Antimicrobial and antibiofilm activity of human recombinant H1 histones against bacterial infections

Abstract

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

Keywords: Galleria mellonella; antimicrobial peptides; biofilm; proteins; toxicity; treatment.

Fig 1

Bacterial viability assessment of P. aeruginosa PAO1 planktonic cultures after H1 histone treatment. (A) Live and dead staining using Syto 9 (green live cells) and propidium iodide (red dead cells) dyes after 16 h of treatment. Histones were used at the MIC₅₀ concentration of PAO1: H1.0 at 55 µg/mL, H1.0 CTD 58 µg/mL, H1.2 at 111 µg/mL, and H1.4 at 46 µg/mL. Cpx was used at 2 µg/mL. Fluorescence images were processed with Image J. The scale bar of 10 µm is consistent for all cases. (B) Quantification of live and dead bacteria by particle counting with Image J. Numbers after ↓ symbol indicate the percentage of reduction in bacterial total cell number compared to the untreated control. Error bars display mean and standard deviation from at least four replicates. Differences in the number of total cells among groups were analyzed by a one-way ANOVA analysis with Tukey’s multiple comparison test (****, P value < 0.0001). Letter a represents a statistical difference of total cell number in the Cpx group compared to the control, H1.0 and H1.2 with a P value < 0.0001, and H1.0 CTD and H1.4 with a P value < 0.001. (C) Percentage of viability of bacterial cells {[live bacteria/(live bacteria + dead bacteria)] × 100}, where live bacteria is the number of particles counted in the green channel (Syto 9 stained) and dead bacteria is the number of particles counted in the red channel (propidium iodide stained). Numbers after ↓ symbol indicate the percentage of reduction compared to the control. Error bars display mean and standard deviation from at least four replicates. Differences among groups were analyzed by a one-way ANOVA analysis with Tukey’s multiple comparison test (*, P value < 0.05; **, P value < 0.01; ****, P value < 0.0001). Letter a represents a statistical difference in the viability percentage of the Cpx group compared to the control with a P value < 0.0001, H1.0 CTD with a P value < 0.001, and H.12 with a P value < 0.05.

Fig 2

H1 histones precipitation assay after incubation of PAO1 planktonic cells. (A) Coomassie-stained SDS-PAGE gel of supernatant and pellet fraction from bacterial cultures, histones, and bacterial cultures treated with histones after centrifugation. Red arrows indicate the band corresponding to the respective histone subtype. (B) Immunoblot and post-transfer Coomassie staining of bands found in the supernatant and pellet fraction from bacterial cultures, histones, and bacterial cultures treated with histones after centrifugation. +, presence; –, absence; S, supernatant; P, pellet.

Fig 3

Assessment of membrane alterations of PAO1 cells after histone treatment. Bacteria were stained with FM-464 (red) and DAPI (blue) dyes to visualize the membrane and genetic material, respectively. Histones were used at the MIC₅₀ of PAO1. Fluorescence images were processed with Image J software. Scale bars correspond to 10 µm in large images and 2 µm in zoomed images. White arrows point to gaps in cell membranes.

Fig 4

Antibiofilm activity of histones against PAO1 biofilms. (A) Biomass reduction of static biofilms after histone treatment. Error bars display mean and standard deviation of at least three replicates. Data differences with respect to the control group were analyzed by a one-way ANOVA analysis with Dunnett’s multiple comparison test (*, P value < 0.05; ***, P value < 0.001. (B) Confocal microscopy visualization of continuous flow biofilms after treatment with H1.0 and H1.4. Biofilms were stained with Syto 9 and propidium iodide to dye live cells in green and dead cells in red. Z-stacks from confocal images with their corresponding orthogonal views are displayed. The scale bar of 10 µm is consistent across all images. Confocal image analysis of the (C) percentage reduction in biomass, (D) percentage reduction in average thickness and (E) percentage increase in dead biomass after treatment of flow biofilms with H1.0 and H1.4. Numbers after the ↓ symbol indicate the percentage of reduction compared to the control, whereas numbers after the ↑ symbol indicate the percentage of increase compared to the control. Error bars display mean and standard deviation of at least three replicates. Data differences with respect to the control group were analyzed by a one-way ANOVA analysis with Šidák’s multiple comparison test (*, P value < 0.05; **, P value < 0.01; ***, P value < 0.001).

Fig 5

In vivo evaluation of H1 histone activity against G. mellonella infected with PAO1. Kaplan-Meier survival curves of G. mellonella larvae infected with PAO1 and treated with histones alone or in combination with Cpx. Control larvae were injected with PBS. Larval mortality was monitored for 16–24 h post-injection with observations conducted hourly. Significant differences in treatment efficacy compared to the PAO1-PBS-treated group were assessed using a log-rank test (*, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001). The results depicted in this figure represent an experiment replicated three times with consistent outcomes, each condition involving eight larvae.