DOI: 10.5061/dryad.547d7wm5t
title: Data from: Oxidative killing of encapsulated and nonencapsulated Streptococcus pneumoniae by lactoperoxidase-generated hypothiocyanite

description: 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 (H2O2). LPO
 oxidizes thiocyanate using H2O2 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 H2O2 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 H2O2
 source) had enhanced susceptibility to hypothiocyanite compared o its
 wild-type strain. Overall, results shown here indicate that numerous
 pneumococcal strains are susceptible to LPO-generated hypothiocyanite.
description: <i>Bacterial killing
 measured by colony counting</i> Components of
 the LPO-based antibacterial system were used as described previously (8).
 Briefly, the following concentrations were used: 6.5 µg/ml LPO, 400 µM
 SCN<sup>-</sup>, 5 mM glucose and 0.1 U/mL glucose oxidase.
 The reaction volume was set to 120 µL. Catalase (700 U/mL) was also used
 when indicated to inhibit the system by scavenging
 H<sub>2</sub>O<sub>2</sub>. The components were
 assembled in a sterile 96-well microplate in triplicates with the bacteria
 being added last at a maximal concentration of 5x10<sup>5
 </sup>CFU/ml. The plates were then placed in a 37°C incubator with
 5% CO<sub>2</sub>. After 6 hours of incubation, 40 µL was
 spread onto BAP in triplicate and incubated at 37°C with 5%
 CO<sub>2</sub>. After 24 hours, the colonies were counted and
 CFU/mL was determined. Agar plates exposed to only the assay medium
 without <i>Spn</i> were always used to ensure that no
 potential contaminants were detected. A time 0 condition was also counted
 to make sure that bacterial death was due to OSCN<sup>-</sup>
 and not related to an unknown variable, and thatno significant changes in
 bacterial numbers were observed in samples containing only bacteria during
 the duration of the experiments. All the reagents were ordered from
 Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise.
   <i>Bacteriostatic activity
 measured by a microplate-based growth assay</i> The
 bacteriostatic activity of OSCN<sup>-</sup> was measured by a
 microplate-based assay described previously (23). Briefly, the components
 (mentioned in the cell-free assay) were assembled in a sterile 96-well
 plate with the bacteria being added last. Bacterial growth was measured in
 a microplate spectrophotometer [Eon (BioTek Instruments Inc., Winooski,
 VT, USA) or Varioskan Flash (Thermo Scientific, Rochester, NY)] on the
 basis of following increases in OD as a measure of bacterial density. This
 method enables fast and very reproducible measurement of bacterial growth
 (23). <i>Spn</i> strains were grown at 37°C for 14 hours, and
 OD at 600 nm was measured every 3 minutes. Each sample was run in
 triplicate. The time required for the positive control
 (<i>Spn</i> alone) to reach an OD of 0.4 (exponential growth
 phase) was used as the reference point for all other conditions, and OD
 values of other samples were compared to this. All the
 <i>Spn</i> strains used in this work were tested individually
 for their suitability for this method.   <i>The commercial
 1<sup>st</sup> line™ immune support
 product</i> <i>1<sup>st</sup> line™ </i>is an over-the-counter product that is marketed as an immune supplement (distributed by Profound Products). This product uses a proprietary technology to keep OSCN<sup>-</sup> stable for a longer period of time allowing for a better antimicrobial effect. To our knowledge, this product is the only commercially available product producing OSCN<sup>-</sup>. We tested this product in conjunction with our previously described cell-free system. Briefly, 0.1 g of LPO was reconstituted in 25 mL of HBSS. 750 µL of H<sub>2</sub>O<sub>2</sub> solution was added to a 15 mL conical tube followed by the addition of 700 U/mL of catalase. The solution was incubated for 10 minutes to allow catalase to scavenge all H<sub>2</sub>O<sub>2</sub> present. 12.5 mL of LPO solution was then added to each tube and mixed. Following this step, 750 µL of SCN<sup>-</sup> was also added to each sample and mixed thoroughly. Finally, 750 mL poly aluminum chloride was administered to each solution and mixed well. Samples were then incubated for 30 minutes at room temperature allowing the generation of OSCN<sup>-</sup>. By the end of this incubation time, the solution separates into two distinct phases. The top, clear phase containing OSCN<sup>-</sup> was used for experiments while the pelleted precipitate was discarded.   <i>Bacterial H<sub>2</sub>O<sub>2</sub> production</i> Generation of H<sub>2</sub>O<sub>2</sub> in bacterial suspension was measured by the ROS-Glo<sup>TM</sup> luminescence kit following the manufacturer’s instructions (Promega Corporation, Madison, WI, USA). This sensitive assay enables specific and direct detection of low amounts of H<sub>2</sub>O<sub>2</sub>. TIGR4 wild-type or <i>D</i><i>spxB</i> bacteria (5x10<sup>6</sup>/ml) suspended in HBSS buffer were incubated at 37 ºC for 30 minutes, followed by centrifugation to collect supernatants for analysis of H<sub>2</sub>O<sub>2</sub> production. Ros-GLo<sup>TM</sup> reagent was added to bacterium-free supernatants and luminescence was read using a Varioskan Flash microplate luminometer (Thermo Scientific, Rochester, NY). The assay was run in triplicates. Results are expressed as relative luminescence units (RLU).   <i>Quantitation of </i>OSCN<sup>-</sup><i> generation</i> <b>          </b>Production of OSCN<sup>-</sup> was assessed using the photometric 5-thio-2-nitrobenzoic acid (TNB) oxidation assay (24). OSCN<sup>-</sup> converts TNB that absorbs light at 412 nm, into a colorless disulfide (5,5’-dithio-bis-[2-nitrobenzoic acid]) (DNTB, Ellman’s reagent). OSCN<sup>-</sup> production is measured as decrease in OD at 412 nm and is calculated based on the Lambert-Beer Law and the absorption coefficient e<sub>412</sub> = 14,100 M<sup>-1</sup> cm<sup>-1 </sup><sup>(25)</sup>. OSCN<sup>-</sup> production is expressed as concentration of OSCN<sup>-</sup> produced in the volume of the cell-free system under different conditions in 30 minutes.