Blackwell Science, LtdOxford, UKCMICellular Microbiology1462-5814Blackwell Publishing Ltd, 200463269275Original ArticleC. Vuong et al.PIA protects from phagocytosis and antibacterial peptides
Cellular Microbiology (2004) 6(3), 269–275
doi:10.1111/j.1462-5822.2004.00367.x
Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system Cuong Vuong,1 Jovanka M. Voyich,1 Elizabeth R. Fischer,2 Kevin R. Braughton,1 Adeline R. Whitney,1 Frank R. DeLeo1 and Michael Otto1* Laboratories of 1Human Bacterial Pathogenesis and 2 Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th Street, Hamilton, MT 59840, USA.
The skin commensal and opportunistic pathogen Staphylococcus epidermidis is the leading cause of nosocomial and biofilm-associated infections. Little is known about the mechanisms by which S. epidermidis protects itself against the innate human immune system during colonization and infection. We used scanning electron microscopy to demonstrate that the exopolysaccharide intercellular adhesin (PIA) resides in fibrous strands on the bacterial cell surface, and that lack of PIA production results in complete loss of the extracellular matrix material that has been suggested to mediate immune evasion. Phagocytosis and killing by human polymorphonuclear leucocytes was significantly increased in a mutant strain lacking PIA production compared with the wild-type strain. The mutant strain was also significantly more susceptible to killing by major antibacterial peptides of human skin, cationic human b-defensin 3 and LL37, and anionic dermcidin. PIA represents the first defined factor of the staphylococcal biofilm matrix that protects against major components of human innate host defence.
only in immunocompromised individuals or after damage to the epithelium. Most infections with S. epidermidis occur on indwelling medical devices and typically involve the formation of biofilms (Vuong and Otto, 2002). A biofilm represents a structured, high-density population of cells embedded in a heterogeneous matrix, which protects sequestered bacteria from antibiotics and the effects of the human immune system (Costerton et al., 1999). Although this general picture of biofilm-mediated protection is widely accepted, the specific factors and mechanisms contributing to protection from innate host defence are mostly unknown (Stewart and Costerton, 2001). In S. epidermidis, the extracellular matrix, often called ‘slime’, has been shown to mediate protection against polymorphonuclear leucocyte (PMN) phagocytosis (Johnson et al., 1986). However, the antiphagocytic properties have not been attributed to a specific molecule. Staphylococcus epidermidis produces an extracellular polysaccharide named polysaccharide intercellular adhesin (PIA), which is a positively charged homopolymer of b-1,6-linked N-acetylglucosamine (NAG) residues (Mack et al., 1996). PIA is produced by the ica gene cluster, which comprises the icaA, icaD, icaB, icaC and icaR genes (Heilmann et al., 1996a). It is an essential factor for staphylococcal biofilm formation and causes haemagglutination and bacterial aggregation (Heilmann et al., 1996a; Mack et al., 1999). PIA contributes significantly to virulence in animal models of catheter infection (Rupp et al., 1999a,b). In an epidemiological study, the presence of the genes responsible for PIA production correlated significantly with infection originating from indwelling medical devices (Galdbart et al., 2000). These previous studies led us to the hypothesis that PIA is involved in the protection against innate host defences.
Introduction
Results
Staphylococcus epidermidis is a commensal microorganism of human skin and the most frequent cause of hospital-acquired infections. S. epidermidis causes infection
PIA is an integral and essential factor of the extracellular matrix of S. epidermidis
Summary
Received 4 September, 2003; revised 21 November, 2003; accepted 25 November, 2003. *For correspondence. E-mail
[email protected]; Tel. (+1) 406-363-9283; Fax (+1) 406-3759677.
Previous macroscopic studies have shown that PIA is essential for intercellular adhesion and biofilm formation (Heilmann et al., 1996a). However, little is known about the subcellular distribution of PIA and its role in the formation of extracellular matrix at a submacroscopic level.
