Key role of poly--dl-glutamic acid in immune evasion and virulence of Staphylococcus epidermidis
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《临床调查学报》
1Laboratory of Human Bacterial Pathogenesis and
2Microscopy Core Facility, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Hamilton, Montana, USA.
Abstract
Coagulase-negative staphylococci, with the leading species Staphylococcus epidermidis, are the predominant cause of hospital-acquired infections. Treatment is especially difficult owing to biofilm formation and frequent antibiotic resistance. However, virulence mechanisms of these important opportunistic pathogens have remained poorly characterized. Here we demonstrate that S. epidermidis secretes poly--DL-glutamic acid (PGA) to facilitate growth and survival in the human host. Importantly, PGA efficiently sheltered S. epidermidis from key components of innate host defense, namely antimicrobial peptides and neutrophil phagocytosis, and was indispensable for persistence during device-related infection. Furthermore, PGA protected S. epidermidis from high salt concentration, a key feature of its natural environment, the human skin. Notably, PGA was synthesized by all tested strains of S. epidermidis and a series of closely related coagulase-negative staphylococci, most of which are opportunistic pathogens. Our study presents important novel biological functions for PGA and indicates that PGA represents an excellent target for therapeutic maneuvers aimed at treating disease caused by S. epidermidis and related staphylococci.
Introduction
Over the past decade, Staphylococcus epidermidis has become the most prevalent pathogen involved in nosocomial infections (1). Usually an innocuous commensal microorganism on human skin, this member of the coagulase-negative group of staphylococci can cause severe infection after penetration of the epidermal protective barriers of the human body. In the US alone, S. epidermidis infections on in-dwelling medical devices, which represent the main type of infection with S. epidermidis, cost the public health system approximately $1 billion per year (1). Importantly, S. epidermidis is frequently resistant to common antibiotics (1). It is, therefore, essential to find new molecular targets for drug development against S. epidermidis infections. The formation of surface-attached cellular agglomerations, so-called biofilms, is a major determinant of S. epidermidis pathogenicity (2). Biofilm formation is believed to contribute significantly to antibiotic resistance and protection from innate host defense (3). Resistance of S. epidermidis biofilms to some antibiotics might, in part, be due to a status of generally reduced metabolism (4). Yet, by and large, the specific mediators of biofilm resistance and immune evasion in S. epidermidis are unknown.
Poly--DL-glutamic acid (PGA) is an anionic, extracellular polymer, in which the -amino and -carboxy groups of D- or L-glutamic acid are linked by isopeptide bonds (5). PGA is produced primarily by Bacillus strains but also infrequently by other strains of bacteria, archaebacteria, and some eukaryotes (5). On the whole, the biological role of PGA has remained unclear. Presumably, it serves as an external C and N reserve or protects from environmental factors such as high osmolarity (5). In Bacillus anthracis, a PGA capsule has a hitherto unique role in sheltering the bacteria from phagocytosis (6). Homologs of the cap locus for PGA biosynthesis are found in a restricted number of human pathogens, including the spirochaete Leptospira interrogans, which causes the waterborne disease leptospirosis (7), and Fusobacterium nucleatum, which causes periodontal disease (8). However, it is unknown whether PGA is produced and plays a role in virulence in these organisms.
Here we demonstrate PGA production in S. epidermidis and related coagulase-negative staphylococci. We constructed an isogenic cap mutant strain of S. epidermidis to analyze the role of PGA in protection from environmental factors and innate host defense and as a virulence determinant in an animal infection model. Our study indicates an important function of PGA during both the commensal and infectious lifestyles of S. epidermidis and presents PGA production as the first widespread mechanism of immune evasion in this principal opportunistic pathogen.
Results
The S. epidermidis genome contains the genes that code for PGA synthesis.
To survive on the human skin and during infection of in-dwelling medical devices, S. epidermidis must have mechanisms to circumvent human innate host defense. Only recently, we discovered that the biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA) protects S. epidermidis from major mechanisms of innate host defense (9). However, PIA is restricted to a subpopulation of S. epidermidis, and therefore, a ubiquitous principle protecting S. epidermidis from innate host defense has remained elusive. We searched the recently published S. epidermidis genome (10) for gene loci potentially involved in such protection. We found that, in contrast to its relative S. aureus, S. epidermidis has the cap locus that codes for production of the anionic exopolymer PGA. The S. epidermidis cap genes show high similarity to those of B. anthracis and B. subtilis (Figure 1A). Of note, in contrast to B. subtilis and several human pathogens that have parts of the cap genes, the genetic organization of the cap locus of B. anthracis is well conserved in S. epidermidis (Figure 1B).
The S. epidermidis cap gene locus drives production of surface-attached PGA.
To analyze the role of the cap genes in S. epidermidis, we constructed a deletion mutant (S. epidermidis cap), in which the entire cap locus was replaced by a spectinomycin resistance cassette. To complement the mutant, we cloned the cap operon with its natural promoter in a plasmid and transformed the mutant with that plasmid (pRBcapBCAD). First, we determined PGA production in the mutant, wild-type, and complemented strains. Real-time PCR data indicated that the cap genes in S. epidermidis are expressed (data not shown). Accordingly, immunodetection of PGA in cell surface extracts of the wild-type and complemented strains demonstrated that PGA is synthesized by S. epidermidis and attached to the bacterial cell surface (Figure 2A). The PGA production level in the complemented strain was 147% of that of the wild-type strain. There was no detectable signal in the cap deletion strain or in any samples obtained from culture filtrates (Figure 2A and data not shown). Scanning electron microscopy with immunogold labeling confirmed that PGA is located on the S. epidermidis cell surface (Figure 2B). Next, we purified the immunoreactive material and performed quantitative and qualitative analyses to verify that it constitutes PGA. In contrast to B. anthracis PGA, which is entirely composed of D-glutamic acid (11), S. epidermidis PGA consisted of about equal amounts of D- and L-glutamic acid (Figure 2C). Notably, PGA production in S. epidermidis was far less than that reported for B. anthracis (1.2 x 10–6 g/l for S. epidermidis versus 5–10 g/l for B. anthracis; ref. 12). Taken together, our data demonstrate that the cap locus in S. epidermidis drives production of a surface-attached form of DL-PGA.
