Plant-expressed pyocins for control of Pseudomonas aeruginosa

Abstract

The emergence, persistence and spread of antibiotic-resistant human pathogenic bacteria heralds a growing global health crisis. Drug-resistant strains of gram-negative bacteria, such as Pseudomonas aeruginosa, are especially dangerous and the medical and economic burden they impose underscore the critical need for finding new antimicrobials. Recent studies have demonstrated that plant-expressed bacteriocins of the colicins family can be efficient antibacterials against all major enteropathogenic strains of E. coli. We extended our studies of colicin-like bacteriocins to pyocins, which are produced by strains of P. aeruginosa for ecological advantage against other strains of the same species. Using a plant-based transient expression system, we expressed six different pyocins, namely S5, PaeM, L1, L2, L3 and one new pyocin, PaeM4, and purified them to homogeneity. Among these pyocins, PaeM4 demonstrated the broadest spectrum of activity by controlling 53 of 100 tested clinical isolates of P. aeruginosa. The activity of plant-made pyocins was confirmed in the agar drop, liquid culture susceptibility and biofilm assays, and in the Galleria mellonella animal infection model.

Engineering bacteriocin‐mediated resistance against the plant pathogen Pseudomonas syringae

“…Previously, bacteriocins that are active against human pathogens have been expressed in N. benthamiana leaves and in leafy green vegetables (Schulz et al , ; Paškevičius et al , ; Schneider et al , ). To express PL1 in planta , a construct that encodes PL1 with an N‐terminal 4 × c‐Myc tag was cloned into a Ti binary vector and transiently expressed in leaves of N. benthamiana using agroinfiltration.…”

“…We are not aware of any prior reports of attempts to express bacteriocins in planta as a strategy to confer resistance against plant pathogenic bacteria. Bacteriocins with activities against E. coli , Salmonella and Pseudomonas aeruginosa have been expressed in plants but with the objective of using these as a means of treating bacterial infections in humans (Schulz et al , ; Paškevičius et al , ; Schneider et al , ). The resulting successful demonstration that active bacteriocins can be expressed in planta suggests that PL1 could also be expressed in planta in an active form to protect plants against Ps infection.…”

LlpB represents a second subclass of lectin‐like bacteriocins

“…Among Gram‐negative bacteria, bacteriocins from Escherichia coli (colicins) and Pseudomonas aeruginosa (pyocins) serve as model systems for studying receptor binding, cell import mechanisms and toxin‐immunity interactions (Cascales et al ., ; Papadakos et al ., ; Ghequire and De Mot, ; Chassaing and Cascales, ). These compounds are potent antibacterials and their use in food and therapeutic applications is currently being investigated (Schulz et al ., ; Paškevičius et al ., ; Scholl, ; Schneider et al ., ). Major advantages of bacteriocins include biodegradability, selective killing and eligibility (of some bacteriocins) for large‐scale production in plants (Behrens et al ., ; Ghequire and De Mot, ).…”

Techno‐economic analysis of a plant‐based platform for manufacturing antimicrobial proteins for food safety

“…11,14,15,[22][23][24] In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. Plant-based platforms have the potential for producing market-relevant volumes of AMPs at competitive costs, because they do not require expensive bioreactors and culture media.…”

“…We have previously reported expression levels as high as 3 g/kg plant fresh weight (FW). 11,14,15,[22][23][24] In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. We used laboratoryscale results and working process knowledge from pilot and commercial processes to develop a process simulation model using SuperPro Designer ® to assess the commercial viability of the production platform and to identify economic "hotspots" to help guide future research and development.…”