The therapeutic potential of thiocyanate and hypothiocyanous acid against pulmonary infections

Abstract

Hypothiocyanous acid (HOSCN) is an endogenous oxidant produced by peroxidase oxidation of thiocyanate (SCN⁻), an ubiquitous sulfur-containing pseudohalide synthesized from cyanide. HOSCN serves as a potent microbicidal agent against pathogenic bacteria, viruses, and fungi, functioning through thiol-targeting mechanisms, independent of currently approved antimicrobials. Additionally, SCN⁻ reacts with hypochlorous acid (HOCl), a highly reactive oxidant produced by myeloperoxidase (MPO) at sites of inflammation, also producing HOSCN. This imparts both antioxidant and antimicrobial potential to SCN⁻. In this review, we discuss roles of HOSCN/SCN⁻ in immunity and potential therapeutic implications for combating infections.

Graphical Abstract

Introduction

Acute respiratory infections (ARIs) are a major global health concern, leading to significant hospitalizations and mortality. ARIs have consistently ranked among the primary causes of death, resulting in an annual global loss of up to 3 million lives between 2010 and 2019, as reported by WHO ¹. ARIs consistently represent approximately 5% of the total annual global deaths, as highlighted in WHO reports for 2010 and 2019 ². In low-income countries, ARIs are a prominent cause of death, accounting for an estimated 10–25% of all deaths among children under 5 ^{2, 3, 4} (WHO 2014b). Furthermore, in resource-poor settings, ARIs contribute significantly to avoidable deaths (WHO 2007). Worldwide, ARIs were the leading cause of disease burden, as measured by disability-adjusted life years (WHO 2004). ARIs are caused by a variety of viruses and bacteria ^{2, 5}. Despite the availability of therapies that can mitigate infection severity and reduce mortality, many respiratory infections remain refractory to treatment due to the absence of effective drugs ⁶. Vaccines are accessible for certain respiratory pathogens such as influenza virus, SARS-CoV-2 or Streptococcus pneumoniae, but their efficacy is limited by antigenic drift. Notably, low-income countries often lack access to working medications ^{7, 8, 9}, and available antiviral drugs exhibit efficacy primarily during early disease stages ¹⁰. Antibiotics resistance is another growing global concern¹¹. Several bacterial pathogens causing respiratory infections, including Pseudomonas aeruginosa (priority 1), Staphylococcus aureus (priority 2) and S. pneumoniae (priority 3) are listed by WHO in 2017 as global antibiotic-resistant priority pathogens for which new antibiotics are urgently needed ¹². Hypothiocyanous acid (HOSCN) and its precursor thiocyanate (SCN⁻) represent promising candidates, which is the subject of this review.

Thiocyanate (SCN⁻) and hypothiocyanous acid

Thiocyanate (SCN⁻), a pseudohalide anion carrying a single negative charge localized at the sulfur atom, is present in various human secretions such as saliva, milk, airway lining fluid and blood plasma ^{13, 14}. Human plasma typically contains SCN⁻ levels ranging from 10 to 300 μM ^{15, 16, 17} whereas its concentrations in saliva can reach mM levels, 10-fold higher than serum values ^{15 18 19, 20}. SCN⁻ is derived from cyanide by sulfurtransferases, including mitochondrial rhodanese and cytosolic mercaptopyruvate sulfurtransferase, and does not possess direct antimicrobial properties (Figure 1) ²¹. Effective transport of SCN⁻ across airway epithelial cells and its concentration in the airway surface liquid relies on the basolateral sodium-iodide-symporter (NIS), while CFTR, TMEM16A, and pendrin can transport SCN⁻ apically into the airway lumen ^{18, 22}. In air-liquid cultures of respiratory epithelial cells, SCN⁻ is predominately transported through the transcellular route rather than the paracellular route ¹⁸. This transport can be reversibly inhibited by competitive inhibitors of NIS in the basolateral compartment and CFTR inhibitors in the apical compartment ¹⁸.

Figure 1. The origin and function of SCN⁻ and HOSCN in the airways.