The cap gene locus and PGA production are ubiquitous among S. epidermidis strains.
Some virulence factors in staphylococci, such as PIA, are mostly restricted to invasive strains (13, 14). In contrast, all 74 S. epidermidis strains of clinical and commensal origin that we investigated had the cap gene locus as shown by analytical PCR (data not shown) and were positive for PGA by immuno-dot blot analysis (Figure 3), indicating that PGA production is ubiquitous in S. epidermidis. Remarkably, there was no significant difference in PGA production between commensal and clinical strains (Figure 3). We therefore hypothesized that PGA is important for the survival of S. epidermidis as a skin commensal organism and during infection.
PGA contributes to resistance of S. epidermidis to high salt concentration.
We first determined whether PGA impacts survival of S. epidermidis under conditions found in its natural habitat, i.e., the human skin. The environment on human skin is characterized by high and varying salt concentration. S. epidermidis can withstand very high salt concentrations of up to approximately 2 M NaCl, but the reasons for this extraordinary resistance are not completely clear (13). Growth and viability of the cap mutant strain were significantly impaired at high concentrations of NaCl (shown for 2 M NaCl in Figure 4, A and B), whereas growth at physiological salt concentration was similar for the wild-type and mutant strains (data not shown). Furthermore, quantitative real-time PCR analysis using a capB probe (Figure 4C) and immuno-dot blots (data not shown) demonstrated that S. epidermidis upregulates cap expression in response to high NaCl concentration. Additionally, in most strains of our collection, PGA production was greater at high NaCl concentration (Figure 3). These data suggest that PGA contributes to survival of S. epidermidis on human skin.
S. epidermidis PGA protects from key components of innate host defense.
Next, we investigated whether PGA contributes to S. epidermidis virulence. Unlike its more aggressive relative S. aureus, S. epidermidis does not have a large arsenal of virulence factors (1). Rather, it causes relatively silent, chronic infections during which resistance against attacks by the innate immune system is of special importance to bacterial survival (1). Therefore, we analyzed whether cap expression in S. epidermidis affects resistance to key components of innate host defense against bacterial infections, namely antibacterial peptides and neutrophil phagocytosis. The cap mutant strain had significantly reduced resistance to 2 representative antibacterial peptides from human skin and neutrophil specific granules, LL-37 and human ?-defensin 3 (Figure 5, A and B) and significantly increased susceptibility to phagocytosis by human neutrophils (Figure 5C). The phagocytosis rate was 42% higher with the mutant strain compared to the wild-type strain, which is a relatively large difference compared with what we observe with other single pathogen factors. These findings indicate that PGA plays a critical role in protecting S. epidermidis from the microcidal effects of innate host defense components.
PGA is indispensable for S. epidermidis persistence on in-dwelling medical devices in an animal infection model.
The role of PGA in protection from innate host defense components suggested that PGA facilitates pathogen survival during S. epidermidis infection. To test this hypothesis, we compared persistence of wild-type and isogenic cap mutant strains in a mouse model of subcutaneous catheter infection. Importantly, biofilm formation on plastic material and intercellular aggregation in vitro did not differ between wild-type and cap mutant strains (data not shown), indicating that colonization and persistence in the animal model were not due to differences in the physicochemical interaction with the catheter material. Most mice (5 of 8) infected with the wild-type strain had significant numbers of bacteria (approximately 3 x 104–6 x 104 CFU) on implanted catheters after 1 week of infection, whereas all 7 mice infected with the cap mutant strain had completely cleared the infection (Figure 5D). This pronounced difference indicates that PGA is a key factor for the success of S. epidermidis in device-associated infection, which represents the predominant type of disease caused by this organism.
A group of coagulase-negative staphylococci related to S. epidermidis also produces PGA.
We know from publicly available genome sequencing data that the cap locus is absent from S. aureus. To investigate whether other staphylococcal species have the genetic information for PGA production, we tested a series of staphylococcal strains for the presence of the cap genes by DNA-DNA hybridization using capB and capD probes and a cocktail of probes from all 4 cap genes. Of 22 strains, representing 16 different species and subspecies, genomic DNA from 11 strains hybridized with the probes (Table 1). Except for S. saprophyticus strains, all strains with a signal in the Southern blot also produced surface-attached PGA, which was demonstrated using anti-PGA antisera (Table 1). Although there are some intraspecies differences, it is remarkable that all phylogenetically related members of the S. epidermidis group (S. epidermidis, S. capitis, S. warneri, S. saccharolyticus, S. caprae, S. hominis, and S. haemolyticus) (15) have the ability to produce PGA, whereas it is far less distributed among other species (Table 1).
Discussion
We are only beginning to understand strategies used by pathogenic bacteria to evade the efficient bactericidal mechanisms of innate host defense (16). To survive in its natural habitat, i.e., on human skin, S. epidermidis needs to protect itself from antibacterial peptides of human epithelial cells. Furthermore, to persist during human infection, it must have efficient protection from ingestion by neutrophils and associated bactericidal mechanisms. Here we present PGA as the first widespread mechanism of immune evasion in S. epidermidis. Notably, we demonstrate a hitherto unknown role for PGA in resistance to cationic antimicrobial peptides. Further, PGA was critical for the establishment of chronic infection by S. epidermidis. The difference in persistence of the cap mutant and wild-type strains was more pronounced than with any other virulence determinant of S. epidermidis that has been investigated by an animal infection model. Inasmuch as PGA production in S. epidermidis is approximately 1 million times lower than in B. anthracis, one can conclude that the efficiency of PGA-mediated protection from innate host defense in the case of S. epidermidis is extraordinary. The mechanism of protection is unclear. Presumably, it is based on the strong anionic properties of PGA. For example, PGA may bind antibacterial peptides, which are usually cationic (17), and thus prevent them from reaching their target, the cytoplasmic membrane. However, protection by PGA may also be mediated, for example, by binding of PGA to other surface polymers, which might enhance the protective effect.
