Harnessing the Power of Our Immune System: The Antimicrobial and Antibiofilm Properties of Nitric Oxide

Abstract

Nitric oxide (NO) is a free radical of the human innate immune response to invading pathogens. NO, produced by nitric oxide synthases (NOSs), is used by the immune system to kill microorganisms encapsulated within phagosomes via protein and DNA disruption. Owing to its ability to disperse biofilm-bound microorganisms, penetrate the biofilm matrix, and act as a signal molecule, NO may also be effective as an antibiofilm agent. NO can be considered an underappreciated antimicrobial that could be levied against infected, at-risk, and hard-to-heal wounds due to the inherent lack of bacterial resistance, and tolerance by human tissues. NO produced within a wound dressing may be an effective method of disrupting biofilms and killing microorganisms in hard-to-heal wounds such as diabetic foot ulcers, venous leg ulcers, and pressure injuries. We have conducted a narrative review of the evidence underlying the key antimicrobial and antibiofilm mechanisms of action of NO for it to serve as an exogenously-produced antimicrobial agent in dressings used in the treatment of hard-to-heal wounds.

Keywords: antimicrobial; biofilm; hard-to-heal; nitric oxide; wound.

Figure 1

(A) Bacteria trapped within the phagosome of a phagocyte of our immune response experience a series of attacks from different RNS produced within the “redox cauldron” such as peroxynitrite [38]. NOS acts as a source of NO for the phagosome, which, due to the acidic environment, reacts to form different RNS. The reaction with carbon dioxide (CO₂) forms nitrosoperoxocarbonate, which further reacts with NO to form either N₂O₃ or NO₂. Peroxidase also plays a key role within the phagosome, catalysing the reaction of NO into NO₂⁻ and then into NO_2, which interacts with the trapped bacterium. (Adapted and modified from Wink et al., 2011 [35]). (B) Free nitrite is used by myeloperoxidase in the generation of hypochlorous acid (HOCl) within neutrophils as part of the immune response, which produces NO₂ as a byproduct. Nitrite further reacts with HOCl to produce the RNS nitryl chloride (NO₂Cl), which, similar to OONO⁻, nitrates tyrosine within proteins [33,39].

Figure 3

(A) Cyclic-diguanylate-guanosine monophosphate (c-di-GMP) within the bacterial cell regulates biofilm aggregation of bacteria. As the concentration of c-di-GMP increases, biofilm formation increases. It is thought that c-di-GMP binds to protein regulators of the dispersal proteins (such as proteins for flagellum movement), inactivating them. (B) NO binds to the haem groups of NO sensing receptor proteins [97] on the outside of the cell, activating them; this induces an upregulation of phosphodiesterase (PDE). PDE binds to and degrades c-di-GMP-releasing protein regulators; the resulting signal cascade ends with the activation of the dispersal proteins [103,104].

Figure 2

A summary of the broad-spectrum antimicrobial mechanisms of action of NO and RNS within a bacterium cell. (A) NO freely diffuses through the cell membrane due to its small size and low charge [28]. (B) NO reacts with sulphur-containing thiol groups on outer cell wall-embedded proteins, deactivating them [48]. (C) NO deactivates proteins embedded within the bacteria on the cell wall, targeting sulphur-containing tyrosine groups to deactivate them [48]. (D) The iron–sulphur-containing proteins are one of the primary targets of NO. The reaction with NO, facilitated by H+, produces two dinitrosyl iron complexes (DNICs) molecules and two free sulphur molecules. The DNICs can further react with oxygen to form OONO⁻ [49]. (E) Peroxynitrite reacts with DNA strands, resulting in deamination and oxidative damage, which will eventually lead to DNA cleavage. Without a functional RNR enzyme producing more dNTP molecules, the damaged DNA is no longer repaired or replaced [50]. (F) Ribonucleotide reductase (RNR) is a catalyst for the conversion of nucleotides (NTP) into deoxynucleotides (dNTP), which are used for DNA repair and replication. NO has strong reactivity towards the tyrosyl free radicals within RNR, which changes the structure of the enzyme, inhibiting it [51].