Ligand-Signaled Upregulation of Enterococcus faecalis ace Transcriptio
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《感染与免疫杂志》
Division of Infectious Diseases, Department of Internal Medicine
Center for the Study of Emerging and Re-emerging Pathogens
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030
ABSTRACT
Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46°C, mirroring the finding that adherence was observed in 46°C, but not 37°C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.
INTRODUCTION
Enterococcus faecalis, a common colonizer of the human intestinal tract, is also known to cause clinical disease, including bacteremia, urinary tract infections, and up to 15% of all cases of bacterial endocarditis (17). The ability of E. faecalis to colonize vascular tissue is thought to occur by adhesin-ligand interactions between its surface determinants and host proteins at the sites of endovascular injuries. To promote this colonization, E. faecalis possesses a number of predicted surface proteins with a characteristic immunoglobulin-like fold, which are called MSCRAMMs (for microbial surface components recognizing adhesive matrix molecules) (25). MSCRAMMs of staphylococci and streptococci have been reported to play a major role in adherence and colonization in animal models and, presumably, in humans (3), and it is likely that the same is true for enterococci.
Knowledge of the factors that influence the ability of E. faecalis to colonize host tissues is beginning to emerge. During the past decade, E. faecalis has been shown in various studies using different methodologies to adhere to one or more host extracellular matrix (ECM) proteins such as collagen types I and IV (CI and CIV), laminin (LN), fibronectin, lactoferrin, vitronectin, and thrombospondin (6). Using a standard in vitro adherence assay, Xiao et al. (29) reported that adherence of E. faecalis to collagen and LN was seen only after growth under a nonphysiological stress condition (i.e., at 46°C). Seemingly in contrast to this observation, Tomita et al. (27) recently demonstrated collagen and LN adherence phenotypes of several E. faecalis clinical isolates by using a microscopic technique; however, this assay appears to be more sensitive than adherence studies that assess the percentage of bacteria bound.
By searching for homologues of known adhesins, Rich et al. (22) identified a gene in E. faecalis subsequently named ace (for adhesin of collagen from E. faecalis) and localized the specific CI binding property of Ace to the A domain based on biochemical evidence. Our further genetic analyses with an isogenic mutant demonstrated that Ace mediates the conditional (i.e., after growth at 46°C) adherence of E. faecalis to CIV and LN (19), in addition to dentin, a stabilized form of collagen (10). The study by Tomita et al. (27) that scored transposon insertion mutants of E. faecalis tissue-specific adhesive clinical strain also found that ace knockouts lacked CIV and LN adherence.
Our subsequent analyses of the ace gene from E. faecalis strains recognized that this gene is ubiquitous (2) and occurs in at least four different forms due to variation in the number of repeats of the B domain (20). Conditional in vitro production of Ace by different strains, detected by using polyclonal anti-Ace antibodies, was correlated with the conditional adherence of these E. faecalis strains to collagen and LN (20). Most recently, a role for Ace as a virulence factor was shown by using an arthritis model by expressing it in a surrogate host; in that study, Ace-expressing Staphylococcus aureus showed increased arthritogenic potential, to a level similar to that of S. aureus expressing Cna, a collagen-binding S. aureus homologue of Ace (30).
It is well known that bacteria can alter the expression of certain genes upon binding and replicating on a substrate, possibly via the mediation of various environment signals including collagen (1, 4, 9, 28). Previous studies have suggested that physiologically relevant cues, such as serum, may increase the adhesion of E. faecalis to heart cells (7, 8), to cultured renal tubular cells (11), and to CI (13). However, the specific signals that are sensed in serum remain largely unknown. An exploratory study by Shepard and Gilmore (24) compared mRNA levels of predicted E. faecalis virulence factors in cultures grown in serum, urine, or laboratory medium and identified environment- and growth-phase-specific variations in several virulence-related genes, including ace. However, an effect of these gene expression changes on phenotype(s) has yet to be elucidated. The present study was designed to examine ace transcription during in vitro growth conditions that mimic more physiological ones. Here, by measuring the levels of ace mRNA using quantitative reverse transcription-PCR (qRT-PCR), we demonstrate the upregulation of ace transcription when E. faecalis cultures were grown in the presence of CIV. Surface-localized Ace was detectable after growth in the presence of CIV, and it was correlated with increased adherence to CIV and LN.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The E. faecalis strains used in the present study include OG1RF (18, 19) TX5256 (ace disruption mutant of OG1RF) (19); two endocarditis isolates, TX0052 and MC02152 (15, 20); and a urine isolate, MD9 (20). These strains were chosen based on clearly different pulsed-field gel electrophoresis patterns and different geographical origins. The ace mutant was constructed previously (19) by cloning an intragenic ace fragment in the suicide vector pTEX4577 containing aph(3')-IIIa (26). E. faecalis were routinely grown in brain heart infusion (BHI; Difco Laboratories, Detroit, Mich.) at 37°C, unless a different condition is specified.
ECM proteins and collagenase digestion. Bovine CI was purchased from Cohesion Technologies, Inc. (Palo Alto, Calif.), human-placenta-derived CIV was from Sigma Chemical Co. (St. Louis, Mo.), fibrinogen was from Enzyme Research Laboratories (South Bend, Ind.), and bovine serum albumin (BSA) was from MP Biomedicals, Inc. (Irvine, Calif.).
To eliminate collagen for certain reactions, CIV was suspended (0.5 mg/ml) in 50 mM Tris-HCl (pH 7.0) containing 40 mM CaCl2 and then incubated at 10°C for 18 h with Clostridium histolyticum collagenase (Sigma Chemical Co.) at a substrate/enzyme ratio of 10:1 (14). The reaction mixture was dialyzed against 0.25% (wt/vol) acetic acid at 4°C for 4 days with five changes, freeze-dried, and resuspended in 0.25% (wt/vol) acetic acid.
Gene expression analysis. (i) Extraction of total RNA. Total RNA was isolated from E. faecalis cultures by using an RNeasy minikit (QIAGEN, Valencia, Calif.) according to the protocol of the supplier with some modifications. A lysozyme solution at 10 mg/ml was used instead of 3 mg/ml for the lysis step. Total RNA (20 to 40 μg) was treated three times with 20 U of RQ1 DNase (Promega Corp., Madison, Wis.) for 30 min at 37°C, and the DNase was removed by using the RNeasy minikit. The RNA concentration was determined by using a spectrophotometer. Part of each sample was electrophoresed through an agarose-formaldehyde gel in morpholinepropanesulfonic acid buffer as previously described (23).
(ii) RT-PCR. Total RNA (between 5 ng to 250 ng) was reverse transcribed with ace specific primers (AceMF, 5'-ACGATTGAAGGAGTGACTAACACA-3'; AceMR; 5'-AAGTGTAACGGACGATAAAGGAAG-3') using the SuperScript One-Step RT-PCR with a Platinum Taq kit (Invitrogen Corp., Carlsbad, Calif.) according to the manufacturer's instructions. As an internal control, a 528-bp fragment of gdh (encoding GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) was amplified by using the gdhF (5'-AGTGGCGCACTAAAAGATATGG-3') and gdhR (5'-AGTTGTATTGAACCCTTGACCG-3') primers. Reactions without reverse transcriptase were performed as controls to detect DNA contamination in the total RNA preparations.
