Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation.

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.

[1]  Arne Skerra,et al.  The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting alpha-helices can determine specific association of two EF-hand proteins. , 2007, Journal of molecular biology.

[2]  J. Palefsky,et al.  Oxidation of methionine 63 and 83 regulates the effect of S100A9 on the migration of neutrophils in vitro , 2007, Journal of leukocyte biology.

[3]  T. Vogl,et al.  Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations. , 2006, Biochimica et biophysica acta.

[4]  J. Palefsky,et al.  S100A8 Triggers Oxidation-sensitive Repulsion of Neutrophils , 2006, Journal of dental research.

[5]  T. Vogl,et al.  Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity. , 2006, Journal of molecular biology.

[6]  J. Palefsky,et al.  Inhibition of Human Papillomavirus Type 16 E7 Phosphorylation by the S100 MRP-8/14 Protein Complex , 2005, Journal of Virology.

[7]  S. Ghavami,et al.  Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions , 2004, Journal of leukocyte biology.

[8]  P. Fidel.  History and new insights into host defense against vaginal candidiasis. , 2004, Trends in microbiology.

[9]  P. Rouleau,et al.  Proinflammatory Activities of S100: Proteins S100A8, S100A9, and S100A8/A9 Induce Neutrophil Chemotaxis and Adhesion 1 , 2003, The Journal of Immunology.

[10]  P. Vignais,et al.  The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils. , 2002, European journal of biochemistry.

[11]  W. Nacken,et al.  Interaction of S100A8/S100A9-arachidonic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells. , 2001, Biochemistry.

[12]  M. Selsted,et al.  Human Neutrophil-Mediated Nonoxidative Antifungal Activity against Cryptococcus neoformans , 2000, Infection and Immunity.

[13]  W. Chazin,et al.  Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14). , 2000, The Journal of infectious diseases.

[14]  F. Odds.  Pathogenic fungi in the 21st century. , 2000, Trends in microbiology.

[15]  W. Nacken,et al.  Zinc binding reverses the calcium‐induced arachidonic acid‐binding capacity of the S100A8/A9 protein complex , 1999, FEBS letters.

[16]  M. Raftery,et al.  Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8* , 1999, The Journal of Biological Chemistry.

[17]  M. Pfaller,et al.  Trends in Antifungal Use and Epidemiology of Nosocomial Yeast Infections in a University Hospital , 1999, Journal of Clinical Microbiology.

[18]  Ronald N. Jones,et al.  National surveillance of nosocomial blood stream infection due to species of Candida other than Candida albicans: frequency of occurrence and antifungal susceptibility in the SCOPE Program. SCOPE Participant Group. Surveillance and Control of Pathogens of Epidemiologic. , 1998, Diagnostic microbiology and infectious disease.

[19]  T. C. White,et al.  Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid , 1997, Journal of bacteriology.

[20]  S. Yui,et al.  Growth‐inhibitory and apoptosis‐inducing activities of calprotectin derived from inflammatory exudate cells on normal fibroblasts: regulation by metal ions , 1997, Journal of leukocyte biology.

[21]  R. Lehrer,et al.  In vitro candidastatic properties of the human neutrophil calprotectin complex. , 1993, Journal of immunology.

[22]  R. Moritz,et al.  Identification of a chemotactic domain of the pro-inflammatory S100 protein CP-10. , 1993, Journal of immunology.

[23]  E. Lingaas,et al.  Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin , 1990, The Lancet.

[24]  P. Hessian,et al.  The heterodimeric complex of MRP-8 (S100A8) and MRP-14 (S100A9). Antibody recognition, epitope definition and the implications for structure. , 2001, European journal of biochemistry.