Antibacterial Efficacy of R-Type Pyocins towards Pseudomonas aeruginosa in a Murine Peritonitis Model

Abstract

R-type pyocins are high-molecular-weight bacteriocins carried within the chromosomes of some bacterial species, such as Pseudomonas aeruginosa, and almost certainly evolved from lysogenic bacteriophages of the Myoviridae family. They contain no head structures and no DNA and are used as defense systems, usually against other strains of the same bacterial species. They bind with their tail fibers to targeted bacterial surface molecules and then kill by inserting a core or needle that dissipates the bacterial membrane potential. Their mechanism of action, high bactericidal potency (one pyocin particle can kill one bacterium), and focused spectrum suggest that R-type pyocins could be developed as antibacterial agents. In a lethal mouse peritonitis model, submicrogram quantities of pyocin prevent death from 90% lethal dose inocula of a pyocin-sensitive, clinical isolate of P. aeruginosa. We show here the dose response curves, treatment windows, or periods of response after infection and the several-log-unit acute reduction of bacterial load in blood and spleen samples, suggesting that R-type pyocins have several characteristics that one would expect from an effective therapeutic.Many Pseudomonas species possess the genes for one or both of the two types of high-molecular-weight pyocins, the R type and the F type (17, 28). Both are carried in the bacterial genome. The R-type pyocin locus consists of 16 open reading frames including 10 structural genes plus regulatory and chaperone genes (24, 25). Morphologically and genetically, the Rtype pyocins resemble the tails of Myoviridae bacteriophages but have no head structure and thus no nucleic acid content (9,15,16,23). They are thought to have evolved from the tail structure of a P2 bacteriophage-related ancestor; but they are not simply defective phages. There are meaningful physical and chemical stability differences, such as differences in protease and acid resistance, between pyocins and phage tails as the former have been adapted for their role as defensive bactericidal agents (14,22). However, similar to bacteriophages, pyocins bind to specific "receptors" on target bacteria and penetrate their membranes with a "core," or needle-like structure (31). In contrast to bacteriophages, pyocins have no material to inject and their penetrating core remains patent. As an immediate consequence, the bacterium is killed by dissipation of its membrane potential, a bactericidal event that can result from the binding of a single pyocin particle (8,27,31). An R-type pyocin particle can act only once; once it kills a cell, it cannot go on to kill others. Bacteriophages and R-type pyocins share common ancestry, but in contrast to bacteriophages, pyocins kill without directly causing bacterial cell lysis, a feature likely desirable for treating systemic infections (7, 33).Francois Jacob discovered and first described pyocins as high-molecular-weight bacteriocins (11). Over subsequent decades, much effort has been expended studying Pseudomonas aeruginosa pyocin morphology, structure,...

Novel High-Molecular-Weight, R-Type Bacteriocins of Clostridium difficile

“…1). However, even in the best producer strains, the particles were released in low quantities in lysates, quantities too low for antibacterial activity to be detected using typical assays developed for this class of bacteriocins (30). To overcome this limitation and test for bactericidal activity, we made lysates of two producer strains, CD4 and CD16, by growing cells to early log phase and inducing them with mitomycin C. Lysates were then concentrated, and the diffocin particles were purified by high-speed centrifugation (see Materials and Methods).…”

“…Killing was observed by a clearing of bacterial growth at the spot where the diffocin was applied. Killing activity was also assessed in a survival titration assay based on the method described for R-type pyocins (29,30). Bactericidal events based on the survival titration assay are expressed as killing units.…”

“…Furthermore, no means of prophylaxis is available except for care facility and personal hygiene, such as thorough hand washing by those persons exposed to the spores, especially hospital staff. Studies showing that phage tail-like R-type bacteriocins can be used orally and parentally to treat bacterial infections (6,12,21,29,30) have prompted us to identify similar entities specific for C. difficile for possibile use in the treatment or prevention of CDI.…”

Within-host competitive interactions as a mechanism for the maintenance of parasite diversity

“…In fact, the health benefits of many probiotics are thought to be due in part to their effectiveness in interference competition [100]. Interest in using bacteriocins as an alternative to traditional, broadspectrum antibiotics is growing [101][102][103]. Historically, the narrow killing range of bacteriocins limited their utility; however, faster diagnostic tests, the ability to engineer bacteriocins to target novel sites [104], and our awareness of the consequences of using broad-spectrum antibiotics has made the therapeutic use of bacteriocins more viable.…”

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

“…Pyocins kill bacteria by first binding to a cell surface structure via tail fibers, followed by contraction of the sheath and insertion of their hollow core through the cell envelope resulting in depolarization of the cytoplasmic membrane and cell death [67]. Pyocin has been shown to increase survival and reduce bacterial counts in a lethal murine experimental Pseudomonas peritonitis model [68]. The bactericidal specificity of pyocins is conferred by their tail fibers encoded by prf15.…”