WT) P. gingivalis (strain 33277), consistent with previous reports (12). A t test on bioluminescence data showed that induction by strains 33277 and CLS was significant at a P level of <0.005 ().
AI-2 production by P. gingivalis is growth phase dependent. While AI-2 signaling is generally considered to comprise a quorum-sensing system, in a number of species, including A. actinomycetemcomitans, S. mutans, E. coli, Clostridium perfringens, and Salmonella serovar Typhimurium, maximal AI-2 signal can be detected during the mid-exponential growth phase (22, 48, 52, 74). Thus, the growth phase dependence of luxS transcription and AI-2 signal activity was investigated in P. gingivalis. As reported by the V. harveyi BB170 bioluminescence assay, AI-2 production was highest in cultures grown to early-exponential phase and declined through late-exponential phase (Fig. 2A). The pattern of AI-2 signal activity was consistent with the levels of luxS mRNA (Fig. 2B), although AI-2 signal activity has a longer half-life than luxS mRNA. Similarly, Burgess et al. (9) reported that levels of the LuxS protein increased throughout growth even though AI-2 activity was maximally detected at mid-exponential phase. Quantitative RT-PCR of the luxS mRNA showed that levels of luxS message were 3- to 4-fold higher at the early- and mid-exponential growth stages than at the late-exponential phase of growth. Furthermore, luxS message decreased fivefold between the late-exponential and stationary phases of growth. Collectively, the results suggest that the LuxS signaling system of P. gingivalis plays its most important role during the early stages of growth, when cell density is low and nutrients may be plentiful. In this context it is important to consider that LuxS also plays a role in the activated methyl cycle, where maximal levels of methionine and S-adenosylmethionine are required for optimal growth.
FIG. 2. AI-2 activity and luxS expression in P. gingivalis are dependent on growth phase. (A) Fold activation of bioluminescence induced by culture supernatant from the P. gingivalis wild-type strain 33277 (WT) at early (OD600, 0.5)-, mid (OD600, 0.7)-, or late (OD600, 1.0)-exponential phase. Differences among growth phases were significant at a P level of <0.01 () (t test). (B) Expression levels of luxS mRNA in the WT at early-, mid-, and late-exponential phase and stationary phase (Stat.) as measured by quantitative RT-PCR (see the text for details). Expression levels are presented as cDNA copy number per microgram of RNA.
LuxS signaling influences expression of hemin acquisition genes in P. gingivalis. Previous studies provided preliminary evidence that LuxS signaling in P. gingivalis plays a role in regulating genes involved in hemin acquisition (9, 12). P. gingivalis strain 33277 possesses several putative and defined mechanisms for hemin and iron uptake, and representative genes from each of these pathways are listed in Table 3. To investigate the role of LuxS in the regulation of hemin and iron uptake pathways in P. gingivalis, differential expression of these genes was investigated by quantitative RT-PCR. A number of genes involved in hemin acquisition were found to be differentially expressed in LS compared to the parent strain (Fig. 3). At early-exponential phase, when LuxS expression is maximal, two genes encoding outer-membrane-expressed heme-binding proteins (HmuR and FetB) showed significantly increased expression in LS. The levels of induction of both hmuR and fetB declined at late-exponential phase, when there is less luxS message, although hmuR expression remained significantly higher in the LS mutant than in the parental strain. Thus, hmuR and fetB appear to be negatively regulated by LuxS signaling. Conversely, tlr, encoding another TonB-linked heme-binding receptor, is positively regulated by LuxS; this gene showed decreased expression in the LS mutant in early-exponential growth. Differential expression of kgp was also observed, with a threefold decrease in expression in LS in early-exponential phase and a threefold increase in expression during late-exponential phase. The correlation between mRNA levels and Kgp activity was explored using a lysine-specific protease assay. The LS mutant exhibited 32.5% less Kgp protease activity than the parental strain (P = 0.00035) when grown to early-exponential phase. These data are in good agreement with results of Burgess et al. (9), who reported a 30% decrease in Kgp activity in a luxS-null mutant of P. gingivalis W50. By contrast, there was no significant difference in expression of rgpA between LS and parent strains. While both the arginine-specific (Rgp) and lysine-specific (Kgp) gingipains can degrade heme-bound proteins and liberate heme (72), only Kgp is thought to act as a hemophore, binding to heme and hemoglobin and delivering heme to outer membrane receptors (29). Both positive and negative regulation of kgp by LuxS signaling are indicative of multiple levels of control, as has also been demonstrated by other groups (34, 53, 63, 78). Collectively, these results demonstrate that LuxS has a varied and nuanced relationship with hemin uptake, with different hemin uptake genes regulated individually as P. gingivalis progresses through its growth cycle.
FIG. 3. Hemin uptake genes are differentially expressed in the LS mutant. Gene expression was measured by quantitative RT-PCR on wild-type (WT) or LS cultures grown to early (OD600, 0.5)- or late (OD600, 1.0)-exponential phase. Fold change was calculated by dividing the copy number of the gene transcript (per microgram of RNA) in the LS mutant by the copy number in the WT. The 16S rRNA and fimA genes were used as controls. Significant differences (P < 0.05 by t test) are labeled with asterisks.
Differential expression of Fe2+ acquisition genes in the LS mutant. In addition to hemin uptake, P. gingivalis can also acquire inorganic Fe2+, potentially through the two homologues of a ferrous iron transport protein, FeoB1 and FeoB2 (Table 3). The hemin and iron uptake pathways are interconnected through the action of FetB, which can remove iron from heme compounds prior to uptake (55). Furthermore, regardless of the uptake mechanism, intracellular iron can be stored in ferritin. Quantitative RT-PCR was used to investigate whether LuxS also has a role in the control of inorganic iron uptake and storage. LuxS signaling negatively regulated the expression of ftn (encoding ferritin) and feoB1 compared to parental levels (Fig. 4). Conversely, there was a low-level positive regulation of feoB2 by LuxS. Recent evidence suggests that feoB2 is not involved in iron transport and may in fact play a novel role in manganese transport (16). These data suggest that LuxS signaling is important for the acquisition of iron not only in the form of hemin but also in the form of Fe2+.
FIG. 4. Iron uptake and storage genes are differentially expressed in the LS mutant. Gene expression was measured by quantitative RT-PCR on wild-type (WT) or LS cultures grown to mid-exponential phase. Fold change was calculated by dividing the copy number of the gene transcript (per microgram of RNA) in the LS mutant by the copy number in the WT. The 16S rRNA and fimA genes were used as controls. Significant differences (P < 0.05 by t test) are labeled with asterisks.
trans-Complementation of LS restored parental expression levels of hmuR. In order to ensure that differential gene expression in the LS mutant was a direct effect of disruption of the luxS gene, the trans-complemented strain CLS was utilized. Quantitative RT-PCR was performed on RNAs extracted from parent, LS, and CLS cultures grown to mid-exponential phase to measure the levels of luxS and hmuR transcripts. hmuR was selected for this experiment because, among the genes tested, hmuR demonstrates the largest change in expression in response to the LuxS signal, thus increasing the dynamic range of the assay. As shown in Fig. 5A, the luxS mRNA level was approximately ninefold higher in CLS than in the parental strain. The increase in luxS expression in CLS most likely reflects the presence of multiple copies of the pTCOW plasmid. Nonetheless, an increase in the luxS transcript level does not necessarily correspond to equally increased levels of AI-2 production, since LuxS enzyme activity is limited by the availability of its substrate (6). The complemented isotype showed decreased expression of hmuR (threefold) compared to that for LS (Fig. 5B), confirming that LuxS signaling has a negative regulatory effect on this gene. Expression of hmuR did not completely return to wild-type levels, corroborating data presented above in Fig. 1 and 5 that suggest that an increase in the monocistronic luxS transcript level in trans cannot fully restore LuxS function.
FIG. 5. Complementation of the LS mutant with the wild-type (WT) luxS gene restores expression of luxS (A) and alters expression of hmuR (B). Expression of luxS mRNA in CLS was significantly higher (P < 0.05 by t test) than that in either the WT or the LS mutant. Expression of hmuR mRNA in CLS was significantly lower (P < 0.01 by t test) than that in the LS mutant. The transcript copy number (per microgram of RNA) was determined by real-time PCR for the WT, CLS, or LS strain grown to mid-exponential phase. Error bars, standard deviations (n = 6).
