Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity.

Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection - induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone's antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects.

Actin enables the antimicrobial action of LL-37 peptide in the presence of microbial proteases.

Host defense peptides play an important host-protective role by their microcidal action, immunomodulatory functions, and tissue repair activities. Proteolysis is a common strategy of pathogens used to neutralize host defense peptides. Here, we show that actin, the most abundant structural protein in eukaryotes, binds the LL-37 host defense peptide, protects it from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis, and enables its antimicrobial activity despite the presence of the proteases. Co-localization of LL-37 with extracellular actin was observed in necrotized regions of samples from oral lesions. Competition assays, cross-linking experiments, limited proteolysis, and mass spectrometry revealed that LL-37 binds by specific hydrophobic interactions to the His-40-Lys-50 segment of actin, located in the DNase I binding loop. The integrity of the binding site of both LL-37 and actin is a prerequisite to the binding. Our results demonstrate that actin, presumably released by dead cells and abundant in infected sites, might be utilized by the immune system to enhance spatio-temporal immunity in an attempt to arrest infection and control inflammation.

Oxygen deprivation affects the antimicrobial action of LL-37 as determined by microplate real-time kinetic measurements under anaerobic conditions.

Some agents, including Escherichia coli and group A Streptococcus pyogenes cause infections in oxygen depleted sites. LL-37 is a human host defence peptide shown previously to play an important role in controlling infections caused by these bacteria. However, the effect of oxygen levels on the antimicrobial activity of LL-37 remains obscure. In order to test the effect of oxygen (or lack thereof) on LL-37's activity against E. coli and S. pyogenes, a method for adapting commonly used microtiter plates for real-time growth-kinetic (and growth-inhibition) measurements under anaerobic conditions was developed. Using the proposed method, anaerobic conditions were attained in the microplate within 30 min and were maintained for at least five days. Anaerobiosis was further confirmed by comparing the growth of two anaerobic oral species (Porphyromonas gingivalis and Fusobacterium nucleatum) in anaerobic compartments of microtiter plates versus aerobic ones. Both species grew only in the anaerobic compartments of the plates as indicated by the growth curves generated. The sensitivities of E. coli and S. pyogenes to LL-37 were tested under anaerobic conditions and compared to those in aerobic ones. The oxygen facultative E. coli grew to a higher density under aerobic conditions and its sensitivity to LL-37 was increased under anaerobiosis. The microaerophilic pathogen S. pyogenes grew faster and to a higher density under anaerobic conditions and was much more resistant to LL-37 under oxygen deprivation. Our results suggest that resistance to antimicrobial agents of microbes infecting anaerobic-microaerophilic sites should be tested under oxygen-restricted conditions.