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

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 to its wild-type strain. Overall, results shown here indicate that numerous pneumococcal strains are susceptible to LPO-generated hypothiocyanite.

Introduction

Streptococcus pneumoniae (Spn) is a leading cause of bacterial infections such as otitis media, pneumonia, septicemia and meningitis [1, 2]. Colonization can occur at any point in a person’s life but occurs most commonly in young children where Spn prevalence reaches over 50% in hosts 2–3 years old [3]. Worldwide, Spn is a major cause of infant mortality with 1.2 million deaths reported every year [2, 4]. Current pneumococcal vaccines target the capsular polysaccharide of Spn, but these vaccines only provide serotype-specific protection against less than one third of circulating serotypes [5]. Spn infections can also be controlled with antibiotics, but widespread antibiotic use has led to accelerated antibiotic resistance in Spn [6]. These challenges have led to the need for novel therapeutics and a better understanding of Spn interactions with the host.

The airway epithelium represents one of the largest physical and immune barriers against airborne microbes such as Spn [7]. Lactoperoxidase (LPO) is a heme peroxidase found in the airway surface liquid (ASL) where it performs its antimicrobial activity [8]. The LPO-based antimicrobial system requires two other components to function properly. First, LPO needs a source of H2O2 to catalyze the reaction. In the human airways, H2O2 is mainly supplied by the NADPH oxidase Dual oxidase 1, Duox1 [9-11]. LPO then uses H2O2 to oxidize the pseudohalide thiocyanate (SCN-) which is abundantly present in the ASL into the antimicrobial ion hypothiocyanite (OSCN-) [9, 12]. The LPO-based system has previously been shown to be an effective in vitro neutralizer of a wide variety of viruses [13-15] and bacteria [8, 16]. Interestingly, even though Spn represents an enormous health burden, the effectiveness of the LPO-based system against Spn has not been tested so far.

Due to the relevance of Spn in public health combined with the emergence of antibiotic resistance, the LPO-based system could provide valuable insight and a possible new therapeutic option for management of Spn infections. We hypothesized that the LPO-based system is effective at killing Spn in vitro. We found that both encapsulated and nonencapsulated pneumococci were susceptible to OSCN—mediated killing in a cell-free experimental system.

Materials and methods

Bacteria

Spn strains EF3030 (encapsulated serotype 19F) [17], EF3030 Δcap (isogenic, nonencapsulated mutant strain) [18], MNZ41 (nonencapsulated) [19], TIGR4 (encapsulated serotype 4) [20], TIGR4ΔspxB (isogenic mutant deficient in pyruvate oxidase) [21] and TIGR Δcap (isogenic mutant deficient in capsule formation generated by the same strategy as EF303 Δcap) [18], D39 (encapsulated serotype 2) and its isogenic, capsule-free mutant, D39 Δcap [22] were inoculated on sheep blood agar plates (BAP) and incubated at 37°C in 5% CO2. After incubation, bacteria were collected and harvested by centrifugation at 10,000g for 5 minutes, washed twice with Hank’s balanced salt solution (HBSS), and suspended in HBSS. Bacterial density was then determined by measuring optical density (OD) at 600 nm. The bacterial density was set to 0.6, which is representative of 109 CFU/mL Spn that was confirmed by performing serial dilutions, plating bacteria on BAP and counting colonies. Bacteria were prepared this way for both, bacterial killing and bacteriostatic measurements. The identities of the Spn strains were confirmed by 16S rRNA Gene Sequencing (Genewiz, South Plainfield, NJ, USA). Optochin-sensitivity of the Spn strains used was also confirmed for each experiment using BD BBL™ Taxo™ P Discs (Fisher Scientific, Pittsburgh, PA, USA).

Bacterial killing measured by colony counting

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-, 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 H2O2. The components were assembled in a sterile 96-well microplate in triplicates with the bacteria being added last at a maximal concentration of 5x105 CFU/ml. The plates were then placed in a 37°C incubator with 5% CO2. After 6 hours of incubation, 40 μL was spread onto BAP in triplicate and incubated at 37°C with 5% CO2. After 24 hours, the colonies were counted and CFU/mL was determined. Agar plates exposed to only the assay medium without Spn 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- and not related to an unknown variable, and that no 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.

Bacteriostatic activity measured by a microplate-based growth assay

The bacteriostatic activity of OSCN- 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]. Spn 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 (Spn 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 Spn strains used in this work were tested individually for their suitability for this method.

The commercial 1st line™ immune support product

1 line™ 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- 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-. 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 H2O2 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 H2O2 present. 12.5 mL of LPO solution was then added to each tube and mixed. Following this step, 750 μL of SCN- was also added to each sample and mixed thoroughly. Finally, 750 μL 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-. By the end of this incubation time, the solution separates into two distinct phases. The top, clear phase containing OSCN- was used for experiments while the pelleted precipitate was discarded.

Bacterial H2O2 production

Generation of H2O2 in bacterial suspension was measured by the ROS-GloTM luminescence kit following the manufacturer’s instructions (Promega Corporation, Madison, WI, USA). This sensitive assay enables specific and direct detection of low amounts of H2O2. TIGR4 wild-type or ΔspxB bacteria (5x106/ml) suspended in HBSS buffer were incubated at 37 ºC for 30 minutes, followed by centrifugation to collect supernatants for analysis of H2O2 production. Ros-GLoTM 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).

Quantitation of OSCN- generation

Production of OSCN- was assessed using the photometric 5-thio-2-nitrobenzoic acid (TNB) oxidation assay [24]. OSCN- converts TNB that absorbs light at 412 nm, into a colorless disulfide (5,5’-dithio-bis-[2-nitrobenzoic acid]) (DNTB, Ellman’s reagent). OSCN- production is measured as decrease in OD at 412 nm and is calculated based on the Lambert-Beer Law and the absorption coefficient ε412 = 14,100 M-1 cm-1 [25]. OSCN- production is expressed as concentration of OSCN- produced in the volume of the cell-free system under different conditions in 30 minutes.

