Characterization and Engineering Studies of a New Endolysin from the <i>Propionibacterium acnes</i> Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity
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) fr...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-05-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/10/8523 |
| Summary: | 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 <i>Propionibacterium</i> bacteriophage PAC1 was characterized. The enzyme (<i>Pa</i>Ami1) was cloned into a T7 expression vector and expressed in <i>E. coli</i> BL21 cells. Kinetics analysis using turbidity reduction assays allowed the determination of the optimal conditions for lytic activity against a range of Gram-positive and negative human pathogens. The peptidoglycan degradation activity of <i>Pa</i>Ami1 was confirmed using isolated peptidoglycan from <i>P. acnes</i>. The antibacterial activity of <i>Pa</i>Ami1 was investigated using live <i>P. acnes</i> cells growing on agar plates. Two engineered variants of <i>Pa</i>Ami1 were designed by fusion to its N-terminus two short antimicrobial peptides (AMPs). One AMP was selected by searching the genomes of <i>Propionibacterium</i> bacteriophages using bioinformatics tools, whereas the other AMP sequence was selected from the antimicrobial peptide databases. Both engineered variants exhibited improved lytic activity towards <i>P. acnes</i> and the enterococci species <i>Enterococcus faecalis</i> and <i>Enterococcus faecium</i>. The results of the present study suggest that <i>Pa</i>Ami1 is a new antimicrobial agent and provide proof of concept that bacteriophage genomes are a rich source of AMP sequences that can be further exploited for designing novel or improved endolysins. |
|---|---|
| ISSN: | 1661-6596 1422-0067 |