A) Several peroxidases are present in the respiratory tract: myeloperoxidase (MPO, neutrophils), lactoperoxidase (LPO, airway epithelium), eosinophil peroxidase (EPO, eosinophils) and peroxidasin (PXN, extracellular matrix). These are each capable of producing HOSCN, while MPO alone is capable of producing HOCl. B) In neutrophils, the NADPH oxidase enzyme complex generates superoxide anions (O₂^.−) in the phagosome or extracellular space. Superoxide dismutates to form H₂O₂, and epithelial enzymes such as dual oxidase (DUOX) 1/2 also contribute directly to H₂O₂ formation from NADPH and oxygen. Extracellular MPO, released from neutrophils by exocytosis or NETosis, generates HOCl from H₂O₂ and Cl⁻. SCN⁻ reacts with HOCl, replacing the oxidant with HOSCN. SCN⁻ is also able to directly react with Compound I of MPO, directly forming HOSCN and preventing HOCl formation. HOSCN has antimicrobial potential for bacteria, viruses and fungi, outlined in this review. C) The formation of SCN⁻ from CN⁻ is mediated by mercaptopyruvate sulfurtransferases in the cytosol and by rhodanese, via the thiosulfate sulfur donor, in mitochondria.

SCN⁻ serves an essential role in the innate immune system as the precursor of the antimicrobial hypothiocyanous acid (HOSCN) (Figure 1) ²³. HOSCN, with a pK_a of 4.85, predominantly exists as ionized hypothiocyanite (OSCN⁻) under physiological conditions ²⁴. The oxygen in OSCN⁻ becomes protonated at lower pH. Protonated OSCN⁻ reacts rapidly with thiol groups, which are of high abundance in cells ²⁵. Thus, HOSCN likely does not diffuse far from its site of generation in most physiological environs. This localizes HOSCN nearby the chordata peroxidases chiefly responsible for its production: lactoperoxidase (LPO), myeloperoxidase (MPO), eosinophil peroxidase (EPO), thyroid peroxidase (TPO), and peroxidasins (PXNs).

LPO is a 78.5 kDa monomeric protein consisting of a single polypeptide chain. After reducing H₂O₂ to water and forming compound I, LPO accepts two electrons from SCN⁻ to produce HOSCN/OSCN⁻ (Figure 1) ^{26, 27}. By generating HOSCN, LPO exerts bacteriostatic and bactericidal activities ²⁸. Other chordata peroxidases and peroxidasins catalyze the formation of HOSCN efficiently under physiological conditions using a similar mechanism to that of LPO (Figure 1) ^{29 29, 30, 31}. MPO can catalyze the formation of hypochlorous (HOCl) and hypobromous acid (HOBr) from chloride or bromide, respectively ³². MPO production of HOCl has been associated with irreversible tissue damage, e.g. production of chlorotyrosine in proteins ^{32, 33}. HOCl and HOBr are stronger oxidants than HOSCN, capable of generating irreversible oxidative products of biomolecules ^{34, 35, 36}. SCN⁻ can decrease HOCl and HOBr concentrations by outcompeting chloride and bromide substrates, leading to the generation of HOSCN, and by non-enzymatically scavenging HOCl and HOBr, thereby producing HOSCN ^{35, 36, 37}. Reactivity of SCN⁻ with HOCl/HOBr and prevention of their formation, in both cases yielding HOSCN, confers antioxidant and cytoprotective properties to SCN⁻ which have been extensively reviewed elsewhere ^{15, 38, 39, 40}. Importantly, the dose makes the poison: these reviews include review of potential toxicity of either thiocyanate or HOSCN, when concentrations of either are too high.

HOSCN production requires a source of H₂O₂, typically an NADPH oxidase such as dual oxidases (DUOXs) ^{41, 42, 43, 44}. Both DUOX1 and DUOX2 can provide H₂O₂ for the LPO system ⁴⁵. DUOX1 is expressed mainly in the upper airway epithelium ⁴⁶, while DUOX2 is expressed mainly in the salivary glands ⁴⁷. We have shown that in mice Duox1 supports the immune response during influenza A lung infection ⁴⁸, but is dispensable during lung infection with Mycobacterium tuberculosis ⁴⁹. As Duox1 is linked to HOSCN production, one interpretation of these results is that DUOX/peroxidase-mediated production of HOSCN is critical to immunity against certain respiratory pathogens, like influenza A virus.