Statistical analysis

Significance among multiple samples was calculated using One-way or Two-Way ANOVA followed by Tukey’s or Sidak's multiple comparison post-hoc tests. Significance between two samples was calculated using Mann-Whitney’s test. Statistical analysis was performed using Prism 6 for Windows version 6.07 software. *, p<0.05; **, p<0.01; ***, p<0.001.

Results

LPO-derived hypothiocyanite kills a diverse array of Spn strains

To explore the effects of OSCN- on Spn, we used our previously established cell-free in vitro system that generates OSCN- at levels comparable to those measured in human airways [26]. This system utilizes the enzyme glucose oxidase, which oxidizes its substrate glucose to produce D-gluconolactate [27]. H2O2 is a byproduct of the reaction and allows us to mimic the nature of H2O2 production in vivo by Dual oxidases [8, 26]. Previously, we have successfully used this experimental system to show that LPO-generated OSCN- inactivates a wide range of influenza strains [13, 27]. Using this H2O2/LPO/SCN- cell-free system, we wanted to determine the effectiveness of OSCN- against Spn. Physiologically relevant levels of each component of the system were utilized: 400 μM SCN- [28] and 6.5 μg/ml LPO [11]. H2O2 production by glucose oxidase was set to a rate of 0.01 U/ml, which is similar to what is seen in primary normal human bronchial epithelial (NHBE) cells by Duox [13, 29]. We tested both encapsulated (TIGR4, EF3030) and nonencapsulated (MNZ41) Spn strains, and we observed that the cell-free H2O2/LPO/SCN- system effectively killed all three strains of Spn (Fig 1A). Fig 1B shows that OSCN- is only produced when all components of the cell-free system are added. OSCN- is generated reproducibly to achieve a final OSCN- concentration of 41.2 ± 4.2 μM (mean ± S.E.M., n = 4) (Fig 1B). Since H2O2 alone is capable of killing Spn [30], we exposed EF3030 Spn to the same levels of glucose oxidase without SCN- or LPO to ensure our results were OSCN—mediated. The results show that Spn survival is not impaired by H2O2 alone (Fig 1C).

Fig 1

LPO-derived hypothiocyanite kills Spn in vitro.

(A) OSCN—mediated Spn killing was tested in the cell-free system against three different bacterial strains: EF3030 encapsulated serotype 19F (n = 5), MNZ41 nonencapsulated (n = 5) and TIGR4 encapsulated serotype 4 (n = 4). Bacteria were incubated for 6 hrs with or without OSCN- generated by the LPO/SCN-/H2O2 system and bacterial killing was quantified by colony counts on BAP. Each dot represents a separate, individual experiment and their mean is also shown, Mann-Whitney test. (B) OSCN- is only produced in the cell-free system when all of its components (LPO, SCN-, H2O2) are present. H2O2 is provided by the enzymatic reaction of glucose oxidase with glucose, not in a bolus-like fashion. Mean+/-S.E.M., n = 4, Two-way ANOVA and Sidak's multiple comparisons test. (C) Spn EF3030 growth is only inhibited by OSCN- and not by H2O2. OSCN- was generated in presence of the complete cell-free system (LPO+SCN-+glucose/GO) while H2O2 was produced by the glucose/GO system in the absence of LPO and SCN-. Bacterial growth was assessed using the microplate-based growth assay (n = 4). Each symbol represents a separate, individual experiment and their mean+/- S.E.M. is also shown, two-way ANOVA and Sidak's multiple comparisons test. Each experiment was done in biological triplicates and each biological replicate was tested in technical triplicates. (Ns, not significant; *, p<0.05; **, p<0.01. CFU, colony-forming unit; LPO, lactoperoxidase; OSCN-, hypothiocyanite; SCN-, thiocyanate; Spn, Streptococcus pneumoniae.

Catalase prevents the antimicrobial action of OSCN- on Spn

To further confirm our findings that OSCN- is antimicrobial against Spn, we utilized a kinetic assay to measure bacterial growth in presence of an inhibitor of the H2O2/LPO/SCN- cell-free system. Catalase is an enzyme found in almost all living organisms that catalyzes the decomposition of H2O2 to water and oxygen [31]. The use of catalase eliminates H2O2 and thereby OSCN- in our cell-free system (Fig 2A), rendering the cell-free system nonfunctional while also ensuring that neither SCN- nor LPO have an antimicrobial effect alone, independent of OSCN- formation. Results shown in Fig 2B indicate that Spn bacteria exposed to OSCN- have inhibited bacterial growth compared to those treated with catalase or unexposed to OSCN-. Both Spn strains tested show the same trend, where OSCN- treatment significantly reduces bacterial growth (p<0.0001) and addition of catalase entirely rescues this effect (p<0.0001) (Fig 2B). The nonencapsulated Spn strain, MNZ41, could not be tested in this assay because it did not grow in liquid cultures used under these experimental conditions. Taken together, these data show for the first time in an in vitro model that OSCN- has a catalase-sensitive, antimicrobial action against different strains of Spn.

Fig 2

Catalase rescues Spn from OSCN—mediated growth inhibition.

(A) Addition of catalase to the cell-free system blocks OSCN- generation measured by the DTNB assay. Error bars represent standard errors of the means, n = 4. Mann-Whitney test. (B) Catalase inhibits the bacteriostatic effect of OSCN- on TIGR4 and EF3030 Spn strains (n = 4). Each symbol represents a separate, individual experiment and their mean is also shown, Two-way ANOVA, Tukey’s multiple comparison test. Each experiment was done in biological triplicates and each biological replicate was tested in technical triplicates. Ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. CAT, catalase.

LPO-derived OSCN- inhibits Spn growth in a SCN- concentration-dependent manner

Physiological levels of SCN- in the airways have been measured to be around 400 μM [28]. We decided to test SCN- in a supra- and superphysiological concentration range between 40 μM-4 mM to determine if killing of one of the Spn strains, EF3030, can be enhanced. Our data show that supraphysiological levels (40 μM) of SCN- kill Spn EF3030 in a robust manner (Fig 3 and 3B). Increasing the SCN- concentration showed a dose-dependent response, where bacterial killing continued to increase, all the way up to 4 mM of SCN- (Fig 3). From this data we conclude that the reported physiological SCN- concentration is sufficient to support the antibacterial activity of the LPO system against Spn.

