The mechanism of calprotectin's candidastatic activity appears to involve zinc chelation.

Calprotectin is a calcium binding protein of the S-100 protein family, expressed variably in myeloid and epithelial cells [I]. It is particularly abundant in granulocytes constituting up to 60% of cytosolic protein [2]. Its complex expression in monocytes, where it is present at 40 fold lower amounts than neutrophls, appears to be related to early stages of monocytdmacrophage differentiation [3]. In man, plasma calprotectin concentration increases by 40I30 fold during bacterial infection from a normal level of < I mg/L. Recently, calprotectin has been shown to have a potent microbiostatic activity in vitro. This activity occurs at physiological concentrations of calprotectin and is reported to be reversed by zinc [4, 51. The exact mechamsm of tlus activity remains unclear. Here we investigated this putative zinc reversible microbiostatic activity and attempted to delineate its mechanism of action against C. al6icans. Calprotectin was purified from crude cytosolic extracts of human neutrophils and its concentration was measured by ELISA [6]. Candidastatic activity was investigated in four distinct experiments which were each carried out three times under conditions in which trace element contamination was minimised. C.ul6icuns growth was assessed by the 24 h change in total viable counts. Calprotectin significantly inhibited the growth of C.al6icans by 10100 fold compared to controls, at concentrations greater than 250 pg/ml, p<0.05 (Fig. la). IOpM ZnSOq completely reversed calprotectin's activity at the test concentration, p<0.05 (Fig. Ib). Zinc concentrations greater than 10 pM were found to inhibit growth (results not shown). C.al61cuns growth was almost completely inhibited in sab which had beem incubated with calprotectin for 24 hours prior to inoculation (Fig.2a). C.ul6icuns growth remained inhibited in calprotectin free ultrafiltrates derived from this sab and supplementation of these ultrafiltrates with zinc renewed growth. Determination of total zinc in these ultrafiltrates (Fig.2b) indicated significantly greater loss from sab which had been incubated with calprotectin compared to controls, p<0.05 (Fig. 2b). The results indicate strongly that the mechanism of calprotectin's candidastatic activity involves zinc chelation resulting in deprivation of this essential nutrient. Consistent with this hypothesis is the finding that while the zinc requirement for yeasts and fungi pM) is higher than that for bacteria pM) [7], the MIC of calprotectin is considerably lower [S]. Also, Sohnle et al. [ 5 ] demonstrated that zinc mdated reversal of calprotectins activity requires a low zinc:calprotectin ratio, on a molar basis. In most microorganisms, apart from some yeasts, the apparent absence of any mechanism to sequester zinc indicates a possible sensitivity on their part to the proposed strategy [6]. The growth inhibitory effect of abscess fluid has been attributed to calprotectin [5]. As a candidate site of action for calprotectin in vivo, we have found calprotectin concentrations of 1-20 mg/ml in blood stained abscess fluid drained from human abdominal abscesses (unpublished observation). In conclusion, the current findings implicate zinc chelation as a novel potentially important host defence function of an abundant neutrophil protein, calprotectin. -