Biochemical characterization of murine calprotectin and the host-pathogen competition for manganese

Abstract

Microorganisms need to acquire metal ion nutrients when they attempt to colonize the host milieu. The competition for transition metal ions between a host and an invading pathogen constitutes an important aspect of innate immunity and microbial pathogenesis. The host deploys the metal-sequestering antimicrobial protein calprotectin (CP) to sites of infection to withhold transition metal ions. The goals of this thesis are to characterize the biochemical, and Mn(Il)-binding properties of the murine orthologue of calprotectin (mCP) as well as to evaluate the molecular details of the competition for Mn(II) between calprotectin and Mn(II) transport proteins from pathogenic bacteria. In the first part of this thesis, we provide initial biochemical characterization of mCP, supporting a role of this protein in transition metal sequestration and antibacterial activity. We demonstrate that this protein is a heterodimer than can undergo Ca(Il)-induced tetramerization.

We further show that mCP can bind a range of first row transition metal ions and displays antibacterial species against a panel of bacterial species. In the second part of this thesis, we characterize the Mn(Il)-binding properties of mCP, revealing Ca(Il)-dependent Mn(II) affinity at a hexahistidine site that bears a remarkable resemblance to the Mn(Il)-sequestering site in human CP (hCP). We use biochemical assays and electron paramagnetic resonance (EPR) spectroscopy to elucidate the Mn(ll)-coordinating residues of mCP. Altogether, we find that mCP possesses a much lower Ca(II) sensitivity than human CP, a fact that may have consequences in vivo. In the final portion of this thesis, we use biochemical assays and EPR spectroscopy to monitor the competition for Mn(II) between hCP and the bacterial Mn(II) transport proteins MntC and PsaA.

We show that in the presence of excess Ca(II), hCP rapidly outcompetes these proteins for Mn(II), revealing the notably high Mn(II) affinity of hCP and giving molecular credence to the role of CP in sequestering Mn(II) in vivo.