On the Occurrence and Multimerization of Two-Polypeptide Phage Endolysins Encoded in Single Genes

Pinto, Daniela; Gonçalo, Raquel; Louro, Mariana; Silva, Marta Sousa; Hernandez, Guillem; Cordeiro, Tiago N.; Cordeiro, Carlos; São-José, Carlos

doi:10.1128/spectrum.01037-22

Cited by 5 publications

(15 citation statements)

References 36 publications

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“…LysP108 features only an amidase attached to a C-terminal SH3b domain. LysSP15-1 has been predicted to also feature an internal translational start site akin to Ply2638A (Pinto et al 2022). (B) Molecular surfaces of all four amidase domains colored according to their electrostatic surface potential generated by APBS (±5 kT/e); red, negatively charged; white, neutral; and blue, positively charged regions.…”

Section: Resultsmentioning

confidence: 99%

“…Endolysins targeting Gram-positive bacteria frequently possess in-frame, internal translational start sites (iTSSs), resulting in the expression of two endolysin isoforms (Pinto et al 2022). These iTSS-harboring endolysins can be categorized into two types: Type I, which have an iTSS between two EADs, leading to the production of an enzymatic short variant (SV) isoform, consisting of a single EAD and C-terminal CBD, along with the full-length (FL) endolysin (Catalao et al 2011, Abaev et al 2013); and Type II, which have an iTSS between the last EAD and CBD, resulting in the co-expression of the CBD alone alongside the FL endolysin (Proenca et al 2015, Dunne et al 2016, Zhou et al 2020).…”

Section: Introductionmentioning

confidence: 99%

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Heterodimerization of Endolysin Isoforms During Bacterial Infection by Staphylococcal Phage φ2638A

Zinsli,

Sobieraj,

Ernst

et al. 2024

Preprint

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Bacteriophage endolysins targeting Gram-positive bacteria typically feature a modular architecture of one or more enzymatically active domains (EADs) and cell wall binding domains (CBDs). Several endolysins also feature internal translational start sites (iTSSs) that produce short variant (SV) isoforms alongside the full-length (FL) endolysin. While the lytic activity of endolysins and their isoforms has been extensively studied as exogenous agents, the purpose behind producing the SV isoform during the phage infection cycle remains to be explored. In this study, we used staphylococcal phage φ2638A as a model to determine the interplay between its full-length endolysin, Ply2638A, and its SV isoform during phage infection. X-ray crystallography structures and AlphaFold-generated models enabled elucidation of individual functions of the M23 endopeptidase, central amidase, and SH3b domains of Ply2638A. Production of the SV isoform (amidase and SH3b) was confirmed during phage infection and shown to form a heterodimer complex with Ply2638A via inter-amidase domain interactions. Using genetically engineered phage variants, we show that production of both isoforms provides an advantage during phage infection as phages producing only one isoform presented impaired lytic activity, which could be partly restored through recombinant protein complementation of the missing isoform. Importantly, when applied as an antimicrobial protein againstStaphylococcus aureusin culture, the activity of Ply2638A remained constant regardless of SV isoform complementation. Drawing from our findings, we propose that SV isoform production provides its biological advantage upon endolysin entry to the periplasmic space to ensure optimal peptidoglycan degradation prior to cell wall lysis and progeny phage release.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Heterodimerization of Endolysin Isoforms During Bacterial Infection by Staphylococcal Phage φ2638A

Zinsli,

Sobieraj,

Ernst

et al. 2024

Preprint

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“…The absence of LysP2 activity could be related to a putative multimeric nature of the enzyme. Whereas many endolysins produced by phages that infect Gram positive bacteria are monomeric, some require the assembly of additional domains which are synthesized from internal translational start sites within the endolysin gene, a situation more common than previously anticipated (Pinto et al, 2022). Inspection of the E. coli codon-optimized lysP2 gene revealed the presence of an internal ATG start codon but the consensus ribosome binding site had been lost (see Supplementary Table S1).…”

Section: Discussionmentioning

confidence: 99%

Cell wall modifications that alter the exolytic activity of lactococcal phage endolysins have little impact on phage growth

Escobedo

Pipaon²,

Rendueles³

et al. 2023

Front. Microbiol.