Fig 3

Spn growth is inhibited in a thiocyanate dose-dependent manner.

(A) Increasing the concentration of SCN- in the cell-free system leads to improved antimicrobial effect of OSCN- against Spn EF3030. Bacteria were incubated for 6 hrs with LPO, glucose oxidase, glucose and different concentrations of SCN-, and bacterial growth was followed by the microplate-based growth assay (n = 4). Each symbol represents a separate, individual experiment, mean+/-S.E.M. One-way ANOVA and Tukey’s multiple comparison test. (B) Representative kinetics of EF3030 growth curves. Each experiment was done in biological triplicates and each biological replicate was tested in technical triplicates. ***, p<0.001; ns, not significant. Dotted lines indicate the OD background of the growth medium without bacteria that was not subtracted in these experiments.

Commercially available 1st line™ effectively kills Spn via OSCN-

The H2O2/LPO/SCN- cell-free system has proven to be effective at killing Spn in vitro. A drawback of this system is, however, that OSCN- has a very short life span (less than 30 minutes after it has been generated) requiring it to constantly be produced in vitro in order to test its effect on microbes. This is why we utilized glucose oxidase, not bolus-like addition of H2O2, to allow a steady production of H2O2 and OSCN- [27]. As the next step, we took advantage of and tested a commercially available product that also generates OSCN- and claims to keep it stable for much longer. This product, 1 line™, utilizes a stabilizing molecule to allow OSCN- to persist for over 12 hours. We compared the bacteriostatic effect of the 1 line™ product on Spn EF3030 and TIGR4. The results demonstrate that OSCN- generated by 1st line™ resulted in robust inhibition of Spn growth (Fig 4 and 4B). The addition of catalase during the generation of OSCN- with 1 line™ also inhibited the antimicrobial action of OSCN- (Fig 4), similar to what was previously shown in our cell-free system (Fig 2). These results provide further evidence that OSCN- is solely responsible for the inhibition of Spn growth.

Fig 4

OSCN- generated using commercially available 1st line™ effectively inhibits Spn growth.

The 1st line™ product efficiently inhibits Spn growth: (A) TIGR4 (n = 5) and (B) EF3030 (n = 4) bacterial strains. Catalase reversed this effect partially (TIGR4) or fully (EF3030). Bacterial growth was measured by the microplate-based growth assay. Each symbol represents a separate, individual experiment, mean+/-S.E.M. One-way ANOVA, Tukey’s multiple comparison test. Each experiment was done in biological triplicates and each biological replicate was tested in technical triplicates. **, p<0.01; ***, p<0.001; ns, not significant. CAT, catalase.

The bacterial capsule is not protective against OCSN-

The bacterial capsule provides protection for Spn against threats of the environment including attacks by the immune system [32, 33]. We next asked whether the capsule provides protection for Spn against OSCN-. To answer this question, we exposed isogenic, capsule-deficient strains of Spn D39, EF3030 and TIGR4, and their corresponding, encapsulated, parental wild-type counterparts to OSCN- in the cell-free experimental system and measured bacterial killing by CFU counting. Results in Fig 5 show that the capsule-free mutants were also susceptible to OSCN- on all three backgrounds, similar to their encapsulated control strains. Catalase was also partially or fully effective in preventing the bactericidal effect of OSCN- against all strains tested (Fig 5). Therefore, we conclude that the capsule provides no protection against the anti-pneumococcal action of OSCN-.

Fig 5

LPO-derived hypothiocyanite kills both encapsulated and nonencapsulated Spn strains.

OSCN—mediated killing of Spn was tested in the cell-free system using 1st line TM against three encapsulated strains of Spn (TIGR4, D39 and EF3030) and their capsule-deficient, isogenic mutants (Δcap) in 4–5 independent experiments: n = 4 for TIGR4 and D39 Δcap, n = 5 for the other strains. Bacteria were incubated for 6 hrs with or without OSCN- generated by the LPO/SCN-/H2O2 system in presence or absence of catalase and bacterial killing was quantified by colony counts on BAP. Each symbol represents a separate, individual experiment, mean+/-S.E.M. is shown. ANOVA and Holm-Sidak's multiple comparisons test. Each experiment was done in biological triplicates and each biological replicate was tested in technical triplicates. Ns, not significant; *, p<0.05; **, p<0.01, ***, p<0.001. CFU, colony-forming unit; LPO, lactoperoxidase; OSCN-, hypothiocyanite; SCN-, thiocyanate; Spn, Streptococcus pneumonia; CAT, catalase.

The mechanism of action of OSCN- is independent of lytA-mediated autolysis

Autolysis is a form of programmed cell death in bacteria including Spn that plays a roles in genetic exchange between bacterial cells, in eliminating damaged cells and has also been implicated in mediating the effects of antibiotics [34]. The lytA genes encodes a major autolysin (N-acetylmuramoyl-l-alanine amidase) in Spn that is a cell wall-degrading enzyme located in the cell envelope [35, 36]. Based on these, we postulated that lytA-mediated autolysis could represent the anti-pneumococcal mechanism of action of OSCN-. To test this hypothesis, we exposed lytA-competent and lytA-deficient D39 Spn strains to OSCN- in the cell-free system generated by 1st line™ and measured bacterial survival by colony counting. As the results in Fig 6 show, 1st line™ not only inhibits bacterial growth of Spn but also kills this bacterium in an OSCN—dependent manner. Fig 6 also shows that the lytA-deficient mutant was also killed efficiently by OSCN- indicating that OSCN- does not initiate lytA-mediated autolysis in Spn.

Fig 6

LytA-deficiency does not protect Spn against OSCN—mediated killing.