Isotypes possessing native luxS but exhibiting increased luxS mRNA levels exhibit decreased hmuR and increased tlr expression. Complementation of a genetic mutation is a useful strategy to confirm the function of that gene. Nonetheless, our results suggested that changes in luxS promoter activity, timing of expression, or expression levels of other genes cotranscribed with luxS can subtly affect LuxS functionality in P. gingivalis. Similarly, De Keersmaecker et al. (18) found that the intrinsic promoter activity of luxS in Salmonella serovar Typhimurium affected LuxS-dependent biofilm formation. Thus, we sought an additional means to corroborate the role of LuxS in the regulation of hemin uptake-associated genes. By screening P. gingivalis mutants in two-component systems, we identified a gene, gppX, that suppresses the transcriptional activity of luxS. GppX is a sensor kinase recently found to be required for maturation and localization of the P. gingivalis gingipains (34). Two independent gene disruption mutants, GPPX-M and GPPX-E (kindly provided by F. Yoshimura, Aichi-Gakuin University), exhibited elevated AI-2 activity and >2-fold-higher levels of luxS mRNA compared to those for the parental strain (Fig. 6A). Hence, the GppX mutants allowed investigation of the effects of altered luxS expression without disruption of the luxS gene or its promoter. Expression of the two different TonB-linked heme-binding receptors, HmuR and Tlr, was determined by quantitative RT-PCR. Figure 6B shows that both GPPX-M and GPPX-E had decreased expression of hmuR (4.7- and 4.9-fold lower than that for the parental strain, respectively) yet increased expression of tlr (3.1- and 3.4-fold more than that for the parent strain, respectively). For both hmuR and tlr, this was the opposite effect to that observed after disruption of luxS. Thus, these data support the evidence presented in Fig. 3 and 5 that LuxS signaling suppresses hmuR expression and promotes tlr expression, although the formal possibility remains that GppX can directly regulate both hmuR and tlr. It is also noteworthy that GppX has homology (e–35) with the LuxQ AI-2 sensor kinase of Vibrio. However, the phenotype of the gppX mutants would tend to exclude a role for GppX as an AI-2 sensor kinase in P. gingivalis. These results do, however, demonstrate a role for the GppX two-component pathway in the control of expression of the luxS gene.
FIG. 6. (A) Culture supernatants from P. gingivalis strains GPPX-M and GPPX-E demonstrate increased (P < 0.05 by t test) bioluminescence (in counts per second [CPS]) in V. harveyi BB170 compared to the wild type (WT). (B) GPPX-M and GPPX-E exhibit increased expression of luxS and tlr, and decreased expression of hmuR, compared to the WT. Quantitative RT-PCR was performed on RNA from the WT, GPPX-M, or GPPX-E at mid-exponential phase. Fold change was calculated by dividing the copy number of the gene transcript (per microgram of RNA) in the GPPX mutants by the copy number in the WT. The 16S rRNA gene was used as an internal control (not shown). All fold differences were significant at a P level of <0.05 (t test).
Treatment of luxS-null mutants with partially purified AI-2 restores parental levels of hemin uptake gene expression. The biosynthetic pathway leading to AI-2 production is part of the activated methyl pathway (6, 62, 80). Indeed, Winzer et al. (80) have argued that AI-2 is not a signal molecule per se but rather an extracellular by-product of S-ribosylhomocysteine recycling and that changes in gene expression due to inactivation of luxS are the result of defects in methionine metabolism. To begin to distinguish between these possibilities for P. gingivalis, we partially purified extracellular AI-2 from culture supernatants of the parental strain. Cultures of LS in late-lag phase were treated with AI-2 or a buffer control, and samples of RNA were extracted. No difference in the growth rate of LS in the presence of AI-2 was observed. The effects of AI-2 on expression of hmuR and tlr were determined by quantitative RT-PCR analysis. Within 5 min, LS cultures treated with AI-2 exhibited >2-fold suppression of hmuR mRNA levels. Furthermore, after 30 min, the hmuR copy number detected from cultures treated with AI-2 was threefold less than that from buffer-treated cultures (Fig. 7A). Conversely, tlr expression was significantly higher (2.6-fold) in cultures treated with AI-2 than in those treated with buffer alone after 30 min (Fig. 7B). There was no significant difference in expression of either heme-binding receptor gene following 60 min of treatment with AI-2, indicating either signal depletion or degradation at this time point. The fimA gene, used as a control, did not show any AI-2-induced changes in expression (Fig. 7C). As an additional control, the AI-2 fraction was purified from the LS strain, which does possess some residual AI-2 activity, presumably due to nonenzymatic formation of AI-2 signal. As shown in Fig. 7D, parental signal induced expression of tlr almost twofold compared to signal from the mutant, a difference similar to that observed in whole cells of the parent and the mutant. The divergent regulation of hmuR and tlr indicates that the activity of the AI-2 fraction is not the result of a nonspecific metabolic effect on the LS cells. These data, along with earlier results, provide strong evidence that hemin uptake mechanisms are regulated by an external AI-2 signal produced by the action of LuxS. Furthermore, it is apparent that the AI-2 signal allows P. gingivalis to switch between different mechanisms of hemin and iron uptake as dictated by environmental conditions.
FIG. 7. Purified AI-2 regulates expression of hmuR and tlr. AI-2 or control NaPO4 buffer was added to LS cultures (OD600, 0.3) and incubated for the additional times indicated. (A to C) Expression levels (per microgram of RNA) of hmuR (A), tlr (B), and fimA as a control (C) were determined by quantitative RT-RCR. (D) Fold induction of tlr in the LS strain by AI-2 purified from the WT compared to the AI-2 fraction from the LS strain. Error bars, standard deviations (n = 6). Asterisk, P < 0.05 (by t test) for cultures treated with AI-2 compared to cultures treated with buffer or the AI-2 fraction from the LS strain.
LuxS signaling plays a role in growth of P. gingivalis under conditions of limited hemin. The ability of LuxS to regulate aspects of hemin uptake led us to postulate that the LS mutant would be defective in growth in hemin-limited medium, where the inability to optimally acquire hemin could have adverse effects on growth rate. To test this hypothesis, we first demonstrated that there was no difference between the growth profiles of the parental strain and the LS mutant strain in the presence of excess hemin (Fig. 8A). In contrast, although the growth of both isotypes was impaired under hemin-limiting conditions, the doubling time of the LS cultures in exponential phase was approximately 2 h longer than that of the parent, and the final OD attained by the LS mutant was 1.5-fold less than that of the parent (Fig. 8B). These data support our conclusions that in the absence of the LuxS signaling system, P. gingivalis is unable to fine-tune hemin uptake mechanisms, and thus survival under hemin-limiting conditions is diminished.
FIG. 8. Growth of the LS mutant is impaired under conditions of hemin limitation. Shown are growth curves of wild-type P. gingivalis (WT) and the LS mutant in the presence of optimal (5-μg ml–1) (A) or limited (0.05-μg ml–1) (B) hemin concentrations. For the limited-hemin condition, the cells were depleted of stored hemin by two subcultures in the absence of hemin. Overnight cultures were inoculated into fresh medium containing the appropriate concentration of hemin. Error bars, standard deviations (n = 3).
DISCUSSION
The ability of pathogenic bacteria to obtain iron from their human hosts is an important factor contributing to their ultimate survival or elimination. In the periodontal pocket, the favored niche of P. gingivalis, iron and heme-containing proteins are available through the crevicular fluid (a serum exudate) and during episodes of gingival bleeding. Levels of iron and heme-containing proteins will thus fluctuate, and organisms with a multiplicity of uptake pathways under tight regulatory control can be predicted to have a selective advantage. Lacking siderophores, P. gingivalis employs specific outer membrane receptors, lipoproteins, and proteases to acquire heme and iron. In this report we show that uptake pathways for iron in the form of both hemin and inorganic Fe2+ are controlled by the LuxS/AI-2 signaling system in P. gingivalis ATCC 33277, a strain with demonstrated pathogenic activity in the rat bone loss model of periodontal disease (36, 58). AI-2 is, therefore, likely to be important for the survival and pathogenicity of P. gingivalis in the periodontal environment.