(iii) Real-time qRT-PCR. Amplification, detection, and real-time analysis were performed by using the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, Calif.). Primers designed to produce amplicons of equivalent length were selected by using Primer Express software (Applied Biosystems). The primer pairs used in qRT-PCR included AceQF1 (5'-GGAGAGTCAAATCAAGTACGTTGGTT-3')-AceQR1 (5'-TGTTGACCACTTCCTTGTCGAT-3') and 23S-rRNAF (5'-GTGATGGCGTGCCTTTTGTA-3')-23S-rRNAR (5'-CGCCCTATTCAGACTCGCTTT-3'). For each sample, cDNA synthesis and PCR amplification were performed in a two-step process. For cDNA synthesis, 4 μg of total RNA was added to 20-μl reaction solution containing 40 U of RNase OUT, and RT reactions were performed with random primers and SuperScript II reverse transcriptase (Invitrogen). The reaction was stopped by heating at 70°C for 15 min. After the RNA complementary to the cDNA was removed with E. coli RNase H (Invitrogen), the cDNA was purified by using QiaQuick PCR purification kit (QIAGEN). The resulting cDNA diluted up to 500 times was used for subsequent PCR amplification with the appropriate forward and reverse gene specific primers and the SYBR Green PCR Master Mix kit (Applied Biosystems). The following PCR conditions were used: 10 min at 95°C for the initial denaturation, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All PCR fragments yielded a single band on an agarose gel. Relative quantification of gene expression was performed by using 23S rRNA mRNA as the internal standard. The CT method (12) was used to calculate the relative amount of specific RNA present in a sample, from which the fold induction of transcription of the gene was estimated by comparing to the values of OG1RF grown in BHI. The data were expressed as the mean ± the standard deviation. The statistical significance was determined by using the Student unpaired t test. Amplifications were performed on four independent RNA samples from each milieu.
Immunofluorescence microscopy. E. faecalis cells either cultured in the presence or absence of ECM proteins in BHI or grown in 40% horse serum in BHI were washed thrice with Dulbecco phosphate-buffered saline (D-PBS) without CaCl2 and MgCl2 and then resuspended in D-PBS to a final optical density at 600 nm (OD600) of 0.5. Next, 400 μl of cell suspensions were applied to Lab-Tek-II chamber slides (Nalge Nunc International Corp., Naperville, Ill.) that were coated with poly-D-lysine (Sigma) and incubated for 30 min at room temperature with shaking at 100 rpm. After four washes with D-PBS, chamber slides were blocked with 1 ml of D-PBS containing 2% BSA at room temperature for 45 min, washed once with D-PBS, and then incubated with 400 μl of a 1:3,000 dilution for anti-Ace polyclonal serum (19) for 1 h. After four washes with D-PBS, attached bacteria in chamber slides were incubated with 400 μl of rhodamine red-labeled goat anti-rabbit immunoglobulin G (1:1,000 dilution) (Molecular Probes, Eugene, Oreg.) at room temperature for 1 h in the dark. After four washes, excess D-PBS was removed and a coverslip was mounted. The slides were examined by epifluorescence microscopy using an Olympus BX51 microscope with a x100 oil immersion objective lens (Olympus, Tokyo, Japan). Digital images were acquired by using an Olympus DP-70 digital camera. Preimmune serum was used as a negative control.
Adherence assay. Adherence of E. faecalis to CIV- and LN-precoated six-well plates (Becton Dickinson Biosciences, Bedford, Mass.) was determined by using an Olympus BX51 microscope. Each well of ECM-precoated plates were blocked with 5 ml of 0.2% BSA in PBS, incubated at 4°C for 2 h, and then washed with PBS three times. Cell pellets from E. faecalis cells either cultured in the presence or in the absence of ECM proteins were washed two times in PBS and resuspended in 0.1% Tween 80-0.1% BSA in PBS. The cell density was adjusted to an OD600 of 0.2, and 1 ml of bacteria was added into each well, followed by incubation at room temperature for 2 h with gentle shaking at 70 rpm. Wells were washed three times with 0.1% Tween 80-0.1% BSA in PBS. The numbers of bacterial cells that adhered to the surface of the ECM were counted from 10 randomly chosen fields of vision. Each experiment was performed four times. Statistical analysis was determined by using the Mann-Whitney test, and the differences were considered significant when the P value was <0.05.
RESULTS AND DISCUSSION
The pathogenicity of E. faecalis may be associated with the expression of specific bacterial MSCRAMMs in response to environmental cues. Ace is one such protein with the typical characteristics of an MSCRAMM, exhibiting CI, CIV, and LN binding (19). Although Ace was not detectable on Western blots after growth in routine laboratory growth conditions, Ace-specific antibodies were found in sera obtained from patients with E. faecalis endocarditis, indicating that Ace is expressed in vivo during infection in humans and not just at 46°C in vitro (20). However, it has not previously been shown whether the in vitro conditional (46°C) Ace expression is mediated at the transcriptional or posttranscriptional level. In order to determine whether, in fact, the ace gene is transcribed during in vitro growth of E. faecalis strain OG1RF at 37°C in BHI, semiquantitative RT-PCR analysis was performed.
Minimal transcription of ace after growth in vitro in BHI. Total RNA of OG1RF grown at 37°C in BHI was isolated during mid-exponential phase, late-exponential phase, entry into stationary phase, and 5 h after the cultures entered stationary phase (Fig. 1A), and the levels of ace and gdh (housekeeping control) mRNA were analyzed. The ace mRNA was barely detected at the mid-exponential and late exponential phases, as well as at entry into stationary phase, and was not detectable in cells at stationary phase (Fig. 1B and C, lanes 2 to 5). As anticipated, the control gdh gene was constitutively expressed in all phases, although, at stationary phase, gdh mRNA levels were slightly reduced (Fig. 1B and C, lanes 6 to 9). This result suggests that ace transcription is very low during standard in vitro growth conditions. We next analyzed the RNA of OG1RF grown at 46°C isolated at mid-exponential phase and at entry into stationary phase (Fig. 1A). Increased ace mRNA was detected at both growth phases (Fig. 1B and C, lanes 15 and 16) and the ace RT-PCR band intensities were almost comparable to control gdh RT-PCR band intensities at all of the concentrations of total RNA tested (Fig. 1B and C, lanes 17 and 18). These results are in agreement with our previous findings of detectable levels of Ace from mutanolysin surface extracts (19) of OG1RF grown at 46°C.
ace transcription in E. faecalis OG1RF is upregulated after growth in the presence of collagen. Since there is a precedent from other gram-positive organisms of upregulation of specific genes upon exposure to the ligand of their products (9), we explored this possibility by investigating ace mRNA levels from bacteria grown in the presence of different ECM proteins. Initially, BHI-broth-grown, mid-exponential-growth-phase (OD600 = 0.7 to 0.8) cultures of OG1RF were supplemented with 0.03 and 0.3 mg of CIV/ml and cultured further for various postexposure times (15 min, 30 min, 45 min, 1 h, 2 h, and 3 h). RT-PCR analysis of these total RNA preparations showed that the addition of CIV for 30 min at a concentration of 0.3 mg/ml resulted in increased ace mRNA amounts from the minimal levels seen without CIV and that the maximum increase was seen at 1 h (data not shown). To test the effects of different ECM proteins on ace transcription, mid-exponential-growth-phase cultures were divided into aliquots and incubated for 1 h (about two generation times) in the presence or absence of 0.3 mg/ml of CI, CIV, fibrinogen, or BSA. As depicted in Fig. 2A, the addition of CI caused a clear increase in the ace transcript levels (lane 3), and the addition of CIV (to which Ace has maximum affinity) resulted in much higher levels of ace mRNA (lane 4), whereas the addition of fibrinogen or BSA had no effect on ace mRNA levels (lanes 5 and 6). Furthermore, we have also demonstrated that CIV-mediated ace induction of OG1RF is observed only in actively growing culture, but not in the cells suspended in PBS (data not shown), as they are for the adherence assay. Importantly, enhancement of ace mRNA levels observed in the presence of CIV occurred in a dose-dependent manner that paralleled the amount of CIV added (Fig. 2B, lanes 4 to 8). These results indicate that differential ace gene expression depends on the milieu and that the presence of collagen may act as a signal for ace regulation.
