Abstract
Histones are essential elements of chromatin structure and gene regulation in eukaryotes. An unexpected attribute of these nuclear proteins is their antimicrobial activity. A framework for histone release and function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps, a specialized cell death process in which DNA-based structures containing histones are extruded to ensnare and kill bacteria. Investigating the susceptibility of various Gram-positive pathogens to histones, we found high-level resistance by one leading human pathogen, group A Streptococcus (GAS). A screen of isogenic mutants revealed that the highly surface-expressed M1 protein, a classical GAS virulence factor, was required for high-level histone resistance. Biochemical and microscopic analyses revealed that the N-terminal domain of M1 protein binds and inactivates histones before they reach their cell wall target of action. This finding illustrates a new pathogenic function for this classic GAS virulence factor, and highlights a potential innate immune evasion strategy that may be employed by other bacterial pathogens.
Highlights
In 1884, Albrecht Kossel discovered that DNA inside the nucleus is associated with proteins, naming these proteins histones[1]
Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) agonist and canonical stimulator of neutrophil extracellular traps (NETs), for 4 h and we found a robust release of DNA by using the cell-impermeable, fluorescent DNA dye Sytox Green (Fig. 1A) and histones coupled to immunohistochemistry (IHC) for pooled histones (H1-H4) or western immunoblot for histone H2A (Fig. 1B)
At 4 h post-PMA stimulation, neutrophil elastase (NE) (Fig. 1C), histone mixture (H1-H4) (Fig. 1D) and histone H2A (Fig. 1E) all co-localized with extracellular DNA in NETs
Summary
In 1884, Albrecht Kossel discovered that DNA inside the nucleus is associated with proteins, naming these proteins histones[1]. In 2004, the Zychlinsky group discovered a novel function of neutrophils termed neutrophil extracellular traps (NETs), wherein neutrophils extrude their DNA to forms a lattice network capable of ensnaring bacteria and exposing them to a high concentration of antimicrobial peptides and proteases[8]. The process of NETosis elaborates abundant quantities of histones into the extracellular milieu, estimated at 2.5 mg histones per 109 neutrophils, such that histones comprise 70% of the protein content within the NET architecture[15]. Besides NETs, other myeloid cell lineages including mast cells[16], macrophages[17], basophils[18] and eosinophils[19] can deploy histones within DNA-based extracellular traps (ETs). We investigated the role of neutrophil-derived extracellular histones in defense against Gram-positive pathogens. We found a robust release of histones by neutrophils exposed to several medically important Gram-positive pathogens. Mutational and biochemical analyses further reveal that M1 protein acts through a mechanism of direct binding and inactivation
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