The relationship between AI-2 signaling and iron/hemin uptake involves independent regulation of distinct uptake mechanisms. AI-2 was found to negatively regulate expression of hmuR, fetB, feoB1, and ftn, whereas tlr and kgp are positively regulated by AI-2. HmuR is a TonB-linked outer membrane receptor and has been reported to be required for both hemoglobin and hemin utilization in P. gingivalis. Inactivation of the hmuR gene impairs growth with hemoglobin or hemin as the sole iron source (64). Furthermore, hmuR conferred the ability to bind hemin and hemoglobin on E. coli (64), and purified recombinant HmuR has been shown to bind both free and protein-bound hemin (54). Tlr is also a TonB-linked outer membrane receptor. The reported difference between the functions of Tlr and HmuR is that tlr-null mutants can grow in optimal, but not limiting, hemin concentrations (1), whereas hmuR-null mutants show diminished growth even in optimal hemin concentrations (64). Thus, HmuR may play a more vital role in hemin uptake than Tlr when hemin supplies are plentiful, whereas Tlr is more important for scavenging hemin when supplies are scarce, as proposed first by Aduse-Opoku et al. (1). FetB (IhtB) is a hemin binding lipoprotein that is located in an iron transport operon along with a third TonB-linked outer membrane receptor, IhtA (15). FetB can also function as a chelatase that may remove iron from heme prior to uptake by P. gingivalis (15). Fe2+ is transported through the inner membrane by the ion transport protein FeoB1 (16) and deposited in ferritin. Proteases such as Kgp can degrade host iron- and heme-containing proteins and can possibly act as hemophores, shuttling heme back to outer membrane receptors (55).
The results of the current study would thus allow us to propose the following broadly based model. During early-exponential growth in vivo, before the onset of periodontitis, hemin and iron levels are low, and LuxS signaling suppresses the production of HmuR, FetB, and FeoB1 in order to conserve energy. Consequently, less ferritin is required for iron storage. Under these conditions, hemin scavenging is accomplished by Tlr, and Kgp is also upregulated to liberate additional heme from host proteins. Subsequently, as the P. gingivalis community becomes established and metabolically stable, tissue destruction in the periodontal pockets advances and more heme/iron is available. P. gingivalis cells in this state have reduced AI-2 expression, lifting the repression of hmuR, fetB, and feoB1 and decreasing expression of tlr, which is no longer necessary for hemin uptake. This model predicts that a LuxS-deficient mutant of P. gingivalis would not show a proliferation-related phenotype under hemin-replete conditions, since upregulation of Tlr is not required. Conversely, growth of a LuxS mutant would be impaired during hemin limitation, because in the absence of AI-2 the mutant would not be able to upregulate Tlr. This predicted phenotype was indeed exhibited by our LuxS mutant, increasing confidence in the proposed model. It should also be stressed, however, that there exist additional levels of control of hemin uptake in P. gingivalis. A Fur homologue is present in P. gingivalis (55), and a putative Fur box exists upstream of the hmuYR operon, indicating that hmuR is also regulated by Fur (64). It has also been demonstrated that hemin binding and transport in P. gingivalis can be induced by hemin (8, 28, 35, 38, 64) and that hemin can also be stored on the cell surface as μ-oxo bisheme dimer layers (68). Such redundancy in hemin uptake and storage mechanisms in P. gingivalis may facilitate tight control and ensure adaptability to environmental conditions in vivo. Moreover, the presence of multiple levels of control may allow relatively small changes in individual gene expression to be amplified through the regulatory network. In addition, in the dense biofilm that is the habitat of P. gingivalis, signals derived from other organisms may also be detected by P. gingivalis. The extent to which hemin and iron uptake genes are regulated by heterologous AI-2 is currently under investigation in our laboratory.
A relationship exists between iron/heme uptake and virulence in P. gingivalis. Iron starvation results in attenuation of virulence in a mouse model (37), while culture in excess hemin enhances the virulence of P. gingivalis (46). Moreover, a mutant of P. gingivalis that has diminished hemin binding displays reduced virulence (67). While there is general agreement that the virulence factors of P. gingivalis that are affected by hemin levels include hemin binding and protease production, the nature of this regulation is less clear. Some studies find that hemin binding and proteolytic activity are increased by excess hemin (10, 66), whereas others report an increase in these virulence factors under hemin limitation (30). Many factors, including strain differences, could account for these discrepancies. However, the results presented here intimate that variations in levels of AI-2 production by the strains under the experimental conditions adopted in these previous studies would impact protease activity, hemin binding, and ultimately virulence.
In P. gingivalis, luxS transcription and AI-2 signaling activity were highest during the early-exponential-growth phase. Thus, the term "quorum sensing" is not strictly accurate to describe AI-2 signal activity in P. gingivalis. Furthermore, luxS expression in P. gingivalis is transcriptionally regulated, whereas in other organisms, such as Salmonella serovar Typhimurium, luxS expression is constitutive (6), although AI-2 production can be regulated by environmental conditions (75). In addition to LuxS, Pfs is required for AI-2 biosynthesis (62). Pfs catalyzes both the formation of S-ribosylhomocysteine (the substrate for LuxS) from S-adenosylhomocysteine to release adenine and the production of 5'-methylthioribose from 5'-methylthioadenosine, also releasing adenine. In Salmonella serovar Typhimurium, pfs is transcriptionally regulated by environmental cues and the transcriptional profile correlates with the pattern of AI-2 production (6). In P. gingivalis, where luxS is cotranscribed with the upstream pfs gene, luxS mRNA levels have been shown to differ according to osmolarity (12), indicating that luxS can be transcriptionally controlled along with pfs. In the present study, the two-component system GppX was found to negatively regulate luxS transcription in P. gingivalis. GppX is a hybrid-type two-component system containing both a sensor domain and a regulator domain (34). Recently, Carter et al. (11) reported that a two-component signal transduction system comprising the transcriptional regulator rolA and the sensor kinase rolB negatively regulates luxS expression in Clostridium difficile. Hence, two-component systems may be used by a variety of bacteria to control luxS expression, and this may be of particular utility in situations where AI-2 does not appear to be used to report cell number. In P. gingivalis, GppX can also regulate proteolytic activity and GppX mutants have reduced Kgp function (34). Since GppX also controls luxS, and LuxS can control kgp, it is clear that the regulatory network in which LuxS participates is multilayered and extensively interconnected.
The current results clarify and extend the previous differential display study of an insertion-duplication luxS mutant (12), which revealed several hemin uptake-related genes that were either activated or repressed in the absence of LuxS. Since the publication of that pregenomic study, several genes have been more precisely annotated. With relevance to the current results, the hemR gene that was reported by Chung et al. (12) as downregulated in the LuxS mutant was originally thought to be a homologue of hmuR. However, hemR contains regions homologous to both hmuR and the protease gene prtT, and hemR is now thought to be unique to P. gingivalis strain 53977 (55). Since the sequence detected by Chung et al. (12) was homologous to the protease region of hemR, these results are not inconsistent with the current study. It is also noteworthy that in the completed genome database, the gene for a hypothetical protein (TIGR PG0499) is immediately downstream of luxS, as opposed to the putative homologue of TraJ present in the incomplete database at the time of the Chung et al. study (12).
An association between LuxS and iron acquisition has been observed with other bacteria. Fong et al. (21) reported that LuxS regulates genes involved in iron uptake in A. actinomycetemcomitans and that a luxS mutant of A. actinomycetemcomitans grows poorly under iron-depleted conditions. Similarly, in V. vulnificus, mutation of luxS appears to cause an in vivo iron assimilation defect (39). The opposite phenotype is exhibited by a Mannheimia haemolytica luxS mutant that can outcompete the parental strain under conditions of iron restriction (77). Thus, a number of organisms may utilize AI-2 signaling to control aspects of iron/heme uptake. Interestingly, cell-free supernatants from A. actinomycetemcomitans can complement gene expression in a LuxS mutant of P. gingivalis (22). Whether or not there may be functional specificity among AI-2 signals from different bacteria, however, awaits further study.
Dental plaque is a multispecies biofilm containing a variety of species or phylotypes of bacteria that number in the hundreds (41). Inter- and intraspecies communication plays an important role in the temporal and spatial development of this dense and diverse community (20, 40, 47). In addition to its role in regulation of hemin uptake proteins, LuxS is also required for the development of heterotypic biofilms of P. gingivalis and S. gordonii in hemin-free buffer (47). Hence, the role of LuxS/AI-2 in P. gingivalis is likely to extend beyond that of control of hemin acquisition.