The remainder of this review emphasizes the therapeutic potential of SCN⁻ and HOSCN in infections. It is not within the scope of this review to provide a comprehensive list of all in vitro studies on the antimicrobial actions of HOSCN; this topic has been reviewed elsewhere ^{50, 51}. Instead, this report specifically addresses in vitro research that contributes to a better understanding of in vivo observations reported in the literature. This discussion encompasses both the in vivo antimicrobial and anti-inflammatory benefits of SCN⁻ and HOSCN, as well as limitations and future research directions required to make SCN⁻/HOSCN therapy a reality for fighting infections in humans.

Antiviral activity of HOSCN

HOSCN exhibits virucidal activity against several pathogenic viruses, including herpes simplex virus 1 (HSV-1), respiratory syncytial virus (RSV), echovirus 11 and influenza viruses ^{50, 52, 53}. HOSCN oxidizes thiol moieties of protein cysteines important for virion function, such as host cell binding ⁴⁸. Thus, HOSCN’s antiviral effects are likely mediated by extracellular interactions with virions, though the possibility of additional intracellular mechanisms remains.

Salivary HOSCN, among the most concentrated in the body, likely plays a role in salivary antiviral immunity by complementing serum-derived antibodies, especially IgG and IgM, to neutralize HSV ⁵⁴. Activation of salivary peroxidase activity has been associated with 70–80% reduction in the incidence of aphthous ulcers, which are linked to HSV and varicella zoster infections ^{55, 56, 57}. HOSCN exhibits its highest effectiveness in HSV control under acidic pH conditions, favoring its protonated state and showing the importance of salivary pH ⁵⁸. Radiotherapy for head and neck cancer may lead to latent HSV-1 reactivation due to impaired peroxidase function in the saliva, decreasing the steady-state concentration of HOSCN to levels insufficient for herpes virus control ^{59, 60, 61}.

Our work has demonstrated the inhibitory effect of HOSCN against diverse strains of influenza virus in vitro ^{62, 63, 64, 65}. Recent findings underscore the in vivo significance of the HOSCN-generating antiviral system, revealing its direct targeting of influenza virus and impairment of its attachment and entry into host cells, resulting in reduced infection severity in vivo in a murine model of influenza lung infection ⁴⁸. Additionally, in vivo oral administration of HOSCN in combination with the drug amantadine (currently restricted by the FDA due to emerging drug resistance) demonstrated efficacy against lethal A/PR8 (H1N1) and A/HK/68 (H3N2) influenza A virus lung infections in mice ⁵². Similarly, in vitro treatment with HOSCN, either alone or in combination with amantadine or oseltamivir, was effective against various influenza strains ^{52, 62, 63}.

HOSCN, in combination with lactoferrin, has been evaluated against SARS-CoV-2, the causative agent of the COVID-19 pandemic ⁶⁶. In this study, 100 μM HOSCN demonstrated the ability to inhibit at least 50% of SARS-CoV-2 replication in vitro ⁶⁶. Coronavirus spike proteins contain cysteine-rich regions, and the spike protein of SARS-CoV-2 possesses the most cysteine-rich cytoplasmic tail among related pathogenic coronaviruses, rendering it susceptible to oxidation by HOSCN. Given that S-acylation of cysteine residues has been identified as crucial for infectivity of SARS-CoV-2 in vitro, it is plausible that HOSCN inhibits the S-acylation of critical cysteines ⁶⁷.

Antibacterial activity of HOSCN

Similar to viral pathogens, numerous bacterial species have been identified as susceptible to HOSCN in vitro ^{50, 51}. More recently, clinical isolates of P. aeruginosa and Staphylococcus aureus from cystic fibrosis (CF) patients were shown to be killed by HOSCN in vitro, arguably with some isolates exhibiting varying levels of susceptibility ⁶⁸. Both nonencapsulated and encapsulated Streptococcus pneumoniae strains were also found to be susceptible to HOSCN, independent of the bacterial capsule, suggesting that HOSCN may penetrate the capsule to react with intracellular targets within the bacterium ^{11, 69, 70}. However, S. pneumoniae has also been shown to resist HOSCN via specific oxidoreductases ⁷¹. More research is needed to resolve these contrasting observations.

HOSCN may be a more potent antimicrobial in combination with other agents. Inhaled HOSCN and lactoferrin (LF) in combination with antibiotics such as aztreonam or tobramycin reduced biofilm formation of Pseudomonas aeruginosa and was not cytotoxic to airway epithelial cells ⁷². HOSCN/lactoferrin also decreased levels of four strains of P. aeruginosa below detection limits and reduced the microbial load in sputum from patients with CF⁷³. SCN⁻ analogs also hold therapeutic potential. The SCN⁻ analog selenocyanate demonstrated greater potency in vitro than SCN⁻ itself in killing CF pathogens including P. aeruginosa, Burkholderia multivorans and methicillin-resistant Staphylococcus aureus ^{68, 74}.

