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Originally published In Press as doi:10.1074/jbc.M411374200 on October 22, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54881-54886, December 24, 2004
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A Crucial Role for Exopolysaccharide Modification in Bacterial Biofilm Formation, Immune Evasion, and Virulence*

Cuong Vuong, Stanislava Kocianova, Jovanka M. Voyich, Yufeng Yao, Elizabeth R. Fischer{ddagger}, Frank R. DeLeo, and Michael Otto§

From the Laboratory of Human Bacterial Pathogenesis and {ddagger}Microscopy Core Facility, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840

Received for publication, October 5, 2004 , and in revised form, October 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Biofilms play an important role in many chronic bacterial infections. Production of an extracellular mixture of sugar polymers called exopolysaccharide is characteristic and critical for biofilm formation. However, there is limited information about the mechanisms involved in the biosynthesis and modification of exopolysaccharide components and how these processes influence bacterial pathogenesis. Staphylococcus epidermidis is an important human pathogen that frequently causes persistent infections by biofilm formation on indwelling medical devices. It produces a poly-N-acetylglucosamine molecule that emerges as an exopolysaccharide component of many bacterial pathogens. Using a novel method based on size exclusion chromatography-mass spectrometry, we demonstrate that the surface-attached protein IcaB is responsible for deacetylation of the poly-N-acetylglucosamine molecule. Most likely due to the loss of its cationic character, non-deacetylated poly-acetylglucosamine in an isogenic icaB mutant strain was devoid of the ability to attach to the bacterial cell surface. Importantly, deacetylation of the polymer was essential for key virulence mechanisms of S. epidermidis, namely biofilm formation, colonization, and resistance to neutrophil phagocytosis and human antibacterial peptides. Furthermore, persistence of the icaB mutant strain was significantly impaired in a murine model of device-related infection. This is the first study to describe a mechanism of exopolysaccharide modification that is indispensable for the development of biofilm-associated human disease. Notably, this general virulence mechanism is likely similar for other pathogenic bacteria and constitutes an excellent target for therapeutic maneuvers aimed at combating biofilm-associated infection.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exopolysaccharide (EPS)1 is a key component of the biofilm matrix in many biofilm-forming bacteria and may be composed of various sugar polymers (1). It has an important role in immune evasion and tolerance toward antibacterial agents. By far most known EPS molecules are neutral or polyanionic (2). Enzymatic alteration of EPS is believed to significantly change its physico-chemical properties and, thus, biofilm structure. However, particularly in Gram-positive bacteria, EPS-modifying enzymes and the relationship between the composition of EPS and its biological function have remained poorly characterized.

The Gram-positive bacterium Staphylococcus epidermidis is the most prevalent pathogen involved in hospital-acquired infections (3). The costs related to infections caused by S. epidermidis in the hospital setting are enormous and represent a major health care burden. Most infections caused by S. epidermidis occur after the insertion of indwelling devices such as catheters or prosthetic heart valves. In these cases, the ability of S. epidermidis to form biofilms represents the most important virulence determinant (3). In a biofilm, the bacteria are dramatically less susceptible to antibiotic treatment and attacks by innate host defense. For these reasons, S. epidermidis biofilm-associated infections are very difficult to eradicate.

PIA, a homopolymer of {beta}-1,6-linked N-acetylglucosamine (GlcNAc) residues, is located in fibrous strands on the S. epidermidis cell surface, where it serves as an essential factor involved in biofilm formation (4). Importantly, it protects the pathogen from innate host defense (5). PIA production is crucial for virulence in animal infection models (6-8) and is encoded by the ica gene locus, which consists of the icaA, icaD, icaB, and icaC genes (9). IcaA and IcaD form a UDP-GlcNAc-transferase located in the cellular membrane (10). Another putative membrane protein, IcaC, is required for the formation of longer polymers and might be involved in the export of the growing PIA chain (10). PIA represents a very unusual EPS molecule, as some GlcNAc residues become deacetylated, which produces a positive net charge of the polymer. The basis of deacetylation is unclear. PIA is also produced in Staphylococcus aureus and some strains of Escherichia coli (11, 12). Furthermore, gene clusters similar to the ica locus are found in other species of the genus Staphylococcus and in a range of human and plant pathogens, such as Yersinia pestis, Yersinia enterocolitica, Xanthomonas axonopodis, Pseudomonas fluorescens, and Bordetella pertussis (12-14). In Y. pestis, for example, ica homolog-dependent biofilms are crucial for vector transmission from fleas to humans (15). Thus, it is likely that PIA production represents an important virulence determinant in a series of infectious diseases.