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Bacteriophages are a nuisance in the production of fermented dairy products driven by starter bacteria and strategies to reduce the risk of phage infection are permanently sought. Bearing in mind that the bacterial cell wall plays a pivotal role in host recognition and lysis, our goal was to elucidate to which extent modifications in the cell wall may alter endolysin activity and influence the outcome of phage infection in Lactococcus. Three lactococcal endolysins with distinct catalytic domains (CHAP, amidase and lysozyme) from phages 1,358, p2 and c2 respectively, were purified and their exolytic activity was tested against lactococcal mutants either overexpressing or lacking genes involved in the cell envelope stress (CES) response or in modifying peptidoglycan (PG) composition. After recombinant production in E. coli, Lys1358 (CHAP) and LysC2 (muramidase) were able to lyse lactococcal cells in turbidity reduction assays, but no activity of LysP2 was detected. The degree of PG acetylation, namely C6-O-acetylation and de-N-acetylation influenced the exolytic activity, being LysC2 more active against cells depleted of the PG deacetylase PgdA and the O-acetyl transferase OatA. On the contrary, both endolysins showed reduced activity on cells with an induced CES response. By measuring several growth parameters of phage c2 on these lactococcal mutants (lytic score, efficiency of plaquing, plaque size and one-step curves), a direct link between the exolytic activity of its endolysin and phage performance could not be stablished.

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“…Likewise, a truncated version of the staphylococcal phage 2638A endolysin ORF007 containing one of two CDs and a CWBD forms a heterodimer with the full-length protein . Lastly, Pinto et al recently performed a bioinformatics search of iTSS sites in putative endolysins and experimentally confirmed at least four new multimeric endolysins, including LysP7951, which adopts a 1:5 CD:CWBD stoichiometry . All of the above enzymes fold into their multimeric state at the level of protein translation.…”

Section: Discussionmentioning

confidence: 99%

“…65 Lastly, Pinto et al recently performed a bioinformatics search of iTSS sites in putative endolysins and experimentally confirmed at least four new multimeric endolysins, including LysP7951, which adopts a 1:5 CD:CWBD stoichiometry. 66 All of the above enzymes fold into their multimeric state at the level of protein translation. Only the pneumococcal enzymes (i.e., Cpl-1, Pal, and LytA) are produced and purified as monomers and require binding to a ligand mimetic of epitopes on the bacterial surface to induce multimerization.…”

Section: ■ Discussionmentioning

confidence: 99%

Understanding the Molecular Basis for Homodimer Formation of the Pneumococcal Endolysin Cpl-1

et al. 2023

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The rise of multi-drug-resistant bacteria that cannot be treated with traditional antibiotics has prompted the search for alternatives to combat bacterial infections. Endolysins, which are bacteriophage-derived peptidoglycan hydrolases, are attractive tools in this fight. Several studies have already demonstrated the efficacy of endolysins in targeting bacterial infections. Endolysins encoded by bacteriophages that infect Gram-positive bacteria typically possess an N-terminal catalytic domain and a C-terminal cell-wall binding domain (CWBD). In this study, we have uncovered the molecular mechanisms that underlie formation of a homodimer of Cpl-1, an endolysin that targets Streptococcus pneumoniae. Here, we use site-directed mutagenesis, analytical size exclusion chromatography, and analytical ultracentrifugation to disprove a previous suggestion that three residues at the N-terminus of the CWBD are involved in the formation of a Cpl-1 dimer in the presence of choline in solution. We conclusively show that the C-terminal tail region of Cpl-1 is involved in formation of the dimer. Alanine scanning mutagenesis generated various tail mutant constructs that allowed identification of key residues that mediate Cpl-1 dimer formation. Finally, our results allowed identification of a consensus sequence (FxxEPDGLIT) required for choline-dependent dimer formationa sequence that occurs frequently in pneumococcal autolysins and endolysins. These findings shed light on the mechanisms of Cpl-1 and related enzymes and can be used to inform future engineering efforts for their therapeutic development against S. pneumoniae.

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On the Occurrence and Multimerization of Two-Polypeptide Phage Endolysins Encoded in Single Genes

Cited by 5 publications

References 36 publications

Heterodimerization of Endolysin Isoforms During Bacterial Infection by Staphylococcal Phage φ2638A

Heterodimerization of Endolysin Isoforms During Bacterial Infection by Staphylococcal Phage φ2638A

Cell wall modifications that alter the exolytic activity of lactococcal phage endolysins have little impact on phage growth

Understanding the Molecular Basis for Homodimer Formation of the Pneumococcal Endolysin Cpl-1

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