Effect of pH on ace expression. During the course of our experiments, we noticed that the final pH of the culture medium was reduced from pH 7.3 to pH 6.4 or 6.7 upon the addition of CIV or CI, respectively. To test whether low extracellular pH could itself affect expression of ace in the absence of collagen, we measured the expression of ace in BHI medium buffered to the same pH values using acetic acid or HCl, respectively. At this pH, there was no significant difference in the level of ace mRNA (Fig. 2B, lanes 3 and 10), although there is a slight decrease in the final OD of the cultures after growth in this buffered medium. This result indicates that the increase in ace transcripts seen in collagen-supplemented medium is not due to the change in pH.
Intact collagen peptides but not collagenase-derived digests induce ace expression. To investigate whether induction of ace transcripts was associated with the structure and/or sequence of collagen peptides or with its primary repeat tripeptides (Gly-X-Y), RT-PCR was carried out by using cultures incubated with collagenase-digested CIV. No change in transcript levels of ace was observed, and the levels remained similar to those for BHI-grown cells (Fig. 2B, lane 9), suggesting that induction of the ace transcripts was associated with intact collagen peptides.
A serum-rich environment affects the expression of ace gene. In an effort to identify other physiological conditions that upregulate ace expression, we tested ace transcript amounts after growth in BHI supplemented with serum. As shown in Fig. 2A, lane 7, growth in 40% horse serum increased the levels of ace mRNA. This observation is in agreement with a previous study (24) that showed a 3.3-fold increase in ace mRNA abundance upon growth in serum compared to growth in 2xYT (yeast extract, tryptone, sodium chloride) medium. Of note, growth in serum resulted in the formation of aggregates both in OG1RF and in TX5256 (ace mutant), indicating that this phenomenon is not dependent upon Ace.
Quantification of ace transcript induction. To measure the fold differences in ace transcription, real-time qRT-PCR was used. The transcription level of 23S rRNA was not significantly affected by growth under any of the conditions tested and, hence, was used for normalization. Comparisons were made between values obtained from the total RNA of E. faecalis cultured in the presence CIV, fibrinogen, or growth at 46°C relative to those from BHI cultures grown at 37°C. Thus, ace mRNA after growth in BHI at 37°C was normalized to a value of 1 as the baseline for comparison. The results showed that there is an 18-fold increase in the ace mRNA levels in cultures grown in BHI containing CIV compared to cells grown in BHI alone (Fig. 3). As anticipated from semiquantitative RT-PCR, the levels of ace mRNA was not altered either in the presence of fibrinogen or by pH. Furthermore, ace mRNA levels of 46°C grown cells was observed to be 2.3-fold more than the levels of cells grown in the presence of CIV (41-fold versus BHI).
Modulation of Ace expression by collagen type IV in diverse E. faecalis strains. To determine whether increased ace mRNA is correlated with Ace protein expression, E. faecalis OG1RF cells were grown in conditions similar to those described above, except that cells were grown for four generations after supplementation with ECM proteins. Cells were then stained with polyclonal anti-Ace antibodies, followed by rhodamine red-labeled secondary antibody; these anti-Ace antibodies have been previously shown to specifically react with Ace on Western blots (20). Analysis of surfaces of cells by fluorescence microscopy showed that anti-Ace serum stained 100% of cells grown in the presence of CI and CIV (Fig. 4D and F) but did not stain the cells grown in the presence of fibrinogen (Fig. 4H). Surface staining was also observed in cells grown in 40% horse serum in BHI (Fig. 4J) and in cells grown at 46°C (Fig. 4L). As expected, Ace antibodies failed to stain the surfaces of the similarly grown ace disruption mutant of OG1RF (TX5256) (data not shown).
Immunofluorescence-based localization of Ace was further carried out with three diverse clinical E. faecalis strains. For the endocarditis strain MC02152, staining was seen on some cells grown in BHI at 37°C (Fig. 4N), a finding consistent with this strain having previously been shown to produce low amounts of Ace (seen as a weak band in Western blot analyses) (20) after growth in BHI at 37°C. However, upon culturing of this strain in the presence of CIV, 100% of the cells were stained with anti-Ace (Fig. 4P). Like OG1RF, TX0052 (endocarditis isolate) and MD9 (urine isolate) cell surfaces showed conditional staining (i.e., after culturing these strains in BHI containing CIV but not after culture in BHI alone). The finding of CIV-induced Ace expression in four of four diverse strains tested suggests this may be a common phenomenon among E. faecalis.
Collagen- and laminin-binding ability of E. faecalis as a function of upregulated ace expression. E. faecalis OG1RF and its isogenic ace disruption mutant, TX5256, were tested for their ability to adhere to immobilized CIV and LN by using microscopy. The cells of OG1RF grown in BHI in the presence of CIV, but not in those grown in BHI alone, adhered to CIV (Fig. 5A) and LN (Fig. 5B), whereas the ace mutant (TX5256) was completely defective in adherence to CIV and LN regardless of its growth conditions. This corroborates our earlier observation that Ace mediates the adherence of E. faecalis OG1RF grown at 46°C to immobilized CIV and LN. Thus, these results demonstrate that E. faecalis adhesion is enhanced directly in response to a host matrix-derived peptide signal. This mechanism of modulation of E. faecalis-collagen interaction is different from the mechanisms reported in S. aureus (5) and E. faecium (21) to alter their collagen adherence.
After growth in 40% horse serum, wild-type OG1RF adhered to immobilized CI, CIV, and LN in our standard radioactive adherence assay (19). Serum-induced CI adherence results are consistent with an earlier study with E. faecalis strain JH2-2 (13). As anticipated, the ace mutant of OG1RF (TX5256) showed markedly reduced adherence to CIV (from 18.1 to 4.2%) and LN (9.2 to 1.2%) compared to the wild type. However, adherence to CI was only partially reduced (from 47.3 to 31.4%), raising the possibility of the presence of an additional collagen-binding protein with high affinity to CI in E. faecalis OG1RF. Our recent observation with a salB mutant of OG1RF that exhibited only CI and fibronectin binding after growth at 37°C (16), corroborates there being a difference between CI and CIV adherence. Furthermore, immunofluorescence microscopy studies with Ace antibodies confirmed that the salB mutant of OG1RF lacks Ace on its surface (data not shown).
Although the ability to adhere to the most abundant host protein, collagen, would intuitively appear to be beneficial to E. faecalis during colonization, it may be of a selective disadvantage during growth in the environment, during transmission or dissemination of infection, and also during chronic infection to avoid the immune response. Therefore, we speculate that E. faecalis may have developed this strategy of programmed response to express proteins such as Ace.
