The P. aeruginosa effector Tse5 forms membrane pores disrupting the membrane potential of intoxicated bacteria

Abstract

The type VI secretion system (T6SS) of Pseudomonas aeruginosa injects effector proteins into neighbouring competitors and host cells, providing a fitness advantage that allows this opportunistic nosocomial pathogen to persist and prevail during the onset of infections. However, despite the high clinical relevance of P. aeruginosa, the identity and mode of action of most P. aeruginosa T6SS-dependent effectors remain to be discovered. Here, we report the molecular mechanism of Tse5-CT, the toxic auto-proteolytic product of the P. aeruginosa T6SS exported effector Tse5. Our results demonstrate that Tse5-CT is a pore-forming toxin that can transport ions across the membrane, causing membrane depolarisation and bacterial death. The membrane potential regulates a wide range of essential cellular functions; therefore, membrane depolarisation is an efficient strategy to compete with other microorganisms in polymicrobial environments.

A Dueling-Competent Signal-Sensing Module Guides Precise Delivery of Cargo Proteins into Target Cells by Engineered Pseudomonas aeruginosa

“…To employ TPP-controlled dueling as a synthetic biology module, we postulate that the strong antibacterial activity of the H1-T6SS may be an unwanted feature for a general delivery tool. To detoxify the H1-T6SS, we systematically introduced chromosomal mutations to the catalytic sites of seven effectors (Tse1 C30A , Tse2 VK‑AA , Tse3 E250Q , Tse4 G176V , Tse6 D396A , Tse7 HH‑AA , and Tse8 S186A ), and the gene encoding the membrane-damaging effector Tse5 was deleted. , The resultant mutant, lacking all eight effector activities, was named DUEC hereafter for simplicity. Time-lapse fluorescence microscopy analysis shows that DUEC cells exhibited active H1-T6SS assembly and dueling that are comparable to wild-type cells (Figure A).…”

“…T6SS is a double-tubular contractile weapon commonly employed by Gram-negative bacteria to secrete toxic effectors and kill competitors in diverse environments upon direct contact. − The outer sheath contracts and releases a substantial amount of energy that drives the inner tube and its associated spike complex and effectors deep into the cytosol of neighboring cells, including Gram-negative and Gram-positive bacteria and fungi. − The sheath length likely dictates the working distance of T6SS given that delivery into a target cell is mostly contact-dependent with few exceptions. − Notably, receiving cells are not killed by penetration of the incoming tube but by the codelivered effectors. ,, These effectors may not only dictate functions but also directly contribute to T6SS assembly because deletion of multiple effector genes abolishes T6SS assembly in several species. ,, Similar to the spike complex proteins, VgrG and PAAR, some effectors can also serve as carriers when fused with cargo proteins. ,,, Therefore, rather than deleting effector genes, an effective strategy has been developed in Vibrio cholerae and Aeromonas dhakensis to use catalytic mutations of effectors to detoxify the T6SS while maintaining the delivery function. ,, However, it is challenging to apply this strategy in much more complex systems such as the T6SS in P. aeruginosa, which has three independent T6SS gene clusters (H1-, H2-, and H3-T6SS), each secreting a specific set of effectors. ,− Eight effectors (Tse1–8) of H1-T6SS with different modes of secretion have been identified, ,− but whether they contribute to T6SS assembly and can serve as cargo carriers has not been explored.…”

Acquisition of T6SS Effector TseL Contributes to the Emerging of Novel Epidemic Strains of Pseudomonas aeruginosa

“…Numerous T6SS-dependent effectors have been predicted by comparative genomic analysis in a broad range of bacteria ( 13 ). The characterization of T6SS effectors of P. aeruginosa , the most predominant opportunistic pathogens in nosocomial infections ( 20 , 22 ), is mainly focused on certain model strains such as PAO1 and PA14 ( 23 , 24 ).…”

Killing in the name of: T6SS structure and effector diversity

“…The effector is a double-stranded DNA deaminase A (DddA) from Burkholderia cenocepacia. DddA has been shown instrumental to kill several bacterial species including P. aeruginosa, P. putida and other Burkholderia species through chromosomal degradation and DNA replication arrest [110]. In bacteria resistant to DddA killing, this effector assumes the role of a powerful and direct mutagen.…”

“…In bacteria resistant to DddA killing, this effector assumes the role of a powerful and direct mutagen. Its mechanism of action involves the deamination of cytosine in double-stranded DNA, resulting in its conversion to uracil [110,112]. These C-to-U transitions occur throughout the genome and accelerate the acquisition of rifampicin-resistant mutants through changes in the rpoB gene that encodes the β-subunit of the RNA polymerase.…”