In the past year, we have focussed on mechanisms that determine the interaction of staphylococci with innate host defense: the contribution of the global regulators agr and luxS, and the elucidation of the biological role of the phenol-soluble (PSM peptides).? ? agr ?We could show that in addition to its role in biofilm formation, quorum-sensing control plays a crucial role in the interaction with innate host defense. Specifically, quorum-sensing had a significant impact on bacterial resistance to antimicrobial peptides and reactive oxygen species. To gain a better understanding of the basis of the observed phenotype, the agr quorum-sensing regulon of S. epidermidis was characterized by a genome-wide analysis of gene expression. Taken together, the results of this study indicate that quorum-sensing regulation in staphylococci has important, previously unknown functions that contribute to protection from mechanisms of human innate host defense?and, therefore, to the pathogen?s survival in the human host.? ? luxS ?The luxS gene is responsible for the biosynthesis of autoinducer-2 (AI-2), a quorum-sensing signal that is well conserved among many bacteria. Whether staphylococci use AI-2 signaling and which phenotypes are influenced by AI-2-dependent regulation in staphylococci is unknown. We constructed an isogenic luxS mutant strain of a biofilm-forming clinical isolate of S. epidermidis and, in collaboration with Dr. Gao?s group in Shanghai, demonstrated that luxS signaling appears to be functional in S. epidermidis. The mutant strain showed increased biofilm formation in vitro and enhanced virulence in a rat model of biofilm-associated infection. Enhanced production of the biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA) in the mutant strain is presumably the major cause of the observed phenotype. Inasmuch as deletion of agr or luxS both result in increased formation of biofilms, our studies indicate a general scheme of quorum-sensing regulation of biofilm development in staphylococci.? ? Novel PSMs in S. epidermidis - Detection and analysis of PSMs in S. aureus. Using LC-MS, we were able to elucidate the PSM peptide compositions of S. aureus and S. epidermidis. PSMs form amphipathic alpha helices, which is reflected by their surfactant-like behavior and strong interaction with reversed phase columns. For the 6 PSM peptides of S. epidermidis we demonstrated a helicity using circular dichroism studies. Notably, PSM production was high in the hyper-virulent community-acquired MRSA strains MW2, LAC and MnCop compared to standard hospital-acquired strains (COL, MRSA252). The high virulence in animal infection models and extraordinary success of these strains in causing disease even in healthy adults has not yet been linked conclusively to specific virulence factor. A recent study performed in collaboration with Dr. DeLeo ruled out the possibility that the Panton-Valentine leukocidin (PVL), which has been regarded as a main contributor to the virulence of CA-MRSA, contributes to the pathogenicity of those strains. In contrast, our results suggest that the PSMs of S. aureus have considerable pro-inflammatory capacity and might significantly contribute to the virulence of CA-MRSA.? ? The role of PSMs in biofilm development. Based on previous preliminary results and the surfactant-like properties of the PSM peptides, we hypothesize that PSM peptides play an important role during biofilm maturation, which most likely consists in biofilm structuring and detachment. These processes are vital for the bacteria, as they introduce fluid channels that deliver nutrients to deeper layers of the biofilm, and cause dissemination during biofilm-associated infection. To verify this hypothesis and address the specific roles of PSM peptides in biofilm development, we had all PSM peptides synthesized and constructed expression vectors for the single PSM peptides of S. epidermidis and S. aureus. Using confocal laser scanning microscopy, we showed that an agr deletion strain, which has a pleiotropic change in gene expression compared to the wild-type strain and does not produce detectable amounts of any PSM, forms a more compact and thicker biofilm than the wild-type strain and shows less channel formation. Importantly, a PSM beta deletion strain shows a phenotype, which is almost as pronounced as in the agr deletion strain, suggesting that the PSM beta peptides are crucial in forming the specific structuring seen in the wild-type biofilm and are main effectors of the quorum-sensing influence on biofilm development.

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National Institute of Allergy and Infectious Diseases (NIAID)
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Dastgheyb, Sana; Parvizi, Javad; Shapiro, Irving M et al. (2015) Effect of biofilms on recalcitrance of staphylococcal joint infection to antibiotic treatment. J Infect Dis 211:641-50
Otto, Michael (2011) Panton-Valentine leukocidin antibodies for the treatment of MRSA skin infections? Expert Rev Anti Infect Ther 9:389-92
Otto, Michael (2010) Staphylococcus colonization of the skin and antimicrobial peptides. Expert Rev Dermatol 5:183-195
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