Except for the role that PGA has in protecting B. anthracis from phagocytosis, the biological function of PGA, particularly in noninfectious organisms, has remained speculative. It is known that several halophilic bacteria synthesize PGA (5). Therefore, it has been assumed that PGA serves to protect from high osmolarity. In our study, we show for the first time that PGA contributes to resistance against high salt concentration. It has been speculated that binding of water molecules by negatively charged PGA protects bacteria from dehydration (18). However, the low level of PGA production suggests that mechanisms other than direct binding of water by PGA might be involved in the protective effect in S. epidermidis.
Importantly, PGA production did not affect the aggregation behavior or biofilm formation of S. epidermidis, demonstrating that our observations are not due to secondary effects of cell aggregation. However, recent results that we obtained by microarray analysis of gene expression in S. epidermidis biofilms indicated that the cap locus is upregulated in biofilms (4). PGA may thus contribute to the overall resistance of S. epidermidis biofilms to innate immunity, although it does not appear to directly influence biofilm formation.
The S. epidermidis cap locus shows high similarity to that of B. anthracis. The genetic information for PGA production in B. anthracis is located on a plasmid (pXO2) (19, 20) and shows high genetic mobility (21). It might have been transferred between B. anthracis and staphylococci and from one strain of Staphylococcus to another. Coinfection of humans or human ancestors may represent a likely scenario for these events. Of note, most of the staphylococcal strains that produce PGA are opportunistic human pathogens. Consistent with its role in S. epidermidis, PGA might thus contribute to survival of those strains in the human host, although this notion remains to be demonstrated.
In conclusion, PGA appears to play a key role for the survival of S. epidermidis in the human host during colonization and infection. Remarkably, though produced in very small amounts, PGA contributes significantly to the pathogenicity of S. epidermidis, which underscores the efficiency of PGA-mediated protection. PGA may represent an excellent target for drug or vaccine development against infection with S. epidermidis and possibly other staphylococcal pathogens. Anti-PGA antibodies have been suggested for the treatment and prophylaxis of B. anthracis infections. Although PGA has a generally low immunogenicity, recent studies have shown that anti-PGA antibodies efficiently protect from anthrax infection in animal models (22-25). Our data indicate that they might also be valuable for the treatment of chronic infections by S. epidermidis and certain other pathogenic staphylococci.
Methods
Bacterial strains, growth conditions, and basic molecular biology methods.
The clinical isolate S. epidermidis 1457 (26) was used in this study. Bacteria were grown in tryptic soy broth (TSB) unless otherwise noted. Antibiotics were used at the following concentrations: chloramphenicol, 10 μg/ml; spectinomycin, 100 μg/ml; and ampicillin, 100 μg/ml. Cultures were incubated at 37°C with shaking at 200 rpm. DNA manipulation, isolation of plasmid DNA, and transformation of Escherichia coli were performed using standard procedures. Staphylococcal plasmid DNA was prepared with the Qiagen Plasmid Midi Kit as described (27). S. epidermidis was transformed by electroporation as described (28). PCRs were performed with Ready-To-Go PCR Beads (Amersham Biosciences) as recommended by the manufacturer. DNA was sequenced using Big Dye Terminator cycle sequencing (version 3.0) on an ABI3700 sequencer (Applied Biosystems). Nucleotide sequences were analyzed using the program Vector NTI Suite (InforMax). Primers for DNA amplifications by PCR (Table 2) were purchased from Sigma Genosys. The 74 strains used for epidemiological studies were obtained from N. El Solh (Institut Pasteur, Paris, France) and are predominantly nonclonal (29).
Real-time PCR.
RNA isolation was performed using a FastPrep BLUE Kit (Q-BioGene Inc.). Oligonucleotide primers and probes were designed using Primer Express 2.0 software (Applied Biosystems). The probe used for analysis of cap expression was located within the capB gene. TaqMan analysis was performed in a 384-well MicroAmp Optical using a 7900 Sequence Detector (Applied Biosystems). Standard curves were determined with purified chromosomal template DNA at concentrations ranging from 0.001 ng/ml to 10 ng/ml. Assays were performed in triplicate using cDNA samples, and 16S rRNA as control, with a standard cycle protocol.
Southern blot analysis.
Equal amounts of EcoRV-digested genomic DNA were separated by gel electrophoresis on a 0.7% agarose gel and visualized by ethidium bromide staining. DNA was transferred onto a nylon membrane (GE Osmonics Labstore) and probed with digoxigenin-labeled PCR product amplified from the capB or capD genes of S. epidermidis 1457 or with a cocktail of probes amplified from the capA, capB, capC, and capD genes. Prehybridization, hybridization, posthybridization, and immunologic detection were performed as described in the technical update accompanying the nonradioactive DNA Labeling and Detection Kit (Roche Applied Science). For immunologic detection, the membrane was incubated with a 1:5,000 dilution of sheep anti-digoxigenin Fab fragments conjugated to alkaline phosphatase. Probe-target hybrids were detected using the chromogenic substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate.
Immuno-dot blot assay.
Surface-attached PGA was released from the cell surface by boiling bacteria for 30 minutes at 100°C or by autoclaving. PGA was then purified by acid precipitation as described (12). Aliquots (3 μl) of the samples were spotted on a nitrocellulose membrane, air-dried, and PGA was detected with anti-PGA antiserum using a scanner and Total Lab Version 2003 software (Nonlinear USA). The assay was calibrated by serial dilution of the most intensive sample. The value detected in the wild-type strain was set to 100% and production values in the other strains were expressed relative to that value. Anti-PGA antiserum (kindly provided by R. Schneerson, National Institute of Child Health & Human Development, NIH, Bethesda, Maryland, USA) was developed against PGA from Bacillus pumilus. For Staphylococcus strains other than S. epidermidis, a strain with a signal higher than that of the background of the S. epidermidis cap mutant was considered a PGA producer.
