DS0507 - Biotechnologies  et valorisation des bio-ressources :

Biosynthetic, structural and functional characterization of Rum C peptides, a family of bacteriocins as viable alternative to conventional antibiotics – RUMBA

RumC antimicrobial peptides, a new weapon in the fight against antibiotic resistance

Biosynthetic, structural and functional characterizations of RumC peptides, a family of bacteriocins as a viable alternative to conventional antibiotics

Antibiotic resistance, a major public health challenge

Since the discovery of penicillin in 1928, humans have widely developed and used antibiotics to combat microbial infections. However, inappropriate and excessive use of these molecules has led to the emergence of pathogens resistant to all classes of antibiotics. According to the World Health Organization, the world is moving towards an era where common infections will kill again. The WHO predicts that multi-resistant pathogens will cause the death of 10 million people a year by 2050, surpassing the number of cancer-related deaths. There is therefore an urgent need to develop innovative strategies and find new molecules, ideally natural ones, to overcome the phenomena of resistance. A promising alternative concerns antimicrobial peptides, which have been extensively studied in recent years and some of which are currently in clinical development. In this context, bacteria are a treasure trove of multiple classes of naturally occurring antimicrobial peptides, more commonly known as bacteriocins.

Our research focuses on Ruminococcus gnavus, a bacterium present in the digestive tract of about 90% of the healthy human. The strain R. gnavus E1 produces 5 bacteriocins, the ruminococcins C1 to C5 (RumC1-5). Because of their production in the intestinal tract by a predominant member of the gut microbiota and their activity against enteropathogens, RumC bacteriocins may be of particular interest in health. The boundaries of the proposal range from the production of the RumC peptides to their efficacy in animals. The project combines a wide range of expertise covering microbiology, biochemistry, chemistry and structural biology. Our objectives were to understand the biosynthetic pathways of the RumC peptides, to characterize these compounds and finally to develop new RumC based molecules. The key steps of the project include the in vivo and in vitro production of the RumC peptides, the identification of the molecular and structural modifications of the bacteriocins and the biological activity of these compounds on resistant and multi-resistant pathogens. Moreover, an important point was to gain insights into the mode of action of the RumC peptides.

The RUMBA project allowed the production in the laboratory by heterologous expression of the RumC1 peptide, of natural origin, to resolve its structure and evaluate its therapeutic potential. The three-dimensional structure of RumC1 reveals a very compact original double hairpin motif, not described so far. This structure confers very interesting characteristics, which are often lacking in antimicrobial peptides to be considered in clinical development, notably a high resistance to physiological conditions and to various physico-chemical treatments. These points are crucial for the large-scale production of RumC1 and the exploitation of its full therapeutic potential. RumC1 has key properties for a drug candidate to treat intestinal infections, notably because it is effective at very low doses against antibiotic-resistant clinical intestinal pathogens and is active in conditions mimicking the intestinal environment. In addition, RumC1 has shown no toxicity to intestinal tissues and could be administered orally.

The successful achievement of the RUMBA project allows us to consider RumC bacteriocins as promising and innovative molecules in the war against antibiotic resistance. Indeed, the results obtained provide a solid basis to take RumC bacteriocins one step further towards their therapeutic use either alone or in combination with other antimicrobial agents, for human or animal health. Implementing this direction will increase, at short or medium term, the panel of new molecules and strategies to fight against antibiotic resistance. This will most likely afford new opportunities for human, animal and environmental wellbeing in the “One Health” concept for the benefit of society.