OSCN—mediated killing of the lytA-deficient D39 Spn strain (ΔlytA) was tested in the cell-free system (n = 5). Bacteria were incubated for 6 hrs with or without OSCN- generated by the 1st line™ and bacterial killing was quantified by colony counts on BAP. Each symbol represents a separate, individual experiment, mean+/-S.E.M. is shown. Mann-Whitney test. Each experiment was done in biological triplicates and each biological replicate was tested in technical triplicates. Ns, not significant; *, p<0.05. CFU, colony-forming unit; LPO, lactoperoxidase; SCN-, thiocyanate; Spn, Streptococcus pneumoniae.

Spn-derived H2O2 provides some protection against OSCN—mediated killing

Interestingly, not only human cells but Spn itself is capable of producing H2O2 [37]. Spn-generated H2O2 could interfere with the killing effect of OSCN-. Spn-generated H2O2 could provide additional H2O2 for OSCN- generation by LPO leading to improved bacterial killing or it could prime bacteria against oxidative stress resulting in impaired Spn killing by OSCN-. To explore these possibilities, we tested a mutant TIGR4 Spn strain (ΔspxB) deficient in pyruvate oxidase, the main H2O2 producer in Spn [21], for susceptibility to OSCN-. As expected, the spxB-deficient TIGR4 strain had an H2O2 generation that was reduced by 72.5±0.9% (mean ± SEM, n = 2) compared to the wild-type TIGR4 counterpart (Fig 7A). The TIGR4ΔspxB mutant and its parental strain were exposed to OCSN- produced by 1st line™, and bacterial killing and growth were evaluated with both, CFU-based counting and microplate-based bacterial growth assays. The TIGR4ΔspxB mutant was also susceptible to the antimicrobial effect of OSCN- that was partially inhibited by catalase (Fig 7B). To better present the antibacterial action of OSCN-, we next defined and calculated “susceptibility to OSCN-” as the decrease in log10 of the colony counts. CFU count results obtained at 6 hours of incubation reached the level of significance (p = 0.029) (Fig 7C). Overall, we conclude that pyruvate oxidase provides improvement in Spn survival following OSCN- exposure.

Fig 7

Pyruvate oxidase-deficiency increases susceptibility of Spn to OSCN-.

(A) H2O2 generation was quantitated in wild-type and spxB-deficient (ΔspxB) TIGR4 Spn strains using the Ros-GLoTM luminescence H2O2 quantitation kit. Mean, n = 2. (B) OSCN—mediated (1st line™) killing of the TIGR4 ΔspxB Spn strain was measured by the microplate-based growth assay in presence or absence of catalase (n = 5). Each symbol represents a separate, individual experiment, mean+/-S.E.M. is shown. One-way ANOVA, Tukey’s multiple comparison test. (C) Killing by OSCN- generated via 1st line™ of wild-type and TIGR4 ΔspxB Spn strains was compared at 6 hrs via CFU counting (n = 4) and “susceptibility to OSCN-”was calculated as the decrease in log10 CFU upon exposure to OSCN-. Each symbol represents a separate, individual experiment, mean+/-S.E.M. is shown. Mann-Whitney’s test. Each experiment was done in biological triplicates and each biological replicate was tested in technical triplicates. ***, p<0.001; *, p<0.05; ns, not significant. RLU, relative luminescence unit; CAT, catalase.

Discussion

While the LPO-based system has been shown to be effective at killing numerous species of bacteria and viruses in vitro, no report to date studied the interaction between this system and Spn. Spn first encounters epithelial cells at the apical surface of the nasal cavity, a region that is a hotbed of defense mechanisms. The LPO-based system kills microbes in the extracellular space before they enter the epithelium and establish infection [8]. We were able to demonstrate that OSCN- kills Spn effectively. The ability of OSCN- to kill a variety of microbes in the extracellular spaces makes it a very interesting innate immune mechanism to study.

It is possible that Spn stimulates one of the main cellular sources of H2O2, Duox1, since we had previously shown that bacterial ligands of P. aeruginosa participate in Duox1 activation [12]. Spn has been shown to trigger H2O2 production in airway epithelial cells in a pneumolysin-independent but lytA-dependent manner [38]. We utilized the enzyme catalase, that converts H2O2 into H2O and O2, to inhibit the production of OSCN-. This was necessary because Spn is a catalase-negative bacterium and is capable of producing its own H2O2 [39]. Spn-derived H2O2 is sufficient to mediate bactericidal activity of other bacteria and to stimulate DNA damage and apoptosis in epithelial cells leading to tissue damage during infection [40]. It was found that in the absence of common antioxidant proteins, Spn utilizes pyruvate oxidase (SpxB) that has a dual role of creating H2O2, and protecting itself from oxidative damage [41]. Pyruvate oxidase, the main H2O2 source in Spn [21], has been found to be important to initiate oxidative attacks on host cells [40]. Our data indicate that SpxB provides a moderate protection against the oxidative attack of OCSN-. Our results are in line with previous findings of other groups where SpxB was determined to be useful against oxidative stress of different origins [41-43]. H2O2 generation by SpxB in Spn likely enhances its antioxidant capacity and thereby improves its defense against oxidative stress. Our results indicate a new, protective consequence of SpxB expression in Spn. The in vivo relevance of this finding remains to be confirmed in animal models.

Spn utilizes a wide spectrum of virulence factors, one of the most important ones being the polysaccharide capsule that forms the outermost layer of the bacteria [44]. This capsule provides protection against phagocytosis, complement components, mucus and spontaneous or antibiotic-induced autolysis [45]. Some Spn strains, however, do not possess a capsule [46, 47]. Our results show that both encapsulated and nonencapsulated Spn strains are susceptible to OSCN-. Thus, it is likely that OSCN- is able to penetrate the capsule and interact with the cell wall or other internal bacterial components. Interestingly, catalase rescued OSCN—mediated Spn killing more efficiently when the capsule was absent, in case of TIGR4 and D39 (Fig 5). This difference seems to be strain-dependent as this effect was less pronounced in case of EF3030 (Fig 5). While the reason for this remains unclear, it could be related to differences in H2O2 generation or in inhibition of catalase by the capsule or other microbial factors among tested strains.

OSCN- has a wide microbial target spectrum [8]. Its mechanism of action likely involves oxidative attack on one or more microbe-specific molecules or cellular mechanisms that essentially will lead to a bactericidal action. Previous studies have shown that OSCN- is oxidizing bacterial sulfhydryls [48-50], thereby inhibiting bacterial respiration [51], but no further research has been published to support this possible mechanism of action.