Construction of an isogenic cap deletion mutant and cap-complementing vector.
To delete capBCAD in S. epidermidis 1457, PCR-amplified regions flanking the cap locus and a spectinomycin resistance cassette were cloned into plasmid pBT2 (30), yielding plasmid pBTcap, which was used for allelic replacement as described (31). The proper integration of the resistance gene marker spc was verified by direct sequencing of the genomic DNA at the borders of the PCR-derived regions. Lack of cap transcript in the cap mutant strain was verified by real-time PCR (data not shown). S. epidermidis 1457 in which capBCAD was deleted was named S. epidermidis cap. To complement for capBCAD in S. epidermidis cap, capBCAD genes were cloned into plasmid pRB474 (30). The resulting plasmid was named pRBcapBCAD.
Purification of PGA and detection of d- and l-glutamic acid.
Cultures were grown in TSB medium supplemented with 1 M NaCl to induce PGA production. PGA was first purified as described above. Then, PGA samples were further purified by ion exchange chromatography using a RESOURCE Q 6 ml column (Amersham Biosciences) on an AKTA Purifier 10 (Amersham Biosciences) and a gradient from 0.2% acetic acid to 0.2% acetic acid/1 M NaCl in 20 column volumes at a flow rate of 5 ml/min. Fractions with positive reaction in an immuno-dot blot were combined, dialyzed against water, lyophilized, resuspended in 6 M HCl, and hydrolyzed at 110°C for 24 hours. Samples were lyophilized again and dissolved in 200 μl of water. Ten microliters of the samples were then injected onto a Chirobiotic T column (Astec) using 0.1% triethylammonium acetate (pH 4.0) in 80% ethanol as elution buffer at a flow rate of 0.4 ml/min. Chromatography was performed using an Agilent 1100 series HPLC connected to a VL Trap mass spectrometer. The extracted ion chromatograms at 146 Da, the mass of glutamic acid, were used to determine the amounts of D- and L-glutamic acid by peak integration in comparison to pure D- and L-glutamic acid.
Isolation of human polymorphonuclear leukocytes and phagocytosis experiments.
Human polymorphonuclear leukocytes (PMNs) were isolated from heparinized venous blood of healthy individuals with a standard method (32). All studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects of NIAID. Cell preparations contained approximately 99% PMNs and all reagents used contained <25.0 pg/ml endotoxin. Phagocytosis of S. epidermidis by human PMNs was analyzed by flow cytometry with a previously described method (32). Briefly, bacteria were cultured to stationary growth phase, washed in PBS, and labeled with fluorescein-5-isothiocyanate for 15 minutes. PMNs (106/100 μl) and bacteria (2 x 107/100 μl) were combined in wells of serum-coated 96-well round-bottom microtiter plates. Plates were incubated for 30 minutes at 37°C and the degree of phagocytosis was determined by flow cytometry.
Peptide bacterial killing assays.
S. epidermidis cultures were harvested, washed with PBS buffer, and resuspended in 10 mM sodium phosphate buffer (pH 7.0). Bacterial killing assays were performed using a final concentration of approximately 105 S. epidermidis cells in each sample. Antimicrobial peptides were dissolved in the following solutions: human ?-defensin 3, 10 mM acetic acid, and LL-37, 10% acetonitrile with 0.1% trifluoroacetic acid. The bacteria were exposed to a range of antimicrobial peptide concentrations (0, 5, 10, 20, 30, and 40 μg/ml). An equal volume of the respective peptide dilution buffer was applied to control samples. Samples were incubated at 37°C for 2 hours and appropriate dilution series of the samples were plated on TSB agar. Survivor S. epidermidis cells were enumerated after 24 hours of incubation at 37°C. The percentage of killed S. epidermidis was calculated using the formula (1 – [CFU peptide / CFUcontrol]) x 100.
Scanning immunoelectron microscopy.
Fifty-microliter aliquots of S. epidermidis cultures were washed with PBS buffer. Cells were resuspended in 200 μl of anti-PGA antiserum and incubated at 37°C with agitation at 400 rpm for 12 hours. Samples were washed with PBS and pellets were subsequently incubated with goat anti-rabbit IgG conjugated with 20 nm of gold (BB International) at 37°C with agitation at 400 rpm for 2 hours. Following antibody labeling, the cell suspensions were attached to coverslips, fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, and post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate. Samples were washed with distilled water, dehydrated in a graded ethanol series, critical-point dried under CO2 with a Bal-Tec model cpd 030 drier (Balzers), mounted on aluminum studs, and sputter-coated with 100 angstroms of chromium in a model IBS/TM200S ion beam sputterer (South Bay Technologies) prior to viewing at 10 kV on a Hitachi S-4500 field emission scanning electron microscope (Hitachi) in backscatter imaging mode.
Murine model of device-related infection.
Female Balb/c mice were used in a model of subcutaneous implanted device-related infection as described (33). Two catheter pieces of 1-cm length were placed under the skin of the dorsum of each animal. CFUs on catheters were counted before insertion and were in the range of 2 x 105 on all implanted catheters. CFU on excised catheters and surrounding tissues were counted after 1 week of infection. All studies were performed in accordance with a protocol approved by the Animal Care and Use Committee of Rocky Mountain Laboratories, NIAID.
Statistics and DNA sequence analysis.
Statistical analysis was performed using GraphPad Prism version 4.0. DNA sequences were compared using Clustal W software.
Acknowledgments
The authors would like to thank Rachel Schneerson for providing anti-PGA antisera, Nevine El Solh for S. epidermidis strains, Aaron B. Carmody for technical assistance, and Donald Gardner, Michael Parnell, and Ralph Larson for help with animal studies.
Footnotes
Nonstandard abbreviations used: PGA, poly--dl-glutamic acid; PIA, polysaccharide intercellular adhesin; PMN, polymorphonuclear leukocyte; TSB, tryptic soy broth.