Here we describe the role of the IcaB protein in PIA biosynthesis and S. epidermidis pathogenesis. We show that IcaB is located on the S. epidermidis cell surface and involved in the introduction of positive charges in the PIA polymer by deacetylation of GlcNAc moieties. Notably, the presence of deacetylated PIA was essential for biofilm formation, immune evasion, adhesion to epithelial cells, and virulence in an animal model of implant infection.Go


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  		TABLE I
Oligonucleotides used in this study Restriction sites are underlined.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Growth Conditions—The clinical isolate S. epidermidis 1457 (22) was grown in tryptic soy broth medium containing 0.5% of filtered glucose, except for overexpression studies with xylose induction, for which basic medium (1% Tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% K2HPO4) was used. Cultures were incubated at 37 °C with shaking at 140 rpm, unless otherwise noted.

Immuno-dot-blot Assay—To quantify PIA production, corresponding amounts of S. epidermidis cells and culture supernatants were used. Surface-located PIA was extracted by incubating the cells in 0.5 M EDTA, pH 8.0 (final volume, 1:50 of cultures) for 5 min at 100 °C. Staphylococcal supernatants were concentrated about 50-fold by centrifugal filter devices (Amicon Ultrafree-MC, YM-10). 3-µl aliquots of the samples were spotted on a nitrocellulose membrane and air-dried, and PIA was detected using anti-PIA antiserum as described previously (23) and a scanner and Total Lab Version 2003 software (Nonlinear USA, Durham, NC).

Construction of an Isogenic icaB Deletion Mutant, an icaB-complementing Vector, and icaB Overexpression Vectors—To delete icaB in S. epidermidis 1457, PCR-amplified regions flanking the icaB gene and an erythromycin resistance cassette were cloned into plasmid pBT2 (24), yielding plasmid pBT{Delta}icaB, which was used for allelic replacement as described (25). The proper integration of the resistance gene marker ermB was verified by direct sequencing of the genomic DNA at the borders of the PCR-derived regions. S. epidermidis 1457, in which icaB was deleted, was named S. epidermidis {Delta}icaB. To complement for icaB in S. epidermidis {Delta}icaB, the icaB gene under control of the icaA promoter was cloned into plasmid pRB473 (24). The resulting plasmid was named pRBicaB. S. epidermidis {Delta}icaB (pRB473) was used for comparison with the complemented strain S. epidermidis {Delta}icaB (pRBicaB). To overexpress icaB, the icaB gene was amplified using mutagenizing primers introducing BamHI and MluI sites at the 5' and 3' ends, respectively. The PCR product was cleaved with BamHI and MluI and cloned into BamHI/MluI-cleaved vector pTX15, yielding pTXicaB. Plasmids pTXicaB and pTX16 as control were transformed into S. epidermidis {Delta}icaB and wild-type strains.

