The Multifunctional Sactipeptide Ruminococcin C1 Displays Potent Antibacterial Activity In Vivo as Well as Other Beneficial Properties for Human Health

Abstract

The world is on the verge of a major antibiotic crisis as the emergence of resistant bacteria is increasing, and very few novel molecules have been discovered since the 1960s. In this context, scientists have been exploring alternatives to conventional antibiotics, such as ribosomally synthesized and post-translationally modified peptides (RiPPs). Interestingly, the highly potent in vitro antibacterial activity and safety of ruminococcin C1, a recently discovered RiPP belonging to the sactipeptide subclass, has been demonstrated. The present results show that ruminococcin C1 is efficient at curing infection and at protecting challenged mice from Clostridium perfringens with a lower dose than the conventional antibiotic vancomycin. Moreover, antimicrobial peptide (AMP) is also effective against this pathogen in the complex microbial community of the gut environment, with a selective impact on a few bacterial genera, while maintaining a global homeostasis of the microbiome. In addition, ruminococcin C1 exhibits other biological activities that could be beneficial for human health, as well as other fields of applications. Overall, this study, by using an in vivo infection approach, confirms the antimicrobial clinical potential and highlights the multiple functional properties of ruminococcin C1, thus extending its therapeutic interest.

Keywords: Clostridium perfringens; RiPP; Ruminococcus gnavus E1; antibiotics; antimicrobial resistance; microbiome; peritonitis infection; sactipeptide.

Figure 1

In vivo antibacterial efficacy of RumC1. Mice were challenged intraperitoneally with C. perfringens CP24. After 0.5, 1, and 4 h post-infection (hpi), vancomycin and RumC1 were injected intraperitoneally at 200 mg/kg and 0.1, 1, or 10 mg/kg, respectively, or PBS was injected (control). The groups were composed of five to seven animals. (A) The survival of mice was followed over 48 h. The survival of mice that received RumC1 at 0.1 mg/kg does not appear on the figure as their survival had the same evolution as the control mice. (B) The health and physical condition of mice was measured over time. High scores are representative of impaired health and physical condition. Criteria measured and observed to determine this score are listed on Figure S2 (C). The weight of mice that were still alive at 24 and 48 hpi was measured. (D,E) C. perfringens loads found in the intraperitoneal cavity (D) or in the spleen (E) were measured by plating at the time of death or at the end of the study for the surviving mice (i.e., 48 hpi). Normally distributed data were analyzed using analysis of variance to compare the effects between the different groups, followed by a Bonferroni test to compare the treated groups two by two (** p-value < 0.01, *** p-value < 0.001).

Figure 2

Clearance of C. perfringens by RumC1 in a complex microbial community. The cecal contents of broiler chickens were supplemented with C. perfringens CP24 at 106 CFU/mL (+), or not (−), and then with RumC1 at 5 × MIC of C. perfringens CP24 or left untreated (control). Microbial fermentation was then performed in anaerobic conditions for 24 h at 39 °C. The composition of the microbiota from each condition was obtained by 16S rRNA sequencing. Each treatment was done on five replicates. (A) Detection of the 16S rRNA gene sequence of CP24 and its relative abundance in the microbial community. (B) Venn diagram representing the number of shared or unshared ASVs between the control group (neither CP24 nor RumC1), the cecal contents inoculated with CP24 (group CP, no RumC1), and the cecal contents supplemented with RumC1 (group RumC1, no CP24). (C) Diversity index table of the control and RumC1 groups shown in (B), SEM = standard error of the mean; a, b: different letters denote significant differences. (D) Principal coordinate analysis of the untreated and RumC1-treated cecal contents inoculated with CP24 or not. % in axis shows the degree of explanation (axis 1 explains 51.7% of the variance). (E) Major genera and clusters with relative abundance >0.1% composing the microbial communities of untreated and RumC1-treated cecal contents. Inoculation of CP24 does not affect these distributions in each group (control vs. RumC1).

Figure 3

Main ASVs impacted by RumC1 treatment and their correlation with SCFA production. Cecal contents of broiler chickens were supplemented with C. perfringens CP24 at 10⁶ CFU/mL and treated with RumC1 at 5 × MIC of C. perfringens CP24 or left untreated (control). (A) The ASVs significantly (i.e., Pearson > 0.8 and p-value < 0.05) impacted by RumC1 are shown on the figure as well as their correlation with metabolite production using a color gradient from red (poor producer) to green (good producer), respectively. (B) SCFA production and fermentation parameters for each group were statistically analyzed by ANOVA test. SEM = standard error of the mean. Slope, maximum pressure, and pH were the parameters measured during the fermentation. Slope = rate of gas production in the exponential phase, max pressure = maximum gas production in 24 h, pH = final pH after 24 h of fermentation.

Figure 4

Anti-biofilm activity of RumC1. The inhibition of biofilm formation (A) or the disruption of the biofilm (B) was evaluated on B. subtilis ATCC 6633. This strain was grown in a Calgary biofilm device (CBD) in TSB (tryptic soy broth) for 48 h in the presence (A) or in the absence (B) of RumC1. (B) After 48 h of growth, the pegs from the CBD were transferred to a new 96-well plate containing fresh media supplemented with RumC1 and incubated for another additional 24 h. (A,B) Biofilm formation on the pegs from the CBD was evaluated with crystal violet staining as described in the Materials and Methods section. OD means Optical Density. Each experiment was repeated in independent triplicates.

Figure 5

Host beneficial activities of RumC1. (A) Evaluation of the anti-inflammatory effect of RumC1. HeLa eLUCidate TLR4/IL-8 cells were incubated with IL-1β, LPS from E. coli or P. aeruginosa, or without any pro-inflammatory stimulus, and then treated with increasing concentrations of RumC1. Luciferase luminescence, induced by pro-inflammatory transcription factors, was measured. Results are expressed as the percentage of maximum response measured without RumC1. (B) Percentage of gap closure during a wound healing assay on HaCaT (human keratinocytes cells) cells in the absence or the presence of RumC1. Percentages of closure were measured four days after the gap formation and treatment. FBS (fetal bovine serum) at 10% was used as a positive control. All experiments were performed in independent triplicates.