The gram-positive bacterium Staphylococcus epidermidis is the most common pathogen in hospital-acquired infections. The costs related to infections caused by Staphylococcus epidermidis in the hospital setting are enormous (more than 1 billion dollars/year) 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. In a biofilm, the bacteria are dramatically less susceptible to antibiotic treatment and to attacks by human immune defenses. For these reasons, S. epidermidis infections are very difficult to eradicate. To provide the scientific basis for the development of drugs interfering with biofilm formation, we are investigating the molecular biology, biochemistry, and epidemiology of biofilm formation in S. epidermidis. This includes studying specific factors contributing to biofilm formation, their regulation, and the interaction of biofilm-forming S. epidermidis strains with the host. : 1. Microarray analysis of gene expression in the biofilm state Gene expression profiling in a biofilm compared to planktonic cells was determined to investigate the specific physiological status of cells in a biofilm. To this end, we analyzed the S. epidermidis genome sequence published by TIGR, annotated the genome, and constructed a microarray comprising all putative genes with a length of > 60 bp. Importantly, our results demonstrated that a biofilm represents a status of generally reduced metabolism. Interestingly, genes responsible for defense mechanisms tended to be expressed at higher levels in a biofilm. Cells in a biofilm are known to be protected against attacks by the human innate immune system and against antibiotics, but the basis for this protection is not well understood. Our microarray data give valuable insight into the mechanism of protection in a biofilm. 2. Control of biofilm formation by global regulatory systems We focused our research on the role of the agr quorum-sensing system in biofilm formation. We could show that agr mutants show increased biofilm formation in S. epidermidis, demonstrating a negative influence of quorum-sensing on biofilm formation in this organism. As a consequence, an isogenic mutant of the agr quorum-sensing system showed increased colonization of indwelling medical devices in an animal infection model. Furthermore, agr mutants were found to occur naturally and significantly more frequently among strains isolated from infection of indwelling medical devices. Our results demonstrate a novel role for quorum-sensing control of biofilm formation in S. epidermidis. Our results also help to re-evaluate the proposed use of so-called quorum-sensing blockers as potential therapeutics against staphylococcal infection. 3. PIA PIA is the main determinant responsible for intercellular adhesion, which is considered the second step in biofilm formation. We detected a novel role of PIA in immune evasion. We found that PIA protects against phagocytosis and killing by human PMNs and also protects against cationic and anionic antibacterial peptides. PIA is the first defined factor to be found in S. epidermidis that protects against major mechanisms of the human innate immune system. PIA is a homopolymer of partially de-acetylated N-acetyl-glucosamine units. The basis and biological function of deacetylation is unknown. We could demonstrate that a protein, IcaB, is localized on the S. epidermidis surface and is involved in PIA deacetylation, most likely representing the PIA deacetylase. Of note, deacetylation of PIA was crucial for biofilm formation, tissue colonization, immune evasion processes, and virulence in an animal infection model. IcaB is the first factor found that modifies bacterial exopolysaccharide to ensure patthogen success in biofilm-associated infection. 4. Role of a poly-gamma-glutamate capsule in S. epidermidis We demonstrated that S. epidermidis, as the only human pathogen other than Bacillus anthracis, produces a capsule-like structure of poly-gamma-glutamate (PGA) to shelter the bacteria from phagocytosis. Additionally, PGA protected S. epidermidis from antibacterial peptides of human skin and high salt concentration. PGA in S. epidermidis thus has a hitherto unique role in facilitating survival in both the natural habitat of the bacteria and during infection. In contrats to PIA, which is only produced by some S. epidermidis strains, PGA was synthesized by all tested strains of commensal and clinical origin, representing the first widespread mechanism of immune evasion discovered in S. epidermidis. Importantly, an animal model indicated a crucial role for PGA in S. epidermidis device-related infection, suggesting that PGA represents an excellent target for therapeutic maneuvers aimed at treating disease caused by S. epidermidis. 5. Phenol-soluble modulins (PSM): role in immune modulation and biofilm formation PSMs are small, amphipathic peptides with strong immuno-modulatory properties. We have expressed the individual peptide components of PSM and determined their pro-inflammatory capacities in collaboration with S. Klebanoff (University of Washington). We found that PSM production is wide-spread and that the relative proportion of the individual components is constant. We determined quorum-sensing control of the production of PSM and found an extremely tight regulation of PSM production by the agr quorum-sensing system, resulting in the complete absence of PSM and PSM-like peptides in agr mutants. We propose that quorum-sensing control of PSM production helps to provoke a well-balanced level of inflammation that contributes to bacterial survival. Production of PSM seems to significantly determine the pro-inflammatory capacity of S. epidermidis, as they are tightly regulated by quorum-sensing and quorum-sensing mutants also showed a dramatic reduction of inflammatory properties such as production of cytokines and induction of PMN chemotaxis. In our microarray analysis of gene expression in biofilms, we found that PSMs were dramatically down-regulation in biofilms. We further investigated the role of these peptides in biofilm formation using over-expressed and purified peptides. Our results suggest an important role of the PSM peptides not only as immuno-modulatory compounds, but also in influencing biofilm structure, possibly in the not yet well understood process of detachment of cell clusters from a biofilm, a process believed to be of major importance to the spread of an infection in vivo.
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