Analysis of PIA Deacetylation by Size-exclusion Chromatography-Mass Spectrometry (SEC/ESI-MS)—PIA samples for SEC/ESI-MS were obtained as described above. Purified PIA used for preliminary studies was obtained from the supernatant of an S. aureus PIA (PNAG)-over-producing strain (S. aureus MN8m, kindly provided by G. Pier, Harvard Medical School, Boston, MA) and treated for varying times up to 24 h with 12 N HCl to achieve chemical deacetylation. Completely acetylated PIA was separated from other molecules on a Superdex 200 10/300 GL column (Amersham Biosciences), whereas partially or completely deacetylated PIA was separated on a Jordi PolarPac WAX 10,000 Å 300 x 7.8-mm column (Alltech, Deerfiel, IL). All samples were run on both columns. SEC/ESI-MS was performed at a flow rate of 1 ml/min using 0.2% acetic acid on an Agilent 1100 system coupled to a Trap VL mass spectrometer.

Biofilm Assay—In vitro biofilm assays were performed in polystyrene microtiter plates as described previously (23). Briefly, microtiter plates (96 U-button polystyrene wells (Greiner, Longwood, FL)) were incubated at 37 °C for 24 h without shaking. Biofilm formation was made visible by staining S. epidermidis cells with 0.1% safranin (Sigma-Aldrich) for 30 s. S. epidermidis biofilm formation in microtiter wells was quantified using a Safire microtiter plate reader (Tecan, U. S. Inc., Research Triangle Park, NC) with Magellan Version 3.00 software. The reader was set to the multiple read mode (circle pattern, 6 x 6 number of reads), and absorbance was measured at 490 nm.

Human Cell Culture and Adherence Assay—A2058 human skin epithelial cells (ATCC CRL-11147) were used to compare the level of adherence of S. epidermidis wild-type and icaB mutant strains as described (26). Experiments were performed in quadruplicate, and results were expressed for each experiment as the mean number of S. epidermidis cells per A2058 cell ± S.E.

Isolation of Human PMNs and Phagocytosis Experiments—PMNs were isolated from heparinized venous blood of healthy individuals with a standard method (27). All studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, NIAID. Cell preparations contained >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 (27).

Peptide Bacterial Killing Assays—Assays were performed as described previously (5). Briefly, ~105 S. epidermidis cells were exposed to a range of antimicrobial peptide concentrations at 37 °C for 2 h, and an appropriate dilution series of the samples were plated on tryptic soy broth agar. Survivor S. epidermidis cells were enumerated after 24 h of incubation at 37 °C. The percentage of killed S. epidermidis was calculated using the equation (1-(colony-forming unitsH2O2/colony-forming unitsControl)) x 100.

Immunodetection of IcaB—IcaB-specific antisera were developed by Sigma Genosys against a mixture of five synthetic BSA-conjugated peptides from the IcaB sequence. The antisera were blocked with cell extracts of S. epidermidis {Delta}icaB. Immunoblots were incubated for 8 h with blocked anti-IcaB antiserum. An anti-rabbit-IgG-horseradish peroxidase conjugate was used for detection with an ECL system (Amersham Biosciences).

Scanning Electron Microscopy—For ultrastructural preservation of the PIA structure, samples were prepared as described by Fassel and Edmiston (28) with previously described modifications (5).

Mouse Model of Device-related Infection—Female Balb/c mice were used in a model of subcutaneous implanted device-related infection according to Kadurugamuwa et al. (19) and Rupp et al. (7). Two catheter pieces of 1 cm in length were placed under the skin of the dorsum of each animal. Colony-forming units on catheters were counted before insertion and were in the range of 2 x 105 on all implanted catheters. Colony-forming units on excised catheters and surrounding tissues were counted after 1 week of infection.