In summary, upregulation of ace gene transcription in the presence of collagen type IV in E. faecalis OG1RF was demonstrated. Although we have yet to delineate the precise mechanism, we have confirmed that expression of Ace, and thereby collagen and laminin adherence, occurs under more physiological conditions than growth at 46°C. Ace induction by other environmental conditions, such as growth in serum and growth at high temperature, suggests the possibility that ace gene expression may be regulated by different mechanisms. Conservation of this induction mechanism in four of four different strains tested suggests that this may be a common programmed response in E. faecalis. Furthermore, understanding of this host matrix protein-associated triggering of a microbial pathogenic response may have important future clinical implications for this emerging multiresistant pathogen.
ACKNOWLEDGMENTS
We thank Karen Jacques-Palaz for technical assistance.
This study was supported by NIH grant R37 AI 47923 from the Division of Microbiology and Infectious Diseases to B.E.M.
FOOTNOTES
Corresponding author. Mailing address: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School at Houston, 6431 Fannin St., MSB 2.112, Houston, TX 77030. Phone: (713) 500-6745. Fax: (713) 500-6766. E-mail: bem.asst@uth.tmc.edu.
REFERENCES
1. Almeida, R. A., D. A. Luther, and S. P. Oliver. 1999. Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells. FEMS Microbiol. Lett. 178:81-85.
2. Duh, R. W., K. V. Singh, K. Malathum, and B. E. Murray. 2001. In vitro activity of 19 antimicrobial agents against enterococci from healthy subjects and hospitalized patients and use of an ace gene probe from Enterococcus faecalis for species identification. Microb. Drug Resist. 7:39-46.
3. Foster, T. J., and M. Hook. 1998. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 6:484-488.
4. Gilbert, F. B., D. A. Luther, and S. P. Oliver. 1997. Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells. FEMS Microbiol. Lett. 156:161-164.
5. Gillaspy, A. F., C. Y. Lee, S. Sau, A. L. Cheung, and M. S. Smeltzer. 1998. Factors affecting the collagen binding capacity of Staphylococcus aureus. Infect. Immun. 66:3170-3178.
6. Gilmore, M. S., P. S. Coburn, S. R. Nallapareddy, and B. E. Murray. 2002. Enterococcal virulence, p. 301-354. In M. S. Gilmore, D. Clewell, P. Courvalin, G. M. Dunny, B. E. Murray, and L. Rice (ed.), The enterococci: pathogenesis, molecular biology, antibiotic resistance, and infection control. American Society Microbiology, Washington, D.C.
7. Guzman, C. A., C. Pruzzo, G. LiPira, and L. Calegari. 1989. Role of adherence in pathogenesis of Enterococcus faecalis urinary tract infection and endocarditis. Infect. Immun. 57:1834-1838.
8. Guzman, C. A., C. Pruzzo, M. Plate, M. C. Guardati, and L. Calegari. 1991. Serum-dependent expression of Enterococcus faecalis adhesins involved in the colonization of heart cells. Microb. Pathog. 11:399-409.
9. Heddle, C., A. H. Nobbs, N. S. Jakubovics, M. Gal, J. P. Mansell, D. Dymock, and H. F. Jenkinson. 2003. Host collagen signal induces antigen I/II adhesin and invasin gene expression in oral Streptococcus gordonii. Mol. Microbiol. 50:597-607.
10. Hubble, T. S., J. F. Hatton, S. R. Nallapareddy, B. E. Murray, and M. J. Gillespie. 2003. Influence of Enterococcus faecalis proteases and the collagen-binding protein, Ace, on adhesion to dentin. Oral Microbiol. Immunol. 18:121-126.
11. Kreft, B., R. Marre, U. Schramm, and R. Wirth. 1992. Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells. Infect. Immun. 60:25-30.
12. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–CT method. Methods 25:402-408.
13. Love, R. M. 2001. Enterococcus faecalis: a mechanism for its role in endodontic failure. Int. Endod. J. 34:399-405.
14. Love, R. M., M. D. McMillan, and H. F. Jenkinson. 1997. Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides. Infect. Immun. 65:5157-5164.
15. Malathum, K., K. V. Singh, G. M. Weinstock, and B. E. Murray. 1998. Repetitive sequence-based PCR versus pulsed-field gel electrophoresis for typing of Enterococcus faecalis at the subspecies level. J. Clin. Microbiol. 36:211-215.
16. Mohamed, J. A., F. Teng, S. R. Nallapareddy, and B. E. Murray. 2006. Pleiotrophic effects of two Enterococcus faecalis sagA-like genes, salA and salB, which encode proteins that are antigenic during human infection, on biofilm formation and binding to collagen type I and fibronectin. J. Infect. Dis. 193:231-240.
17. Murray, B. E. 1990. The life and times of the Enterococcus. Clin. Microbiol. Rev. 3:46-65.
18. Murray, B. E., K. V. Singh, R. P. Ross, J. D. Heath, G. M. Dunny, and G. M. Weinstock. 1993. Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. J. Bacteriol. 175:5216-5223.
19. Nallapareddy, S. R., X. Qin, G. M. Weinstock, M. Hook, and B. E. Murray. 2000. Enterococcus faecalis adhesin, Ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I. Infect. Immun. 68:5218-5224.
20. Nallapareddy, S. R., K. V. Singh, R. W. Duh, G. M. Weinstock, and B. E. Murray. 2000. Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of Ace during human infections. Infect. Immun. 68:5210-5217.
21. Nallapareddy, S. R., G. M. Weinstock, and B. E. Murray. 2003. Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family. Mol. Microbiol. 47:1733-1747.
22. Rich, R. L., B. Kreikemeyer, R. T. Owens, S. LaBrenz, S. V. Narayana, G. M. Weinstock, B. E. Murray, and M. Hook. 1999. Ace is a collagen-binding MSCRAMM from Enterococcus faecalis. J. Biol. Chem. 274:26939-26945.
23. Sambrook, J., E. F. Fritsch, and J. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
24. Shepard, B. D., and M. S. Gilmore. 2002. Differential expression of virulence-related genes in Enterococcus faecalis in response to biological cues in serum and urine. Infect. Immun. 70:4344-4352.
25. Sillanpaa, J., Y. Xu, S. R. Nallapareddy, B. E. Murray, and M. Hook. 2004. A family of putative MSCRAMMs from Enterococcus faecalis. Microbiology 150:2069-2078.
26. Singh, K. V., X. Qin, G. M. Weinstock, and B. E. Murray. 1998. Generation and testing of mutants of Enterococcus faecalis in a mouse peritonitis model. J. Infect. Dis. 178:1416-1420.
27. Tomita, H., and Y. Ike. 2004. Tissue-specific adherent Enterococcus faecalis strains that show highly efficient adhesion to human bladder carcinoma T24 cells also adhere to extracellular matrix proteins. Infect. Immun. 72:5877-5885.
28. Vriesema, A. J., H. Beekhuizen, M. Hamdi, A. Soufan, A. Lammers, B. Willekens, O. Bakker, A. G. Welten, M. H. Veltrop, J. S. van De Gevel, J. Dankert, and S. A. Zaat. 2000. Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells. Infect. Immun. 68:1765-1772.
29. Xiao, J., M. Hook, G. M. Weinstock, and B. E. Murray. 1998. Conditional adherence of Enterococcus faecalis to extracellular matrix proteins. FEMS Immunol. Med. Microbiol. 21:287-295.
30. Xu, Y., J. M. Rivas, E. L. Brown, X. Liang, and M. Hook. 2004. Virulence potential of the staphylococcal adhesin CNA in experimental arthritis is determined by its affinity for collagen. J. Infect. Dis. 189:2323-2333.(Sreedhar R. Nallapareddy, and Barbara E.)