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Fig. 1. Immuno dot-blot of PIA production. PIA produced in S. epidermidis wild-type and ica– mutant strains was detected on the cell surface (sf) and in the supernatant (sn) of cultures with an immuno dot-blot using PIA-specific antiserum.
We first determined PIA content in subcellular fractions of S. epidermidis wild-type and ica– mutant strains to determine whether PIA is retained on the cell surface in the wild-type strain and to confirm its absence in the mutant strain (Fig. 1). As expected, the wild-type strain
produced PIA, which was located on the cell surface and not in the cell supernatant. Moreover, PIA was not produced by the ica– mutant strain (Fig. 1). We note that there was decreased intercellular adhesion and clumping in the ica– mutant, consistent with previous studies (Heilmann et al., 1996b; data not shown). We next used scanning electron microscopy (SEM) to visualize ultrastructural differences between the wild-type and mutant strains (Fig. 2). Importantly, we discovered that wild-type S. epidermidis produced extracellular fibrous material not present on the ica– mutant (Fig. 2A and C). We also performed immunoelectron microscopy and labelled PIA on the wild-type and mutant strains (Fig. 2B and D). PIA co-localized specifically with the fibrous material on the bacterial cell surface and is therefore an integral component of that structure (Fig. 2B and D). Collectively, our results demonstrate that PIA is an essential, surfacelocated component of the S. epidermidis extracellular matrix.
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Fig. 2. Ultrastructural analysis of S. epidermidis. Scanning electron and immunoelectron microscopy images of the wild-type (A and B) and ica– mutant (C and D) strains of S. epidermidis. PIA was immunogold labelled using aPIA-antisera (B and D). Magnification 25 000¥. The scale bar represents 0.6 mm. © 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 269–275
PIA protects from phagocytosis and antibacterial peptides 271 PIA increases resistance to cationic and anionic human antibacterial peptides Antibacterial peptides such as cathelicidin/hCAP18 (LL-37), human b-defensin 3 (hBD3) and dermcidin are secreted by skin epithelial cells or sweat glands (Turner et al., 1998; Harder et al., 2001; Schittek et al., 2001). Thus, these peptides represent the first form of innate host defence against bacterial infection. In addition, cathelicidins and defensins are part of the oxygen-independent bactericidal mechanism used by human PMNs (Schroder, 1999). To determine whether PIA produced by S. epidermidis confers resistance to antibacterial peptides, we tested the susceptibility of the wild-type and ica– mutant strains to LL-37, hBD3 and dermcidin (Fig. 3). All peptides had significantly higher bactericidal activity towards the ica– mutant strain at physiological salt concentration and mildly acid pH, resembling the conditions on human skin (Fig. 3). Importantly, these findings demonstrate that PIA protects S. epidermidis from the microbicidal effects of antibacterial peptides (Fig. 3). Although our finding that these peptides had significant antistaphylococcal activity is consistent with previous studies (Harder et al., 2001), these data are the
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Fig. 3. Killing of S. epidermidis by LL-37, dermcidin and hBD3. Antibacterial activity of each peptide (hBD3, A and D; LL-37, C and E; dermcidin, C and F) was determined by incubating wild-type and ica– mutant strains of S. epidermidis with the indicated concentrations of peptide for 2 h at 37∞C in 10 mM phosphate buffer, pH 6.5/150 mM NaCl (left column, A–C) or 10 mM phosphate buffer, pH 7.0 (right column, D–F). S. epidermidis survival was determined with the formula 1 – (cfuH2O2/cfuControl) ¥ 100. Results are the mean ± SD of six measurements. © 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 269–275
