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Published on: June 12, 2019
The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity
Adriano M Gigante1, Francisco Olivença1, Maria João Catalão1
1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal.
Abstract:
Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.
Insights
The N-terminus of mycobacteriophage Ms6 LysB protein binds to peptidoglycan (PG), a crucial step for phage release. This binding interaction is essential for the proper lysis of mycobacterial cell walls during the phage lytic cycle.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophages are viruses that infect bacteria and complete their lytic cycle by lysing the host cell.
- Phage lysis involves specialized proteins, including holins and endolysins, that disrupt the bacterial cell envelope.
- Mycobacteriophages, which infect mycobacteria, possess a unique lysis protein, LysB, involved in outer membrane detachment.
Purpose of the Study:
- To investigate the function of the N-terminal region of mycobacteriophage Ms6 LysB.
- To determine if the N-terminus of Ms6 LysB interacts with peptidoglycan (PG).
- To elucidate the role of this interaction in the phage lysis process.
Main Methods:
- Construction and testing of a fusion protein (Ms6LysBPGBD-EGFP) containing the N-terminal region of Ms6 LysB and enhanced green fluorescent protein.
- Cell binding assays using various mycobacterial species and other bacteria treated with SDS or Ms6 LysB.
- Pulldown assays with purified peptidoglycan from different bacterial species (M. smegmatis, E. coli, P. aeruginosa, B. subtilis).
- Infection assays using an Ms6 mutant with a truncated LysB protein lacking the N-terminal 90 amino acids.
Main Results:
- The Ms6LysBPGBD-EGFP fusion protein demonstrated binding to mycobacterial cells pretreated with SDS or Ms6 LysB.
- Ms6 LysB and Ms6LysBPGBD-EGFP exhibited binding to purified peptidoglycan from M. smegmatis, E. coli, P. aeruginosa, and B. subtilis, specifically to the A1γ chemotype.
- An Ms6 mutant lacking the N-terminal 90 amino acids of LysB resulted in abrupt cell lysis, indicating the importance of this region.
Conclusions:
- The N-terminus of mycobacteriophage Ms6 LysB possesses a peptidoglycan-binding domain (PGBD).
- This PG-binding capability is crucial for the efficient lysis of the host cell envelope by Ms6 LysB.
- The findings contribute to understanding the diverse mechanisms of bacteriophage-mediated cell lysis.