Center for the Study of Emerging and Re-emerging Pathogens
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030
ABSTRACT
Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46°C, mirroring the finding that adherence was observed in 46°C, but not 37°C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.
INTRODUCTION
Enterococcus faecalis, a common colonizer of the human intestinal tract, is also known to cause clinical disease, including bacteremia, urinary tract infections, and up to 15% of all cases of bacterial endocarditis (17). The ability of E. faecalis to colonize vascular tissue is thought to occur by adhesin-ligand interactions between its surface determinants and host proteins at the sites of endovascular injuries. To promote this colonization, E. faecalis possesses a number of predicted surface proteins with a characteristic immunoglobulin-like fold, which are called MSCRAMMs (for microbial surface components recognizing adhesive matrix molecules) (25). MSCRAMMs of staphylococci and streptococci have been reported to play a major role in adherence and colonization in animal models and, presumably, in humans (3), and it is likely that the same is true for enterococci.
Knowledge of the factors that influence the ability of E. faecalis to colonize host tissues is beginning to emerge. During the past decade, E. faecalis has been shown in various studies using different methodologies to adhere to one or more host extracellular matrix (ECM) proteins such as collagen types I and IV (CI and CIV), laminin (LN), fibronectin, lactoferrin, vitronectin, and thrombospondin (6). Using a standard in vitro adherence assay, Xiao et al. (29) reported that adherence of E. faecalis to collagen and LN was seen only after growth under a nonphysiological stress condition (i.e., at 46°C). Seemingly in contrast to this observation, Tomita et al. (27) recently demonstrated collagen and LN adherence phenotypes of several E. faecalis clinical isolates by using a microscopic technique; however, this assay appears to be more sensitive than adherence studies that assess the percentage of bacteria bound.
By searching for homologues of known adhesins, Rich et al. (22) identified a gene in E. faecalis subsequently named ace (for adhesin of collagen from E. faecalis) and localized the specific CI binding property of Ace to the A domain based on biochemical evidence. Our further genetic analyses with an isogenic mutant demonstrated that Ace mediates the conditional (i.e., after growth at 46°C) adherence of E. faecalis to CIV and LN (19), in addition to dentin, a stabilized form of collagen (10). The study by Tomita et al. (27) that scored transposon insertion mutants of E. faecalis tissue-specific adhesive clinical strain also found that ace knockouts lacked CIV and LN adherence.
Our subsequent analyses of the ace gene from E. faecalis strains recognized that this gene is ubiquitous (2) and occurs in at least four different forms due to variation in the number of repeats of the B domain (20). Conditional in vitro production of Ace by different strains, detected by using polyclonal anti-Ace antibodies, was correlated with the conditional adherence of these E. faecalis strains to collagen and LN (20). Most recently, a role for Ace as a virulence factor was shown by using an arthritis model by expressing it in a surrogate host; in that study, Ace-expressing Staphylococcus aureus showed increased arthritogenic potential, to a level similar to that of S. aureus expressing Cna, a collagen-binding S. aureus homologue of Ace (30).
It is well known that bacteria can alter the expression of certain genes upon binding and replicating on a substrate, possibly via the mediation of various environment signals including collagen (1, 4, 9, 28). Previous studies have suggested that physiologically relevant cues, such as serum, may increase the adhesion of E. faecalis to heart cells (7, 8), to cultured renal tubular cells (11), and to CI (13). However, the specific signals that are sensed in serum remain largely unknown. An exploratory study by Shepard and Gilmore (24) compared mRNA levels of predicted E. faecalis virulence factors in cultures grown in serum, urine, or laboratory medium and identified environment- and growth-phase-specific variations in several virulence-related genes, including ace. However, an effect of these gene expression changes on phenotype(s) has yet to be elucidated. The present study was designed to examine ace transcription during in vitro growth conditions that mimic more physiological ones. Here, by measuring the levels of ace mRNA using quantitative reverse transcription-PCR (qRT-PCR), we demonstrate the upregulation of ace transcription when E. faecalis cultures were grown in the presence of CIV. Surface-localized Ace was detectable after growth in the presence of CIV, and it was correlated with increased adherence to CIV and LN.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The E. faecalis strains used in the present study include OG1RF (18, 19) TX5256 (ace disruption mutant of OG1RF) (19); two endocarditis isolates, TX0052 and MC02152 (15, 20); and a urine isolate, MD9 (20). These strains were chosen based on clearly different pulsed-field gel electrophoresis patterns and different geographical origins. The ace mutant was constructed previously (19) by cloning an intragenic ace fragment in the suicide vector pTEX4577 containing aph(3')-IIIa (26). E. faecalis were routinely grown in brain heart infusion (BHI; Difco Laboratories, Detroit, Mich.) at 37°C, unless a different condition is specified.
ECM proteins and collagenase digestion. Bovine CI was purchased from Cohesion Technologies, Inc. (Palo Alto, Calif.), human-placenta-derived CIV was from Sigma Chemical Co. (St. Louis, Mo.), fibrinogen was from Enzyme Research Laboratories (South Bend, Ind.), and bovine serum albumin (BSA) was from MP Biomedicals, Inc. (Irvine, Calif.).
To eliminate collagen for certain reactions, CIV was suspended (0.5 mg/ml) in 50 mM Tris-HCl (pH 7.0) containing 40 mM CaCl2 and then incubated at 10°C for 18 h with Clostridium histolyticum collagenase (Sigma Chemical Co.) at a substrate/enzyme ratio of 10:1 (14). The reaction mixture was dialyzed against 0.25% (wt/vol) acetic acid at 4°C for 4 days with five changes, freeze-dried, and resuspended in 0.25% (wt/vol) acetic acid.
Gene expression analysis. (i) Extraction of total RNA. Total RNA was isolated from E. faecalis cultures by using an RNeasy minikit (QIAGEN, Valencia, Calif.) according to the protocol of the supplier with some modifications. A lysozyme solution at 10 mg/ml was used instead of 3 mg/ml for the lysis step. Total RNA (20 to 40 μg) was treated three times with 20 U of RQ1 DNase (Promega Corp., Madison, Wis.) for 30 min at 37°C, and the DNase was removed by using the RNeasy minikit. The RNA concentration was determined by using a spectrophotometer. Part of each sample was electrophoresed through an agarose-formaldehyde gel in morpholinepropanesulfonic acid buffer as previously described (23).
(ii) RT-PCR. Total RNA (between 5 ng to 250 ng) was reverse transcribed with ace specific primers (AceMF, 5'-ACGATTGAAGGAGTGACTAACACA-3'; AceMR; 5'-AAGTGTAACGGACGATAAAGGAAG-3') using the SuperScript One-Step RT-PCR with a Platinum Taq kit (Invitrogen Corp., Carlsbad, Calif.) according to the manufacturer's instructions. As an internal control, a 528-bp fragment of gdh (encoding GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) was amplified by using the gdhF (5'-AGTGGCGCACTAAAAGATATGG-3') and gdhR (5'-AGTTGTATTGAACCCTTGACCG-3') primers. Reactions without reverse transcriptase were performed as controls to detect DNA contamination in the total RNA preparations.