Conflict of interest: The authors have declared that no conflict of interest exists.
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2Microscopy Core Facility, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Hamilton, Montana, USA.
Abstract
Coagulase-negative staphylococci, with the leading species Staphylococcus epidermidis, are the predominant cause of hospital-acquired infections. Treatment is especially difficult owing to biofilm formation and frequent antibiotic resistance. However, virulence mechanisms of these important opportunistic pathogens have remained poorly characterized. Here we demonstrate that S. epidermidis secretes poly--DL-glutamic acid (PGA) to facilitate growth and survival in the human host. Importantly, PGA efficiently sheltered S. epidermidis from key components of innate host defense, namely antimicrobial peptides and neutrophil phagocytosis, and was indispensable for persistence during device-related infection. Furthermore, PGA protected S. epidermidis from high salt concentration, a key feature of its natural environment, the human skin. Notably, PGA was synthesized by all tested strains of S. epidermidis and a series of closely related coagulase-negative staphylococci, most of which are opportunistic pathogens. Our study presents important novel biological functions for PGA and indicates that PGA represents an excellent target for therapeutic maneuvers aimed at treating disease caused by S. epidermidis and related staphylococci.
Introduction
Over the past decade, Staphylococcus epidermidis has become the most prevalent pathogen involved in nosocomial infections (1). Usually an innocuous commensal microorganism on human skin, this member of the coagulase-negative group of staphylococci can cause severe infection after penetration of the epidermal protective barriers of the human body. In the US alone, S. epidermidis infections on in-dwelling medical devices, which represent the main type of infection with S. epidermidis, cost the public health system approximately $1 billion per year (1). Importantly, S. epidermidis is frequently resistant to common antibiotics (1). It is, therefore, essential to find new molecular targets for drug development against S. epidermidis infections. The formation of surface-attached cellular agglomerations, so-called biofilms, is a major determinant of S. epidermidis pathogenicity (2). Biofilm formation is believed to contribute significantly to antibiotic resistance and protection from innate host defense (3). Resistance of S. epidermidis biofilms to some antibiotics might, in part, be due to a status of generally reduced metabolism (4). Yet, by and large, the specific mediators of biofilm resistance and immune evasion in S. epidermidis are unknown.
Poly--DL-glutamic acid (PGA) is an anionic, extracellular polymer, in which the -amino and -carboxy groups of D- or L-glutamic acid are linked by isopeptide bonds (5). PGA is produced primarily by Bacillus strains but also infrequently by other strains of bacteria, archaebacteria, and some eukaryotes (5). On the whole, the biological role of PGA has remained unclear. Presumably, it serves as an external C and N reserve or protects from environmental factors such as high osmolarity (5). In Bacillus anthracis, a PGA capsule has a hitherto unique role in sheltering the bacteria from phagocytosis (6). Homologs of the cap locus for PGA biosynthesis are found in a restricted number of human pathogens, including the spirochaete Leptospira interrogans, which causes the waterborne disease leptospirosis (7), and Fusobacterium nucleatum, which causes periodontal disease (8). However, it is unknown whether PGA is produced and plays a role in virulence in these organisms.
Here we demonstrate PGA production in S. epidermidis and related coagulase-negative staphylococci. We constructed an isogenic cap mutant strain of S. epidermidis to analyze the role of PGA in protection from environmental factors and innate host defense and as a virulence determinant in an animal infection model. Our study indicates an important function of PGA during both the commensal and infectious lifestyles of S. epidermidis and presents PGA production as the first widespread mechanism of immune evasion in this principal opportunistic pathogen.
Results
The S. epidermidis genome contains the genes that code for PGA synthesis.
To survive on the human skin and during infection of in-dwelling medical devices, S. epidermidis must have mechanisms to circumvent human innate host defense. Only recently, we discovered that the biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA) protects S. epidermidis from major mechanisms of innate host defense (9). However, PIA is restricted to a subpopulation of S. epidermidis, and therefore, a ubiquitous principle protecting S. epidermidis from innate host defense has remained elusive. We searched the recently published S. epidermidis genome (10) for gene loci potentially involved in such protection. We found that, in contrast to its relative S. aureus, S. epidermidis has the cap locus that codes for production of the anionic exopolymer PGA. The S. epidermidis cap genes show high similarity to those of B. anthracis and B. subtilis (Figure 1A). Of note, in contrast to B. subtilis and several human pathogens that have parts of the cap genes, the genetic organization of the cap locus of B. anthracis is well conserved in S. epidermidis (Figure 1B).
The S. epidermidis cap gene locus drives production of surface-attached PGA.
To analyze the role of the cap genes in S. epidermidis, we constructed a deletion mutant (S. epidermidis cap), in which the entire cap locus was replaced by a spectinomycin resistance cassette. To complement the mutant, we cloned the cap operon with its natural promoter in a plasmid and transformed the mutant with that plasmid (pRBcapBCAD). First, we determined PGA production in the mutant, wild-type, and complemented strains. Real-time PCR data indicated that the cap genes in S. epidermidis are expressed (data not shown). Accordingly, immunodetection of PGA in cell surface extracts of the wild-type and complemented strains demonstrated that PGA is synthesized by S. epidermidis and attached to the bacterial cell surface (Figure 2A). The PGA production level in the complemented strain was 147% of that of the wild-type strain. There was no detectable signal in the cap deletion strain or in any samples obtained from culture filtrates (Figure 2A and data not shown). Scanning electron microscopy with immunogold labeling confirmed that PGA is located on the S. epidermidis cell surface (Figure 2B). Next, we purified the immunoreactive material and performed quantitative and qualitative analyses to verify that it constitutes PGA. In contrast to B. anthracis PGA, which is entirely composed of D-glutamic acid (11), S. epidermidis PGA consisted of about equal amounts of D- and L-glutamic acid (Figure 2C). Notably, PGA production in S. epidermidis was far less than that reported for B. anthracis (1.2 x 10–6 g/l for S. epidermidis versus 5–10 g/l for B. anthracis; ref. 12). Taken together, our data demonstrate that the cap locus in S. epidermidis drives production of a surface-attached form of DL-PGA.