Antifungal activity of HOSCN

SCN⁻ exhibits in vitro antifungal properties against several species. Early exploratory reports indicated the susceptibilities of several fungal species to HOSCN including Aspergillus niger, Pencillium chrysogeum, Aspergillus flaws, Alternaria sp., Trichoderma sp., Corynespora cassiicola, Phytopthora meadii, Claviceps sp., and Corticium salmonicolor ^{75, 76}. Candida albicans has also displayed susceptibility to LPO-generated HOSCN in vitro ⁷⁷. Normal saliva contains LF and LPO, which provide a modest candidacidal effect ^{78, 79}. The combination of HOSCN with LF showed fungicidal effect against C. albicans. HOSCN can inhibit various fungal functions, including amino acid and sugar transport, respiration, and glycolysis ⁸⁰. Alterations in the fungal membrane can enhance the efficacy of HOSCN by exposing vital cellular components ⁸¹.

HOSCN and LF synergistically exert their antifungal action, with lactoferrin altering the fungal membrane and enhancing HOSCN uptake by fungal cells ⁸². HOSCN has been found effective in inhibiting the growth of Saccharomyces cerevisiae, Aspergillus niger, Rhodutorula rubra, Byssochlamys fulva and Mucor rouxii in apple juice ⁸³. In addition to HOSCN, LPO is also directly capable of degrading certain fungal toxins, including aflatoxin B1 and G1, with aflatoxin G1 being inactivated 1.5 times more effectively than aflatoxin B1 ⁸⁴. Additionally, the LPO system can oxidize alpha-amanitin, a potent hepatotoxin produced by the Amanita phalloides fungus ^{85, 86}.

Molecular and cellular targets of HOSCN in pathogens

Although the antimicrobial properties of HOSCN have been reported for various microorganisms ⁵⁰, the precise molecular mechanisms and targets underlying its antimicrobial action remain incompletely understood. Clarifying the microbicidal mechanism of action of HOSCN is relevant for its potential future use in HOSCN/SCN⁻ therapy. These mechanisms are likely to vary across different pathogens due to the broad reactivity of oxidants with diverse molecules. Early studies reported key cellular processes being targeted by HOSCN in bacteria, primarily of the Streptococcus genus (Table 1). For example, HOSCN reversibly inhibited glucose transport in Streptococcus agalactiae. HOSCN modified thiol groups in the bacterial cell membrane which led to glucose transport blockage ⁸⁷. Another study showed that glucose uptake was impaired in bacteria when cows were prophylactically treated with HOSCN and immunized with colostral proteins from S. sobrinus or S. mutants ⁸⁸. HOSCN was generated at pH 5.5 or 6.5, by bovine milk lactoperoxidase, KSCN and H₂O₂ ⁸⁸. In the saliva, the growth of Streptococcus mitis was inhibited when treated with HOSCN due to valine uptake inhibition ⁸⁹. After HOSCN treatment, the growth of Streptococcus cremoris was inhibited reversibly due to the inhibition of oxygen uptake and glycolysis ⁹⁰. HOSCN treatment targeted glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the glycolytic pathway in the following bacterial strains Streptococcus sanguis, S. mitis, S. mutans, and S. salivarius (Table 1) ⁹¹. In E. coli, HOSCN reversibly blocked bacterial growth due to oxidation of bacterial thiol groups ⁹². Moreover, HOSCN can inhibit the urease activity of H. pylori, an activity required for the bacterium to withstand the stomach’s acidic environment ⁹³. In Streptococcus pneumoniae, two genes have been identified in a mutant screen that make the bacterium sensitive to HOSCN: hrcA and ctsR ⁹⁴ (Table 1.). Both genes are heat shock protein repressors ^{95, 96} and their activation by HOSCN could lead to reduced expression of heat shock proteins yielding a weaker shield against HOSCN-mediated oxidative stress and damage. The activity of hrcA has been reported to be redox-sensitive and mediated by reduction-induced oligomerization via cysteine residues ⁹⁷.