ACKNOWLEDGMENTS
We thank B. Bassler, N. Shoemaker, and F. Yoshimura for providing strains and plasmids.
This work was supported by NIDCR (R01 DE14605).
FOOTNOTES
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Department of Periodontics, Endodontics and Dental Hygiene, School of Dentistry, University of Louisville, Louisville, Kentucky 40292
ABSTRACT
The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe2+ ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.
INTRODUCTION
A number of bacterial intercommunication systems have been identified that are based on the production and detection of external signals and that enable coordinated responses throughout a population. Quorum sensing is a system by which a bacterial population can monitor its cell density through release of specific signaling molecules called autoinducers. As the population grows, autoinducers accumulate in the local environment until a critical detection threshold level is reached, at which point changes in gene expression are triggered. In this manner bacterial populations can synchronize expression of specific genes required for community survival under the prevailing environmental conditions (2, 27, 81).
Gram-negative bacteria frequently use acylated homoserine lactones (AHLs) as autoinducers, and this signal is classified as autoinducer 1 (AI-1). In the archetypal quorum-sensing system of the marine symbiotic bacterium Vibrio fischeri, AHL is synthesized by the LuxI protein, and transcriptional activation of the target gene is mediated by LuxR (26). In gram-positive bacteria, quorum sensing is achieved through peptide autoinducers that modulate two-component phosphorelay cascades (19). Both the AI-1 and peptide signaling systems are considered species specific and control a wide range of biological activities including bioluminescence, sporulation, biofilm formation, conjugation, motility, competence, and antibiotic production (13, 17, 25, 51, 61, 73). By contrast, the AI-2 system, originally identified in Vibrio harveyi, is considered non-species specific and may function as a universal bacterial language (81). AI-2 is produced through the action of the LuxS enzyme, which cleaves S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). It is now suspected that AI-2 may comprise a family of structures generated from rearrangements of DPD (49, 69). In V. harveyi, AI-2 is detected by LuxPQ, which channel information to the phosphotransferase protein LuxU. LuxU, in turn, transfers the signal to LuxO, a 54-dependent transcriptional activator (4, 23, 24). Other bacterial species may process AI-2 by different mechanisms; for example, in Salmonella enterica serovar Typhimurium, AI-2 is internalized via the Lsr ABC transporter (76).
The luxS gene is widespread among both gram-positive and gram-negative bacteria, many of which are pathogens (79). Culture supernatants from organisms possessing luxS are generally able to stimulate bioluminescence in a sensor 1– sensor 2+ reporter strain of V. harveyi (3). Despite the cross-species-reactive nature of AI-2, the cellular processes controlled by AI-2 differ among organisms. Some examples include type III secretion in enterohemorrhagic and enteropathogenic Escherichia coli (71), motility in E. coli and Helicobacter pylori (44, 70), biofilm formation in Vibrio cholerae, H. pylori, Streptococcus gordonii, and Streptococcus mutans (7, 14, 32, 47, 48, 84), protease and hemolysin production in Vibrio vulnificus and Streptococcus pyogenes (39, 45), and leukotoxin production and iron uptake in Actinobacillus actinomycetemcomitans (22). Thus, individual organisms appear capable of adapting LuxS signaling for their own particular needs.
Porphyromonas gingivalis is an important human pathogen involved in the initiation and progression of periodontal disease. P. gingivalis can colonize numerous surfaces and form biofilms in the oral cavity but especially thrives in the deep periodontal pockets that develop as periodontal disease progresses (50). P. gingivalis is able to invade gingival epithelial cells and persist intracellularly for extended periods (43, 82). Virulence factors of the organism include fimbriae and other surface adhesins, as well as an abundance of proteinases that can degrade host proteins including immune components and iron-sequestering compounds (42, 57). P. gingivalis possesses the luxS gene and produces functional AI-2 (9, 12). Furthermore, in an early demonstration of the cross-species nature of AI-2, P. gingivalis was shown to respond to heterologous AI-2 produced by A. actinomycetemcomitans (22). LuxS-deficient mutants of P. gingivalis have been found to produce lower levels of proteases (9) and to exhibit altered expression of genes involved in hemin uptake (12). P. gingivalis shows a strong preference for iron in the form of hemin and expresses a number of heme/hemin uptake systems (55). These hemin uptake mechanisms include the TonB-linked outer membrane hemin binding proteins HmuR and Tlr and the hemin binding lipoprotein FetB (IhtB). P. gingivalis also possesses numerous operons encoding ABC transport systems and permeases, in order to transport iron sources across the cytoplasmic membrane (65). Strategies employed by P. gingivalis for the uptake of Fe2+ include the use of the ferrous iron transport protein FeoB (16).
In the present study, we undertook a systematic investigation of the role of LuxS in the transcriptional regulation of P. gingivalis hemin and Fe2+ acquisition mechanisms. Quantitative real-time reverse transcription-PCR (RT-PCR) was used to determine differential expression of these genes in wild-type and luxS mutant isotypes. Further confirmation of the involvement of LuxS and AI-2 signal was demonstrated by complementing the luxS-null mutant in trans with the intact luxS gene and by treatment of the mutant strain with purified AI-2. Expression of luxS was found to be controlled by the GppX two-component signal transduction pathway. Additionally, growth of the LuxS mutant was impaired under conditions of hemin limitation.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. P. gingivalis, E. coli, and V. harveyi strains, along with plasmids utilized in this study, are listed in Table 1. P. gingivalis 33277 and its derivatives LS, CLS, GPPX-M, and GPPX-E were grown anaerobically (85% N2, 10% H2, 5% CO2) at 37°C in sTSBY medium (Trypticase soy broth [BBL] supplemented with yeast extract [1 mg ml–1], hemin [5 μg ml–1], and menadione [1 μg ml–1]). For bioluminescence assays, P. gingivalis was cultured in AI bioassay (AB) medium (0.3 M NaCl, 0.05 M MgSO4, 0.2% Casamino Acids, 10 mM KH2PO4, 1 mM L-arginine, 1% glycerol [pH 7.5]) (31) modified by the addition of 50% sTSBY. E. coli DH5 and SURE (Stratagene) strains were grown aerobically with shaking in Luria-Bertani (LB) broth (Difco) or on LB agar at 37°C. The V. harveyi reporter strain BB170 (sensor 1– sensor 2+) was kindly provided by B. Bassler (Princeton University) and was grown aerobically in AB medium at 30°C. Antibiotics, when necessary, were used at the following concentrations: for P. gingivalis, erythromycin at 15 μg ml–1 and tetracycline at 1 μg ml–1; for E. coli, erythromycin at 30 μg ml–1 and ampicillin at 100 μg ml–1.
DNA manipulation. General recombinant DNA techniques were performed as described by Sambrook et al. (60) unless otherwise noted and in accordance with the manufacturers' recommendations for DNA isolation, restriction enzyme digestion (Promega), ligation (New England Biolabs), and plasmid purification (QIAGEN). For Southern blotting, DNA probes were labeled and hybridization detected using the Gene Images AlkPhos direct labeling and detection system (Amersham Biosciences). Standard PCR experiments were performed using the Taq polymerase system (Eppendorf). For cloning and sequencing experiments, a high-fidelity enzyme, PfuTurbo (Stratagene), was used. All primers and their sequences are listed in Table 2. PCR products that required sequencing were cloned into pUC19, and nucleotide sequencing was performed using M13 primers by the University of Florida Sequencing Core with an ABI Prism 377XL automated DNA sequencer.
Construction of P. gingivalis luxS mutant and complemented mutant strains. A luxS gene mutant in P. gingivalis was constructed by allelic replacement. A DNA sequence containing 96 bp of the 5' end of luxS and 784 bp upstream of the ATG initiation codon was amplified from chromosomal DNA by PCR using primers 5'UP784-LS and 3'LS-96, which introduced EcoRI and BamHI sites, respectively. A DNA sequence containing 162 bp of the 3' end of luxS and 678 bp downstream of the luxS stop codon was amplified using primers 5'LS-324 and 3'DOWN678LS, which introduced BamHI and PstI sites, respectively. Both fragments were ligated into the multiple cloning site of pUC19 in E. coli SURE cells (Stratagene) to create plasmid pLS (Table 1), with a 226-bp section (nucleotides 97 to 323) missing from the luxS gene. A BamHI fragment containing ermF was cloned into the BamHI site between the two luxS fragments in pLS to create pLS::ermF (Table 1). Plasmid pLS::ermF was linearized with ScaI and introduced into P. gingivalis 33277 by electroporation. A double-crossover recombination event was selected for by plating on sTSBY agar containing erythromycin. Mutation was confirmed by PCR using primers 5'luxS-1 and 3'luxS-466 and by Southern blotting with luxS and ermF gene probes. The resulting mutant was designated LS.
