Oxidative killing of encapsulated and nonencapsulated Streptococcus pneumoniae by lactoperoxidase-generated hypothiocyanite

Abstract

Streptococcus pneumoniae (Pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Despite recent successes due to pneumococcal vaccination and antibiotic use, Pneumococcus remains a significant medical problem. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase (LPO), thiocyanate anion and hydrogen peroxide (H 2 O 2). LPO oxidizes thiocyanate using H 2 O 2 into the final product hypothiocyanite that has antimicrobial effects against a wide range of microorganisms. However, hypothiocyanite's effect on Pneumococcus has never been studied. Our aim was to determine whether hypothiocyanite can kill S. pneumoniae. Bactericidal activity was measured in a cell-free in vitro system by determining the number of surviving pneumococci via colony forming units on agar plates, while bacteriostatic activity was assessed by measuring optical density of bacteria in liquid cultures. Our results indicate that hypothiocyanite generated by LPO exerted robust killing of both encapsulated and nonencapsulated pneumococcal strains. Killing of S. pneumoniae by a commercially available hypothiocyanite-generating product was even more pronounced than that achieved with laboratory reagents. Catalase, an H 2 O 2 scavenger, inhibited killing of pneumococcal by hypothiocyanite under all circumstances. Furthermore, the presence of the bacterial capsule or lytA-dependent autolysis had no effect on hypothiocyanite-mediated killing of pneumococci. On the contrary, a pneumococcal mutant deficient in pyruvate oxidase (main bacterial H 2 O 2 source) had enhanced susceptibility to hypothiocyanite compared to its wild-type strain. Overall, results shown here indicate that numerous pneumococcal strains are susceptible to LPO-generated hypothiocyanite.

Microbicidal Activity of Hypothiocyanite against Pneumococcus

“…NADPH oxidases are among the main sources of endogenous ROS in the body. In the airways, dual oxidases 1 and 2 (DUOX1 and DUOX 2) are major sources of H 2 O 2 that is used to convert SCN- to OSCN- in the presence of LPO [ 8 , 9 ]. This family of transmembrane proteins transfer an electron from NADPH to an FAD cofactor followed by a transfer to a heme group which generates the final oxidative product [ 24 , 25 , 26 ].…”

“…This is due to the fact that antibiotics have specific targets (i.e., the cell wall, nucleic acid synthesis or inhibition of metabolic pathways) [ 33 ]. Contrary to these traditional targets, OSCN- has the ability to oxidatively target a wide range of species, including bacteria [ 9 , 10 , 11 , 12 ], viruses [ 13 , 14 , 15 , 16 ] and fungi [ 17 ], due to its nonspecific nature using ROS. However, one major drawback of OSCN- is its short half-life [ 34 ], making it a difficult substance to work with, both in a laboratory setting as well as in vivo.…”

“…in vitro [ 12 ]; however, we were the first laboratory to show that it is also effective against Spn . In our recent work, we studied the effects of OSCN- on different serotypes of Spn , specifically those that contain or lack a protective capsule [ 9 ]. Using a cell-free in vitro system containing LPO/SCN-/H 2 O 2 , we were able to show that SCN- was converted to OSCN- and there was effective killing of both encapsulated and non-encapsulated variants of Spn .…”

“…One of the antimicrobial substances used by the airway epithelium is the antimicrobial oxidizing agent, hypothiocyanite (OSCN-). In the airways, thiocyanate (SCN-) is oxidized by H 2 O 2 in the presence of lactoperoxidase (LPO) to form OSCN- [ 8 , 9 ]. This system has been shown to have in vitro microbicidal activity against a wide variety of pathogens including bacteria [ 9 , 10 , 11 , 12 ], viruses [ 13 , 14 , 15 , 16 ] and fungi [ 17 ].…”

“…In the airways, thiocyanate (SCN-) is oxidized by H 2 O 2 in the presence of lactoperoxidase (LPO) to form OSCN- [ 8 , 9 ]. This system has been shown to have in vitro microbicidal activity against a wide variety of pathogens including bacteria [ 9 , 10 , 11 , 12 ], viruses [ 13 , 14 , 15 , 16 ] and fungi [ 17 ]. A comprehensive table of such organisms can be found in our review [ 12 ].…”