Table 1.

Molecular and cellular targets of the antimicrobial action of HOSCN.

HOSCN effect	Species	Refs.
Bacteria
Inhibition of glycolysis (hexokinase, GAPDH and 6- phosphogluconate dehydrogenase and aldolase) and glucose uptake	S. agalactiae, S. cremoris, S. pyogenes, S. sangius, S. mitis, S. mutans, S. salavarius, S. sobrinus	87, 88, 89, 90, 91, 143
Inhibition of oxygen uptake (electrochemical proton gradient and respiratory dehydrogenase)	E. coli	92, 143
Inactivation of urease that compromises the ability of the bacterium to adapt to low pH	H. pylori	93
Viruses
Inhibition of virus binding to the airway epithelium (possible target: cysteines in hemagglutinin)	Influenza A and B viruses	48

While more is known about the antibacterial mechanism of action of HOSCN, it has remained largely unknown how HOSCN inactivates viruses. We have shown that the binding of influenza A and B viruses to the host airway epithelium is the step in the viral infection cycle inhibited by HOSCN in vitro ⁴⁸. When several influenza virus strains were exposed to HOSCN in vitro, subsequent steps of the viral replication cycle were inhibited: virus binding, endosomal uptake, viral RNA synthesis and overall virus replication ⁴⁸. HOSCN did not affect the virions’ neuraminidase activity or the ability of the host cells to protect themselves against influenza infection ⁴⁸. The molecular basis for this action of HOSCN remains to be explored but modifications of cysteine residues in hemagglutinin, the viral surface protein responsible for virus binding to host cells ⁹⁸, is the primary suspect mechanism ⁴⁸.

In general, HOSCN’s oxidizing potential targeting cysteines in proteins key to microbial metabolism, protection and survival is suspected to form the molecular basis of its broad-ranging antimicrobial activity ^{25, 50}. HOSCN readily reacts with numerous biomolecules containing thiols and selenols, including glutathione (GSH), GAPDH, and thioredoxin reductase (TrxR) ^{25, 99, 100, 101}. Oxidoreductases like TrxR and GSH reductase can repair HOSCN oxidation products by donating electrons from NADPH, a product of the pentose phosphate pathway, linking redox state to glucose metabolism ¹⁰⁰. The initial products of HOSCN’s reaction with thiol groups are thiosulfenyl thiocyanates (RS-SCN), which can further undergo hydrolysis to sulfenic acid intermediates or reduction by thiols to form disulfides, depending on steric constraints ^{102, 103, 104}. These intermediates may also form heterodimeric disulfides, such as S-glutathionylated cysteine residues ^{102, 103, 104}. Whether HOSCN plays a crucial role in specific glutathionylation events that regulate cellular signaling remains unclear ¹⁰⁵. Lower pH can enhance the rate of oxidation by HOSCN, depending on the thiol ¹⁰¹. Under physiological pH, OSCN⁻ predominates over HOSCN, limiting its reactivity ²⁴. At high concentrations, HOSCN can also modify residues lacking thiols, such as tryptophan (Trp), histidine and tyrosine ¹⁰⁶. While HOSCN can oxidize Trp to 2-hydroxytryptophan on proteins in vitro, it cannot oxidize free Trp or Trp-containing peptides ¹⁰³, It is uncertain whether Trp oxidation by HOSCN occurs under physiological conditions ¹⁰⁷.

Currently, there is not a specific biomarker for HOSCN. Increased HOSCN levels correlate with thiol oxidation, but this can result from many other processes ¹⁰⁸. HOBr and HOCl rapidly react with multiple functional groups in biological molecules ^{109, 110, 111}, whereas HOSCN selectively targets thiols (RSH, cysteine), selenols and selenoethers ^{24, 101, 112}. As thiol oxidation by HOSCN yields reducible products, HOSCN decreases damage to biological targets caused by HOCl or HOBr ¹¹³. Interestingly, HOSCN or a related radical species (possibly SCN^•, OSCN^−•, or (SCN)₂^−•) formed by MPO can induce peroxidation of plasma lipids and isolated LDL, which can be blocked by ascorbate ^{111, 114, 115}. However, there is no evidence of HOSCN reactivity with lipid double bonds ^{110, 111}, and the reactivity of HOSCN with lipids in the presence of thiols remains uncertain. Furthermore, HOSCN does not react with isolated nucleosides, isolated DNA, or DNA within intact cellular systems ^{112, 113}. Thus, it is plausible that the majority of HOSCN’s antimicrobial activity is thiol-mediated. However, it remains important for organism specific reactions to become elucidated.

