Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity
Abstract:The emergence of multidrug-resistant (MDR) bacteria has risen rapidly, leading to a great threat to global public health. A promising solution to this problem is the exploitation of phage endolysins. In the present study, a putative N-acetylmuramoyl-L-alanine type-2 amidase (NALAA-2, EC 3.5.1.28) from Propionibacterium bacteriophage PAC1 was characterized. The enzyme (PaAmi1) was cloned into a T7 expression vector and expressed in E. coli BL21 cells. Kinetics analysis using turbidity reduction assays allowed t… Show more
“…The acquired results suggest an efficient bactericidal activity and allowed the determination of the minimum bactericidal concentration (MBC) and the half-maximal lethal concentration (LC 50 ) of Ami1 against S. aureus , S. epidermidis , and E. coli DH5a, after 1.30 h, 3 h, and 6 h of incubation (Table 1 ). The bactericidal activity of Ami1 falls within the range expected for highly active endolysins that have been recently reported (Guo et al 2017 ; Wu et al 2018 ; Plotka et al 2019 ; Varotsou et al 2023 ; Premetis et al 2023b ). Fig.…”
Section: Resultssupporting
confidence: 81%
“…Towards this direction, the utilization of enzybiotics, a specific class of enzymes that exhibit the potency to treat bacterial infections (Labrou 2019 ; Song et al 2020 ), is a very promising and innovative approach (Harhala et al 2021 ; Varotsou et al 2023 ). The antimicrobial potential of enzybiotics lies heavily on their ability to hydrolyze PG, a fundamental structural element of the bacterial cell wall, causing cell lysis and therefore its death (Young 1992 ; Fischetti 2001 ).…”
The misuse and overuse of antibiotics have contributed to a rapid emergence of antibiotic-resistant bacterial pathogens. This global health threat underlines the urgent need for innovative and novel antimicrobials. Endolysins derived from bacteriophages or prophages constitute promising new antimicrobials (so-called enzybiotics), exhibiting the ability to break down bacterial peptidoglycan (PG). In the present work, metagenomic analysis of soil samples, collected from thermal springs, allowed the identification of a prophage-derived endolysin that belongs to the N-acetylmuramoyl-L-alanine amidase type 2 (NALAA-2) family and possesses a LysM (lysin motif) region as a cell wall binding domain (CWBD). The enzyme (Ami1) was cloned and expressed in Escherichia coli, and its bactericidal and lytic activity was characterized. The results indicate that Ami1 exhibits strong bactericidal and antimicrobial activity against a broad range of bacterial pathogens, as well as against isolated peptidoglycan (PG). Among the examined bacterial pathogens, Ami1 showed highest bactericidal activity against Staphylococcus aureus sand Staphylococcus epidermidis cells. Thermostability analysis revealed a melting temperature of 64.2 ± 0.6 °C. Overall, these findings support the potential that Ami1, as a broad spectrum antimicrobial agent, could be further assessed as enzybiotic for the effective treatment of bacterial infections.
Key points
• Metagenomic analysis allowed the identification of a novel prophage endolysin
• The endolysin belongs to type 2 amidase family with lysin motif region
• The endolysin displays high thermostability and broad bactericidal spectrum
“…The acquired results suggest an efficient bactericidal activity and allowed the determination of the minimum bactericidal concentration (MBC) and the half-maximal lethal concentration (LC 50 ) of Ami1 against S. aureus , S. epidermidis , and E. coli DH5a, after 1.30 h, 3 h, and 6 h of incubation (Table 1 ). The bactericidal activity of Ami1 falls within the range expected for highly active endolysins that have been recently reported (Guo et al 2017 ; Wu et al 2018 ; Plotka et al 2019 ; Varotsou et al 2023 ; Premetis et al 2023b ). Fig.…”
Section: Resultssupporting
confidence: 81%
“…Towards this direction, the utilization of enzybiotics, a specific class of enzymes that exhibit the potency to treat bacterial infections (Labrou 2019 ; Song et al 2020 ), is a very promising and innovative approach (Harhala et al 2021 ; Varotsou et al 2023 ). The antimicrobial potential of enzybiotics lies heavily on their ability to hydrolyze PG, a fundamental structural element of the bacterial cell wall, causing cell lysis and therefore its death (Young 1992 ; Fischetti 2001 ).…”
The misuse and overuse of antibiotics have contributed to a rapid emergence of antibiotic-resistant bacterial pathogens. This global health threat underlines the urgent need for innovative and novel antimicrobials. Endolysins derived from bacteriophages or prophages constitute promising new antimicrobials (so-called enzybiotics), exhibiting the ability to break down bacterial peptidoglycan (PG). In the present work, metagenomic analysis of soil samples, collected from thermal springs, allowed the identification of a prophage-derived endolysin that belongs to the N-acetylmuramoyl-L-alanine amidase type 2 (NALAA-2) family and possesses a LysM (lysin motif) region as a cell wall binding domain (CWBD). The enzyme (Ami1) was cloned and expressed in Escherichia coli, and its bactericidal and lytic activity was characterized. The results indicate that Ami1 exhibits strong bactericidal and antimicrobial activity against a broad range of bacterial pathogens, as well as against isolated peptidoglycan (PG). Among the examined bacterial pathogens, Ami1 showed highest bactericidal activity against Staphylococcus aureus sand Staphylococcus epidermidis cells. Thermostability analysis revealed a melting temperature of 64.2 ± 0.6 °C. Overall, these findings support the potential that Ami1, as a broad spectrum antimicrobial agent, could be further assessed as enzybiotic for the effective treatment of bacterial infections.
