Resistance of Streptococcus pneumoniae to Hypothiocyanous Acid Generated by Host Peroxidases

Abstract

Streptococcus pneumoniae is a serious human respiratory pathogen. It generates hydrogen peroxide (H 2 O 2 ) as part of its normal metabolism, yet it lacks enzymes that remove this oxidant. Here we show that lactoperoxidase and myeloperoxidase, two host enzymes present in the respiratory tract, convert bacterial H 2 O 2 into HOSCN that S. pneumoniae can resist. We found that incubation of S. pneumoniae with myeloperoxidase in chloride-rich buffer killed the bacteria due to formation of toxic hypochlorous acid (HOCl). However, the addition of physiological concentrations of thiocyanate protected the bacteria. Similarly, S. pneumoniae remained viable in the presence of lactoperoxidase and thiocyanate even though the majority of bacterial H 2 O 2 was converted to hypothiocyanous acid (HOSCN). S. pneumoniae and Pseudomonas aeruginosa , another respiratory pathogen, were similarly sensitive to H 2 O 2 and HOCl. In contrast, S. pneumoniae tolerated much higher doses of HOSCN than P. aeruginosa . When associated with neutrophil extracellular traps (NETs), S. pneumoniae continued to generate H 2 O 2 , which was converted to HOCl by MPO present on NETs. However, there was no loss in bacterial viability because HOCl was scavenged by the NET proteins. We conclude that at sites of infection, bacteria will be protected from HOCl by thiocyanate and extracellular proteins including those associated with NETs. Resistance to HOSCN may give S. pneumoniae a survival advantage over other pathogenic bacteria. Understanding the mechanisms by which S. pneumoniae protects itself from HOSCN may reveal novel strategies for limiting the colonization and pathogenicity of this deadly pathogen.

A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid

“…Initial insight came from a study on H 2 O 2 -producing oral streptococci, where some species were shown to have an uncharacterized HOSCN oxidoreductase activity that correlated with their ability to survive HOSCN (26). We noted that S. pneumoniae survives HOSCN exposure much better than the opportunistic and HOSCN reductase-negative respiratory pathogen P. aeruginosa (20), which unlike S. pneumoniae, does not colonize and rarely causes disease in healthy people. We have now identified and characterized a HOSCN reductase in S. pneumoniae that contributes to the ability of this deadly pathogen to tolerate HOSCN.…”

“…We recently reported that S. pneumoniae is relatively tolerant of HOSCN, when compared with another respiratory microbe Pseudomonas aeruginosa ( 20 ). This raises the question as to what mechanisms are responsible for HOSCN tolerance.…”

Hypothiocyanite and host–microbe interactions

“…Therefore, the ability of P. aeruginosa , a major pathogen of the CF lung (Greenwald & Wolfgang, 2022), to respond to and survive stress caused by HOSCN is important to understand. P. aeruginosa is substantially more sensitive to HOSCN than S. pneumoniae (Shearer, Kaldor, et al, 2022), and is more sensitive at pH 6.8 than at pH 7.4 (Chandler et al, 2013), consistent with protonation of HOSCN being an important determinant of its toxicity for bacteria.…”

“…HOSCN is much less reactive than HOCl (Pattison et al, 2012), which may reduce the impact of cell density in HOSCN stress assays. Experiments with S. pneumoniae suggest that bacteria react only slowly with HOSCN (Shearer, Kaldor, et al, 2022; Shearer, Pace, et al, 2022), suggesting that even bolus addition of HOSCN may result in a relatively slow flux of oxidant into the cells.…”

“…S. pneumoniae , like some oral streptococci, produces abundant H 2 O 2 (Carlsson et al, 1983). While the LPO‐SCN − system is capable of killing S. pneumoniae (Gingerich et al, 2020), this species is more HOSCN‐resistant than other tested lung pathogens (Shearer, Kaldor, et al, 2022). HOSCN resistance in this bacterium is partially due to the ability of S. pneumoniae to import glutathione and glutathionylated proteins after HOSCN treatment, and mutants lacking either the glutathione transporter GshT or glutathione reductase Gor are significantly more sensitive to both bolus addition of HOSCN in minimal salts solution and to the LPO‐SCN − system in rich media (Shearer, Paton, et al, 2022).…”