The lantibiotic gallidermin acts bactericidal against Staphylococcus epidermidis and Staphylococcus aureus and antagonizes the bacteria-induced proinflammatory responses in dermal fibroblasts

Abstract

Antimicrobial resistance needs to be tackled from new angles, and antimicrobial peptides could be future candidates for combating bacterial infections. This study aims to investigate in vitro the bactericidal effects of the lantibiotic gallidermin on Staphylococcus epidermidis and Staphylococcus aureus, possible cytotoxic effects and its impact on host-microbe interactions. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of gallidermin were determined, and cytotoxicity and proinflammatory effects of gallidermin on fibroblasts, red blood cells (RBCs) and in whole blood were investigated. Both MIC and MBC for all four tested strains of S. epidermidis was 6.25 μg/ml. Both MIC and MBC for methicillin-sensitive S. aureus was 12.5 μg/ml and for methicillin-resistant S. aureus (MRSA) 1.56 μg/ml. Gallidermin displayed no cytotoxic effects on fibroblasts, only a high dose of gallidermin induced low levels of CXCL8 and interleukin-6. Gallidermin hemolyzed less than 1% of human RBCs, and did not induce reactive oxygen species production or cell aggregation in whole blood. In cell culture, gallidermin inhibited the cytotoxic effects of the bacteria and totally suppressed the bacteria-induced release of CXCL8 and interleukin-6 from fibroblasts. We demonstrate that gallidermin, expressing low cell cytotoxicity, is a promising candidate for treating bacterial infections caused by S. epidermidis and S. aureus, especially MRSA.

Keywords: Streptococcus; antimicrobial resistance; bacteriocin; cytokines; fibroblasts; gallidermin.

Figure 1

Gallidermin inhibits the growth of Staphylococcus epidermidis and Staphylococcus aureus. Minimal inhibitory concentration (MIC) after treatment with gallidermin (Gdm) were determined for four strains of S. epidermidis: biofilm‐negative ATCC 12228, biofilm‐positive RP62A, strain N15 isolated from a healthy person, and strain 117 isolated from a prosthetic hip infection (a). MIC and MBC were also determined for two strains of S. aureus; methicillin‐sensitive MSSA ATCC 29213 and methicillin‐resistant MRSA CCUG 35601 (b). Gallidermin was diluted in a 96‐well microtiter plate with concentrations ranging from 0.19 to 100 μg/ml. Bacteria (10⁵ CFU/100 μl) were added and the suspensions was incubated at 37°C for 24 hr. Optical density (OD) was read at 620 nm after 24 hr. MIC correlates to the lowest concentration of gallidermin that inhibits bacterial growth. Negative control without bacteria (−C) and positive control with bacteria but without gallidermin (+C) are also shown. Data are presented as mean values with SEM indicated. n = 3

Figure 2

Morphological effects of gallidermin on dermal fibroblasts. Human primary dermal fibroblasts (50,000 cells/well) were treated with various concentrations of gallidermin (25–400 μg/ml) for 24 hr, followed by images taken at a magnification of 200x. Fibroblasts without gallidermin served as negative control (−C). Representative images are shown from one experiment. n = 3

Figure 3

The effects of gallidermin on viability, cytotoxicity and immune responses of dermal fibroblasts. Human primary dermal fibroblasts (50,000 cells/well) were untreated (−C) or treated with various concentrations of gallidermin (25–400 μg/ml) for 24 hr. Viability was determined as uptake of neutral red by viable cells (a). As a measure of cell cytotoxicity, the absorbance values of LDH release were measured and analyzed as the relative change from the negative control (nontreated fibroblasts) set to one (b). Secretion of the proinflammatory mediator CXCL8 was determined with ELISA and showed a significant increase when fibroblasts were treated with 400 μg/ml gallidermin, whereas lower concentrations had no effects on CXCL8 (c). IL‐6 displayed a similar pattern, i.e. only 400 μg/ml gallidermin significantly elevated IL‐6 (d). Data are presented as mean values with SEM indicated. Statistically significant differences were determined by using one‐way ANOVA with Bonferroni post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). n = 3