The RUMBA project has resulted in the publication of 2 articles in high-impact journals, the writing of a book chapter, and 6 oral and 8 poster communications at national and international conferences. The references of the 2 articles are indicated below:
Chiumento S., et al., (2019). Ruminococcin C, a promising antibiotic produced by a human gut symbiont. Science Advances, 5, eaaw9969.
Characterization of RumC1, a new member of the sactipeptide family shows a unique pattern of post-translational modifications. The sequential mode of activation involves of a human enzyme. The publication highlights the therapeutic potential of RumC1 in terms of antimicrobial activity and safety for human cells.
Roblin C., et al., (2020). The unusual structure of Ruminococcin C1 antimicrobial peptide confers clinical properties. Proc. Natl. Acad. Sci., doi/10.1073/pnas.2004045117.
RumC1 shows a strong antimicrobial activity on a large panel of human clinical pathogens, which are resistant to the major antibiotics currently used in therapy. We demonstrate the lack of toxicity of RumC1 for human intestinal tissues. The compact structure of RumC1 confers stability to gastrointestinal conditions and various physico-chemical parameters, which is necessary for a future pharmaceutical development.

Bacterial infections represent a serious threat for human health in general and the growth and viability of livestock in particular. Infected animals also constitute a reservoir of pathogens able to affect humans through environmental spreading and food chain contamination. The massive use of antibiotics in livestock, even for non-therapeutic applications, has worsened the situation and has led to the emergence of resistant and multi-resistant strains. In order to limit the increasing use of conventional antibiotics prone to cause the appearance of resistances and to treat infections of humans and animals, there is an urgent need for new molecules exhibiting antimicrobial activities even against resistant pathogens. Such new antimicrobial molecules could either be used in preventive strategies to limit livestock infection or in therapeutic treatment in case of confirmed infection. These new generations of antimicrobial molecules will help fighting against resistant and multi-resistant bacteria, which is one of the major challenges in human and animal health.
Bacteriocins, a diverse group of proteinaceous, ribosomally synthesized antimicrobial compounds produced by bacteria, are part of this new generation of antimicrobial agents. They are considered as important alternatives to traditional antibiotics for humans and animals. Some of them are naturally modified peptides proven to be active against pathogenic bacteria, including multi-resistant strains and do not seem associated to the emergence of resistance.
The present project aims to establish RumC bacteriocins from Ruminococcus gnavus as a viable alternative to conventional antibiotics. Indeed, as a result of their production in the intestinal tract by a predominant member of the gut microbiota and their activity against enteropathogens such as Clostridium perfringens, RumC bacteriocins may be of particular interest both in human and animal health. Toward this goal, our objectives are to understand the very challenging biosynthetic pathway leading to the RumC peptides, to fully characterize these compounds and finally to contribute to the development of new RumC based molecules. The project goes from the in vivo production of the RumC peptides to the assessment of their in vivo activity in animals, including the unambiguous identification of the molecular and structural modifications that account for their biological properties. This program will be undertaken by the four partners of the consortium. More specifically, the first step will concern the optimization of the already described protocol to produce, in vivo, the five RumC peptides. This task will facilitate the purification and the characterization of the mature peptides produced in vivo. This point is essential, since the pattern of the modifications (molecular and structural) will serve as a reference for the in vitro production of the RumC bacteriocins. The next important points are devoted to 1- the characterization of the two Radical-SAM enzymes involved in the post-translational modifications of the RumC peptides. The enzymatic activities of these proteins will be assessed in vitro by using the linear unmodified peptides; 2- assessing the biological activity of the RumC modified peptides either the purified natural molecules or the synthetic ones. This will be done by comparison against pathogenic and indigenous bacterial indicator strains. In addition, the safety and the in vivo activity of RumC peptides on animals will be also tested using both gnotobiotic and poultry models by our industrial partner; 3- engineering studies of the newly synthesized bacteriocins.

Project coordination

Victor DUARTE (Laboratoire de Chimie et Biologie des Métaux)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRSDR12 _ IMM Centre National de la Recherche Scientifique Délégation Provence et Corse _ Institut de Microbilogie de la Méditerranée
ADISSEO France SAS
CNRS DR12_iSm2 Centre National de la Recherche Scientifique délégation Provence et Corse_Institut des Sciences Moléculaires de Marseille
CEA/DRF/BIG/LCBM Laboratoire de Chimie et Biologie des Métaux

Help of the ANR 480,000 euros
Beginning and duration of the scientific project: October 2016 - 48 Months

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