The cap gene locus and PGA production are ubiquitous among S. epidermidis strains.
Some virulence factors in staphylococci, such as PIA, are mostly restricted to invasive strains (13, 14). In contrast, all 74 S. epidermidis strains of clinical and commensal origin that we investigated had the cap gene locus as shown by analytical PCR (data not shown) and were positive for PGA by immuno-dot blot analysis (Figure 3), indicating that PGA production is ubiquitous in S. epidermidis. Remarkably, there was no significant difference in PGA production between commensal and clinical strains (Figure 3). We therefore hypothesized that PGA is important for the survival of S. epidermidis as a skin commensal organism and during infection.
PGA contributes to resistance of S. epidermidis to high salt concentration.
We first determined whether PGA impacts survival of S. epidermidis under conditions found in its natural habitat, i.e., the human skin. The environment on human skin is characterized by high and varying salt concentration. S. epidermidis can withstand very high salt concentrations of up to approximately 2 M NaCl, but the reasons for this extraordinary resistance are not completely clear (13). Growth and viability of the cap mutant strain were significantly impaired at high concentrations of NaCl (shown for 2 M NaCl in Figure 4, A and B), whereas growth at physiological salt concentration was similar for the wild-type and mutant strains (data not shown). Furthermore, quantitative real-time PCR analysis using a capB probe (Figure 4C) and immuno-dot blots (data not shown) demonstrated that S. epidermidis upregulates cap expression in response to high NaCl concentration. Additionally, in most strains of our collection, PGA production was greater at high NaCl concentration (Figure 3). These data suggest that PGA contributes to survival of S. epidermidis on human skin.
S. epidermidis PGA protects from key components of innate host defense.
Next, we investigated whether PGA contributes to S. epidermidis virulence. Unlike its more aggressive relative S. aureus, S. epidermidis does not have a large arsenal of virulence factors (1). Rather, it causes relatively silent, chronic infections during which resistance against attacks by the innate immune system is of special importance to bacterial survival (1). Therefore, we analyzed whether cap expression in S. epidermidis affects resistance to key components of innate host defense against bacterial infections, namely antibacterial peptides and neutrophil phagocytosis. The cap mutant strain had significantly reduced resistance to 2 representative antibacterial peptides from human skin and neutrophil specific granules, LL-37 and human ?-defensin 3 (Figure 5, A and B) and significantly increased susceptibility to phagocytosis by human neutrophils (Figure 5C). The phagocytosis rate was 42% higher with the mutant strain compared to the wild-type strain, which is a relatively large difference compared with what we observe with other single pathogen factors. These findings indicate that PGA plays a critical role in protecting S. epidermidis from the microcidal effects of innate host defense components.
PGA is indispensable for S. epidermidis persistence on in-dwelling medical devices in an animal infection model.
The role of PGA in protection from innate host defense components suggested that PGA facilitates pathogen survival during S. epidermidis infection. To test this hypothesis, we compared persistence of wild-type and isogenic cap mutant strains in a mouse model of subcutaneous catheter infection. Importantly, biofilm formation on plastic material and intercellular aggregation in vitro did not differ between wild-type and cap mutant strains (data not shown), indicating that colonization and persistence in the animal model were not due to differences in the physicochemical interaction with the catheter material. Most mice (5 of 8) infected with the wild-type strain had significant numbers of bacteria (approximately 3 x 104–6 x 104 CFU) on implanted catheters after 1 week of infection, whereas all 7 mice infected with the cap mutant strain had completely cleared the infection (Figure 5D). This pronounced difference indicates that PGA is a key factor for the success of S. epidermidis in device-associated infection, which represents the predominant type of disease caused by this organism.
A group of coagulase-negative staphylococci related to S. epidermidis also produces PGA.
We know from publicly available genome sequencing data that the cap locus is absent from S. aureus. To investigate whether other staphylococcal species have the genetic information for PGA production, we tested a series of staphylococcal strains for the presence of the cap genes by DNA-DNA hybridization using capB and capD probes and a cocktail of probes from all 4 cap genes. Of 22 strains, representing 16 different species and subspecies, genomic DNA from 11 strains hybridized with the probes (Table 1). Except for S. saprophyticus strains, all strains with a signal in the Southern blot also produced surface-attached PGA, which was demonstrated using anti-PGA antisera (Table 1). Although there are some intraspecies differences, it is remarkable that all phylogenetically related members of the S. epidermidis group (S. epidermidis, S. capitis, S. warneri, S. saccharolyticus, S. caprae, S. hominis, and S. haemolyticus) (15) have the ability to produce PGA, whereas it is far less distributed among other species (Table 1).
Discussion
We are only beginning to understand strategies used by pathogenic bacteria to evade the efficient bactericidal mechanisms of innate host defense (16). To survive in its natural habitat, i.e., on human skin, S. epidermidis needs to protect itself from antibacterial peptides of human epithelial cells. Furthermore, to persist during human infection, it must have efficient protection from ingestion by neutrophils and associated bactericidal mechanisms. Here we present PGA as the first widespread mechanism of immune evasion in S. epidermidis. Notably, we demonstrate a hitherto unknown role for PGA in resistance to cationic antimicrobial peptides. Further, PGA was critical for the establishment of chronic infection by S. epidermidis. The difference in persistence of the cap mutant and wild-type strains was more pronounced than with any other virulence determinant of S. epidermidis that has been investigated by an animal infection model. Inasmuch as PGA production in S. epidermidis is approximately 1 million times lower than in B. anthracis, one can conclude that the efficiency of PGA-mediated protection from innate host defense in the case of S. epidermidis is extraordinary. The mechanism of protection is unclear. Presumably, it is based on the strong anionic properties of PGA. For example, PGA may bind antibacterial peptides, which are usually cationic (17), and thus prevent them from reaching their target, the cytoplasmic membrane. However, protection by PGA may also be mediated, for example, by binding of PGA to other surface polymers, which might enhance the protective effect.
