Abstract
Clostridium difficile has been reported as the most common cause of nosocomial diarrhea (antibiotic-associated diarrhea), resulting in significant morbidity and mortality in hospitalized patients. The resistance of the clostridial spores to antibiotics and their side effects on the gut microbiota are two factors related to the emergence of infection and its relapses. Lantibiotics provide an innovative alternative for cell growth inhibition due to their dual mechanism of action (membrane pore-forming and cell wall synthesis inhibition) and low resistance rate. Based on the fact that bacteriocins are usually active against bacteria closely related to the producer strains, a new dual approach combining genome mining and synthetic biology was performed, by designing new lantibiotics with high activity and specificity toward Clostridium. We first attempted the heterologous expression of putative lantibiotics identified following Clostridium genome mining. Subsequently, we designed new hybrid lantibiotics combining the start or end of the putative clostridial peptides and the start or end parts of nisin. The designed peptides were cloned and expressed using the nisin biosynthetic machinery in Lactococcus lactis. From the 20 initial peptides, only 1 fulfilled the requirements established in this work to be considered as a good candidate: high heterologous production level and high specificity/activity against clostridial species. The high specificity and activity observed for the peptide AMV10 makes it an interesting candidate as an alternative to traditional antibiotics in the treatment of C. difficile infections, avoiding side effects and protecting the normal gut microbiota.
Highlights
The genus Clostridium comprises about 150 metabolically diverse species of Gram-positive, endospore-forming anaerobic bacteria that are ubiquitous in virtually all anoxic habitats where organic compounds are present, including soils, aquatic sediments, and the intestinal tracts of animals and humans (Udaondo et al, 2017)
In order to identify new lantibiotics specific and active against pathogenic Clostridium strains as C. difficile, two bioinformatics programs were used
Starting from the premise that bacteriocins are usually active against bacteria closely related to the producing strain (Zacharof and Lovitt, 2012; Yang et al, 2014; Ventura et al, 2015), and using gene mining approaches (Weber et al, 2015; van Heel et al, 2016, 2018, 4; Montalbán-López et al, 2017), we have identified 54 putative lantibiotic sequences after the analysis of more than 560 Clostridium spp. genomes deposited in NCBI
Summary
The genus Clostridium comprises about 150 metabolically diverse species of Gram-positive, endospore-forming anaerobic bacteria that are ubiquitous in virtually all anoxic habitats where organic compounds are present, including soils, aquatic sediments, and the intestinal tracts of animals and humans (Udaondo et al, 2017). It has always been related to the elderly until the start of the new millennium, when numerous studies have described several outbreaks in Europe (Bauer et al, 2011), with more than 150,000 cases per year of C. difficile infection and a 20-fold increase of mortality. Those events have been attributed to the emergence of new and more virulent strains (Mastrantonio and Rupnik, 2018). The development of novel specific antimicrobial compounds against Clostridium spp. turn out to be a necessity and a very relevant line of investigation
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