The LPO-based system presents an interesting therapeutic target, since it is an effective antimicrobial innate mechanism that does not have many drawbacks due to its final product being nontoxic to host cells and its broad activity against a wide range of pathogens [52]. By testing the 1 line™ product (alongside our cell-free method of OSCN- generation), we observed the same efficient Spn killing results. While we experienced similar levels of killing of Spn when comparing our glucose oxidase system and the 1 line™ product, there are some subtle differences that would likely affect the efficacy of in vivo studies. The greatest benefit to the 1 line™ system is the stabilizing aspect of the compound. Allowing OSCN- to persist for over 12 hours would likely increase the efficacy and potency of the system which could be a major advantage over the non-stabilized, natural OSCN- anion. This also allows for higher concentrations of OSCN- to be achieved without having toxicity issues.

While these are encouraging, the study presented here has technical limitations as it was conducted solely in an in vitro, cell-free experimental system. In vivo conditions are obviously much more complex and could represent new challenges to prove the antibacterial efficacy of OSCN-. Duox1-dependent H2O2 production is regulated by several inflammatory and microbial stimuli in vivo and could be targeted by pneumococcal host evasion mechanisms not explored here. The in vivo life span of OSCN- might be influenced by several additional biological factors that could not be tested in the in vitro system studied here. The in vivo administration route and formulation to boost airway production of OSCN- offers numerous possibilities. Overall, these new questions will be answered in future studies using airway epithelial cultures, animal models and human patients.

We determined the effectiveness of OSCN- against Spn in this proof-of-concept study as it had not been reported previously. We were able to demonstrate that OSCN- effectively kills both encapsulated and nonencapsulated strains of Spn in a cell-free system. We also successfully utilized a commercially available product, 1 line™, to demonstrate the therapeutic potential of the LPO system in an in vitro model. The mechanism of anti-pneumococcal action of OSCN- is independent of the bacterial capsule or lytA-mediated autolysis but is opposed by the bacterial oxidant generator SpxB. These studies warrant further research to elucidate the molecular mechanism of antibacterial action of OSCN-.

18 Sep 2019

PONE-D-19-23735

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

PLOS ONE

Dear Dr. Rada,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

Specifically:

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In addition to reviewer 1, I have myself reviewed the manuscript. Although reviewer 1 comments read a bit harsh at times, I have to agree that overall the presented results are merely confirmative of numerous publications that have shown effectiveness of the LPO-isothiocyanate system against streptococci that inhabit the oral cavity and respiratory system.

It would have been more informative if the effectiveness was investigated under more relevant biofilm conditions or in a multispecies setting. Mimicking the mucus environment of the respiratory system could have provided novel insights into killing kinetics. For example, would slow growth and limited diffusion affect the antimicrobial activity?

Further, it is not clear what growth conditions were used for some of the experiments. For example, the growth curve in Fig. 3B, what was the growth medium?

Also, no effect of SpxB produced hydrogen peroxide was observed. Could that be simply due to the fact that under the here used conditions, SpxB is not active?

==============================

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Yours sincerely,

Jens Kreth

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

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Reviewer #1: No

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5. Review Comments to the Author

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Reviewer #1: When one steps away from the hyperbole …

How can the LPO system (LPO + H2O2 + SCN- = HOSCN) be a “novel, anti-pneumococcal therapy” when it is a principal component in the innate defense stratagem of the endocrine system, and there already exists an abundance of literature (100 + papers?) that evidence the effectiveness of the LPO system against streptococci, dating back more than 70 years.

the present paper is at best pedestrian. The claim that this is the first study of the efficacy of the LPO system vs. Streptococcus pneumoniae is false. Use the Google machine.

Comically, the authors test a undefined "commercial product" by Profound Products that “stabilizes” HOSCN. According to the description the “1st Line” product, it also contains as “other” ingredients “hydrogen peroxide, poly aluminum chloride, lactoperoxidase and bentonite (note that no hydrogen peroxide or aluminum is consumed as these are converted by the enzyme in the manufacture of thiocynate (sp) ions).” We note that thiocyanate is not produced by these ingredients, and LPO is inactivated by hydrogen peroxide in the absence of thiocyanate. This reviewer has knowledge this product actually contains iodide, and HOSCN oxidizes iodide to yield iodine, the probable active ingredient.

l.128: The LPO system described effectively generates a bolus of HOSCN. It is not clear if the HOSCN was generated exogenously, then added to the wells.

l.133: It is not at all clear what grown medium was used. Was the medium just HBSS or BAP? If HBSS, this stressor would not realistically reflect the infectious agent in vivo. HOSCN reacts instantly with BAP.

l.193: “… short-lived TNB cannot be purchased and was generated by reducing DTNB with the help of beta-mercaptoethanol”. First, TNB is not short-lived. We have a flask of it that was synthesized more than a decade ago and shows no sign of decomposition. Second, if TNB was synthesized from DTNB in situ using ME, there is a likelihood that unreactive ME remains, which would render the assay inaccurate.

l.237: “H2O2 is provided by the enzymatic reaction of glucose oxidase with glucose, not in a bolus-like fashion.” … the authors clearly do not understand enzyme kinetics. I calculate a half-life of about 18 seconds for this reaction under the conditions employed.

l.249: Yes, catalase, the most efficient enzyme known to man, removes one of the essential components of the LPO system.

l. 278: Figure 3B exhibits a profound ignorance of the LPO system. Under the conditions described in the experimental section, only [SCN-] > 50 uM will produce HOSCN under their reaction conditions. Also, the assay does not test for regrowth (the conditions are likely inhibitory, not cytocital).

l.337: Been shown before.

l.401: HOSCN only attacks sulfhydryl groups.

l.407: So is bleach.

l.411: I am aware of at least two patents for the use of the LPO system in the treatment of lung infections.

I would like to know if the authors have received any financial support from Profound Products.

Do not publish … anywhere.

