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Survivors of Immune System Antimicrobials: How Do Bacteria Detoxify Hypothiocyanite?

Bacteria are known to be adaptive to their environments, but how exactly are they able to thrive in the presence of known antimicrobials?

To survive in the human body, many strains of bacteria, both beneficial and pathogenic, have to withstand the antimicrobials produced by the immune system. But how exactly are bacteria adapted to survive such harsh conditions?

Fortunately, a study conducted by researchers at the University of Alabama at Birmingham Department of Microbiology has discovered how Escherichia coli copes with the toxicity of the antimicrobials hypothiocyanite (OSCN– )/hypothiocyanous acid (HOSCN). The mechanism by which it copes is also observed in Staphylococcus bacteria and other beneficial gut microbes.

According to Assistant Professor Michael Gray at the University of Alabama at Birmingham Department of Microbiology, antimicrobials like those above are released when the human immune system triggers inflammation. “Understanding how bacteria can evade these powerful oxidants, including the hypohalous acids like HOSCN, is crucial to human health,” he said.

His research successfully identified an enzyme that E. coli produces to cope with the antimicrobials OSCN and HOSCN in a hostile inflamed gut environment for its survival. This study allows for new insights into how bacteria, in general, can counteract the constant assaults by the human immune system, which depending on their functions, may cause infection or aid in the functions of the body.

E. coli survives well in patients with inflammatory bowel disease and can outcompete other beneficial gut microbes essential for proper gut health and functioning, thus the researchers propose that HOSCN may be one of the main antimicrobials produced in the gut.

HOSCN has been previously known to be found in airway and saliva secretion as a potent antimicrobial that is relatively non-toxic towards mammalian cells. However, if pathogenic microbes are able to survive the action of this antimicrobial, they will continue to thrive and are likely to result in prolonged infection with more severe outcomes.

The team of researchers, led by Assistant Professor Gray, and assistant professor of chemistry at Western Michigan University, Kalamazoo, reported their findings that the E. coli enzyme (a flavoprotein, capable of catalysing redox reactions) RclA reduces HOSCN to harmless thiocyanate at extremely fast rates, which helps to strongly protect E. coli from the antimicrobial action of HOSCN. They also proposed that HOSCN is the key substrate for RclA, compared to its previously proposed ability in detoxifying reactive chlorine.

With this in mind, the researchers also tested the homologous flavoprotein enzymes produced by Bacteroides thetaiotaomicron, Staphylococcus aureus, and Streptococcus pneumoniaS. aureus and S. pneumonia have been found to thrive in inflamed tissues, like the lungs, where high levels of HOSCN are observed, while B. thetaiotaomicron is known to be a beneficial gut microbe.

The three homologous flavoprotein enzymes—observed to have 47 per cent to 49 per cent similarity in terms of amino acid sequence identity compared to the E. coli-produced RclA—were duplicated in E. coli, where they all displayed strong resistive effects to the antimicrobial activity of HOSCN. In addition, the active sites of the E. coli RclA enzyme and the enzymes from the three other species of bacteria were also found to have high amino acid sequence activity, providing a strong link to back up their ability to detoxify HOSCN.

The antimicrobial activity of HOSCN is due to its ability to oxidise amino acids that contain sulfur, like cysteine, destroying the original structures and functions of bacterial proteins. The RclA enzyme, classified as an oxidoreductase—enzymes that catalyse the transfer of electrons from a donor to acceptor species—have two cysteine residues in their active sites and are proposed to be the reason behind the active sites’ affinity for HOSCN. Both cysteine residues are essential for the proper functioning of the RclA enzyme and NAD(P)H is also involved in the reduction of HOSCN.

“Perhaps our most exciting finding is that homologs of RclA, including from the gut commensal species B. thetaiotaomicron and Limosilactobacillus reuteri and from species implicated in serious lung disease, protect against HOSCN damage to the same degree as E. coli RclA,” Gray said. Professor Gray also mentions that his discovery shows that a large variety of bacteria have evolved to tolerate human immune system antimicrobials, like HCN. “Learning more about the scope of protection provided by this enzyme to pathogenic species will gain us better knowledge on potentially a wide range of diseases, including cystic fibrosis, inflammatory bowel disease and oral diseases,” he continued.

“While we have not yet directly addressed the effect of this enzyme on host colonisation in vivo, we have laid an important foundation for future studies with the data we have gathered here,” Gray said. [APBN]

Source: Meredith et al. (2022). Escherichia coli RclA is a highly active hypothiocyanite reductase. Proceedings of the National Academy of Sciences, 119(30), e2119368119.