For genetic complementation of the mutation in LS, the intact luxS gene was expressed in trans under the control of the P. gingivalis mfa1 promoter (56). The mfa1 regulatory region and the luxS open reading frame were fused by a series of PCR amplifications using primers MFA-HIND, MFA-SPH, LUX-SAL, and LUX-SPH (Table 2), which introduced HindIII, SphI, and SalI sites into directed locations. These PCR products were digested with SphI and ligated for gene fusion. The ligation mixture was subjected to another PCR, to generate a fused DNA fragment, using primers MFA-HIND and LUX-SAL. The fused PCR product was cloned into HindIII and SalI sites of shuttle vector plasmid pT-COW (kindly provided by N. Shoemaker, University of Illinois), to create pT-MLUX, which was introduced into LS by conjugation. Erythromycin- and tetracycline-resistant transconjugants were selected, and the presence of plasmid was confirmed by restriction analysis. The complemented strain was designated CLS.
RNA isolation and cDNA synthesis. Total RNA was isolated from triplicate, independent bacterial cultures. Bacterial cells were lysed using Trizol LS reagent (Invitrogen), and RNA was extracted with phenol-chloroform and precipitated with isopropanol. RNA preparations were washed with 70% ethanol, dissolved in RNase-free H2O, and treated with RNase-free DNase I (Ambion). Treated RNA samples were further purified with a second on-column DNase I treatment using the RNeasy minikit (QIAGEN). The iScript cDNA synthesis kit (Bio-Rad) was used to generate cDNA from RNA (1-μg) templates.
Quantitative real-time PCR. (i) Real-time primers. Primers for real-time PCR were designed using Beacon Designer software, version 2.0, and are listed in Table 2. Predicted product sizes were in the 100- to 200-bp range.
(ii) Preparation of standards. Specific DNA products for each gene under investigation were synthesized from chromosomal DNA using standard PCR methods and visualized by gel electrophoresis to verify that a single specific product had been generated. Each product was purified using the QIAquick PCR purification kit (QIAGEN) and quantified using an Eppendorf BioPhotometer. The DNA product copy number (per microliter) (also called the starting quantity [SQ]) was calculated using the formula of Yin et al. (83) as [(6.023 x 1023 x g of DNA/ml)/(molecular weight of product)]/1,000. The molecular weight of the product is calculated as base pairs x 6.58 x 102 g. A 10-fold dilution series of each DNA standard was prepared for SQs of 108 to 104 copies μl–1. These were used in duplicate in each real-time PCR assay to allow the real-time PCR software to estimate the SQ of that gene in cDNA samples.
(iii) Real-time PCR. The standard DNA dilution series (SQ, 108 to 104 copies μl–1) or cDNA templates (2 μl) were added in duplicate to an iCycler iQ 96-well PCR plate (Bio-Rad). To each well, the following were added: 1 μl each 5' and 3' specific primer (50 pmol each); 12.5 μl iQ SYBRGreen Supermix (Bio-Rad) containing 100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), iTaq DNA polymerase (50 U ml–1), 6 mM MgCl2, 20 nM fluorescein, and stabilizers; and 8.5 μl distilled H2O. The 96-well plate was sealed with optical tape (Bio-Rad), and samples were quantified in the iCycler machine (Bio-Rad) using a standard thermal cycling program. Real-time results were analyzed using iCycler iQ Optical System software, version 3.0a (Bio-Rad). The melt curve profile was analyzed to verify a single peak for each sample, indicating primer specificity.
Kgp protease assay. Lysine protease activity was measured as previously described (9, 59, 68, 72). Briefly, P. gingivalis cultures were resuspended in substrate buffer (140 mM NaCl, 100 mM Tris-HCl [pH 7.5]) containing 0.4 mM -N-acetyl-L-lysine-p-nitroanilide (Ac-Lys-pNA.HCl) (Bachem). Samples were incubated in triplicate at 37°C for 1 h, and the increase in A405 due to release of p-nitroaniline was measured.
Autoinducer bioluminescence assay. P. gingivalis strains were grown overnight in AB-sTSBY (50:50), because the organism does not grow in AB medium. This initial culture was diluted to an optical density at 600 nm (OD600) of 0.1 in fresh AB-sTSBY and grown to appropriate optical densities. Cell-free culture supernatants were prepared by centrifugation at 10,000 x g for 10 min and filtration using 0.22-μm-pore-size MillexGP filter units (Millipore). The ability of these cell-free preparations to induce the AI-2 signaling system in the V. harveyi reporter strain BB170 was determined using the bioluminescence assay previously described (31, 74). Briefly, an overnight culture of BB170 (OD600, 1.0) was diluted 1:1,000 in AB medium, and 900 μl was added to 24-well plates (Costar). Cell-free P. gingivalis culture supernatants were added (100 μl per well) and incubated at 30°C with agitation. The cell-free culture supernatant from BB170 grown in 50:50 AB-sTSBY medium was used as a source of AI-2 in positive-control wells, and sterile medium was used in negative-control wells. Luminescence was measured using a VictorDB plate reader.
Purification of P. gingivalis AI-2. AI-2 was partially purified from P. gingivalis 33277 cultures according to the method of Sperandio et al. (69). Briefly, early-exponential-phase cultures (OD600, 0.5) were centrifuged (5,000 x g, 30 min) and filtered (pore size, 0.22 μm; Millipore). Cell-free supernatants were size fractionated for compounds of <3 kDa using YM-3 Centriprep centrifugal filter devices (Millipore). The crude extracts were freeze-dried, and 200 mg was dissolved in 1.5 ml cold 5 mM NaPO4 buffer (pH 6.2) and passed through a C18 Sep-PakPlus cartridge (Waters). The flowthrough that eluted with 5 mM NaPO4 buffer (pH 6.2) contained AI-2, and activity was confirmed in a bioluminescence assay with the V. harveyi reporter strain BB170.
AI-2 complementation of LS. P. gingivalis LS cultures were grown in sTSBY to late-lag phase (OD600, 0.3). The culture medium was then supplemented with 2.5% (vol/vol) AI-2 extract purified from wild-type 33277. Negative controls were supplemented with 2.5% (vol/vol) 5 mM NaPO4 buffer (pH 6.2) or 2.5% (vol/vol) of the AI-2 fraction from LS cultures. At appropriate time intervals after addition of AI-2 or buffer, bacterial cells were collected by centrifugation and RNA was isolated.
RESULTS
Construction of isogenic luxS-null mutant and complemented mutant strains. To analyze the effects of luxS on the phenotype of P. gingivalis, a deletion mutant strain (LS) was constructed by allelic replacement. To ensure that the observed phenotype of the mutant strain results from disruption of luxS, LS was complemented in trans with the wild-type allele on plasmid pTCOW (strain CLS). Because the luxS gene in P. gingivalis comprises part of an operon with pfs, and no promoter sequences were identified in the upstream region of luxS, the regulatory region of the P. gingivalis mfa1 gene was used to promote transcription of luxS in the CLS complemented strain. LS and CLS were compared to the wild-type parental strain 33277 using the AI-2 bioluminescence assay with the V. harveyi reporter strain BB170 (3). Light production by cell-free culture supernatants from LS was significantly decreased compared to light production by the parental strain (Fig. 1). Bioluminescence activity was restored in CLS, but not to wild-type levels. This may be a consequence of aberrant regulation of luxS transcription from the mfa1 promoter or deficient processing of the monocistronic luxS mRNA resulting from plasmid expression.
FIG. 1. Effects of the luxS mutation (strain LS) and it complementation by the wild-type gene (strain CLS) on AI-2 production, tested using the V. harveyi reporter strain BB170. AI-2 production was evaluated as fold activation (activity induced by test supernatant/activity induced by medium alone). Bioluminescence induced by a homologous V. harveyi culture supernatant (not shown) was more than 1 log unit higher than that for wild-type (WT) P. gingivalis (strain 33277), consistent with previous reports (12). A t test on bioluminescence data showed that induction by strains 33277 and CLS was significant at a P level of <0.005 ().