Statistics—Statistics were performed using GraphPad Prism Version 4.0. (GraphPad Software, Inc., San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IcaB Is Responsible for PIA Deacetylation—In these studies we investigated the mechanism responsible for the formation of deacetylated GlcNAc in PIA and its role in virulence of S. epidermidis. On the basis of sequence similarity to oligosaccharide deacetylases in other organisms, we hypothesized that icaB is involved in the deacetylation of PIA. To determine the role of icaB, we constructed an isogenic icaB mutant of a biofilm-forming, clinical S. epidermidis isolate by allelic replacement with an erythromycin resistance cassette (Fig. 1). Correct insertion of the cassette in the chromosome was confirmed by sequencing of the flanking regions. The absence of the icaB transcript in the icaB mutant and maintained expression of icaC, the only gene located downstream of icaB in the ica operon (Fig. 1), were verified by reverse transcription-PCR (data not shown). PIA deacetylation was analyzed with SEC/ESI-MS. In contrast to previously used protocols (4, 16), this method does not require extensive purification of PIA before analysis, which may lead to precipitation and selective enrichment of specific forms of PIA. PIA from the wild-type strain revealed fragmentation patterns that included ions originating from deacetylated residues (Fig. 2). These ions were not found in PIA from the icaB mutant strain (Fig. 2), indicating that this strain is deficient in PIA deacetylation. In addition, we purified PIA from mutant and wild-type strains and analyzed the degree of deacetylation by a conventional method based on the detection of free amino groups (17). We found that 16.4 ± 9.9% of GlcNAc residues in the wild-type strain were deacetylated, whereas no deacetylation was detected in the icaB mutant strain. These findings indicate that the introduction of deacetylated GlcNAc into PIA occurs by a dedicated mechanism to deacetylate a polymeric PIA precursor. Importantly, our data demonstrate that the IcaB protein is involved in PIA deacetylation. Furthermore, our ESI-MS data provide novel insight into the mechanism of oligosaccharide deacetylases. They indicate that the PIA-deacetylating enzyme modifies GlcNAc residues in PIA in random rather than in a specific distance. This theory is supported by the following observations. First, Glc-NAc oligomer peaks in the wild-type strain appeared in series with varied rather than uniform degrees of deacetylation (Fig. 2). Second, we found fragments of short GlcNAc oligomers that are multiply deacetylated (Fig. 2). For example, the presence of a peak corresponding to 2 glucosamine residues (323 Da) confirms this idea and indicates that the enzyme is capable of deacetylating adjacent GlcNAc residues.



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  		FIG. 1.
Construction of an isogenic icaB mutant of S. epidermidis by allelic replacement. The PIA biosynthetic gene cluster consists of the icaADBC operon and of an adjacent regulatory gene, icaR. In the isogenic mutant strain S. epidermidis {Delta}icaB, the icaB gene of S. epidermidis was replaced by an erythromycin resistance cassette. This cassette lacks a transcriptional terminator and, thus, allows for transcription of icaC located downstream of icaB.

 



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  		FIG. 2.
Analysis of PIA deacetylation by SEC/ESI-MS. Samples of culture filtrates and cell surface fractions of S. epidermidis strains were analyzed by SEC/ESI-MS. Mass spectra averaged over the elution range of PIA are shown. PIA was isolated from the surface of the wild-type strain (WT) and from the culture filtrate of the icaB mutant strain ({Delta}icaB). No PIA was detected on the surface of the icaB mutant strain nor in the culture filtrate of the wild-type strain. Fragmentation of polysaccharide homopolymers results in a series of ESI-MS m/z peaks with a constant mass distance, which is equal to the mass of the monomer (29). Completely acetylated PIA has an expected series of ESI-MS peaks with mass distances of 203 Da, the mass of the GlcNAc moiety (blue arrows). Deacetylation results in additional peaks with a mass defect of multiples of 42 Da, the mass of an acetyl rest (red arrows). PIA from the complemented icaB mutant strain S. epidermidis {Delta}icaB (pRBicaB) produced ESI-MS peaks consistent with normal deacetylation observed in the wild-type strain (not shown).

 
Deacetylation Is Crucial for Attachment of PIA to the Bacterial Cell Surface—Deacetylation introduces positive charges in the otherwise neutral PIA molecule, as free amino groups are exposed that become protonated at neutral and acid pH. We found that the altered chemistry of deacetylated PIA significantly affected its biophysical properties. We have previously shown that PIA is located in fibrous strands on the S. epidermidis surface (5). In contrast to the wild-type strain, PIA from the icaB mutant was released to the culture filtrate (Fig. 3, A and B). Thus, deacetylation is essential for surface localization of PIA. Moreover, completely acetylated PIA from the icaB mutant strain had a much higher tendency to precipitate in aqueous solution compared with partially deacetylated PIA from the wild-type strain. Hence, deacetylation of PIA is important for solubility in an aqueous environment.