Defense mechanisms of pathogens against HOSCN

In 2018, when we published a long, comprehensive review of all the reports up until then demonstrating the antimicrobial action of HOSCN against viruses, bacteria, fungi and parasites ⁵⁰, tolerance mechanisms against HOSCN remained largely undiscovered. Since then, however, a growing body of evidence suggests that bacteria possess mechanisms to reduce the damage by HOSCN (summarized in Table 2). The most information on HOSCN tolerance mechanisms has been reported for Streptococcus pneumoniae. We have observed that HOSCN is capable of killing S. pneumoniae in vitro at physiologically relevant concentrations of SCN⁻ (400 μM) ⁶⁹. In contrast, Shearer et al. reported that S. pneumoniae is resistant to HOSCN in vitro, as well as HOCl in the presence of NETs ⁷¹. Differences in HOSCN sensitivity may, in part, be a result of different exposure durations: 4–6 hours of exposure in the study demonstrating killing ⁶⁹, and 30 minutes in the study showing resistance ⁷¹. Nevertheless, we did observe that spxB, a pyruvate oxidase and the main bacterial H₂O₂ source ¹¹⁶, can protect S. pneumoniae from HOSCN ⁶⁹, a finding later confirmed ⁹⁴. Thus, spxB increases the tolerance of S. pneumoniae towards HOSCN (Table 2). Additionally, glutathione utilization has been shown to protect S. pneumoniae against HOSCN produced by LPO in vitro ¹¹⁷. Bacterial growth in the presence of HOSCN was significantly decreased in case of mutants unable to import glutathione or to recycle oxidized glutathione ¹¹⁷. Furthermore, a new flavoprotein disulfide reductase has been discovered in S. pneumoniae that was named Har (hypothiocyanous acid reductase) ¹¹⁸. Bacterial growth was unaffected by Har deficiency but was inhibited when Har deletion was combined with disruption of glutathione import or recycling, proposing a role of Har in combination with glutathione utilization to protect S. pneumoniae from HOSCN ¹¹⁸. In a genome-wide screen 37 genes associated with HOSCN tolerance have been identified in S. pneumoniae ⁹⁴. Bacterial mutants deficient in these genes were significantly inhibited in their growth in the presence of HOSCN ⁹⁴. Generation of mutants with single-gene deletions of genes involved in redox homeostasis (trxA, sodA and spxB) in S. pneumoniae validated the results of the mutant screen ⁹⁴. TrxA is an oxidoreductase involved in the thioredoxin/thioredoxin reductase system ¹¹⁹. SodA is manganese-containing superoxide dismutase converting superoxide to H₂O₂ and protecting bacteria from superoxide-mediated damage ¹²⁰. As discussed earlier, spxB is a pyruvate oxidase generating bacterial H₂O₂ in S. pneumoniae. Several other hits (manY, manA, relA, thiN, ktrB) of the mutant screen that have not been confirmed with single gene deletion mutants in reports yet could also represent additional mechanisms of HOSCN tolerance of S. pneumonaie ⁹⁴. In Staphylococcus aureus, merA has been identified as an HOSCN reductase, safeguarding bacterial cells ¹²¹.

Table 2.

Defense mechanisms of bacteria against HOSCN.

Bacterial species	References
Streptococcus pneumoniae
spxB, pyruvate oxidase	69, 144
glutathione utilization	117
Har, HOSCN reductase	118
trxA, oxidoreductase in Trx system	144
sodA, superoxide dismutase	144
Additional genes to be confirmed (manY, manA, relA, thiN, ktrB)	144
Staphylococcus aureus
MerA, flavoprotein disulfide reductase	121
Escherichia coli
RclA, highly active HOSCN reductase	122
increased expression of corA, mgtA, tcyP, fruB	124
Pseudomonas aeruginosa
rclX, predicted peroxidasin	125
polyphosphate preventing protein aggregation	126
Induction of RclR and MexT	125, 126
Pyocyanin inhibiting Duox	129, 130