AI-2 production by P. gingivalis is growth phase dependent. While AI-2 signaling is generally considered to comprise a quorum-sensing system, in a number of species, including A. actinomycetemcomitans, S. mutans, E. coli, Clostridium perfringens, and Salmonella serovar Typhimurium, maximal AI-2 signal can be detected during the mid-exponential growth phase (22, 48, 52, 74). Thus, the growth phase dependence of luxS transcription and AI-2 signal activity was investigated in P. gingivalis. As reported by the V. harveyi BB170 bioluminescence assay, AI-2 production was highest in cultures grown to early-exponential phase and declined through late-exponential phase (Fig. 2A). The pattern of AI-2 signal activity was consistent with the levels of luxS mRNA (Fig. 2B), although AI-2 signal activity has a longer half-life than luxS mRNA. Similarly, Burgess et al. (9) reported that levels of the LuxS protein increased throughout growth even though AI-2 activity was maximally detected at mid-exponential phase. Quantitative RT-PCR of the luxS mRNA showed that levels of luxS message were 3- to 4-fold higher at the early- and mid-exponential growth stages than at the late-exponential phase of growth. Furthermore, luxS message decreased fivefold between the late-exponential and stationary phases of growth. Collectively, the results suggest that the LuxS signaling system of P. gingivalis plays its most important role during the early stages of growth, when cell density is low and nutrients may be plentiful. In this context it is important to consider that LuxS also plays a role in the activated methyl cycle, where maximal levels of methionine and S-adenosylmethionine are required for optimal growth.
FIG. 2. AI-2 activity and luxS expression in P. gingivalis are dependent on growth phase. (A) Fold activation of bioluminescence induced by culture supernatant from the P. gingivalis wild-type strain 33277 (WT) at early (OD600, 0.5)-, mid (OD600, 0.7)-, or late (OD600, 1.0)-exponential phase. Differences among growth phases were significant at a P level of <0.01 () (t test). (B) Expression levels of luxS mRNA in the WT at early-, mid-, and late-exponential phase and stationary phase (Stat.) as measured by quantitative RT-PCR (see the text for details). Expression levels are presented as cDNA copy number per microgram of RNA.
LuxS signaling influences expression of hemin acquisition genes in P. gingivalis. Previous studies provided preliminary evidence that LuxS signaling in P. gingivalis plays a role in regulating genes involved in hemin acquisition (9, 12). P. gingivalis strain 33277 possesses several putative and defined mechanisms for hemin and iron uptake, and representative genes from each of these pathways are listed in Table 3. To investigate the role of LuxS in the regulation of hemin and iron uptake pathways in P. gingivalis, differential expression of these genes was investigated by quantitative RT-PCR. A number of genes involved in hemin acquisition were found to be differentially expressed in LS compared to the parent strain (Fig. 3). At early-exponential phase, when LuxS expression is maximal, two genes encoding outer-membrane-expressed heme-binding proteins (HmuR and FetB) showed significantly increased expression in LS. The levels of induction of both hmuR and fetB declined at late-exponential phase, when there is less luxS message, although hmuR expression remained significantly higher in the LS mutant than in the parental strain. Thus, hmuR and fetB appear to be negatively regulated by LuxS signaling. Conversely, tlr, encoding another TonB-linked heme-binding receptor, is positively regulated by LuxS; this gene showed decreased expression in the LS mutant in early-exponential growth. Differential expression of kgp was also observed, with a threefold decrease in expression in LS in early-exponential phase and a threefold increase in expression during late-exponential phase. The correlation between mRNA levels and Kgp activity was explored using a lysine-specific protease assay. The LS mutant exhibited 32.5% less Kgp protease activity than the parental strain (P = 0.00035) when grown to early-exponential phase. These data are in good agreement with results of Burgess et al. (9), who reported a 30% decrease in Kgp activity in a luxS-null mutant of P. gingivalis W50. By contrast, there was no significant difference in expression of rgpA between LS and parent strains. While both the arginine-specific (Rgp) and lysine-specific (Kgp) gingipains can degrade heme-bound proteins and liberate heme (72), only Kgp is thought to act as a hemophore, binding to heme and hemoglobin and delivering heme to outer membrane receptors (29). Both positive and negative regulation of kgp by LuxS signaling are indicative of multiple levels of control, as has also been demonstrated by other groups (34, 53, 63, 78). Collectively, these results demonstrate that LuxS has a varied and nuanced relationship with hemin uptake, with different hemin uptake genes regulated individually as P. gingivalis progresses through its growth cycle.
FIG. 3. Hemin uptake genes are differentially expressed in the LS mutant. Gene expression was measured by quantitative RT-PCR on wild-type (WT) or LS cultures grown to early (OD600, 0.5)- or late (OD600, 1.0)-exponential phase. Fold change was calculated by dividing the copy number of the gene transcript (per microgram of RNA) in the LS mutant by the copy number in the WT. The 16S rRNA and fimA genes were used as controls. Significant differences (P < 0.05 by t test) are labeled with asterisks.
Differential expression of Fe2+ acquisition genes in the LS mutant. In addition to hemin uptake, P. gingivalis can also acquire inorganic Fe2+, potentially through the two homologues of a ferrous iron transport protein, FeoB1 and FeoB2 (Table 3). The hemin and iron uptake pathways are interconnected through the action of FetB, which can remove iron from heme compounds prior to uptake (55). Furthermore, regardless of the uptake mechanism, intracellular iron can be stored in ferritin. Quantitative RT-PCR was used to investigate whether LuxS also has a role in the control of inorganic iron uptake and storage. LuxS signaling negatively regulated the expression of ftn (encoding ferritin) and feoB1 compared to parental levels (Fig. 4). Conversely, there was a low-level positive regulation of feoB2 by LuxS. Recent evidence suggests that feoB2 is not involved in iron transport and may in fact play a novel role in manganese transport (16). These data suggest that LuxS signaling is important for the acquisition of iron not only in the form of hemin but also in the form of Fe2+.
FIG. 4. Iron uptake and storage genes are differentially expressed in the LS mutant. Gene expression was measured by quantitative RT-PCR on wild-type (WT) or LS cultures grown to mid-exponential phase. Fold change was calculated by dividing the copy number of the gene transcript (per microgram of RNA) in the LS mutant by the copy number in the WT. The 16S rRNA and fimA genes were used as controls. Significant differences (P < 0.05 by t test) are labeled with asterisks.
trans-Complementation of LS restored parental expression levels of hmuR. In order to ensure that differential gene expression in the LS mutant was a direct effect of disruption of the luxS gene, the trans-complemented strain CLS was utilized. Quantitative RT-PCR was performed on RNAs extracted from parent, LS, and CLS cultures grown to mid-exponential phase to measure the levels of luxS and hmuR transcripts. hmuR was selected for this experiment because, among the genes tested, hmuR demonstrates the largest change in expression in response to the LuxS signal, thus increasing the dynamic range of the assay. As shown in Fig. 5A, the luxS mRNA level was approximately ninefold higher in CLS than in the parental strain. The increase in luxS expression in CLS most likely reflects the presence of multiple copies of the pTCOW plasmid. Nonetheless, an increase in the luxS transcript level does not necessarily correspond to equally increased levels of AI-2 production, since LuxS enzyme activity is limited by the availability of its substrate (6). The complemented isotype showed decreased expression of hmuR (threefold) compared to that for LS (Fig. 5B), confirming that LuxS signaling has a negative regulatory effect on this gene. Expression of hmuR did not completely return to wild-type levels, corroborating data presented above in Fig. 1 and 5 that suggest that an increase in the monocistronic luxS transcript level in trans cannot fully restore LuxS function.
FIG. 5. Complementation of the LS mutant with the wild-type (WT) luxS gene restores expression of luxS (A) and alters expression of hmuR (B). Expression of luxS mRNA in CLS was significantly higher (P < 0.05 by t test) than that in either the WT or the LS mutant. Expression of hmuR mRNA in CLS was significantly lower (P < 0.01 by t test) than that in the LS mutant. The transcript copy number (per microgram of RNA) was determined by real-time PCR for the WT, CLS, or LS strain grown to mid-exponential phase. Error bars, standard deviations (n = 6).