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Reviewer #1: No

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For journal use only: PONEDEC3

12 Feb 2020

Response to the critiques

Responses to the editor's critiques:

We disagree that our findings are merely confirmative of previous findings. While several Streptococcus species have already been found to be sensitive to OSCN-, no published scientific literature is out there to show this for the clinically most important Streptococcus species, Streptococcus pneumoniae. I know this is surprising but we searched PubMed thoroughly and did not find any publication showing the effectiveness of the LPO-based system in any experimental setup against S. pneumoniae. We actually wrote the most up-to-date review in 2019 on the topic and listed all the 100+ publications studying several microbial species that have been studied for LPO-mediated killing, but S. pneumoniae is not there. We think our data is an important set of information completely suitable to your journal showing that the clinically most relevant Streptococcus species, S. pneumoniae, is sensitive to OCSN- in vitro. This is also important because we have unpublished data showing that some Streptococcus species are not sensitive, so one cannot simply assume that all Streptococcus species are sensitive to OSCN-.

Regarding the criticism of not characterizing the spxB-deficient Spn mutant, there are actually data shown in the manuscript that SpxB is active under our experimental conditions in Fig. 7A as the spxB-deficient mutant had about 80% reduction in H2O2 production compared to the WT strain. The killing experiments were done under the same experimental conditions.

The editor is right, we forgot to add what medium was used in the growth assay. Bacteria were grown on BAP medium. This was added to the revised manuscript.

Response to the reviewer:

Critique: How can the LPO system (LPO + H2O2 + SCN- = HOSCN) be a “novel, anti-pneumococcal therapy” when it is a principal component in the innate defense stratagem of the endocrine system, and there already exists an abundance of literature (100 + papers?) that evidence the effectiveness of the LPO system against streptococci, dating back more than 70 years.

Response: As also detailed above in our answer to the editor, no one documented the effect of this system against S. pneumoniae (Pneumococcus) before. I agree that several other Streptococcal species were published but not S. pneumoniae. We cited them all in our review published in 2019 in the Journal of Microbiology (https://www.ncbi.nlm.nih.gov/pubmed/29858825). So, based on this, using/targeting the OSCN-based mechanism provides theoretically a novel strategy to kill pneumococcus, not other Streptococci. Several mechanisms of the immune system can be and have been targeted to improve bacterial clearance, so the same can be done with the LPO-based system. At the same time, we acknowledge that the results are restricted to an in vitro system.Therapeutic considerations might be overreaching; therefore, we removed this part of the discussion from the revised version.

The reviewer’s comment related to the endocrine system is unclear? We never said the LPO-based system is an endocrine system.

Critique: the present paper is at best pedestrian. The claim that this is the first study of the efficacy of the LPO system vs. Streptococcus pneumoniae is false. Use the Google machine.

Response: We used both the Google and PubMed “machines” and there is no publication to show that OSCN kills Streptococcus pneumoniae. The reviewer is more than welcome to refer to papers showing Spn killing by OSCN in the literature.

Critique: Comically, the authors test a undefined "commercial product" by Profound Products that “stabilizes” HOSCN. According to the description the “1st Line” product, it also contains as “other” ingredients “hydrogen peroxide, poly aluminum chloride, lactoperoxidase and bentonite (note that no hydrogen peroxide or aluminum is consumed as these are converted by the enzyme in the manufacture of thiocynate (sp) ions).” We note that thiocyanate is not produced by these ingredients, and LPO is inactivated by hydrogen peroxide in the absence of thiocyanate. This reviewer has knowledge this product actually contains iodide, and HOSCN oxidizes iodide to yield iodine, the probable active ingredient.

Response: We defined the product clearly in the methods section and only referred to it in the text as commercial product. We can provide any additional information, as much as needed. Of course, the 1st Line product has LPO, H2O2, SCN-, polyaluminum chloride and bentonite in it –as they state in their description. Why would SCN- be produced by these ingredients when it is provided as one of the substrates? This comment was unclear. We measured OSCN- production with this kit and found it to be comparable to the levels obtained by the glucose/glucose oxidase system. SCN- is provided in the kit, so LPO is unlikely to be inactivated by hydrogen peroxide in the absence of thiocyanate – as the reviewer stated it. We cannot comment on the undocumented knowledge the reviewer claims to have on this product. No data are shown or referred to.

Critique: l.128: The LPO system described effectively generates a bolus of HOSCN. It is not clear if the HOSCN was generated exogenously, then added to the wells.

Response: We might have different definitions for “bolus”. When we used the term “bolus”, the compound was not produced by enzymatic reaction but provided already synthesized in an unnaturally high concentration in one moment. HOSCN was generated in the wells, as described in the methods.

Critique: l.133: It is not at all clear what grown medium was used. Was the medium just HBSS or BAP? If HBSS, this stressor would not realistically reflect the infectious agent in vivo. HOSCN reacts instantly with BAP.

Response: HBSS was used in the killing assay as stated in the methods and bacteria were then inoculated on BAP for growth. This experiment uses an in vitro system with its obvious limitations and advantages as a model of the in vivo environment. Similar buffers were used in most of the published papers using such in vitro systems to study the antimicrobial effect of this LPO-based system. The information gathered is useful to document that another bacterial species, S. pneumoniae is also killed by the LPO/SCN- system. How much is this true and reflected in vivo, remains to be clarified not only for S. pneumoniae, but for all the other pathogens published since –paradoxically- the relevance of this antimicrobial system has not yet been established in any mammalian organism including mice and humans. We added more information about the growth conditions of bacteria to the revised manuscript.

Critique: l.193: “… short-lived TNB cannot be purchased and was generated by reducing DTNB with the help of beta-mercaptoethanol”. First, TNB is not short-lived. We have a flask of it that was synthesized more than a decade ago and shows no sign of decomposition. Second, if TNB was synthesized from DTNB in situ using ME, there is a likelihood that unreactive ME remains, which would render the assay inaccurate.

Response: We read several publications that measured OSCN- production by this simple method and all claimed that TNB is not stable over long period of time. We, therefore, decided to purchase DTNB and reduce it with the help of ME, as dozens of related manuscripts did it too.