Except for the role that PGA has in protecting B. anthracis from phagocytosis, the biological function of PGA, particularly in noninfectious organisms, has remained speculative. It is known that several halophilic bacteria synthesize PGA (5). Therefore, it has been assumed that PGA serves to protect from high osmolarity. In our study, we show for the first time that PGA contributes to resistance against high salt concentration. It has been speculated that binding of water molecules by negatively charged PGA protects bacteria from dehydration (18). However, the low level of PGA production suggests that mechanisms other than direct binding of water by PGA might be involved in the protective effect in S. epidermidis.
Importantly, PGA production did not affect the aggregation behavior or biofilm formation of S. epidermidis, demonstrating that our observations are not due to secondary effects of cell aggregation. However, recent results that we obtained by microarray analysis of gene expression in S. epidermidis biofilms indicated that the cap locus is upregulated in biofilms (4). PGA may thus contribute to the overall resistance of S. epidermidis biofilms to innate immunity, although it does not appear to directly influence biofilm formation.
The S. epidermidis cap locus shows high similarity to that of B. anthracis. The genetic information for PGA production in B. anthracis is located on a plasmid (pXO2) (19, 20) and shows high genetic mobility (21). It might have been transferred between B. anthracis and staphylococci and from one strain of Staphylococcus to another. Coinfection of humans or human ancestors may represent a likely scenario for these events. Of note, most of the staphylococcal strains that produce PGA are opportunistic human pathogens. Consistent with its role in S. epidermidis, PGA might thus contribute to survival of those strains in the human host, although this notion remains to be demonstrated.
In conclusion, PGA appears to play a key role for the survival of S. epidermidis in the human host during colonization and infection. Remarkably, though produced in very small amounts, PGA contributes significantly to the pathogenicity of S. epidermidis, which underscores the efficiency of PGA-mediated protection. PGA may represent an excellent target for drug or vaccine development against infection with S. epidermidis and possibly other staphylococcal pathogens. Anti-PGA antibodies have been suggested for the treatment and prophylaxis of B. anthracis infections. Although PGA has a generally low immunogenicity, recent studies have shown that anti-PGA antibodies efficiently protect from anthrax infection in animal models (22-25). Our data indicate that they might also be valuable for the treatment of chronic infections by S. epidermidis and certain other pathogenic staphylococci.
Methods
Bacterial strains, growth conditions, and basic molecular biology methods.
The clinical isolate S. epidermidis 1457 (26) was used in this study. Bacteria were grown in tryptic soy broth (TSB) unless otherwise noted. Antibiotics were used at the following concentrations: chloramphenicol, 10 μg/ml; spectinomycin, 100 μg/ml; and ampicillin, 100 μg/ml. Cultures were incubated at 37°C with shaking at 200 rpm. DNA manipulation, isolation of plasmid DNA, and transformation of Escherichia coli were performed using standard procedures. Staphylococcal plasmid DNA was prepared with the Qiagen Plasmid Midi Kit as described (27). S. epidermidis was transformed by electroporation as described (28). PCRs were performed with Ready-To-Go PCR Beads (Amersham Biosciences) as recommended by the manufacturer. DNA was sequenced using Big Dye Terminator cycle sequencing (version 3.0) on an ABI3700 sequencer (Applied Biosystems). Nucleotide sequences were analyzed using the program Vector NTI Suite (InforMax). Primers for DNA amplifications by PCR (Table 2) were purchased from Sigma Genosys. The 74 strains used for epidemiological studies were obtained from N. El Solh (Institut Pasteur, Paris, France) and are predominantly nonclonal (29).
Real-time PCR.
RNA isolation was performed using a FastPrep BLUE Kit (Q-BioGene Inc.). Oligonucleotide primers and probes were designed using Primer Express 2.0 software (Applied Biosystems). The probe used for analysis of cap expression was located within the capB gene. TaqMan analysis was performed in a 384-well MicroAmp Optical using a 7900 Sequence Detector (Applied Biosystems). Standard curves were determined with purified chromosomal template DNA at concentrations ranging from 0.001 ng/ml to 10 ng/ml. Assays were performed in triplicate using cDNA samples, and 16S rRNA as control, with a standard cycle protocol.
Southern blot analysis.
Equal amounts of EcoRV-digested genomic DNA were separated by gel electrophoresis on a 0.7% agarose gel and visualized by ethidium bromide staining. DNA was transferred onto a nylon membrane (GE Osmonics Labstore) and probed with digoxigenin-labeled PCR product amplified from the capB or capD genes of S. epidermidis 1457 or with a cocktail of probes amplified from the capA, capB, capC, and capD genes. Prehybridization, hybridization, posthybridization, and immunologic detection were performed as described in the technical update accompanying the nonradioactive DNA Labeling and Detection Kit (Roche Applied Science). For immunologic detection, the membrane was incubated with a 1:5,000 dilution of sheep anti-digoxigenin Fab fragments conjugated to alkaline phosphatase. Probe-target hybrids were detected using the chromogenic substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate.
Immuno-dot blot assay.
Surface-attached PGA was released from the cell surface by boiling bacteria for 30 minutes at 100°C or by autoclaving. PGA was then purified by acid precipitation as described (12). Aliquots (3 μl) of the samples were spotted on a nitrocellulose membrane, air-dried, and PGA was detected with anti-PGA antiserum using a scanner and Total Lab Version 2003 software (Nonlinear USA). The assay was calibrated by serial dilution of the most intensive sample. The value detected in the wild-type strain was set to 100% and production values in the other strains were expressed relative to that value. Anti-PGA antiserum (kindly provided by R. Schneerson, National Institute of Child Health & Human Development, NIH, Bethesda, Maryland, USA) was developed against PGA from Bacillus pumilus. For Staphylococcus strains other than S. epidermidis, a strain with a signal higher than that of the background of the S. epidermidis cap mutant was considered a PGA producer.
