Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.

Two newly discovered immune modulators, chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS)and staphylococcal complement inhibitor (SCIN), cluster on the conserved 3 end of ␤-hemolysin (hlb)-converting bacteriophages (␤C-s). Since these ␤C-s also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3 end of ␤C-represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a ␤C-with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5-to-3 orientation and on the 3 end of a ␤C-. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different ␤I-s. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of ␤-hemolysin-converting bacteriophages.Recently, we described two new innate immune modulators of Staphylococcus aureus: staphylococcal complement inhibitor (SCIN) and chemotaxis inhibitory protein of S. aureus (CHIPS). SCIN is a C3 convertase inhibitor, blocking the formation of C3b on the surface of the bacterium and the ability of human neutrophils to phagocytose S. aureus (25). CHIPS is a bacterial chemokine receptor modulator that specifically binds two chemokine receptors. CHIPS attenuates the response of the C5a receptor (C5aR) as well as the formylated peptide receptor of human neutrophils. This results in inhibition of neutrophil chemotaxis and activation in response to C5a and formylated peptides (6,10,20,21). Both SCIN and CHIPS are important virulence factors that protect S. aureus from innate immune defense systems.The SCIN (scn) and CHIPS (chp) genes are found in 6/7 and 3/7 sequenced S. aureus strains, respectively. Analyses of these sequenced genomes showed that both scn and chp are carried by ␤-hemolysin (hlb)-converting bacteriophages (␤C-s), formerly known as double-and triple-converting phages. Doubleconverting bacteriophages were earlier described to negatively convert ␤-hemolysin expression but simultaneously introduce the staphylokinase (SAK) gene. Triple conversion leads to ␤-hemolysin-negative and SAK-and staphylococcal enterotoxin A (SEA)-positive strains. 13 and 42 are examples of, respectively, double-and triple-converting bacteriophages. Both 13 and ...

Panton-Valentine Leukocidin (PVL) is a staphylococcal bicomponent pore-forming toxin linked to severe invasive infections. Target-cell and species specificity of PVL are poorly understood, and the mechanism of action of this toxin in Staphylococcus aureus virulence is controversial. Here, we identify the human complement receptors C5aR and C5L2 as host targets of PVL, mediating both toxin binding and cytotoxicity. Expression and interspecies variations of the C5aR determine cell and species specificity of PVL. The C5aR binding PVL component, LukS-PV, is a potent inhibitor of C5a-induced immune cell activation. These findings provide insight into leukocidin function and staphylococcal virulence and offer directions for future investigations into individual susceptibility to severe staphylococcal disease.

The complement system is pivotal in host defense but also contributes to tissue injury in several diseases. The assembly of C3 convertases (C4b2a and C3bBb) is a prerequisite for complement activation. The convertases catalyze C3b deposition on activator surfaces. Here we describe the identification of staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein that blocks human complement by specific interaction with C4b2a and C3bBb. Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule.

Leukocyte migration is a key event both in host defense against invading pathogens as well as in inflammation. Bacteria generate chemoattractants primarily by excretion (formylated peptides), complement activation (C5a), and subsequently through activation of leukocytes (e.g., leukotriene B4, platelet-activating factor, and interleukin 8). Here we describe a new protein secreted by Staphylococcus aureus that specifically impairs the response of neutrophils and monocytes to formylated peptides and C5a. This chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1-kD protein encoded on a bacteriophage and is found in >60% of clinical isolates. CHIPS reduces the neutrophil recruitment toward C5a in a mouse peritonitis model, even though its activity is much more potent on human than on mouse cells. These findings suggest a new immune escape mechanism of S. aureus and put forward CHIPS as a potential new antiinflammatory therapeutic compound.