first to demonstrate that each kills S. epidermidis. We previously identified mechanisms based on electrostatic repulsion used by Staphylococcus aureus to protect against antibacterial peptides, namely D-alanylation of teichoic acids and incorporation of a specific cationic phospholipid in the cellular membrane (Peschel et al., 1999; 2001). Electrostatic repulsion may also contribute to PIA-mediated protection against the cationic peptides LL-37 and hBD3. This assumption is supported by the finding that the efficacies of hBD3 and LL-37 are higher at low salt conditions, which are favourable to electrostatic repulsion, than those of anionic dermcidin (Fig. 3D–F). On the other hand, PIA also protected against anionic dermcidin with an increased level of protection detected at physiological compared with low salt concentration. These findings, together with the anionic nature of dermcidin, suggest that the mechanism of protection towards dermcidin is not based on electrostatic repulsion. The observed increased protection against dermcidin is of special interest, as the anionic characteristics of dermcidin are very unusual for an antibacterial peptide. Anionic dermcidin might be specifically targeted against microbial factors that confer resistance against the more common cationic antibacterial peptides (Peschel, 2002). Its microbicidal activity appears to be favourably adapted to conditions of human skin (i.e. high salt), which represents the natural habitat of S. epidermidis and the environment in which dermcidin functions (compare conditions with and without NaCl in Fig. 3C and F). In providing protection not only against cationic peptides, but also against dermcidin, PIA seems to be suited specifically to combat the antibacterial peptides found on human skin. PIA inhibits phagocytosis and killing by human PMNs To determine whether PIA facilitates evasion of cellular components of innate host defence, we compared phagocytosis and killing of the wild-type and ica– mutant strains of S epidermidis by human PMNs (Fig. 4). Compared with the wild-type strain, phagocytosis of the ica– mutant was significantly increased (12.2 ± 7.2% increase, P < 0.001, n = 5), demonstrating that PIA contributes significantly to protection against PMN phagocytosis (Fig. 4A). Consistent with that finding, human PMNs killed ª 18% more of the ica– mutant strain (42.6 ± 9.7% and 60.5 ± 15.5% killed for the wild-type and mutant strains respectively; P = 0.04, n = 6) (Fig. 4B). The varied susceptibilities of these two strains to PMN killing probably results from the observed differences in phagocytosis (Fig. 4A) and/or effects of antibacterial peptides (Fig. 3), as susceptibility to hydrogen peroxide, a key proximal reactive oxygen species produced by PMNs, did not differ between wildtype and ica– mutant strains (Fig. 4C).
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results indicate that PIA inhibits killing of S. epidermidis by skin-associated antibacterial peptides and human PMNs. PIA probably serves to sequester bacteria and associated proinflammatory products, acting as a mechanical barrier to block the effects of antibacterial peptides and inhibit PMN phagocytosis. Electrostatic repulsion between cationic antibacterial peptides and PIA probably also contributes to the protective effect of PIA. It is tempting to speculate that the specific characteristics of PIA contribute to the fact that S. epidermidis is the predominant microbe on human skin and in nosocomial infections. Our findings underline the importance of PIA as a virulence factor of S. epidermidis that is involved in the formation of biofilms and protection against innate host defence. Of note, PIA is the first exopolymer of the S. epidermidis cell surface shown to mediate such protection. The ica genes responsible for the production of PIA in other staphylococcal species have been identified (Allignet et al., 2001), although they have yet to be implicated in evasion of host defence. Several other bacterial pathogens express genes homologous to those within the S. epidermidis ica gene cluster, including hms of Yersinia pestis, which is also involved in biofilm formation (Darby et al., 2002). Thus, it is likely that production of PIA represents a general mechanism used by bacteria to protect against the human innate immune system. Experimental procedures Bacterial strains and growth conditions
Fig. 4. PIA promotes S. epidermidis resistance to phagocytosis and killing by human PMNs. A. Phagocytosis of wild-type and ica– mutant strains of S. epidermidis by human PMNs. Results are the mean ± SD of five experiments. B. Killing of S. epidermidis by human PMNs. At 2 h, PMNs were lysed and S. epidermidis were plated on tryptic soy agar. Percentage S. epidermidis killed was calculated using the equation 1 – (cfuPMN+/cfuPMN–) ¥ 100. Results are the mean ± SD of six experiments. C. Sensitivity of S. epidermidis to hydrogen peroxide. S. epidermidis parental wild-type and ica– mutant strains were incubated with varied concentrations of H2O2 for 1 h at 37∞C. S. epidermidis killed was determined by cfu counting and calculated by the formula 1 – (cfuH2O2/cfuControl) ¥ 100.