Figure 4

Hemolysis and reactive oxygen species production in whole blood treated with gallidermin. Human red blood cells (RBCs), diluted to a 30% RBCs fraction in PBS, were treated with gallidermin (Gdm) for 1 hr at 37°C, and optical density was read at 540 nm (a). 1% Triton X‐treated RBCs served as a positive control (+C), giving 100% hemolysis. All tested gallidermin concentrations caused less than 1% of hemolysis. Gallidermin (50 or 200 μg/ml) was added to whole blood and reactive oxygen species (ROS) production was measured as luminol‐amplified chemiluminescence (b). Whole blood without stimulation served as a negative control and is indicated by the dotted line. Whole blood stimulated with fMLP (0.5 μmol/L) served as a positive control (+C). Data are presented as mean values with SEM indicated. Statistically significant differences compared to the negative control were determined by using one‐way ANOVA with Bonferroni post hoc test. n = 3–4

Figure 5

Morphological effects of Staphylococcus epidermidis, Staphylococcus aureus and gallidermin on dermal fibroblasts. S. epidermidis 117, S. epidermidis RP62A, MSSA, or MRSA was incubated with human primary dermal fibroblasts (50,000 cells/well) at MOI:10, in the absence or presence of gallidermin (Gdm), with 6.25 μg/ml for S. epidermidis and 12.5 μg/ml for S. aureus. The cells were incubated for 24 hr at 37°C, whereupon microscopic images were captured with at a magnification of 100x. As a positive control, fibroblasts were stimulated with 6.25 μg/ml (not shown) or 12.5 μg/ml gallidermin and nontreated fibroblasts were used as a negative control (−C). Representative images are shown. n = 3

Figure 6

Gallidermin suppresses the inflammatory responses of Staphylococcus epidermidis‐stimulated fibroblasts. Human primary dermal fibroblasts (50,000 cells/well) were treated with S. epidermidis with or without 6.25 μg/ml gallidermin (Gdm) for 24 hr at 37°C. Cell culture supernatants were analyzed for CXCL8 and IL‐6 released from fibroblasts stimulated with the bacterial strain 117 or RP62A at MOI:1 and MOI:10. Nontreated fibroblasts were used as a negative control (‐C). Strain 117 and RP62A raised the CXLC8 level whereas gallidermin suppressed this induction at MOI:1 (a) as well as MOI:10 (b). IL‐6 was also elevated by 117 and RP62, but was counteracted when gallidermin was added at MOI:1 (c) and MOI:10 (d). Data are presented as mean values with SEM indicated. Statistically significant differences were determined by using one‐way ANOVA with Bonferroni post hoc test (*p < 0.05, **/++p < 0.01, ***/+++p < 0.001). *Statistically significant differences compared to ‐C. +Statistically significant differences compared to corresponding bacterial strain without gallidermin. n = 3

Figure 7

Gallidermin suppresses the inflammatory responses of Staphylococcus aureus‐stimulated fibroblasts. Human primary dermal fibroblasts (50,000 cells/well) were treated with MSSA and MRSA with or without 12.5 μg/ml gallidermin (Gdm) for 24 hr at 37°C. Cell culture supernatants were analyzed for CXCL8 (a, b) and IL‐6 (c, d) released from fibroblasts stimulated with MSSA or MRSA at MOI:1 and MOI:10. Nontreated fibroblasts were used as a negative control (−C). MRSA induced CXCL8 secretion, whereas gallidermin inhibited this induction at MOI:1 (a). At MOI:10, both MSSA and MRSA resulted in high CXCL8 levels although gallidermin suppressed this CXCL8 accumulation from MRSA‐treated fibroblasts (b). The same pattern for MRSA was seen for IL‐6 (c, d). Data are presented as mean values with SEM indicated. Statistically significant differences were determined by using one‐way ANOVA with Bonferroni post hoc test (*p < 0.05, ***/+++p < 0.001). *Statistically significant differences compared to ‐C. +Statistically significant differences compared to corresponding bacterial strain without gallidermin. n = 3