Biology subjects

Gabant, P.

Publications and source records attributed to Gabant, P..

5 recordsLinked to original sources

bioRxiv2026

An Enhanced Split Intein-Mediated Ligation (SIML) Platform for Rapid Discovery and Functional Screening of Circular Bacteriocins

Bacteriocins are ribosomally synthesized antimicrobial peptides with promising applications in biotechnology, particularly in food preservation and animal and human health. Circular bacteriocins are especially attractive due to their head-to-tail cyclized structure, which confers enhanced stability and antimicrobial potency relative to linear peptides. Here, we report an in vitro cell-free protein synthesis system coupled with an enhanced Split Intein-Mediated Ligation platform (IV-CFPS/SIML) for the efficient synthesis of circular bacteriocins through systematic evaluation of cyclization sites and alternative split inteins. Using enterocin AS-48 as a model, we systematically evaluated multiple serine-based cyclization sites in combination with three split inteins, NpuDnaE, Gp41-1, and SspGyrB, to identify configurations supporting efficient splicing and high antimicrobial activity. Gp41-1 emerged as the most effective intein and was subsequently applied to the production of garvicin ML, amylocyclicin, and 27 naturally occurring sequence variants, demonstrating that cyclization site selection, intein identity, and minor sequence variations strongly influence antimicrobial potency and target range. Finally, SIML expression cassettes encoded in pUC-derived vectors enabled in vivo production and functional expression of selected circular bacteriocins in recombinant Escherichia coli. Collectively, these results establish SIML as a versatile platform for in vitro and in vivo production, screening, and functional characterization of known and putative circular bacteriocins.

synthetic biology↗

bioRxiv2024

Multiplexing bacteriocin synthesis to kill and prevent antimicrobial resistance

Antibiotic resistance represents an emergency for global public health. This calls for using alternative drugs and developing innovative therapies based on a clear understanding of their mechanisms of action and resistance in bacteria. Bacteriocins represent a unique class of natural molecules selectively eliminating bacteria. These secreted proteins exhibit a narrower spectrum of activity compared to conventional broad-spectrum antimicrobials by interacting with specific protein and lipid receptors on bacterial cell envelopes. Despite their diverse molecular structures, the commonality of being genetically encoded makes bacteriocins amenable to synthetic biology design. In using cell-free gene expression (CFE) and continuous-exchange CFE (CECFE), we produced controlled combinations (cocktails) of bacteriocins in single synthesis reactions for the first time. A first set of bacteriocin cocktails comprising both linear and circular proteins allowed the targeting of different bacterial species. Other cocktails were designed to target one bacterial species and considering bacteriocins pathways to cross the cell-envelope. Such combinations demonstrated efficient bacterial eradication and prevention of resistance. We illustrate the effectiveness of these bacteriocin mixtures in eradicating various human pathogenic-multiresistant--isolates. Finally, we highlight their potential as targeted and versatile tools in antimicrobial therapy by testing a combination of bacteriocins for treatment in vivo in the animal model Galleria mellonella.

synthetic biology↗

bioRxiv2024

Uncovering the class II-bacteriocin predatiome in salivarius streptococci

Facing the surge of antibiotic resistance, the medical field has a critical need for alternatives to treat bacterial infections. Among these, the use of bacteriocins, ribosomally-synthesized antimicrobial peptides produced by bacteria, is considered to be a promising route. In the human commensal Streptococcus salivarius, the production of unmodified class II bacteriocins is directly controlled at the transcriptional level by the quorum-sensing ComRS system. Here, we used an integrated approach combining bioinformatics and synthetic biology to identify novel bacteriocins from salivarius streptococci active against human pathogens. We developed a bioinformatic pipeline that combines conservation of DNA motifs for genetic regulation and features of bacteriocin primary sequences to uncover cryptic class II bacteriocins. Notably, we discovered more than 50 novel bacteriocin candidates clustered into 21 groups from 100 genomes of S. salivarius. Strain-based analysis of bacteriocin cocktails revealed an important diversity restricted to seven distinct loci, probably resulting from bacteriocin intra- and inter- species exchanges. Using in vitro or in vivo production and synthetic biology tools, we showed that most of them are active against a panel of Gram-positive bacteria, including clinically- relevant pathogens. Overall, this work provides a new search-to-test generic pipeline for the discovery of novel bacteriocins in Gram-positive bacteria that could be used in cocktails for broad applications in the food and biomedical fields. IMPORTANCETo survive in highly challenging environments, streptococci have evolved a competence- predation coupling mechanism for genome plasticity. This developmental process is highly regulated at the transcriptional level, masking the predation killing effects in usual laboratory conditions. Here, we present a general strategy that combines bioinformatics and synthetic biology to unveil class II bacteriocins in streptococci. Its implementation to the beneficial species Streptococcus salivarius revealed around 40 class II salivaricin cocktails explained by the plasticity of seven independent loci. Notably, the salivaricin predatiome includes a subtle blend of fratricins, sobrinicins, and broad-spectrum bacteriocins with overlapping activities against a wide spectrum of low-GC Gram-positive bacteria, including notorious pathogens. Furthermore, most of those bacteriocins are predicted to be variants of a common -hairpin structure, indicating that their mode of action evolved convergently. Finally, the discovery of ca. 50 novel bacteriocins offers perspectives for the rational assembly of potent cocktails active against pathogenic staphylococci, streptococci or enterococci.

