RESUMO
The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn(2+) and Zn(2+) with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn(2+) sequestration. The asymmetry of the CP heterodimer creates a single Mn(2+)-binding site from six histidine residues, which distinguishes CP from all other Mn(2+)-binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn(2+) and Zn(2+) being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn(2+) sequestration. These data establish the importance of Mn(2+) sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.
Assuntos
Imunidade Inata , Complexo Antígeno L1 Leucocitário/química , Complexo Antígeno L1 Leucocitário/imunologia , Manganês/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Calgranulina A/química , Calgranulina A/genética , Calgranulina A/imunologia , Calgranulina B/química , Calgranulina B/genética , Calgranulina B/imunologia , Cristalografia por Raios X , Histidina/química , Interações Hospedeiro-Patógeno/imunologia , Humanos , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/farmacologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia , Zinco/metabolismoRESUMO
Cortical interneurons are critical for information processing, and their dysfunction has been implicated in neurological disorders. One subset of this diverse cell population expresses tyrosine hydroxylase (TH) during postnatal rat development. Cortical TH-immunoreactive neurons appear at postnatal day (P) 16. The number of TH cells sharply increases between P16 and P20 and subsequently decreases to adult values. The absence of apoptotic markers in these cells suggests that the reduction in cell number is not due to cell death but is due to a decline in TH production. Cortical TH cells lack all additional catecholaminergic enzymes, and many coexpress GABA and calretinin, but little else is known about their phenotype or function. Because interneurons containing choline acetyltransferase (ChAT) or vasoactive intestinal peptide (VIP) share characteristics with cortical TH neurons, the coexpression of TH with ChAT or VIP was examined throughout the neocortex at P16, P20, and P30. The proportions of TH cell profiles double-labeled for ChAT or VIP significantly increased between P16 and P30. Based on their proximity to blood vessels, intrinsic cholinergic and VIPergic cells have been hypothesized to regulate cortical microcirculation. Labeling with the gliovascular marker aquaporin-4 revealed that at least half of the TH cells were apposed to microvessels at these ages, and many of these cells contained ChAT or VIP. Cortical TH neurons did not coproduce nitric oxide synthase. These results suggest that increasing proportions of cortical TH neurons express ChAT or VIP developmentally and that a subset of these TH neurons may regulate local blood flow.
