Calprotectin (CP) is an abundant protein in neutrophils that is known to sequester Zn and Mn from pathogens using two distinct metal sites in a process known as nutritional immunity. CP has potent anti-microbial activity against many pathogens, including the fungal pathogen Candida albicans. However, the mechanism by which CP exerts its anti-candida activity is unknown. Recent studies from the Culotta lab have shown that, in a murine model of disseminated candidiasis, C. albicans lesions in the kidneys are associated with profound clearing of Zn, suggestive of Zn withholding from the pathogen, possibly by CP. Surprisingly, Cu may also be withheld from C. albicans during kidney infection as the yeast present in these lesions exhibit mRNA markers of Cu starvation. There is no known role for CP in withholding Cu, but biochemically CP has the capacity to bind Cu. Thus, I began to investigate if CP could be responsible for Cu and/or Zn sequestration from C. albicans. Using laboratory yeast cultures, I have shown that CP can sequester Zn and Cu away from C. albicans and Cu sequestration requires metal binding residues in CP that are distinct from the Zn binding site. I have also shown that C. albicans upregulates Cu or Zn uptake genes in response to the metal limiting environment imposed by CP. Given these results, I hypothesize that CP aids in the fight against C. albicans during kidney infection by sequestering multiple metal ions, including Cu, away from the pathogen. To investigate this, I will carry out the following aims.
Aim 1 : To understand the impact of CP on metal accumulation and metal stress responses in cultures of C. albicans. To gain a better understanding of the mechanism by which CP can sequester Cu versus Zn, I will test the ability of various site specific metal binding mutants of CP to inhibit C. albicans growth in culture and to withhold Cu and Zn from this pathogen. Yeast gene expression will also be analyzed to define any Cu and Zn starvation stress responses as well as any oxidative stress that occurs in C. albicans when CP attacks this pathogen.
Aim 2 : To understand the connection between CP and metal homeostasis in C. albicans during kidney infection. After establishing a role of CP in sequestering both Zn and Cu away from C. albicans in vitro, I will investigate if CP is responsible for the decrease in kidney Cu and Zn observed in the aforementioned murine model of disseminated candidiasis. First, kidneys from infected mice will be examined histologically to tract neutrophil and CP recruitment to sites of fungal lesions. Laser capture microdissection will be used to isolate fungal lesions in the kidney for the analysis of metals and fungal mRNA. By this approach, we can define local changes in Cu and Zn (and other metals) and will identify any fungal stress responses by examining markers of Cu and Zn uptake as well as oxidative stress. These analyses will be carried out in both WT and in CP-deficient mice to determine if CP is responsible for metal withholding from C. albicans during fungal invasion of the kidney. Together, these studies will provide insight into how CP combats C. albicans during the innate immune response, including an unprecedented role for Cu sequestration by CP in nutritional immunity.
In this proposal, we will explore how a protein involved in the immune response, calprotectin, fights infections of Candida albicans, the most prevalent human fungal pathogen, by withholding essential metal ions.
| Besold, Angelique N; Culbertson, Edward M; Nam, Lily et al. (2018) Antimicrobial action of calprotectin that does not involve metal withholding. Metallomics : |
| Liu, Ning-Ning; Uppuluri, Priya; Broggi, Achille et al. (2018) Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence. PLoS Pathog 14:e1007076 |
| Besold, Angelique N; Gilston, Benjamin A; Radin, Jana N et al. (2017) The role of calprotectin in withholding zinc and copper from Candida albicans. Infect Immun : |