(iii) Real-time qRT-PCR. Amplification, detection, and real-time analysis were performed by using the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, Calif.). Primers designed to produce amplicons of equivalent length were selected by using Primer Express software (Applied Biosystems). The primer pairs used in qRT-PCR included AceQF1 (5'-GGAGAGTCAAATCAAGTACGTTGGTT-3')-AceQR1 (5'-TGTTGACCACTTCCTTGTCGAT-3') and 23S-rRNAF (5'-GTGATGGCGTGCCTTTTGTA-3')-23S-rRNAR (5'-CGCCCTATTCAGACTCGCTTT-3'). For each sample, cDNA synthesis and PCR amplification were performed in a two-step process. For cDNA synthesis, 4 μg of total RNA was added to 20-μl reaction solution containing 40 U of RNase OUT, and RT reactions were performed with random primers and SuperScript II reverse transcriptase (Invitrogen). The reaction was stopped by heating at 70°C for 15 min. After the RNA complementary to the cDNA was removed with E. coli RNase H (Invitrogen), the cDNA was purified by using QiaQuick PCR purification kit (QIAGEN). The resulting cDNA diluted up to 500 times was used for subsequent PCR amplification with the appropriate forward and reverse gene specific primers and the SYBR Green PCR Master Mix kit (Applied Biosystems). The following PCR conditions were used: 10 min at 95°C for the initial denaturation, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All PCR fragments yielded a single band on an agarose gel. Relative quantification of gene expression was performed by using 23S rRNA mRNA as the internal standard. The CT method (12) was used to calculate the relative amount of specific RNA present in a sample, from which the fold induction of transcription of the gene was estimated by comparing to the values of OG1RF grown in BHI. The data were expressed as the mean ± the standard deviation. The statistical significance was determined by using the Student unpaired t test. Amplifications were performed on four independent RNA samples from each milieu.
Immunofluorescence microscopy. E. faecalis cells either cultured in the presence or absence of ECM proteins in BHI or grown in 40% horse serum in BHI were washed thrice with Dulbecco phosphate-buffered saline (D-PBS) without CaCl2 and MgCl2 and then resuspended in D-PBS to a final optical density at 600 nm (OD600) of 0.5. Next, 400 μl of cell suspensions were applied to Lab-Tek-II chamber slides (Nalge Nunc International Corp., Naperville, Ill.) that were coated with poly-D-lysine (Sigma) and incubated for 30 min at room temperature with shaking at 100 rpm. After four washes with D-PBS, chamber slides were blocked with 1 ml of D-PBS containing 2% BSA at room temperature for 45 min, washed once with D-PBS, and then incubated with 400 μl of a 1:3,000 dilution for anti-Ace polyclonal serum (19) for 1 h. After four washes with D-PBS, attached bacteria in chamber slides were incubated with 400 μl of rhodamine red-labeled goat anti-rabbit immunoglobulin G (1:1,000 dilution) (Molecular Probes, Eugene, Oreg.) at room temperature for 1 h in the dark. After four washes, excess D-PBS was removed and a coverslip was mounted. The slides were examined by epifluorescence microscopy using an Olympus BX51 microscope with a x100 oil immersion objective lens (Olympus, Tokyo, Japan). Digital images were acquired by using an Olympus DP-70 digital camera. Preimmune serum was used as a negative control.
Adherence assay. Adherence of E. faecalis to CIV- and LN-precoated six-well plates (Becton Dickinson Biosciences, Bedford, Mass.) was determined by using an Olympus BX51 microscope. Each well of ECM-precoated plates were blocked with 5 ml of 0.2% BSA in PBS, incubated at 4°C for 2 h, and then washed with PBS three times. Cell pellets from E. faecalis cells either cultured in the presence or in the absence of ECM proteins were washed two times in PBS and resuspended in 0.1% Tween 80-0.1% BSA in PBS. The cell density was adjusted to an OD600 of 0.2, and 1 ml of bacteria was added into each well, followed by incubation at room temperature for 2 h with gentle shaking at 70 rpm. Wells were washed three times with 0.1% Tween 80-0.1% BSA in PBS. The numbers of bacterial cells that adhered to the surface of the ECM were counted from 10 randomly chosen fields of vision. Each experiment was performed four times. Statistical analysis was determined by using the Mann-Whitney test, and the differences were considered significant when the P value was <0.05.
RESULTS AND DISCUSSION
The pathogenicity of E. faecalis may be associated with the expression of specific bacterial MSCRAMMs in response to environmental cues. Ace is one such protein with the typical characteristics of an MSCRAMM, exhibiting CI, CIV, and LN binding (19). Although Ace was not detectable on Western blots after growth in routine laboratory growth conditions, Ace-specific antibodies were found in sera obtained from patients with E. faecalis endocarditis, indicating that Ace is expressed in vivo during infection in humans and not just at 46°C in vitro (20). However, it has not previously been shown whether the in vitro conditional (46°C) Ace expression is mediated at the transcriptional or posttranscriptional level. In order to determine whether, in fact, the ace gene is transcribed during in vitro growth of E. faecalis strain OG1RF at 37°C in BHI, semiquantitative RT-PCR analysis was performed.
Minimal transcription of ace after growth in vitro in BHI. Total RNA of OG1RF grown at 37°C in BHI was isolated during mid-exponential phase, late-exponential phase, entry into stationary phase, and 5 h after the cultures entered stationary phase (Fig. 1A), and the levels of ace and gdh (housekeeping control) mRNA were analyzed. The ace mRNA was barely detected at the mid-exponential and late exponential phases, as well as at entry into stationary phase, and was not detectable in cells at stationary phase (Fig. 1B and C, lanes 2 to 5). As anticipated, the control gdh gene was constitutively expressed in all phases, although, at stationary phase, gdh mRNA levels were slightly reduced (Fig. 1B and C, lanes 6 to 9). This result suggests that ace transcription is very low during standard in vitro growth conditions. We next analyzed the RNA of OG1RF grown at 46°C isolated at mid-exponential phase and at entry into stationary phase (Fig. 1A). Increased ace mRNA was detected at both growth phases (Fig. 1B and C, lanes 15 and 16) and the ace RT-PCR band intensities were almost comparable to control gdh RT-PCR band intensities at all of the concentrations of total RNA tested (Fig. 1B and C, lanes 17 and 18). These results are in agreement with our previous findings of detectable levels of Ace from mutanolysin surface extracts (19) of OG1RF grown at 46°C.
ace transcription in E. faecalis OG1RF is upregulated after growth in the presence of collagen. Since there is a precedent from other gram-positive organisms of upregulation of specific genes upon exposure to the ligand of their products (9), we explored this possibility by investigating ace mRNA levels from bacteria grown in the presence of different ECM proteins. Initially, BHI-broth-grown, mid-exponential-growth-phase (OD600 = 0.7 to 0.8) cultures of OG1RF were supplemented with 0.03 and 0.3 mg of CIV/ml and cultured further for various postexposure times (15 min, 30 min, 45 min, 1 h, 2 h, and 3 h). RT-PCR analysis of these total RNA preparations showed that the addition of CIV for 30 min at a concentration of 0.3 mg/ml resulted in increased ace mRNA amounts from the minimal levels seen without CIV and that the maximum increase was seen at 1 h (data not shown). To test the effects of different ECM proteins on ace transcription, mid-exponential-growth-phase cultures were divided into aliquots and incubated for 1 h (about two generation times) in the presence or absence of 0.3 mg/ml of CI, CIV, fibrinogen, or BSA. As depicted in Fig. 2A, the addition of CI caused a clear increase in the ace transcript levels (lane 3), and the addition of CIV (to which Ace has maximum affinity) resulted in much higher levels of ace mRNA (lane 4), whereas the addition of fibrinogen or BSA had no effect on ace mRNA levels (lanes 5 and 6). Furthermore, we have also demonstrated that CIV-mediated ace induction of OG1RF is observed only in actively growing culture, but not in the cells suspended in PBS (data not shown), as they are for the adherence assay. Importantly, enhancement of ace mRNA levels observed in the presence of CIV occurred in a dose-dependent manner that paralleled the amount of CIV added (Fig. 2B, lanes 4 to 8). These results indicate that differential ace gene expression depends on the milieu and that the presence of collagen may act as a signal for ace regulation.