Key points
• Metagenomic analysis allowed the identification of a novel prophage endolysin
• The endolysin belongs to type 2 amidase family with lysin motif region
• The endolysin displays high thermostability and broad bactericidal spectrum
“…This observation has analogy to our recent study in which the phage PAP 1-1 also showed lytic activity against C. acnes in a similar dose dependent way 28 . Varotsou et al 50 have also recently reported dose dependent effect of recombinant endolysin originated from a C. acnes phage against several bacterial species. Our further literature research for use of recombinant endolysin against C. acnes failed to yield any other reports.…”
The bacteriophage CAP 10-3 forming plaques against Cutibacterium acnes which causes skin acne was previously isolated from human skin acne lesion. Incomplete whole genome sequence (WGS) of the bacteriophage CAP 10-3 was obtained and it had 29,643 bp long nucleotide with 53.86% GC content. The sequence was similar to C. acnes phage PAP 1-1 with a nucleotide sequence identity of 89.63% and the bacteriophage belonged to Pahexavirus. Bioinformatic analysis of the WGS predicted 147 ORFs and functions of 40 CDSs were identified. The predicted endolysin gene of bacteriophage CAP 10-3 was 858 bp long which was deduced as 285 amino acids (~ 31 kDa). The protein had the highest similarity with amino acid sequence of the endolysin from Propionibacterium phage PHL071N05 with 97.20% identity. The CAP 10-3 endolysin gene was amplified by PCR with primer pairs based on the gene sequence, cloned into an expression vector pET-15b and transformed into Escherichia coli BL21(DE3) strain. The predicted protein band (~ 33 kDa) for the recombinant endolysin was detected in an SDS-PAGE gel and western blot assay. The concentrated supernatant of cell lysate from E. coli BL21(DE3) (pET-15b_CAP10-3 end) and a partially purified recombinant CAP 10-3 endolysin showed antibacterial activity against C. acnes KCTC 3314 in a dose-dependent manner. In conclusion, the recombinant CAP 10-3 endolysin was successfully produced in E. coli strain and it can be considered as a therapeutic agent candidate for treatment of human skin acne.
“…For instance, Islam et al (2022) demonstrated significant enhancement in the bactericidal activity of the chimeric endolysin against multidrug-resistant A. baumannii isolates by fusing cecropin A, a commonly used antimicrobial peptide, with the N-terminus of Ab endolysin. Similarly, PaAmi1, an amidase from Propionibacterium bacteriophage PAC1, exhibited an expanded lytic spectrum when fused with two antimicrobial peptides ( Varotsou et al, 2023 ). In our study, we opted to fuse endolysins with two antimicrobial peptides derived from ApoE, COG133, and ApoE23.…”
The rise of antimicrobial resistance in ESKAPEE pathogens poses significant clinical challenges, especially in polymicrobial infections. Bacteriophage-derived endolysins offer promise in combating this crisis, but face practical hurdles. Our study focuses on engineering endolysins from a Klebsiella pneumoniae phage, fusing them with ApoE23 and COG133 peptides. We assessed the resulting chimeric proteins’ bactericidal activity against ESKAPEE pathogens in vitro. ApoE23-Kp84B (CHU-1) reduced over 3 log units of CFU for A. baumannii, E. faecalis, K. pneumoniae within 1 h, while COG133-Kp84B (CHU-2) showed significant efficacy against S. aureus. COG133-L1-Kp84B, with a GS linker insertion in CHU-2, exhibited outstanding bactericidal activity against E. cloacae and P. aeruginosa. Scanning electron microscopy revealed alterations in bacterial morphology after treatment with engineered endolysins. Notably, CHU-1 demonstrated promising anti-biofilm and anti-persister cell activity against A. baumannii and E. faecalis but had limited efficacy in a bacteremia mouse model of their coinfection. Our findings advance the field of endolysin engineering, facilitating the customization of these proteins to target specific bacterial pathogens. This approach holds promise for the development of personalized therapies tailored to combat ESKAPEE infections effectively.
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