Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans Original paper

What was studied?

This study defines copper-dependent activity against Candida albicans by testing how histatin salivary peptides bind copper and use that copper to kill the fungus. The authors measured peptide–copper binding by spectrophotometric titrations, resolved copper coordination by X-ray absorption spectroscopy, and ran growth and killing assays with and without added copper or chelators. They mapped two copper(I) binding sites of nanomolar affinity and a high-affinity N-terminal copper(II) site, then linked these sites to antifungal potency. When the medium contained extra copper, histatin-5 required a much lower dose to inhibit growth; when a copper-specific chelator removed copper, activity fell. This work connects exact histidine positions to copper binding and to killing strength, and shows how copper in the oral niche can tune antifungal peptide performance.

Who was studied?

The experiments used Candida albicans (strain SC5314) in vitro and a panel of histatin-5–based peptides, including full-length histatin-5, N-terminal fragments, and His→Ala mutants that remove specific histidines. The team challenged C. albicans with peptides under copper-replete, copper-depleted, and baseline conditions, and used a cell-impermeant copper chelator, bathocuproine disulphonate (BCS), to limit copper availability. This design lets the authors link copper access, peptide sequence, and fungal survival with clear readouts of potency and copper binding behavior.

Most important findings

Copper boosted histatin-5 efficacy while copper removal erased it. Adding copper lowered the EC50 from about 5 µM to ~1 µM; BCS co-treatment prevented growth inhibition, while a general chelator (EDTA) weakened but did not abolish activity. Cells grown in copper-rich medium became more sensitive, and simultaneous exposure to BCS blocked killing despite similar cell-associated copper during the short assay, supporting a direct need for accessible extracellular copper during peptide action.
The chemistry explains the biology. Histatin-5 binds copper(II) very tightly at its N-terminus (ATCUN site; Kd ≈ 8 pM), and it hosts two copper(I) sites of ~20 nM affinity anchored by adjacent histidines (a “bis-His” motif). X-ray absorption data indicate a third ligand helps complete the copper(I) site, consistent with His3 participation in one complex.

These distinct sites give histatin-5 the ability to engage both copper redox states, a feature that can amplify oxidative stress within fungal cells and drive killing.
Sequence matters for copper-enabled potency. Truncated N-terminal peptides lacked activity alone but regained strong activity with added copper (EC50 ~5–6 µM). Mutating the key H7,H8 bis-His motif abolished copper-dependent killing, while mutating the distal H18,H19 pair had modest effects, highlighting the proximal bis-His site as the critical anchor for copper(I) and activity. The correlation between copper(I) affinity and antifungal potency across fragments and mutants defines a clean, microbiome-relevant signature: histatin peptides that preserve the bis-His motif and have access to copper show stronger activity against C. albicans.

Key implications

For oral and mucosal care, copper availability can tune innate antifungal defense. Saliva that delivers copper to histatin-5 can strengthen killing of C. albicans, while copper chelation or low copper may blunt that defense. In a microbiome signatures database, clinicians can tag “histatin–copper axis” with features that include the histatin-5 bis-His motif, high-affinity N-terminal copper(II) binding, and a copper-responsive killing phenotype. These markers predict higher peptide efficacy in copper-replete niches and lower efficacy with chelation or deficiency. Therapeutic strategies may include peptide designs that retain the proximal bis-His site or formulations that supply bioavailable copper in a controlled way, while avoiding excess that could injure host tissue. Clinicians should consider copper status, chelator use, and salivary flow when they judge risk for Candida overgrowth and when they evaluate peptide-based adjuncts.