Isolation, characterization, and potential clinical application of the M1-Pseudomonas aeruginosa pyocin

The evolution and persistence of antibiotic resistance is arguably the largest public health concern of the last half-century. Six antimicrobial-resistant (AMR) pathogens were responsible for almost 80% of the 1.27 million deaths directly linked to antibiotic resistance in 2019. Of these six multidrug-resistant bacterial species, Pseudomonas aeruginosa presents a particularly urgent concern given its lethal nature and ability to evade the host’s immune response. Pseudomonas aeruginosa is a nosocomial pathogen capable of causing multidrug-resistant lung infections in patients with cystic fibrosis (CF) as well as chronic wound infections commonly associated with burn victims. The inability to manage such infections and the ever-progressing issue of multidrug resistance necessitates the exploration of alternative methods of treatment. Similar to many other studied human pathogens, P. aeruginosa produces small antimicrobial peptides as a means of intraspecies competition for limited space and resources. For P. aeruginosa, these ribosomally synthesized bacteriocins are referred to as “pyocins”. This group of species-specific bacteriocins are generally classified as either S-, R-, or F-type pyocins depending on their molecular structure. Numerous studies over the last few decades have demonstrated the auspicious potential of these antimicrobial peptides to be used as a means of alternative treatment for infections caused by P. aeruginosa. 
\nFormally classified as an S-type pyocin, PaeM (M1) is a colicin M-like pyocin that targets the biosynthesis of peptidoglycan through interfering with the shuttle carrier molecule, lipid II. M1 pyocin has yet to be fully characterized in its ability to not only eliminate P. aeruginosa in vitro, but also potentially inhibit the production of virulence factors that contribute to the ability of P. aeruginosa to cause lethal disease. In this work, I screened and isolated chromosomal DNA from P. aeruginosa clinical isolate CF532 and cloned the structural gene using the pBAD/Thio-TOPO vector in chemically competent E. coli. The recombinant M1 (rM1) pyocin was overexpressed through an arabinose-induced promoter and purified using nickel affinity column chromatography. Following confirmation of the rM1 pyocin purity through SDS-PAGE, 453 P. aeruginosa clinical isolates obtained from either cystic fibrosis or severely burned patients were screened using a zone of inhibition (ZOI) assay. Each tested strain was determined to be either resistant, sensitive, or partially-sensitive to rM1 pyocin based on the presence and strength of the ZOI. The M1-sensitive strain CF710 was used for analysis in a broth inhibition assay, demonstrating complete inhibition through absorbance (OD600) measurements. Further in vitro characterization of rM1 treatment was done through biofilm inhibition and elimination assays with CF710 using a modified Calgary model that displayed either a complete or significant reduction in the biomass formed depending on the amount of rM1 pyocin used. The reduction in biomass was visualized with the use of confocal laser scanning microscopy (CLSM) through staining with SYTOTM 9 and propidium iodide. A broth elimination assay conducted with the addition of rM1 pyocin to high cell-density cultures displayed a complete elimination in measurable absorbance (OD600), therefore demonstrating target cell lysis as opposed to inhibition of growth. These findings were followed with a lactate dehydrogenase (LDH) assay to note the release of this intracellular enzyme, thus denoting cell lysis. Congruent with the results of the broth elimination experiment, the LDH assay demonstrated an increase in the release of LDH as a result of the addition of rM1 pyocin to a culture of M1-sensitive P. aeruginosa. The ability of rM1 pyocin to inhibit the production or release of pyorubin, a virulence factor that is hypothesized to play a role in the protection of P. aeruginosa against harmful oxidative stress, was demonstrated against both an M1-sensitive and resistant strain. These findings suggest that rM1 pyocin may have additional clinical practicality beyond targeted elimination of sensitive P. aeruginosa infections.

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