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  		FIG. 3.
Subcellular location of PIA and IcaB. A, immuno-dot-blot of PIA. The degree of immunoreactivity with anti-PIA antiserum was measured with a photodetection system and quantitated. The value obtained in the surface sample of the wild-type was set to 100%. Statistics indicated are versus wild-type (for {Delta}icaB), wild-type pRB473 (for {Delta}icaB [pRB473]), and {Delta}icaB pRB473 (for {Delta}icaB [pRBicaB]). Bars represent the mean of eight evaluated dots ± S.E. B, scanning electron microscopy images of S. epidermidis wild-type and isogenic icaB mutant cells. The bar represents 1 µm. C, detection of IcaB by immunoblot in the cell surface protein fraction containing non-covalently bound proteins. IcaB was detected in strains of S. epidermidis and S. epidermidis {Delta}icaB harboring the icaB overexpression plasmid pTXicaB. Plasmid pTX16 is a control plasmid without icaB expression. B and C: WT, S. epidermidis 1457 wild-type strain; {Delta}icaB, isogenic icaB mutant strain.

 
IcaB Is a Surface-bound Protein—We next determined the subcellular location of the IcaB protein using IcaB-specific antisera (Fig. 3C). Notably, IcaB was extracted from the wild-type and icaB-overexpressing strains by boiling cells with sodium dodecyl sulfate, an established method to isolate proteins that are non-covalently attached to the surface of Gram-positive bacteria (18). IcaB was not detected in the culture filtrate or cellular fractions (from which surface-attached proteins were previously released) of these strains nor in any fraction derived from the icaB mutant strain. IcaB had an apparent molecular mass of ~30 kDa, which is in good accordance with the theoretical value of the translational product after cleavage of the putative signal peptide (30.4 kDa). Thus, our data indicate that IcaB is exported and attached to the staphylococcal cell surface by non-covalent interaction. PIA deacetylation, therefore, very likely occurs in the cell surface matrix.

PIA Deacetylation Is Indispensable for Efficient Biofilm Formation and Surface Colonization—To investigate the impact of PIA deacetylation on major virulence mechanisms of S. epidermidis, we first determined colonization of an abiotic surface and human epithelial cells in vitro. Biofilm formation on plastic in vitro was completely abolished in the icaB mutant strain and restored in the complemented mutant (Fig. 4A). Furthermore, adhesion of the icaB mutant to epithelial cells was significantly lower than that of the wild-type (p < 0.0001) and complemented mutant strains (p < 0.0001) (Fig. 4B). These results indicate that PIA deacetylation contributes to efficient colonization of surface matrices relevant to S. epidermidis infection.



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  		FIG. 4.
PIA deacetylation is essential for surface colonization by S. epidermidis. A, biofilm formation on polystyrene microtiter wells. Bars represent the mean of 16 wells ±S.E. B, adhesion to human skin epithelial cells. Bars represent the mean of four independent experiments ± S.E. A and B, statistics indicated are versus wild-type (for {Delta}icaB), wild-type pRB473 (for {Delta}icaB [pRB473]), and {Delta}icaB (pRB473) (for {Delta}icaB [pRBicaB]). WT, S. epidermidis 1457 wild-type strain; {Delta}icaB, isogenic icaB mutant strain.

