Reprogramming Targeted-Antibacterial-Plasmids (TAPs) to achieve broad-host range antibacterial activity.

The emergence and spread of antimicrobial resistance results in antibiotic inefficiency against multidrug resistant bacterial strains. Alternative treatment to antibiotics must be investigated to fight bacterial infections and limit this global public health problem. We recently developed an innovative strategy based on mobilizable Targeted-Antibacterial-Plasmids (TAPs) that deliver CRISPR/Cas systems with strain-specific antibacterial activity, using the F plasmid conjugation machinery for transfer into the targeted strains. These TAPs were shown to specifically kill a variety of Enterobacteriaceae strains, including E. coli K12 and the pathogen strains EPEC, Enterobacter cloacae and Citrobacter rodentium. Here, we extend the host-range of TAPs using the RP4 plasmid conjugation system for their mobilization, thus allowing the targeting of E. coli but also phylogenetically distant species, including Salmonella enterica Thyphimurium, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. This work demonstrates the versatility of the TAP strategy and represents a significant step toward the development of non-antibiotic strain-specific antimicrobial treatments.

Plasmid Transfer by Conjugation in Gram-Negative Bacteria: From the Cellular to the Community Level.

Bacterial conjugation, also referred to as bacterial sex, is a major horizontal gene transfer mechanism through which DNA is transferred from a donor to a recipient bacterium by direct contact. Conjugation is universally conserved among bacteria and occurs in a wide range of environments (soil, plant surfaces, water, sewage, biofilms, and host-associated bacterial communities). Within these habitats, conjugation drives the rapid evolution and adaptation of bacterial strains by mediating the propagation of various metabolic properties, including symbiotic lifestyle, virulence, biofilm formation, resistance to heavy metals, and, most importantly, resistance to antibiotics. These properties make conjugation a fundamentally important process, and it is thus the focus of extensive study. Here, we review the key steps of plasmid transfer by conjugation in Gram-negative bacteria, by following the life cycle of the F factor during its transfer from the donor to the recipient cell. We also discuss our current knowledge of the extent and impact of conjugation within an environmentally and clinically relevant bacterial habitat, bacterial biofilms.

A Type VI Secretion System Trans-Kingdom Effector Is Required for the Delivery of a Novel Antibacterial Toxin in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has evolved multiple strategies to disarm and take advantage of its host. For this purpose, this opportunist pathogen has particularly developed protein secretion in the surrounding medium or injection into host cells. Among this, the type VI secretion system (T6SS) is utilized to deliver effectors into eukaryotic host as well as target bacteria. It assembles into a contractile bacteriophage tail-like structure that functions like a crossbow, injecting an arrow loaded with effectors into the target cell. The repertoire of T6SS antibacterial effectors of P. aeruginosa is remarkably broad to promote environmental adaptation and survival in various bacterial communities, and presumably in the eukaryotic host too. Here, we report the discovery of a novel pair of antibacterial effector and immunity of P. aeruginosa, Tle3 and Tli3. Tli3 neutralizes the toxicity of Tle3 in the periplasm to protect from fratricide intoxication. The characterization of the secretion mechanism of Tle3 indicates that it requires a cytoplasmic adaptor, Tla3, to be targeted and loaded onto the VgrG2b spike and thus delivered by the H2-T6SS machinery. Tla3 is different from the other adaptors discovered so far and defines a novel family among T6SS with a DUF2875. Interestingly, this led us to discover that VgrG2b that we previously characterized as an anti-eukaryotic effector possesses an antibacterial activity as well, as it is toxic towards Escherichia coli. Excitingly Tli3 can counteract VgrG2b toxicity. VgrG2b is thus a novel trans-kingdom effector targeting both bacteria and eukaryotes. VgrG2b represents an interesting target for fighting against P. aeruginosa in the environment and in the context of host infection.