Effect of pH on ace expression. During the course of our experiments, we noticed that the final pH of the culture medium was reduced from pH 7.3 to pH 6.4 or 6.7 upon the addition of CIV or CI, respectively. To test whether low extracellular pH could itself affect expression of ace in the absence of collagen, we measured the expression of ace in BHI medium buffered to the same pH values using acetic acid or HCl, respectively. At this pH, there was no significant difference in the level of ace mRNA (Fig. 2B, lanes 3 and 10), although there is a slight decrease in the final OD of the cultures after growth in this buffered medium. This result indicates that the increase in ace transcripts seen in collagen-supplemented medium is not due to the change in pH.
Intact collagen peptides but not collagenase-derived digests induce ace expression. To investigate whether induction of ace transcripts was associated with the structure and/or sequence of collagen peptides or with its primary repeat tripeptides (Gly-X-Y), RT-PCR was carried out by using cultures incubated with collagenase-digested CIV. No change in transcript levels of ace was observed, and the levels remained similar to those for BHI-grown cells (Fig. 2B, lane 9), suggesting that induction of the ace transcripts was associated with intact collagen peptides.
A serum-rich environment affects the expression of ace gene. In an effort to identify other physiological conditions that upregulate ace expression, we tested ace transcript amounts after growth in BHI supplemented with serum. As shown in Fig. 2A, lane 7, growth in 40% horse serum increased the levels of ace mRNA. This observation is in agreement with a previous study (24) that showed a 3.3-fold increase in ace mRNA abundance upon growth in serum compared to growth in 2xYT (yeast extract, tryptone, sodium chloride) medium. Of note, growth in serum resulted in the formation of aggregates both in OG1RF and in TX5256 (ace mutant), indicating that this phenomenon is not dependent upon Ace.
Quantification of ace transcript induction. To measure the fold differences in ace transcription, real-time qRT-PCR was used. The transcription level of 23S rRNA was not significantly affected by growth under any of the conditions tested and, hence, was used for normalization. Comparisons were made between values obtained from the total RNA of E. faecalis cultured in the presence CIV, fibrinogen, or growth at 46°C relative to those from BHI cultures grown at 37°C. Thus, ace mRNA after growth in BHI at 37°C was normalized to a value of 1 as the baseline for comparison. The results showed that there is an 18-fold increase in the ace mRNA levels in cultures grown in BHI containing CIV compared to cells grown in BHI alone (Fig. 3). As anticipated from semiquantitative RT-PCR, the levels of ace mRNA was not altered either in the presence of fibrinogen or by pH. Furthermore, ace mRNA levels of 46°C grown cells was observed to be 2.3-fold more than the levels of cells grown in the presence of CIV (41-fold versus BHI).
Modulation of Ace expression by collagen type IV in diverse E. faecalis strains. To determine whether increased ace mRNA is correlated with Ace protein expression, E. faecalis OG1RF cells were grown in conditions similar to those described above, except that cells were grown for four generations after supplementation with ECM proteins. Cells were then stained with polyclonal anti-Ace antibodies, followed by rhodamine red-labeled secondary antibody; these anti-Ace antibodies have been previously shown to specifically react with Ace on Western blots (20). Analysis of surfaces of cells by fluorescence microscopy showed that anti-Ace serum stained 100% of cells grown in the presence of CI and CIV (Fig. 4D and F) but did not stain the cells grown in the presence of fibrinogen (Fig. 4H). Surface staining was also observed in cells grown in 40% horse serum in BHI (Fig. 4J) and in cells grown at 46°C (Fig. 4L). As expected, Ace antibodies failed to stain the surfaces of the similarly grown ace disruption mutant of OG1RF (TX5256) (data not shown).
Immunofluorescence-based localization of Ace was further carried out with three diverse clinical E. faecalis strains. For the endocarditis strain MC02152, staining was seen on some cells grown in BHI at 37°C (Fig. 4N), a finding consistent with this strain having previously been shown to produce low amounts of Ace (seen as a weak band in Western blot analyses) (20) after growth in BHI at 37°C. However, upon culturing of this strain in the presence of CIV, 100% of the cells were stained with anti-Ace (Fig. 4P). Like OG1RF, TX0052 (endocarditis isolate) and MD9 (urine isolate) cell surfaces showed conditional staining (i.e., after culturing these strains in BHI containing CIV but not after culture in BHI alone). The finding of CIV-induced Ace expression in four of four diverse strains tested suggests this may be a common phenomenon among E. faecalis.
Collagen- and laminin-binding ability of E. faecalis as a function of upregulated ace expression. E. faecalis OG1RF and its isogenic ace disruption mutant, TX5256, were tested for their ability to adhere to immobilized CIV and LN by using microscopy. The cells of OG1RF grown in BHI in the presence of CIV, but not in those grown in BHI alone, adhered to CIV (Fig. 5A) and LN (Fig. 5B), whereas the ace mutant (TX5256) was completely defective in adherence to CIV and LN regardless of its growth conditions. This corroborates our earlier observation that Ace mediates the adherence of E. faecalis OG1RF grown at 46°C to immobilized CIV and LN. Thus, these results demonstrate that E. faecalis adhesion is enhanced directly in response to a host matrix-derived peptide signal. This mechanism of modulation of E. faecalis-collagen interaction is different from the mechanisms reported in S. aureus (5) and E. faecium (21) to alter their collagen adherence.
After growth in 40% horse serum, wild-type OG1RF adhered to immobilized CI, CIV, and LN in our standard radioactive adherence assay (19). Serum-induced CI adherence results are consistent with an earlier study with E. faecalis strain JH2-2 (13). As anticipated, the ace mutant of OG1RF (TX5256) showed markedly reduced adherence to CIV (from 18.1 to 4.2%) and LN (9.2 to 1.2%) compared to the wild type. However, adherence to CI was only partially reduced (from 47.3 to 31.4%), raising the possibility of the presence of an additional collagen-binding protein with high affinity to CI in E. faecalis OG1RF. Our recent observation with a salB mutant of OG1RF that exhibited only CI and fibronectin binding after growth at 37°C (16), corroborates there being a difference between CI and CIV adherence. Furthermore, immunofluorescence microscopy studies with Ace antibodies confirmed that the salB mutant of OG1RF lacks Ace on its surface (data not shown).
Although the ability to adhere to the most abundant host protein, collagen, would intuitively appear to be beneficial to E. faecalis during colonization, it may be of a selective disadvantage during growth in the environment, during transmission or dissemination of infection, and also during chronic infection to avoid the immune response. Therefore, we speculate that E. faecalis may have developed this strategy of programmed response to express proteins such as Ace.