 
PIA Deacetylation Is Required for Immune Evasion—Immune evasion is believed to be a key virulence mechanism of S. epidermidis. This notion is underscored by the fact that virulence of this pathogen is based on prolonged persistence in relative silence rather than on aggressive virulence factors (3). We have recently shown that PIA protects S. epidermidis from major components of innate host defense (5). To test if deacetylation of PIA plays a major role in immune evasion processes, we evaluated phagocytosis by human neutrophils and resistance to antibacterial peptides of S. epidermidis icaB mutant and wild-type strains. Phagocytosis by human neutrophils was significantly increased at all time points in the icaB mutant strain (Fig. 5A). Inasmuch as neutrophils are the critical effector cells in human innate host response to bacterial infections, the data indicate that PIA deacetylation is important for immune evasion. Furthermore, the icaB mutant strain showed significantly higher sensitivity to the effects of LL-37 and human {beta}-defensin-3, two representative antibacterial peptides from human skin and neutrophil specific granules (Figs. 5, B and C). These findings indicate that IcaB plays a key role in protecting S. epidermidis from the microcidal effects of innate host defense components. Remarkably, the values for the icaB mutant were in the same range as those that we have obtained earlier for a mutant of the same strain devoid of PIA (5). Thus, the role of PIA in immune evasion is largely dependent on deacetylation of GlcNAc residues.



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  		FIG. 5.
PIA deacetylation is important for resistance to innate host defense. A, resistance to PMN phagocytosis. Bars represent the mean of five independent experiments ± S.E. B and C, resistance to human cationic antibacterial peptides. Bars represent the mean of five independent experiments ± S.E. A-C, statistics indicated are versus wild-type (for {Delta}icaB) and {Delta}icaB (for {Delta}icaB [pRBicaB]). WT, S. epidermidis 1457 wild-type strain; {Delta}icaB, isogenic icaB mutant strain.

 
An Isogenic icaB Mutant Shows Impaired Persistence in a Model of Device-related Infection—Persistence of surface-attached bacterial agglomerations, particularly on indwelling medical devices, is considered the main basis of S. epidermidis virulence. To investigate if deacetylation of PIA affects persistence of S. epidermidis during infection of indwelling medical devices, we compared S. epidermidis wild-type and icaB mutant strains in a murine model of subcutaneous catheter infection (Fig. 6). Similarly to described models of device-related infection with S. epidermidis (7, 19), the model includes the insertion of a piece of medical tubing under the skin of the dorsum of the animals. After 1 week of infection, bacteria on the implant were counted. Significantly fewer bacteria were detected at the infection site in the mice infected with the icaB mutant compared with those infected with the wild-type (p = 0.0142). These results indicate that deacetylation of PIA represents an important virulence mechanism during S. epidermidis device-related infection.



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  		FIG. 6.
PIA deacetylation is a key factor during device-related infection by S. epidermidis. Female balb/c mice were infected with equal amounts of bacteria attached to medical tubing. After 1 week of infection, bacteria on the tubing were counted. WT, S. epidermidis 1457 wild-type strain; {Delta}icaB, isogenic icaB mutant strain.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
EPS is one of the main constituents of the biofilm matrix (1). Investigation of the role of EPS in biofilm formation has focused on Gram-negative bacteria, among which EPS of Vibrio cholerae (20) and the Pseudomonas aeruginosa exopolymer alginate have gained much attention (1). Less is known about EPS in gram-positives. Furthermore, there is limited information about the bacterial mechanisms involved in the biosynthesis and modification of EPS components. Specifically, we lack knowledge about how the structure of EPS influences the virulence of human pathogens.

In this report, we investigated how enzymatic modification of EPS in S. epidermidis influences biofilm formation and virulence. SEC/ESI-MS analysis revealed that PIA in an icaB deletion mutant was completely acetylated. The introduction of deacetylated GlcNAc into PIA, therefore, occurs by a dedicated mechanism to deacetylate a polymeric PIA precursor rather than by incorporation of UDP-glucosamine as an alternative substrate of the IcaAD UDP-GlcNAc-transferase. Given that IcaB also has similarity to oligosaccharide deacetylases, our results strongly suggest that the protein is the PIA-deacetylating enzyme. However, this remains to be demonstrated by conversion of acetylated PIA with purified IcaB. Furthermore, our data indicate that (i) deacetylation sites in wild-type PIA are randomly distributed and (ii) deacetylation by the surface-attached IcaB occurs in the cell surface matrix, in contrast to the other steps in PIA biosynthesis, which take place inside the cell (10). Based on our results, we propose a model of PIA biosynthesis, which is shown in Fig. 7.