Discussion It has remained unclear until now how S. epidermidis evades destruction by the human innate immune system during colonization and infection. We demonstrate here that PIA is essential for the formation of extracellular material, which protects against destruction from critical components of the innate immune response. Specifically, our
Staphylococcus epidermidis 1457 and its isogenic ica– mutant S. epidermidis M10 (ermR) (Mack et al., 1994) were grown in tryptic soy broth (TSB) supplemented with 0.5% glucose. Bacterial cultures for each experiment were inoculated from precultures grown overnight at a dilution of 1:100 and incubated at 37∞C with shaking at 120 r.p.m. for 16 h, unless otherwise noted. Erythromycin was used at a final concentration of 5 mg ml-1 to select for S. epidermidis M10 in precultures.
Production of aPIA antiserum Staphylococcus epidermidis cultures were harvested by centrifugation at 3000 g, and pellets were washed with phosphatebuffered saline (PBS buffer: 10 mM sodium phosphate, pH 7.0, 150 mM NaCl). Surface-associated PIA was extracted by incubating the cells in 0.5 M EDTA, pH 8.0 (final volume: 1:50 of bacterial cultures) for 5 min at 100∞C. PIA-containing extracts were dialysed against distilled water for 24 h and subsequently digested with DNase (0.5 mg ml-1 final concentration; Sigma), RNase (0.5 mg ml-1 final concentration; Sigma), lysostaphin (0.5 mg ml-1 final concentration; Biosynexus) and lysozyme (0.5 mg ml-1 final concentration; Sigma) at 37∞C for 16 h, followed by incubation with proteinase K (4 mg ml-1 final concentration; Qiagen) at 37∞C for 16 h. Samples were centrifuged at 28 000 g at 4∞C for 30 min. Clarified supernatants were con© 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 269–275
PIA protects from phagocytosis and antibacterial peptides 273 centrated about fivefold by centrifugal concentrators (Amicon Centriprep YM-10; Millipore) and injected in 10 ml aliquots on to a HiLoad 26/60 Superdex 200 gel filtration column (Amersham Pharmacia Biotech). Sodium phosphate (20 mM), pH 7.0, containing 150 mM sodium chloride was used as a buffer at a flow rate of 3 ml min-1. Most PIA eluted shortly after the exclusion volume. PIA-containing fractions were dialysed against water, lyophilized, dissolved in concentrated hydrochloric acid, neutralized with sodium hydroxide and buffered by 100 mM sodium phosphate buffer, pH 7.0. A solution containing 2 mg of PIA was used to produce rabbit antisera using a standard protocol (Sigma Genosys). To block non-specific binding, aPIA antiserum was diluted 1:100 in TBS (Tris-buffered saline: 10 mM Tris-HCl, pH 7.4, 150 mM NaCl) and incubated with several extracts isolated from S. epidermidis M10 for 16 h with gentle shaking. Precipitated material was sedimented by centrifugation (30 min, 28 000 g, 4∞C), and 1 mM sodium azide was added to the clear supernatant used for further investigation. The following extracts of S. epidermidis M10 were used to block the aPIA antiserum: (i) extract isolated by boiling cells with 0.5 M EDTA for 5 min at 100∞C; (ii) extract isolated by boiling cells in 1% SDS for 5 min at 100∞C; (iii) extract isolated from cells treated with lysostaphin; (iv) crude cell extract prepared by breaking cells with glass beads; and (v) extract obtained from culture medium precipitated by trichloroacetic acid. Extracts were prepared from 50 ml of S. epidermidis M10 cultures or from 200 ml of bacterial supernatant respectively.
the Institutional Review Board for Human Subjects, NIAID. Cell preparations contained >99% PMNs, and all reagents used contained
