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. S. epidermidis, usually a commensal of human skin, may cause septicemia or endocarditis in patients undergoing immunosuppressive therapy, premature newborns, or injection drug users. However, 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. We propose that drugs preventing and/or targeting biofilm formation will be of extraordinary use in anti-staphylococcal therapy, as they will enable the immune system to cope with an infection and increase the efficiency of common antibiotics. 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. Investigation of biofilm formation of Staphylococcus epidermidis has been divided into 4 projects: 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. A low cutoff for orf length was chosen, as one of our main interest was in small peptides assumed to play a major role in biofilm formation. The role of phenol-soluble modulins and similar small peptides in biofilm formation is described below. Importantly, our results demonstrated that a biofilm represents a status of generally reduced metabolism, showing a lower expression of many genes related to """"""""housekeeping"""""""" functions such as those involved in protein synthesis and biosynthetic pathways, for example. 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 and further investigation of specific factors gave 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. We determined decreased expression of the detergent-like peptide delta-toxin and increased expression of the autolysin AtlE involved in initial attachment to hydrophobic surfaces as causes for the observed effect. We are currently expanding the investigation of quorum-sensing influencing biofilm formation by using epidemiological approaches and animal models of infection. Furthermore, the role of quorum-sensing control of biofilm formation is determined using confocal laser scanning microscopy. All of our results demonstrate a novel role of quorum-sensing control of biofilm formation in S. epidermidis as the most important pathogen involved in biofilm-associated infection. 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 to be the second step in biofilm formation. Studies have shown that presence of the ica genes responsible for PIA expression is correlated with catheter infection in humans. ica mutants show a distinctly decreased ability to cause catheter infection in animal models. PIA is a homopolymer of partially de-acetylated N-acetyl-glucosamine units. We investigated the influence of de-acetylation on biofilm formation and on the impact on the effect of the human immune defense. The degree of PIA acetylation determines the charge of PIA. Differences of the charge of PIA, and consequently of bacterial surface charge, are thought to dramatically influence these characteristics. A deletion mutant of the putative de-acetylase gene of S. epidermidis (icaB) was constructed. We have constructed vectors for the over-expression of IcaB and made antisera against IcaB. This allowed us to localize IcaB and propose a new model for IcaB processing to its mature form. We have established novel HPLC-MS-based methods to detect PIA polymers in acetylated and de-acetylated forms and used these methods to investigate the role of IcaB in acetylation of the PIA polymer. Furthermore, the role of PIA in immune evasion was determined. 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 that protects against major mechanisms of the human innate immune system. 4. 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 suggest 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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