Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis.

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N-terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin-resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in-vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N-terminal domain of human CXCR2.

Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis.

Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His6)-tagged proteins in L. lactis. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His6-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His6-tag. This seems to be due to an influence of the His6-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins.

Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.

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.

Pre-existing antibody-mediated adverse effects prevent the clinical development of a bacterial anti-inflammatory protein.

Bacterial pathogens have evolved to secrete strong anti-inflammatory proteins that target the immune system. It was long speculated whether these virulence factors could serve as therapeutics in diseases in which abnormal immune activation plays a role. We adopted the secreted chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) as a model virulence factor-based therapeutic agent for diseases in which C5AR1 stimulation plays an important role. We show that the administration of CHIPS in human C5AR1 knock-in mice successfully dampens C5a-mediated neutrophil migration during immune complex-initiated inflammation. Subsequent CHIPS toxicology studies in animal models were promising. However, during a small phase I trial, healthy human volunteers showed adverse effects directly after CHIPS administration. Subjects showed clinical signs of anaphylaxis with mild leukocytopenia and increased C-reactive protein concentrations, which are possibly related to the presence of relatively high circulating anti-CHIPS antibodies and suggest an inflammatory response. Even though our data in mice show CHIPS as a potential anti-inflammatory agent, safety issues in human subjects temper the use of CHIPS in its current form as a therapeutic candidate. The use of staphylococcal proteins, or other bacterial proteins, as therapeutics or immune-modulators in humans is severely hampered by pre-existing circulating antibodies.