Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection

Abstract

Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein which possesses anti-microbial activity primarily due to its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9 −/− mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro, and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9−/− mice by injecting recombinant calprotectin. Further, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin’s anti-hyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro, or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ΔzafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared to wild-type. Collectively, these studies demonstrate a novel stage - specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin.

Role of Calprotectin in Withholding Zinc and Copper from Candida albicans

“…With this response to CP, C. albicans maintains constant intracellular Zn over numerous cell divisions despite virtually undetectable bioavailable Zn outside the cell. Similar findings with Zn transporters and CP have been reported for the fungal pathogen A. fumigatus (17,18), and upregulation of transition metal import genes is also an effective strategy against CP in bacterial pathogens including Acinetobacter baumannii, S. aureus, and Salmonella (14,16,20,65,66). Aside from Zn, we demonstrate for the first time that CP can withhold Cu from a microbial organism by virtue of its picomolar affinity for Cu(II).…”

“…CP has been well studied for its Zn-withholding properties from various pathogenic organisms, including Gram-positive and Gramnegative bacteria as well as the fungus Aspergillus fumigatus, where a mutant deficient in Zn sensing shows greater susceptibility toward CP in vivo (6,(13)(14)(15)(16)(17)(18). In addition to Zn, CP can withhold Mn from bacterial pathogens in vitro and in vivo (13,14,16,19,20) and can deplete Fe and Ni from specific microbial cultures in vitro (21,22).…”

CD177+neutrophils as functionally activated neutrophils negatively regulate IBD

“…ROS are then generated in an oxidative burst by a NADPH oxidase, and antimicrobial proteins are released, including calprotectin, defensin and lactoferrin 28. Calprotectin, comprising S100A8 and S100A9, exhibits antimicrobial effects on bacteria, fungi and protozoa,46 which is reported to mediate NADPH oxidase activation and ROS production 47 48. Analysis of differential gene expression profiles between CD177 + and CD177 − neutrophils has shown that functional differences were mainly reflected in defence response, inflammatory response, response to ROS and hydrogen peroxide in patients with CD or UC.…”

Immunity against fungi

“…Yet, only ~40% of CGD patients develop aspergillosis during their lifetime, despite daily inhalation of ubiquitous conidia. This observation indicates that nonoxidative mechanisms compensate in the remainder of the Aspergillus-exposed patients; better understanding of these mechanisms is an important future research direction (9,69,71,77,139). Invasive infections by other molds, such as Mucorales and Fusarium, or by commensal Candida yeasts are very uncommon (<5%), whereas invasive infections by endemic dimorphic fungi and Cryptococcus, or CMC do not develop in CGD (137,140).…”