Discovery of novel phage-derived lytic proteins with potential antimicrobial activity against pseudomonas aeruginosa.

Abstract

The escalation of bacterial resistance to existing antibiotics has accelerated alarmingly. This trend is further compounded by the misuse of antibiotics, leading to an intensified global challenge. Identifying novel alternatives to antibiotics to curtail the rise in resistance has led to the discovery of several alternatives, including phage-derived lytic proteins. This study investigates a solution to the urgent need for antibiotic alternatives amidst rising antibiotic resistance. It highlights the untapped potential of phage-derived lytic proteins as antibacterial agents. Sequence-based screening was used to mine an online phage database for novel putative sequences encoding lytic proteins focussing on sequences from University of Kwa-Zulu Natal (UKZN). Four open reading frames (ORFs) displaying domains typical of phage-lytic proteins were selected and synthesised. Phage lytic proteins are derivatives of phage and degrade the peptidoglycan component of the bacterial cell wall and have since gained interest as antimicrobials. Their sizes were 396 bp for Holin, 1341 bp LysA, 1374 bp LysB and 1383 bp Lysin, each with no signal peptide for cytosolic expression. Using PCR, they were subsequently sub-cloned into the pEAQ-HT for expression in Nicotiana benthamiana. The genes were also sub-cloned into pET-30b(+) vector for expression in E. coli bacteria as His6 tagged recombinants with the tag fused to the N- or C-terminal of the proteins. While challenges were experienced with purification, the bacterial system outperformed the plant-based system in recombinant protein production of all four target proteins. Transient expression in N. benthamiana yielded a truncated version of three proteins and no detectable expression of the holin (SM31) protein. Lysin (SM07) was selected for detailed characterisation due to its ancestral lineage to the other proteins and the presence of the potential catalytic domains and catalytic residues for lytic proteins. Lysin (SM07) was purified successfully using IMAC with the His6 tag on the N-terminal. The enzyme demonstrated promising antimicrobial activity against P. aeruginosa with a minimum inhibitory concentration of 0.004 μg/μl with no significant toxicity observed against Vero kidney cells. In a prototype, a surface disinfectant was formulated using SM07 as the sole active ingredient. The spray showed a reduced P. aeruginosa bacterial count of approximately 98.7%. Additionally, a 3D structure was modelled using AlphaFold revealing a cell wall binding domain (CBD), and a catalytic/enzymatic domain possessing a typical catalytic dyad (Glu228 and Glu237). Finding a CBD is unusual in Gram-negative active endolysins, raising intriguing possibilities for future antimicrobial research. This discovery will contribute to combatting nosocomial infections resulting from P. aeruginosa.