In Gram-negative bacteria, a recent study identified in Escherichia coli the flavoprotein RclA that reduces HOSCN to SCN⁻ and protects the bacterium against HOSCN toxicity in vitro ¹²². RclA has previously been implicated in reactive chlorine resistance and is conserved in a variety of epithelium-colonizing bacteria, hinting at the potential significance of its HOSCN-reducing activity in host-microbe interactions ¹²². E. coli mutants lacking rclA display susceptibility to direct HOSCN exposure in a minimal glucose medium ¹²³. RclA homologs from S. aureus, S. pneumoniae and Bacteroides thetaiotaomicron have also demonstrated protective effects against HOSCN when expressed in E. coli ¹²². In E. coli, the LPO-SCN⁻ system was shown to induce the upregulation of genes like corA and mgtA that encode metal ion transporter, cysJ (encoding the alpha subunit of sulfite reductase), tcyP (encoding a cysteine transporter), and fruB (encoding a fructose transport protein). However, the precise contributions of these genes to HOSCN response remain ambiguous ¹²⁴.

In response to HOSCN, P. aeruginosa upregulates the expression of RclX, a predicted peroxiredoxin ¹²⁵. Transcriptional changes in P. aeruginosa PA14 in response to both HOSCN and HOCl have been reported ¹²⁵. Additionally, treatment of P. aeruginosa PA14 with a bolus exposure of HOSCN in minimal glucose medium leads to protein aggregation, triggering the heat shock response and causing membrane damage ¹²⁶. P. aeruginosa produces high levels of polyphosphate, a conserved polymer that binds unfolded proteins, thereby preventing protein aggregation resulting from HOSCN-induced protein unfolding ¹²⁶. Two independent studies investigating the whole genome transcriptomic responses in P. aeruginosa to HOSCN reported significant overlap in their findings. The responses included the induction of protein-stabilizing chaperons, RclR and MexT, regulons and sulfur-containing amino acid metabolism ^{125, 126}. Additionally, HOSCN reductase activity has been observed in chronic colonizing strains of P. aeruginosa from cystic fibrosis patients ¹⁰⁰.

There are additional proposed mechanisms through which microbes could potentially undermine HOSCN’s antimicrobial activities of HOSCN. Rather than directly targeting HOSCN, bacteria might inhibit the function or the expression of proteins involved in HOSCN generation. For instance, our research has demonstrated that pyocyanin, a redox-active secreted toxin of P. aeruginosa known for its proinflammatory effects on the host ^{127, 128}, can inhibit Duox-mediated H₂O₂ production, Duox1 protein expression and HOSCN-mediated killing of P. aeruginosa and Burkholderia cepacia in human respiratory epithelial cell cultures ^{129, 130}.

These emerging findings suggest the existence of ‘redox warfare’ taking place between the innate immune system and inhaled pathogens in the lung. Subsequent investigations are necessary to delve deeper into the responses of various microorganisms to HOSCN across diverse experimental conditions. Such endeavors will not only contribute to a more comprehensive understanding of the mechanisms underlying HOSCN resistance but also facilitate the development of inhibitors targeting these mechanisms to enhance HOSCN-based innate immune responses.

SCN⁻/HOSCN therapy to combat infections

Given the substantial body of literature demonstrating the in vitro antimicrobial effects of HOSCN generated by peroxidases from SCN⁻ and H₂O₂, a pertinent question arises: could this innate immune mechanism be therapeutically enhanced to improve clinical outcomes in infections?

While evidence regarding the in vivo significance and efficacy of this system in eliminating microbes in mammalian organisms remains limited, it is steadily accumulating. For example, our study revealed that mice deficient in Duox1 are more susceptible to influenza A virus lung infection compared to their wild-type counterparts ⁴⁸. Conversely, we also found that Duox1 deficiency does not influence the susceptibility of mice to lung infections caused by another pathogen, Mycobacterium tuberculosis ⁴⁹. Studies have shown a correlation between SCN⁻ concentrations in airway surface liquid and improved lung function in individuals with cystic fibrosis ¹³¹. Furthermore, excess iodide exposure in humans leads to increased salivary levels of iodide and antimicrobial hypoiodous acid ¹³². These findings agree with the notion that peroxidase substrates (SCN⁻, I⁻) are enriched in mucosal secretions in humans, namely saliva and airway lining fluid ⁴⁰. This underscores the potential for oral or intravenous administration of peroxidase substrates to enhance airway defenses against respiratory pathogens in humans.