Construction of an isogenic cap deletion mutant and cap-complementing vector.
To delete capBCAD in S. epidermidis 1457, PCR-amplified regions flanking the cap locus and a spectinomycin resistance cassette were cloned into plasmid pBT2 (30), yielding plasmid pBTcap, which was used for allelic replacement as described (31). The proper integration of the resistance gene marker spc was verified by direct sequencing of the genomic DNA at the borders of the PCR-derived regions. Lack of cap transcript in the cap mutant strain was verified by real-time PCR (data not shown). S. epidermidis 1457 in which capBCAD was deleted was named S. epidermidis cap. To complement for capBCAD in S. epidermidis cap, capBCAD genes were cloned into plasmid pRB474 (30). The resulting plasmid was named pRBcapBCAD.
Purification of PGA and detection of d- and l-glutamic acid.
Cultures were grown in TSB medium supplemented with 1 M NaCl to induce PGA production. PGA was first purified as described above. Then, PGA samples were further purified by ion exchange chromatography using a RESOURCE Q 6 ml column (Amersham Biosciences) on an AKTA Purifier 10 (Amersham Biosciences) and a gradient from 0.2% acetic acid to 0.2% acetic acid/1 M NaCl in 20 column volumes at a flow rate of 5 ml/min. Fractions with positive reaction in an immuno-dot blot were combined, dialyzed against water, lyophilized, resuspended in 6 M HCl, and hydrolyzed at 110°C for 24 hours. Samples were lyophilized again and dissolved in 200 μl of water. Ten microliters of the samples were then injected onto a Chirobiotic T column (Astec) using 0.1% triethylammonium acetate (pH 4.0) in 80% ethanol as elution buffer at a flow rate of 0.4 ml/min. Chromatography was performed using an Agilent 1100 series HPLC connected to a VL Trap mass spectrometer. The extracted ion chromatograms at 146 Da, the mass of glutamic acid, were used to determine the amounts of D- and L-glutamic acid by peak integration in comparison to pure D- and L-glutamic acid.
Isolation of human polymorphonuclear leukocytes and phagocytosis experiments.
Human polymorphonuclear leukocytes (PMNs) were isolated from heparinized venous blood of healthy individuals with a standard method (32). All studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects of NIAID. Cell preparations contained approximately 99% PMNs and all reagents used contained <25.0 pg/ml endotoxin. Phagocytosis of S. epidermidis by human PMNs was analyzed by flow cytometry with a previously described method (32). Briefly, bacteria were cultured to stationary growth phase, washed in PBS, and labeled with fluorescein-5-isothiocyanate for 15 minutes. PMNs (106/100 μl) and bacteria (2 x 107/100 μl) were combined in wells of serum-coated 96-well round-bottom microtiter plates. Plates were incubated for 30 minutes at 37°C and the degree of phagocytosis was determined by flow cytometry.
Peptide bacterial killing assays.
S. epidermidis cultures were harvested, washed with PBS buffer, and resuspended in 10 mM sodium phosphate buffer (pH 7.0). Bacterial killing assays were performed using a final concentration of approximately 105 S. epidermidis cells in each sample. Antimicrobial peptides were dissolved in the following solutions: human ?-defensin 3, 10 mM acetic acid, and LL-37, 10% acetonitrile with 0.1% trifluoroacetic acid. The bacteria were exposed to a range of antimicrobial peptide concentrations (0, 5, 10, 20, 30, and 40 μg/ml). An equal volume of the respective peptide dilution buffer was applied to control samples. Samples were incubated at 37°C for 2 hours and appropriate dilution series of the samples were plated on TSB agar. Survivor S. epidermidis cells were enumerated after 24 hours of incubation at 37°C. The percentage of killed S. epidermidis was calculated using the formula (1 – [CFU peptide / CFUcontrol]) x 100.
Scanning immunoelectron microscopy.
Fifty-microliter aliquots of S. epidermidis cultures were washed with PBS buffer. Cells were resuspended in 200 μl of anti-PGA antiserum and incubated at 37°C with agitation at 400 rpm for 12 hours. Samples were washed with PBS and pellets were subsequently incubated with goat anti-rabbit IgG conjugated with 20 nm of gold (BB International) at 37°C with agitation at 400 rpm for 2 hours. Following antibody labeling, the cell suspensions were attached to coverslips, fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, and post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate. Samples were washed with distilled water, dehydrated in a graded ethanol series, critical-point dried under CO2 with a Bal-Tec model cpd 030 drier (Balzers), mounted on aluminum studs, and sputter-coated with 100 angstroms of chromium in a model IBS/TM200S ion beam sputterer (South Bay Technologies) prior to viewing at 10 kV on a Hitachi S-4500 field emission scanning electron microscope (Hitachi) in backscatter imaging mode.
Murine model of device-related infection.
Female Balb/c mice were used in a model of subcutaneous implanted device-related infection as described (33). Two catheter pieces of 1-cm length were placed under the skin of the dorsum of each animal. CFUs on catheters were counted before insertion and were in the range of 2 x 105 on all implanted catheters. CFU on excised catheters and surrounding tissues were counted after 1 week of infection. All studies were performed in accordance with a protocol approved by the Animal Care and Use Committee of Rocky Mountain Laboratories, NIAID.
Statistics and DNA sequence analysis.
Statistical analysis was performed using GraphPad Prism version 4.0. DNA sequences were compared using Clustal W software.
Acknowledgments
The authors would like to thank Rachel Schneerson for providing anti-PGA antisera, Nevine El Solh for S. epidermidis strains, Aaron B. Carmody for technical assistance, and Donald Gardner, Michael Parnell, and Ralph Larson for help with animal studies.
Footnotes
Nonstandard abbreviations used: PGA, poly--dl-glutamic acid; PIA, polysaccharide intercellular adhesin; PMN, polymorphonuclear leukocyte; TSB, tryptic soy broth.
Conflict of interest: The authors have declared that no conflict of interest exists.
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