microbiology↗

bioRxiv2023

Bacteriocin peer selection for the production of antibiotic selection free biotherapeutic pDNA

Plasmid vectors are well established tools used to genetically engineer bacteria both in the laboratory and at industrial scale. The past few decades have seen a rising interest in the use of plasmid DNA (pDNA) for biotherapeutic applications. This interest is a strong driver for the development of technologies to increase pDNA production at biopharmaceutical scale in terms of decreasing production costs and meeting regulatory requirements. Although cell free technologies are emerging, pDNA vectors are still produced by fermentation in Escherichia coli strains. As plasmids are extra-chromosomic molecules there is a probability of losing a certain ratio within the E. coli population during the fermentation process leading to a decrease of DNA production efficiency. Maintaining pDNA in the population is thus a key element to reach efficient and robust production. Traditionally, antibiotic resistance genes and antibiotics have been used to generate a selective pressure to ensure pDNA stability in the microbial population during the production process. Nowadays, having an antibiotic resistance gene in the pDNA coding sequence represents a limitation both for safety and legal requirements and in terms of production yield. For this reason, we have developed a pDNA antibiotic-free bacteriocin-based selection system, based on the genes involved in the production, processing, secretion and immunity of the bacteriocin microcin V. Our approach is based on the peer pressure exerted by the bacteriocin and does not rely on the addition of any selective agent in the medium to limit population drift and ensure plasmid stability. This novel antibiotic-free approach may be applied to any pDNA vector in different E. coli strains and expands their potential applications in both animal and human health as delivery vectors for biotherapeutics.

synthetic biology↗

bioRxiv2023

Enhancing the antibacterial function of probiotic Escherichia coli Nissle: when less is more

Probiotic bacteria confer multiple health benefits, including preventing the growth, colonisation, or carriage of harmful bacteria in the gut. Bacteriocins are antibacterial peptides produced by diverse bacteria and their production is tightly regulated and coordinated at the transcriptional level. A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from non-native constitutive promoters or in response to host or pathogen signal from synthetic promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We have conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce McC from the wild-type promoter and two mutant promoters that allow, relative to the wild-type promoter, high and low amounts of McC production. This was done by introducing specific changes to the sequence of the wild-type promoter driving transcription of the McC operon, whilst ensuring that the modified promoters respond to native regulation. By studying the transcriptomic responses and antibacterial efficacy of the retrofitted EcN bacteria in a Galleria mellonella infection model of enterohemorrhagic E. coli, we show that EcN bacteria that produce the lowest amount of McC display the highest antibacterial efficacy with little to none undesired collateral impact on their fitness. The results highlight considerations researchers may take into account when retrofitting probiotic bacteria with heterogenous gene products for therapeutic, prophylactic or diagnostic applications. IMPORTRANCEBacteria that resist killing by antibiotics are a major risk to modern medicine. The use of beneficial probiotic bacteria as chassis to make antibiotic-like compounds at the site of infection in the body is emerging as a popular alternative to the use of conventional antibiotics. A potential drawback of engineering probiotic bacteria in this way is that producing antibiotic-like compounds could impart undesired side-effects on the performance of such bacteria and thereby compromise their intended use. This study highlights considerations researchers may take into account when engineering probiotic bacteria for therapeutic, prophylactic or diagnostic applications.

microbiology↗