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The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity.

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.

SUBMITTER: Gigante AM

PROVIDER: S-EPMC8309991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNAala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNAala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNAala genes.ResultsThe three tRNAala genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNAalaU and tRNAalaV containing the attB site, a single base difference was observed between the two species. We observed that the tRNAalaU gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNAalaV gene was used as the target. No integration occurred in the BCG tRNAalaU T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNAala T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNAalaU and tRNAalaV T-loops of the same BCG chromosome.ConclusionSmall changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNAala genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies.

Project description:BackgroundThere are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N.MethodsWe used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe.ResultsBinding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased.ConclusionsThe results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.

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The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity.

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