Isotypes possessing native luxS but exhibiting increased luxS mRNA levels exhibit decreased hmuR and increased tlr expression. Complementation of a genetic mutation is a useful strategy to confirm the function of that gene. Nonetheless, our results suggested that changes in luxS promoter activity, timing of expression, or expression levels of other genes cotranscribed with luxS can subtly affect LuxS functionality in P. gingivalis. Similarly, De Keersmaecker et al. (18) found that the intrinsic promoter activity of luxS in Salmonella serovar Typhimurium affected LuxS-dependent biofilm formation. Thus, we sought an additional means to corroborate the role of LuxS in the regulation of hemin uptake-associated genes. By screening P. gingivalis mutants in two-component systems, we identified a gene, gppX, that suppresses the transcriptional activity of luxS. GppX is a sensor kinase recently found to be required for maturation and localization of the P. gingivalis gingipains (34). Two independent gene disruption mutants, GPPX-M and GPPX-E (kindly provided by F. Yoshimura, Aichi-Gakuin University), exhibited elevated AI-2 activity and >2-fold-higher levels of luxS mRNA compared to those for the parental strain (Fig. 6A). Hence, the GppX mutants allowed investigation of the effects of altered luxS expression without disruption of the luxS gene or its promoter. Expression of the two different TonB-linked heme-binding receptors, HmuR and Tlr, was determined by quantitative RT-PCR. Figure 6B shows that both GPPX-M and GPPX-E had decreased expression of hmuR (4.7- and 4.9-fold lower than that for the parental strain, respectively) yet increased expression of tlr (3.1- and 3.4-fold more than that for the parent strain, respectively). For both hmuR and tlr, this was the opposite effect to that observed after disruption of luxS. Thus, these data support the evidence presented in Fig. 3 and 5 that LuxS signaling suppresses hmuR expression and promotes tlr expression, although the formal possibility remains that GppX can directly regulate both hmuR and tlr. It is also noteworthy that GppX has homology (e–35) with the LuxQ AI-2 sensor kinase of Vibrio. However, the phenotype of the gppX mutants would tend to exclude a role for GppX as an AI-2 sensor kinase in P. gingivalis. These results do, however, demonstrate a role for the GppX two-component pathway in the control of expression of the luxS gene.
FIG. 6. (A) Culture supernatants from P. gingivalis strains GPPX-M and GPPX-E demonstrate increased (P < 0.05 by t test) bioluminescence (in counts per second [CPS]) in V. harveyi BB170 compared to the wild type (WT). (B) GPPX-M and GPPX-E exhibit increased expression of luxS and tlr, and decreased expression of hmuR, compared to the WT. Quantitative RT-PCR was performed on RNA from the WT, GPPX-M, or GPPX-E at mid-exponential phase. Fold change was calculated by dividing the copy number of the gene transcript (per microgram of RNA) in the GPPX mutants by the copy number in the WT. The 16S rRNA gene was used as an internal control (not shown). All fold differences were significant at a P level of <0.05 (t test).
Treatment of luxS-null mutants with partially purified AI-2 restores parental levels of hemin uptake gene expression. The biosynthetic pathway leading to AI-2 production is part of the activated methyl pathway (6, 62, 80). Indeed, Winzer et al. (80) have argued that AI-2 is not a signal molecule per se but rather an extracellular by-product of S-ribosylhomocysteine recycling and that changes in gene expression due to inactivation of luxS are the result of defects in methionine metabolism. To begin to distinguish between these possibilities for P. gingivalis, we partially purified extracellular AI-2 from culture supernatants of the parental strain. Cultures of LS in late-lag phase were treated with AI-2 or a buffer control, and samples of RNA were extracted. No difference in the growth rate of LS in the presence of AI-2 was observed. The effects of AI-2 on expression of hmuR and tlr were determined by quantitative RT-PCR analysis. Within 5 min, LS cultures treated with AI-2 exhibited >2-fold suppression of hmuR mRNA levels. Furthermore, after 30 min, the hmuR copy number detected from cultures treated with AI-2 was threefold less than that from buffer-treated cultures (Fig. 7A). Conversely, tlr expression was significantly higher (2.6-fold) in cultures treated with AI-2 than in those treated with buffer alone after 30 min (Fig. 7B). There was no significant difference in expression of either heme-binding receptor gene following 60 min of treatment with AI-2, indicating either signal depletion or degradation at this time point. The fimA gene, used as a control, did not show any AI-2-induced changes in expression (Fig. 7C). As an additional control, the AI-2 fraction was purified from the LS strain, which does possess some residual AI-2 activity, presumably due to nonenzymatic formation of AI-2 signal. As shown in Fig. 7D, parental signal induced expression of tlr almost twofold compared to signal from the mutant, a difference similar to that observed in whole cells of the parent and the mutant. The divergent regulation of hmuR and tlr indicates that the activity of the AI-2 fraction is not the result of a nonspecific metabolic effect on the LS cells. These data, along with earlier results, provide strong evidence that hemin uptake mechanisms are regulated by an external AI-2 signal produced by the action of LuxS. Furthermore, it is apparent that the AI-2 signal allows P. gingivalis to switch between different mechanisms of hemin and iron uptake as dictated by environmental conditions.
FIG. 7. Purified AI-2 regulates expression of hmuR and tlr. AI-2 or control NaPO4 buffer was added to LS cultures (OD600, 0.3) and incubated for the additional times indicated. (A to C) Expression levels (per microgram of RNA) of hmuR (A), tlr (B), and fimA as a control (C) were determined by quantitative RT-RCR. (D) Fold induction of tlr in the LS strain by AI-2 purified from the WT compared to the AI-2 fraction from the LS strain. Error bars, standard deviations (n = 6). Asterisk, P < 0.05 (by t test) for cultures treated with AI-2 compared to cultures treated with buffer or the AI-2 fraction from the LS strain.
LuxS signaling plays a role in growth of P. gingivalis under conditions of limited hemin. The ability of LuxS to regulate aspects of hemin uptake led us to postulate that the LS mutant would be defective in growth in hemin-limited medium, where the inability to optimally acquire hemin could have adverse effects on growth rate. To test this hypothesis, we first demonstrated that there was no difference between the growth profiles of the parental strain and the LS mutant strain in the presence of excess hemin (Fig. 8A). In contrast, although the growth of both isotypes was impaired under hemin-limiting conditions, the doubling time of the LS cultures in exponential phase was approximately 2 h longer than that of the parent, and the final OD attained by the LS mutant was 1.5-fold less than that of the parent (Fig. 8B). These data support our conclusions that in the absence of the LuxS signaling system, P. gingivalis is unable to fine-tune hemin uptake mechanisms, and thus survival under hemin-limiting conditions is diminished.
FIG. 8. Growth of the LS mutant is impaired under conditions of hemin limitation. Shown are growth curves of wild-type P. gingivalis (WT) and the LS mutant in the presence of optimal (5-μg ml–1) (A) or limited (0.05-μg ml–1) (B) hemin concentrations. For the limited-hemin condition, the cells were depleted of stored hemin by two subcultures in the absence of hemin. Overnight cultures were inoculated into fresh medium containing the appropriate concentration of hemin. Error bars, standard deviations (n = 3).
DISCUSSION
The ability of pathogenic bacteria to obtain iron from their human hosts is an important factor contributing to their ultimate survival or elimination. In the periodontal pocket, the favored niche of P. gingivalis, iron and heme-containing proteins are available through the crevicular fluid (a serum exudate) and during episodes of gingival bleeding. Levels of iron and heme-containing proteins will thus fluctuate, and organisms with a multiplicity of uptake pathways under tight regulatory control can be predicted to have a selective advantage. Lacking siderophores, P. gingivalis employs specific outer membrane receptors, lipoproteins, and proteases to acquire heme and iron. In this report we show that uptake pathways for iron in the form of both hemin and inorganic Fe2+ are controlled by the LuxS/AI-2 signaling system in P. gingivalis ATCC 33277, a strain with demonstrated pathogenic activity in the rat bone loss model of periodontal disease (36, 58). AI-2 is, therefore, likely to be important for the survival and pathogenicity of P. gingivalis in the periodontal environment.