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  		FIG. 7.
Model of PIA biosynthesis. The biosynthesis of PIA in S. epidermidis occurs in three steps. 1) IcaA adds GlcNAc moieties from UDP-GlcNAc to the growing PIA chain. The IcaA-transferase requires IcaD for full activity (10). 2) Presumably, the nascent PIA chain is then exported by IcaC. Although no experimental evidence exists for the transport function of IcaC, this assumption is supported by the fact that IcaC is predicted to be a membrane protein, and an icaC mutant strain produces PIA oligomers of only ~15 residues in length (10). In this model, export of the nascent PIA chain would be required for the formation of longer polymers. 3) Results from this study indicate that PIA is deacetylated after export to introduce positive charges, which are crucial for its biological function. The IcaB protein is involved in this process and likely represents the PIA deacetylase. Surface-attached cationic PIA functions to change the bacterial cell surface charge, which for example leads to increased resistance against cationic antibacterial peptides.

 
Comparison of the icaB mutant with the wild-type strain allowed investigation of the biological role of deacetylation and the introduction of positive charges in the PIA molecule. The icaB mutant strain had a strong defect in colonization and resistance against innate host defense, which was reflected by significantly decreased virulence in a mouse model of catheter-related infection. Notably, many of the biological effects seen in an icaB mutant strain may be related to the fact that non-deacetylated PIA was not retained on the S. epidermidis cell surface. Deacetylated PIA is cationic due to free amino groups (theoretical pK = 6.9) that become protonated at neutral or acidic pH. The cationic character of PIA presumably is essential for the attachment of PIA to the negatively charged bacterial cell surface. By PIA production, the bacteria, thus, can efficiently change the electrostatic properties of their cell surface. This is very likely responsible for the resistance against antibacterial peptides, which are usually cationic (5). In comparison to other known EPS components, which are almost all neutral or negatively charged, PIA might, therefore, be exceptionally well suited for its role in biofilm formation and immune evasion.

In conclusion, we demonstrate that enzymatic modification of PIA by deacetylation is crucial for biofilm formation, immune evasion, and virulence of S. epidermidis. To our knowledge this is the first report to show that modification of a specific EPS component by a dedicated mechanism affects virulence and pathogen success in biofilm-associated infection. Because PIA and homologues of the ica gene cluster have been discovered in a variety of pathogenic and biofilm-forming microorganisms, PIA deacetylation likely represents a widespread mechanism of virulence. Furthermore, PIA biosynthesis is a potential target for anti-biofilm drug development (21). Targeting IcaB function might be of particular interest, as we demonstrate herein that IcaB has a crucial function in biofilm formation and virulence. In addition, IcaB represents the only extracellular protein of the ica system, making it most easily accessible for therapeutics designed to control S. epidermidis infection on indwelling devices.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, NIAID, The National Institutes of Health, 903 S. 4th St., Hamilton, MT 59840. Tel.: 406-363-9283; Fax: 406-375-9677; E-mail: motto{at}niaid.nih.gov.

1 The abbreviations used are: EPS, exopolysaccharide; SEC/ESI-MS, size-exclusion chromatography-mass spectrometry; PMN, polymorpho-nuclear leukocytes. Back


   ACKNOWLEDGMENTS
 
We thank Gerald Pier, Harvard Medical School, for providing PIA/PNAG isolated from S. aureus MN8m, Aaron Carmody for technical assistance, and Donald Gardner, Michael Parnell, and Ralph Larson for help with animal studies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Watnick, P., and Kolter, R. (2000) J. Bacteriol. 182, 2675-2679[Free Full Text]
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