Critique: l.237: “H2O2 is provided by the enzymatic reaction of glucose oxidase with glucose, not in a bolus-like fashion.” … the authors clearly do not understand enzyme kinetics. I calculate a half-life of about 18 seconds for this reaction under the conditions employed.

Response: We actually backed up our decision for using this method of H2O2 production with more meaningful, and for the studied topic, more useful hydrogen peroxide measurements. Adding 1 mM bolus H2O2 to the system -as is done by several investigators in the field- has nothing to do with the nature of H2O2 production by their physiological source, bronchial epithelial cells. We measured and optimized H2O2 production of the glucose/glucose oxidase system so that it mimics the slow but relatively steady nature and amplitude of H2O2 production by airway cells. While it is true that –of course- the rate of H2O2 production slowed over time, it was still detectable at 60 minutes. Despite its deceleration over time, it is still a better approach to model H2O2 generation by airway cells than pouring a bucket load of physiologically irrelevant concentration of H2O2 into our system.

Critique: l.249: Yes, catalase, the most efficient enzyme known to man, removes one of the essential components of the LPO system.

Response: While the effect of catalase is obvious, one cannot simply assume that it will also work in our experimental system and has to show results. Removing a critical component of a system is an essential approach to show how the system works and this approach should rather be appreciated and encouraged by the reviewer to show the H2O2-dependence of the studied mechanism.

Critique: l. 278: Figure 3B exhibits a profound ignorance of the LPO system. Under the conditions described in the experimental section, only [SCN-] > 50 uM will produce HOSCN under their reaction conditions. Also, the assay does not test for regrowth (the conditions are likely inhibitory, not cytocital).

Response: I do not see any ignorance of the LPO system here and in fact the reviewer’s calculations clearly confirm that OSCN- is responsible for the inhibitory effect of microbial growth observed in Fig 3B as the lowest SCN- concentration used is 40 uM. The second observation is not new, at all, as we clearly stated in the manuscript that the growth assay used in figure 3B measures inhibition of bacterial growth and not direct microbicidal activity. We had the CFU assay for that.

Critique: l.337: Been shown before.

Response: No, it has not. Please cite any report from the medical literature and we will immediately retract our submission.

Critique: l.401: HOSCN only attacks sulfhydryl groups.

Response: That’s what we said too, so we are unclear about the point of this critique. In line 401 we referred to the long list of microbes OSCN- has been described to target, if that was confusing.

Critique: l.407: So is bleach.

Response: Natural products of the immune system have been and are being used as part of therapies for a long list of diseases.

Critique: .411: I am aware of at least two patents for the use of the LPO system in the treatment of lung infections.

Response: So are we. We cited them in our last year’s review in Journal of Microbiology. None of them, however, claims to be targeting S. pneumoniae. We refer to its novelty to be used against Pneumococcus, not other bacteria.

Critique: I would like to know if the authors have received any financial support from Profound Products.

Response: No, we have not. We would have acknowledged it in the manuscript if we had according to the policies of PLoS One and any other journal. We used this product because this was the only, purchasable product we could find. We are glad to test any other, commercially available OSCN-producing product in the future.

Submitted filename: Gingerich Rada Plos One - Response to reviewers.docx

Click here for additional data file.

15 May 2020

PONE-D-19-23735R1

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

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: No

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Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

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Reviewer #2: In this manuscript Gingerich and colleagues test an lactoperoxidase(LPO)-based antibacterial system against S. pneumoniae. LPO is a natural antibacterial agent that plays an important role in the innate immune system. LPO has been shown to have some efficacy in treating certain oral diseases. However, as might be expected translation of strong in vitro data to strong activity in vivo is a challenge (because several components are required that do not retain optimal activity in human fluids). Nevertheless, there is some merit to studying the activity of LPO against clinically relevant bacteria. For this submission I am reviewing a previously reviewed version of the manuscript. The reviewer comments and responses were combative, predominately focused on novelty. I have mostly focused on the rigor of the studies, in line with the acceptance guidelines of PLOS ONE. The manuscript is well written, and the experiments are described in a logical manner. The methods are described in good detail and could be reproduced by others. The antibacterial system has dose-dependent efficacy and the authors study several components of S. pneumoniae physiology that could impact efficacy (capsule, autolysis and spxB). I have some minor comments as follows:

Line 409. Change ‘Spxb’ to ‘SpxB’

Discussion. It might be relevant to add a paragraph on limitations/technical challenges that will need to be addressed before the product can be used in vivo – these may well be future experiments that the authors would like to conduct. There is clear antibacterial efficacy in vitro but translation to in vivo efficacy will be a challenge, and something that most readers will contemplate.

Reviewer #3: Overall, this is a scientifically sound study and the conclusions are supported by the presented experiments. The authors have also adequately addressed the concerns raised by previous reviewers. In particular, this reviewer's own PubMed search failed to find any studies that documented the effect of OSCN- specifically against Spn. However, the following comments would help improve the clarity of the data presented in this manuscript:

1. For all figures, it is unclear if the reported data represents technical or biological replicates.

2. For all bacteria killing assays assessed by CFU, the methods section states that "A time zero condition was also counted to make sure that bacterial death was due to OSCN- and not related to an unknown variable". It is agreed that is an important control, but these time zero CFU counts do not appear to be reported in any of the related figures (1A, 5, 6, 7C). Please include this data.

3. In some instances, data derived from the bacteriostatic assay (where growth inhibition is measured by OD in a microtiter plate assay) is incorrectly referred to or discussed as demonstrating "killing", when in fact this assay is measuring growth inhibition. (ex: Figures 2 and 3 legend titles, results line 289).

4. For all figures reporting CFU data, converting the Y axis to log scale would improve resolution of the actual CFU data points that cluster around the "0" Y axis point (ex: when bacteria are incubated in the presence of OSCN-).

5. The data presented in Figure 5 suggest that in most cases, the catalase control worked better in the capsule mutants compared to their parental strains; is there a reason this might be the case?