In summary, upregulation of ace gene transcription in the presence of collagen type IV in E. faecalis OG1RF was demonstrated. Although we have yet to delineate the precise mechanism, we have confirmed that expression of Ace, and thereby collagen and laminin adherence, occurs under more physiological conditions than growth at 46°C. Ace induction by other environmental conditions, such as growth in serum and growth at high temperature, suggests the possibility that ace gene expression may be regulated by different mechanisms. Conservation of this induction mechanism in four of four different strains tested suggests that this may be a common programmed response in E. faecalis. Furthermore, understanding of this host matrix protein-associated triggering of a microbial pathogenic response may have important future clinical implications for this emerging multiresistant pathogen.
ACKNOWLEDGMENTS
We thank Karen Jacques-Palaz for technical assistance.
This study was supported by NIH grant R37 AI 47923 from the Division of Microbiology and Infectious Diseases to B.E.M.
FOOTNOTES
Corresponding author. Mailing address: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School at Houston, 6431 Fannin St., MSB 2.112, Houston, TX 77030. Phone: (713) 500-6745. Fax: (713) 500-6766. E-mail: bem.asst@uth.tmc.edu.
REFERENCES
1. Almeida, R. A., D. A. Luther, and S. P. Oliver. 1999. Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells. FEMS Microbiol. Lett. 178:81-85.
2. Duh, R. W., K. V. Singh, K. Malathum, and B. E. Murray. 2001. In vitro activity of 19 antimicrobial agents against enterococci from healthy subjects and hospitalized patients and use of an ace gene probe from Enterococcus faecalis for species identification. Microb. Drug Resist. 7:39-46.
3. Foster, T. J., and M. Hook. 1998. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 6:484-488.
4. Gilbert, F. B., D. A. Luther, and S. P. Oliver. 1997. Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells. FEMS Microbiol. Lett. 156:161-164.
5. Gillaspy, A. F., C. Y. Lee, S. Sau, A. L. Cheung, and M. S. Smeltzer. 1998. Factors affecting the collagen binding capacity of Staphylococcus aureus. Infect. Immun. 66:3170-3178.
6. Gilmore, M. S., P. S. Coburn, S. R. Nallapareddy, and B. E. Murray. 2002. Enterococcal virulence, p. 301-354. In M. S. Gilmore, D. Clewell, P. Courvalin, G. M. Dunny, B. E. Murray, and L. Rice (ed.), The enterococci: pathogenesis, molecular biology, antibiotic resistance, and infection control. American Society Microbiology, Washington, D.C.
7. Guzman, C. A., C. Pruzzo, G. LiPira, and L. Calegari. 1989. Role of adherence in pathogenesis of Enterococcus faecalis urinary tract infection and endocarditis. Infect. Immun. 57:1834-1838.
8. Guzman, C. A., C. Pruzzo, M. Plate, M. C. Guardati, and L. Calegari. 1991. Serum-dependent expression of Enterococcus faecalis adhesins involved in the colonization of heart cells. Microb. Pathog. 11:399-409.
9. Heddle, C., A. H. Nobbs, N. S. Jakubovics, M. Gal, J. P. Mansell, D. Dymock, and H. F. Jenkinson. 2003. Host collagen signal induces antigen I/II adhesin and invasin gene expression in oral Streptococcus gordonii. Mol. Microbiol. 50:597-607.
10. Hubble, T. S., J. F. Hatton, S. R. Nallapareddy, B. E. Murray, and M. J. Gillespie. 2003. Influence of Enterococcus faecalis proteases and the collagen-binding protein, Ace, on adhesion to dentin. Oral Microbiol. Immunol. 18:121-126.
11. Kreft, B., R. Marre, U. Schramm, and R. Wirth. 1992. Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells. Infect. Immun. 60:25-30.
12. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–CT method. Methods 25:402-408.
13. Love, R. M. 2001. Enterococcus faecalis: a mechanism for its role in endodontic failure. Int. Endod. J. 34:399-405.
14. Love, R. M., M. D. McMillan, and H. F. Jenkinson. 1997. Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides. Infect. Immun. 65:5157-5164.
15. Malathum, K., K. V. Singh, G. M. Weinstock, and B. E. Murray. 1998. Repetitive sequence-based PCR versus pulsed-field gel electrophoresis for typing of Enterococcus faecalis at the subspecies level. J. Clin. Microbiol. 36:211-215.
16. Mohamed, J. A., F. Teng, S. R. Nallapareddy, and B. E. Murray. 2006. Pleiotrophic effects of two Enterococcus faecalis sagA-like genes, salA and salB, which encode proteins that are antigenic during human infection, on biofilm formation and binding to collagen type I and fibronectin. J. Infect. Dis. 193:231-240.
17. Murray, B. E. 1990. The life and times of the Enterococcus. Clin. Microbiol. Rev. 3:46-65.
18. Murray, B. E., K. V. Singh, R. P. Ross, J. D. Heath, G. M. Dunny, and G. M. Weinstock. 1993. Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. J. Bacteriol. 175:5216-5223.
19. Nallapareddy, S. R., X. Qin, G. M. Weinstock, M. Hook, and B. E. Murray. 2000. Enterococcus faecalis adhesin, Ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I. Infect. Immun. 68:5218-5224.
20. Nallapareddy, S. R., K. V. Singh, R. W. Duh, G. M. Weinstock, and B. E. Murray. 2000. Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of Ace during human infections. Infect. Immun. 68:5210-5217.
21. Nallapareddy, S. R., G. M. Weinstock, and B. E. Murray. 2003. Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family. Mol. Microbiol. 47:1733-1747.
22. Rich, R. L., B. Kreikemeyer, R. T. Owens, S. LaBrenz, S. V. Narayana, G. M. Weinstock, B. E. Murray, and M. Hook. 1999. Ace is a collagen-binding MSCRAMM from Enterococcus faecalis. J. Biol. Chem. 274:26939-26945.
23. Sambrook, J., E. F. Fritsch, and J. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
24. Shepard, B. D., and M. S. Gilmore. 2002. Differential expression of virulence-related genes in Enterococcus faecalis in response to biological cues in serum and urine. Infect. Immun. 70:4344-4352.
25. Sillanpaa, J., Y. Xu, S. R. Nallapareddy, B. E. Murray, and M. Hook. 2004. A family of putative MSCRAMMs from Enterococcus faecalis. Microbiology 150:2069-2078.
26. Singh, K. V., X. Qin, G. M. Weinstock, and B. E. Murray. 1998. Generation and testing of mutants of Enterococcus faecalis in a mouse peritonitis model. J. Infect. Dis. 178:1416-1420.
27. Tomita, H., and Y. Ike. 2004. Tissue-specific adherent Enterococcus faecalis strains that show highly efficient adhesion to human bladder carcinoma T24 cells also adhere to extracellular matrix proteins. Infect. Immun. 72:5877-5885.
28. Vriesema, A. J., H. Beekhuizen, M. Hamdi, A. Soufan, A. Lammers, B. Willekens, O. Bakker, A. G. Welten, M. H. Veltrop, J. S. van De Gevel, J. Dankert, and S. A. Zaat. 2000. Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells. Infect. Immun. 68:1765-1772.
29. Xiao, J., M. Hook, G. M. Weinstock, and B. E. Murray. 1998. Conditional adherence of Enterococcus faecalis to extracellular matrix proteins. FEMS Immunol. Med. Microbiol. 21:287-295.
30. Xu, Y., J. M. Rivas, E. L. Brown, X. Liang, and M. Hook. 2004. Virulence potential of the staphylococcal adhesin CNA in experimental arthritis is determined by its affinity for collagen. J. Infect. Dis. 189:2323-2333.(Sreedhar R. Nallapareddy, and Barbara E.)