Surviving Reactive Chlorine Stress: Responses of Gram-Negative Bacteria to Hypochlorous Acid

Abstract

Sodium hypochlorite (NaOCl) and its active ingredient, hypochlorous acid (HOCl), are the most commonly used chlorine-based disinfectants. HOCl is a fast-acting and potent antimicrobial agent that interacts with several biomolecules, such as sulfur-containing amino acids, lipids, nucleic acids, and membrane components, causing severe cellular damage. It is also produced by the immune system as a first-line of defense against invading pathogens. In this review, we summarize the adaptive responses of Gram-negative bacteria to HOCl-induced stress and highlight the role of chaperone holdases (Hsp33, RidA, Cnox, and polyP) as an immediate response to HOCl stress. We also describe the three identified transcriptional regulators (HypT, RclR, and NemR) that specifically respond to HOCl. Besides the activation of chaperones and transcriptional regulators, the formation of biofilms has been described as an important adaptive response to several stressors, including HOCl. Although the knowledge on the molecular mechanisms involved in HOCl biofilm stimulation is limited, studies have shown that HOCl induces the formation of biofilms by causing conformational changes in membrane properties, overproducing the extracellular polymeric substance (EPS) matrix, and increasing the intracellular concentration of cyclic-di-GMP. In addition, acquisition and expression of antibiotic resistance genes, secretion of virulence factors and induction of the viable but nonculturable (VBNC) state has also been described as an adaptive response to HOCl. In general, the knowledge of how bacteria respond to HOCl stress has increased over time; however, the molecular mechanisms involved in this stress response is still in its infancy. A better understanding of these mechanisms could help understand host-pathogen interactions and target specific genes and molecules to control bacterial spread and colonization.

Keywords: Gram-negative bacteria; VBNC; antimicrobial resistance; household bleach; hypochlorous acid; oxidative stress; pathogens; reactive chlorine species; sodium hypochlorite; stress response.

Figure 1

Schematic representation of HOCl production in neutrophils. MPO: myeloperoxidase. O₂⁻: superoxide.

Figure 2

Schematic of HOCl targets in Gram-negative bacterial cells. (a) HOCl easily penetrates the bacterial cell due to its neutrality and attacks (b) several membrane components and processes, including transporters and proteins, such as ATPase, which disrupts ATP production (c) lipids, causing, for example, loss of membrane stability, (d) protein synthesis and proteins, especially the sulfur-containing ones, and (e) DNA, leading to DNA breakage and impairment of DNA synthesis. HOCl: hypochlorous acid; P: phosphate; ADP: adenosine diphosphate; ATP: adenosine triphosphate.

Figure 3

Reaction of HOCl with (a) thiol-containing compounds, (b) amine and amide, and (c) unsaturated fatty acids.

Figure 4

Schematic representation of the stress response triggered by HOCl exposure. HOCl activates (a) chaperones; induces the expression of (b) protein repair systems, especially for those in which cysteine and (c) methionine are the main target amino acids; (d) detoxifying enzymes; (e) DNA repair systems; and (f) induces changes in membrane properties, such as downregulating porins, reducing permeability and increasing hydrophobicity. HOCl: hypochlorous acid; H₂O₂: hydrogen peroxide; Cys: cysteine; Met: methionine; Met-SO: methionine sulfoxide; Met-SO2: methionine sulfone; Msrs: methionine sulfoxide reductases.

Figure 5

Activation of transcriptional factors by HOCl stress (green area). *HypT is the only regulator known to be specifically activated by HOCl. Its activation induces the expression of genes involved in cysteine, methionine, and sulfur biosynthesis, and downregulates genes involved in iron acquisition and homeostasis. NemR, a regulator responsive to HOCl, RES, and methylglyoxal, upregulates genes involved in glyoxylase and N-ethylmaleimide synthesis. RclR is activated by HOCl and N-chlorotaurine and expresses a flavoprotein disulfide reductase, an uncharacterized periplasmic protein, and transmembrane quinone-binding protein. The gray area shows the genes regulated by OxyR and SoxR, the most studied ROS-sensors. HOCl: hypochlorous acid; H₂O₂: hydrogen peroxide.

Figure 6

Possible mechanisms of HOCl biofilm stimulation in Gram-negative bacteria. HOCl induces (a) changes in cell morphology, which enhance the ability to adhere to surfaces; (b) increases the production of extracellular polymeric substance (EPS) matrix, increasing cell tolerance; (c) upregulates the production of c-di-GMP by DCGs; (d) regulates transcriptional regulators involved in HOCl-induced stress response. NaOCl: sodium hypochlorite; HOCl: hypochlorous acid; EPS: extracellular polymeric substance; DCG: diguanylate cyclase; c-di-GMP: cyclic-di-GMP.