There is precedence for using peroxidase substrates in animal models to protect against acute infections. For instance, potassium iodide administration improved outcomes in sheep infected with respiratory syncytial virus ¹³³. In our studies, we demonstrated that nebulized SCN⁻ improves outcomes in mice with P. aeruginosa lung infection ^{21, 134}. Additionally, we found that HOSCN administration improved the outcomes of lethal influenza infection in mice by potentiating the effects of anti-influenza drugs ⁵². This suggests that SCN⁻/HOSCN could be employed as part of combination therapies. HOSCN production in vivo could not only be enhanced by pharmacological administration of SCN⁻ but also by increasing H₂O₂ production generated by Duox1/2 or Nox2. This possibility should also be explored in the future in detail.

The accumulating data suggest that SCN⁻/HOSCN-based therapies could be developed and used in the future to target infections. Given HOSCN’s reactivity and short half-life, direct administration to sites of infection is logical, whereas SCN⁻ functions more as a prodrug and scavenger of stronger oxidants (HOCl, HOBr). Thus, SCN⁻ may prove effective when either administered locally ^{21, 134} or systemically to combat infections.

Conclusions

SCN⁻ and HOSCN have been studied as antimicrobial agents in vitro and in vivo against a wide range of pathogens. They have also been explored as potential treatments for respiratory, gastrointestinal, neurological and cardiovascular diseases, with a history of use as an antihypertensive^{16, 135, 136, 137, 138, 139, 140}. Emerging evidence suggests the safety and efficacy of SCN⁻ and HOSCN in combatting pathogenic infections and inflammation, particularly in short-term lung applications.

By reacting with thiols in protein cysteines important to the function of the pathogen, HOSCN interrupts important steps in mechanisms of pathogen survival including metabolism and host cell entry. Additionally, HOSCN could potentially overcome pathogen resistance to existing antimicrobial drugs. Agents targeting anti-HOSCN pathogen mechanisms represent an entirely new avenue of study that may empower innate immune function against specific organisms.

Ultimately, SCN⁻ and HOSCN hold clear promise for therapy in human lung infections. By limiting HOCl, SCN⁻ has the potential to serve as an antioxidant in inflammatory diseases that feature neutrophilic involvement. HOSCN is expected to control pathogens by reacting with critical protein cysteines. While there are clear reports of potassium SCN⁻ toxicity from the early 20^th century, when this drug was utilized in large doses to be tested as an antihypertensive, far lower doses of SCN⁻ are anticipated to be sufficient for antimicrobial and anti-inflammatory therapy. However, additional studies are needed to confirm both safety and benefit of SCN⁻ and HOSCN in controlling infections, as both molecules have potential for toxicity in sufficiently high doses. The molecular mechanisms of pathogen control reviewed here can guide further HOSCN research into pathogen control. Adjunctive therapies targeting anti-HOSCN resistance mechanisms may also become important, or complementary therapies of SCN⁻ and other anti-pathogen drugs.

Highlights.

• Hypothiocyanous acid (HOSCN) is an endogenous oxidant that demonstrates antimicrobial activities against viruses, bacteria and fungi

• Multiple preclinical studies reported benefits of promoting HOSCN formation in vivo to treat pulmonary infections

• HOSCN is better tolerated by mammalian cells than other chordata peroxidase-derived oxidants, such as hypochlorous acid

• Some bacterial strains are resistant to HOSCN, primarily via similar oxidoreductase mechanisms to those reported in mammalian cells

• HOSCN (or precursor thiocyanate) treatment holds promise for viral, bacterial, and fungal infections, but investigators must equally consider potential toxicity and HOSCN resistance

Abbreviations

Duox

dual oxidase

EPO

eosinophil peroxidase

HOBr

hypobromous acid

HOCl

hypochlorous acid

HOSCN

hypothiocyanous acid

HSV-1

herpes simplex virus 1

H₂O₂

hydrogen peroxide

LF

lactoferrin

LPO

lactoperoxidase

MPO

myeloperoxidase

NIS

sodium iodide symporter

NOX

NADPH Oxidase

OSCN⁻

hypothiocyanite

PTC

papillary thyroid cancer

PXN

peroxidasin

RSV

respiratory syncytial virus

SCN⁻

thiocyanate

TPO

thyroid peroxidase

TrxR

thioredoxin reductase