6. For the experiment in Figure 7 (comparing wildtype and pyruvate oxidase mutant killing by OSCN-), why was the "1st line" product used rather than the in vitro assay used in Figure 1? The fact that the mutant was more susceptible to OSCN- is very interesting. Was this phenotype genetically complemented? Figure 7C is somewhat confusing, why not present the Y axis data as CFU counts?

7. Results line 339: the term "allolysis" (instead of autolysis) is more appropriate when discussing in this context.

**********

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Reviewer #2: Yes: Robert C. Shields

Reviewer #3: No

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18 Jun 2020

Response to the critiques

We thank the editor for allowing a resubmission and re-review of our revised manuscript. We also thank the reviewers for their constructive criticisms of our manuscript that have significantly improved it. We made every effort to address all the comments. We hope that the manuscript is now acceptable for publication.

- Response to the editor:

According to the editor’s request, we made sure that the manuscript meets PLOS ONE’s style requirements, including those for file naming. The single data set that was referred to as “data not shown” in the original, not the revised, version of the manuscript has been removed. No need, therefore, for supplementary data deposition. We are also attaching and uploading the minimal data set as a Supporting Information file requested by your journal.

- Responses to comments of reviewer 2:

Critique: Line 409. Change ‘Spxb’ to ‘SpxB’

Response: Thanks for pointing this out, we made the change in the revised text.

Critique: Discussion. It might be relevant to add a paragraph on limitations/technical challenges that will need to be addressed before the product can be used in vivo – these may well be future experiments that the authors would like to conduct. There is clear antibacterial efficacy in vitro but translation to in vivo efficacy will be a challenge, and something that most readers will contemplate.

Response: We thank the reviewer for bringing this important issue up. According to this, a separate paragraph has been added to the end of the discussion to mention all the limitations of our current study using a cell-free, in vitro experimental system.

- Responses to the comments of reviewer 3:

Critique: For all figures, it is unclear if the reported data represents technical or biological replicates.

Response: We thank the reviewer for this request. We added this information to all the figure legends in the revised manuscript.

Critique: For all bacteria killing assays assessed by CFU, the methods section states that "A time zero condition was also counted to make sure that bacterial death was due to OSCN- and not related to an unknown variable". It is agreed that is an important control, but these time zero CFU counts do not appear to be reported in any of the related figures (1A, 5, 6, 7C). Please include this data.

Response: We thank the reviewer for this comment. While we agree that this data serve as important controls, we consider them quality controls of the assay, therefore, decided not to report them individually at each figure but to expand the related sentence in the description of the CFU killing method to this:

“A time 0 condition was also counted to make sure that bacterial death was due to OSCN- and not related to an unknown variable, and that no significant changes in bacterial numbers were observed in samples containing only bacteria during the duration of the experiments.”

Critique: In some instances, data derived from the bacteriostatic assay (where growth inhibition is measured by OD in a microtiter plate assay) is incorrectly referred to or discussed as demonstrating "killing", when in fact this assay is measuring growth inhibition. (ex: Figures 2 and 3 legend titles, results line 289).

Response: We tried to be consistent with the related nomenclature of “killing” vs “growth inhibition” throughout the manuscript but obviously missed these few occasions. We thank the reviewer for identifying them. The terms “killing” or “killed” were changed in both instances to “growth inhibition”.

Critique: For all figures reporting CFU data, converting the Y axis to log scale would improve resolution of the actual CFU data points that cluster around the "0" Y axis point (ex: when bacteria are incubated in the presence of OSCN-).

Response: We had long discussions in the group whether the linear or log scale results should be presented in the figures with CFU data. We had decided to present the results on linear scale, instead of log scale, because we think it better shows the impressive antibacterial effect of OSCN- against Spn.

Critique: The data presented in Figure 5 suggest that in most cases, the catalase control worked better in the capsule mutants compared to their parental strains; is there a reason this might be the case?

Response: We thank the reviewer for this interesting observation. Indeed, catalase worked better in case of TIGR4 and D39 in the absence of the capsule while this difference was less pronounced in case of EF3030 (Fig. 5). While we do not have any proven explanation for this at this point, we have added a few sentences about this to the discussion pointing to potential explanations.

Critique: For the experiment in Figure 7 (comparing wild0type and pyruvate oxidase mutant killing by OSCN-), why was the "1st line" product used rather than the in vitro assay used in Figure 1? The fact that the mutant was more susceptible to OSCN- is very interesting. Was this phenotype genetically complemented? Figure 7C is somewhat confusing, why not present the Y axis data as CFU counts?

Response: In figure 6 and 7 we did not only want to test the effect of the respective bacterial mutants but also aimed at delivering the first results to show that “1st line” is not only bacteriostatic but also directly kills Spn. This was actually mistakenly stated in the figure 6 legend and now it has been corrected to “1st line”. A sentence was added about the direct Spn-killing effect of 1st line to the figure 6 section of the results.

No, the SpxB-deficient mutant was not complemented.

We would like to keep the “susceptibility to OSCN’” term as introduced in figure 7 as we think it better explains the antimicrobial action of OSCN- than showing the CFU results. We are aware of it that it might get somewhat confusing, therefore we added an extra explanation to the corresponding part of the results section.

Critique: Results line 339: the term "allolysis" (instead of autolysis) is more appropriate when discussing in this context.

Response: We thank the reviewer for the comment. However, we feel in the overall context of the manuscript autolysis is the response we tested and chose not to change the wording. Classically, autolysis is associated with lytA expression and lysis of self, while allolysis is more a term of fratricide (lysis of nearby cells). Allolysis is also associated with the competence system and includes up to seven genes. In our study, we used a specific lytA gene knock out strain to look at the effects of autolysis. We did not look at the impact of differences in competence or fratricide in the in vitro model of Spn killing, so allolysis is not an appropriate term to be used based on our experimental approach.

Submitted filename: Gingerich Rada Plos One - Response to reviewers.docx

Click here for additional data file.

8 Jul 2020

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

PONE-D-19-23735R2

Dear Dr. Rada,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #2: Yes

Reviewer #3: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Reviewer #2: Yes: Robert Shields

Reviewer #3: No

17 Jul 2020

PONE-D-19-23735R2

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

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