The relationship between AI-2 signaling and iron/hemin uptake involves independent regulation of distinct uptake mechanisms. AI-2 was found to negatively regulate expression of hmuR, fetB, feoB1, and ftn, whereas tlr and kgp are positively regulated by AI-2. HmuR is a TonB-linked outer membrane receptor and has been reported to be required for both hemoglobin and hemin utilization in P. gingivalis. Inactivation of the hmuR gene impairs growth with hemoglobin or hemin as the sole iron source (64). Furthermore, hmuR conferred the ability to bind hemin and hemoglobin on E. coli (64), and purified recombinant HmuR has been shown to bind both free and protein-bound hemin (54). Tlr is also a TonB-linked outer membrane receptor. The reported difference between the functions of Tlr and HmuR is that tlr-null mutants can grow in optimal, but not limiting, hemin concentrations (1), whereas hmuR-null mutants show diminished growth even in optimal hemin concentrations (64). Thus, HmuR may play a more vital role in hemin uptake than Tlr when hemin supplies are plentiful, whereas Tlr is more important for scavenging hemin when supplies are scarce, as proposed first by Aduse-Opoku et al. (1). FetB (IhtB) is a hemin binding lipoprotein that is located in an iron transport operon along with a third TonB-linked outer membrane receptor, IhtA (15). FetB can also function as a chelatase that may remove iron from heme prior to uptake by P. gingivalis (15). Fe2+ is transported through the inner membrane by the ion transport protein FeoB1 (16) and deposited in ferritin. Proteases such as Kgp can degrade host iron- and heme-containing proteins and can possibly act as hemophores, shuttling heme back to outer membrane receptors (55).
The results of the current study would thus allow us to propose the following broadly based model. During early-exponential growth in vivo, before the onset of periodontitis, hemin and iron levels are low, and LuxS signaling suppresses the production of HmuR, FetB, and FeoB1 in order to conserve energy. Consequently, less ferritin is required for iron storage. Under these conditions, hemin scavenging is accomplished by Tlr, and Kgp is also upregulated to liberate additional heme from host proteins. Subsequently, as the P. gingivalis community becomes established and metabolically stable, tissue destruction in the periodontal pockets advances and more heme/iron is available. P. gingivalis cells in this state have reduced AI-2 expression, lifting the repression of hmuR, fetB, and feoB1 and decreasing expression of tlr, which is no longer necessary for hemin uptake. This model predicts that a LuxS-deficient mutant of P. gingivalis would not show a proliferation-related phenotype under hemin-replete conditions, since upregulation of Tlr is not required. Conversely, growth of a LuxS mutant would be impaired during hemin limitation, because in the absence of AI-2 the mutant would not be able to upregulate Tlr. This predicted phenotype was indeed exhibited by our LuxS mutant, increasing confidence in the proposed model. It should also be stressed, however, that there exist additional levels of control of hemin uptake in P. gingivalis. A Fur homologue is present in P. gingivalis (55), and a putative Fur box exists upstream of the hmuYR operon, indicating that hmuR is also regulated by Fur (64). It has also been demonstrated that hemin binding and transport in P. gingivalis can be induced by hemin (8, 28, 35, 38, 64) and that hemin can also be stored on the cell surface as μ-oxo bisheme dimer layers (68). Such redundancy in hemin uptake and storage mechanisms in P. gingivalis may facilitate tight control and ensure adaptability to environmental conditions in vivo. Moreover, the presence of multiple levels of control may allow relatively small changes in individual gene expression to be amplified through the regulatory network. In addition, in the dense biofilm that is the habitat of P. gingivalis, signals derived from other organisms may also be detected by P. gingivalis. The extent to which hemin and iron uptake genes are regulated by heterologous AI-2 is currently under investigation in our laboratory.
A relationship exists between iron/heme uptake and virulence in P. gingivalis. Iron starvation results in attenuation of virulence in a mouse model (37), while culture in excess hemin enhances the virulence of P. gingivalis (46). Moreover, a mutant of P. gingivalis that has diminished hemin binding displays reduced virulence (67). While there is general agreement that the virulence factors of P. gingivalis that are affected by hemin levels include hemin binding and protease production, the nature of this regulation is less clear. Some studies find that hemin binding and proteolytic activity are increased by excess hemin (10, 66), whereas others report an increase in these virulence factors under hemin limitation (30). Many factors, including strain differences, could account for these discrepancies. However, the results presented here intimate that variations in levels of AI-2 production by the strains under the experimental conditions adopted in these previous studies would impact protease activity, hemin binding, and ultimately virulence.
In P. gingivalis, luxS transcription and AI-2 signaling activity were highest during the early-exponential-growth phase. Thus, the term "quorum sensing" is not strictly accurate to describe AI-2 signal activity in P. gingivalis. Furthermore, luxS expression in P. gingivalis is transcriptionally regulated, whereas in other organisms, such as Salmonella serovar Typhimurium, luxS expression is constitutive (6), although AI-2 production can be regulated by environmental conditions (75). In addition to LuxS, Pfs is required for AI-2 biosynthesis (62). Pfs catalyzes both the formation of S-ribosylhomocysteine (the substrate for LuxS) from S-adenosylhomocysteine to release adenine and the production of 5'-methylthioribose from 5'-methylthioadenosine, also releasing adenine. In Salmonella serovar Typhimurium, pfs is transcriptionally regulated by environmental cues and the transcriptional profile correlates with the pattern of AI-2 production (6). In P. gingivalis, where luxS is cotranscribed with the upstream pfs gene, luxS mRNA levels have been shown to differ according to osmolarity (12), indicating that luxS can be transcriptionally controlled along with pfs. In the present study, the two-component system GppX was found to negatively regulate luxS transcription in P. gingivalis. GppX is a hybrid-type two-component system containing both a sensor domain and a regulator domain (34). Recently, Carter et al. (11) reported that a two-component signal transduction system comprising the transcriptional regulator rolA and the sensor kinase rolB negatively regulates luxS expression in Clostridium difficile. Hence, two-component systems may be used by a variety of bacteria to control luxS expression, and this may be of particular utility in situations where AI-2 does not appear to be used to report cell number. In P. gingivalis, GppX can also regulate proteolytic activity and GppX mutants have reduced Kgp function (34). Since GppX also controls luxS, and LuxS can control kgp, it is clear that the regulatory network in which LuxS participates is multilayered and extensively interconnected.
The current results clarify and extend the previous differential display study of an insertion-duplication luxS mutant (12), which revealed several hemin uptake-related genes that were either activated or repressed in the absence of LuxS. Since the publication of that pregenomic study, several genes have been more precisely annotated. With relevance to the current results, the hemR gene that was reported by Chung et al. (12) as downregulated in the LuxS mutant was originally thought to be a homologue of hmuR. However, hemR contains regions homologous to both hmuR and the protease gene prtT, and hemR is now thought to be unique to P. gingivalis strain 53977 (55). Since the sequence detected by Chung et al. (12) was homologous to the protease region of hemR, these results are not inconsistent with the current study. It is also noteworthy that in the completed genome database, the gene for a hypothetical protein (TIGR PG0499) is immediately downstream of luxS, as opposed to the putative homologue of TraJ present in the incomplete database at the time of the Chung et al. study (12).
An association between LuxS and iron acquisition has been observed with other bacteria. Fong et al. (21) reported that LuxS regulates genes involved in iron uptake in A. actinomycetemcomitans and that a luxS mutant of A. actinomycetemcomitans grows poorly under iron-depleted conditions. Similarly, in V. vulnificus, mutation of luxS appears to cause an in vivo iron assimilation defect (39). The opposite phenotype is exhibited by a Mannheimia haemolytica luxS mutant that can outcompete the parental strain under conditions of iron restriction (77). Thus, a number of organisms may utilize AI-2 signaling to control aspects of iron/heme uptake. Interestingly, cell-free supernatants from A. actinomycetemcomitans can complement gene expression in a LuxS mutant of P. gingivalis (22). Whether or not there may be functional specificity among AI-2 signals from different bacteria, however, awaits further study.
Dental plaque is a multispecies biofilm containing a variety of species or phylotypes of bacteria that number in the hundreds (41). Inter- and intraspecies communication plays an important role in the temporal and spatial development of this dense and diverse community (20, 40, 47). In addition to its role in regulation of hemin uptake proteins, LuxS is also required for the development of heterotypic biofilms of P. gingivalis and S. gordonii in hemin-free buffer (47). Hence, the role of LuxS/AI-2 in P. gingivalis is likely to extend beyond that of control of hemin acquisition.
ACKNOWLEDGMENTS
We thank B. Bassler, N. Shoemaker, and F. Yoshimura for providing strains and plasmids.
This work was supported by NIDCR (R01 DE14605).
FOOTNOTES
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