High avidity drives the interaction between the streptococcal C1 phage endolysin, PlyC, with the cell surface carbohydrates of Group A <i>Streptococcus</i>

Broendum, Sebastian S.; Williams, Daniel E.; Hayes, Brooke K.; Kraus, Felix; Fodor, James; Clifton, Ben E.; Volbeda, Anne; Codée, Jeroen D. C.; Riley, Blake T.; Drinkwater, N.; Farrow, Kylie A; Tsyganov, Kirill; Heselpoth, Ryan D.; Nelson, Daniel; Jackson, C.J.; Buckle, Ashley M.; McGowan, Sheena

doi:10.1111/mmi.14719

Cited by 10 publications

(19 citation statements)

References 69 publications

(128 reference statements)

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“…First, given that Cpl-1 is predicted to possess five choline binding sites, an avidity effect may increase the apparent low affinity for the pneumococcal surface . A similar hypothesis has been developed for PlyCB, the CWBD of the PlyC endolysin that contains eight binding sites . A second, and even more likely hypothesis, is that other components of the teichoic acid or moieties on the peptidoglycan itself may be necessary for high-affinity binding, as suggested recently for other pneumococcal choline-binding proteins …”

Section: Discussionmentioning

confidence: 72%

“…57 A similar hypothesis has been developed for PlyCB, the CWBD of the PlyC endolysin that contains eight binding sites. 58 A second, and even more likely hypothesis, is that other components of the teichoic acid or moieties on the peptidoglycan itself may be necessary for highaffinity binding, as suggested recently for other pneumococcal choline-binding proteins. 59 There is growing evidence that endolysins and other cell wall hydrolases form multimeric structures more often than previously thought.…”

Section: ■ Discussionmentioning

confidence: 89%

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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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Section: Discussionmentioning

confidence: 72%

Section: ■ Discussionmentioning

confidence: 89%

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

et al. 2023

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“…Our leveraging of a multi-pronged strategy was critical for overcoming technical obstacles inherent to proteins that interact with complex, insoluble molecules, providing access to a class of macromolecular interactions that are not well understood. The biophysical and kinetic parameters of proteins with three-dimensional highly-crosslinked macromolecular substrates 34,35 are dissimilar from those well-established for small, soluble substrates, and future adoption of combinatorial approaches such as ours may improve our understanding of general binding principles across these important enzymes that are ubiquitous across life. Additionally, the ability to quickly generate rich quantitative heatmaps for enzymes across different experimental conditions enables more sophisticated computational analyses.…”

Section: Discussionmentioning

confidence: 99%

Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context

Radkov

Sapiro

Flores

et al. 2022

Preprint

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Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

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“…All domains connect by linker regions, and both the GyH and CHAP domains are enzymatically active. PlyCB forms an octameric ring that interacts with PlyCA via its helical docking domain [ 12 , 13 ]. Here, we sought to understand immunogenicity of PlyC, focusing on the PlyC regions/domains that are targeted by specific IgG antibodies.…”

Section: Introductionmentioning

confidence: 99%

Immunogenicity of Endolysin PlyC

et al. 2022

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Endolysins are bacteriolytic enzymes derived from bacteriophages. They represent an alternative to antibiotics, since they are not susceptible to conventional antimicrobial resistance mechanisms. Since non-human proteins are efficient inducers of specific immune responses, including the IgG response or the development of an allergic response mediated by IgE, we evaluated the general immunogenicity of the highly active antibacterial enzyme, PlyC, in a human population and in a mouse model. The study includes the identification of molecular epitopes of PlyC. The overall assessment of potential hypersensitivity to this protein and PlyC-specific IgE testing was also conducted in mice. PlyC induced efficient IgG production in mice, and the molecular analysis revealed that PlyC-specific IgG interacted with four immunogenic regions identified within the PlyCA subunit. In humans, approximately 10% of the population demonstrated IgG reactivity to the PlyCB subunit only, which is attributed to cross-reactions since this was a naïve serum. Of note, in spite of being immunogenic, PlyC induced a normal immune response, without hypersensitivity, since both the animals challenged with PlyC and in the human population PlyC-specific IgE was not detected.

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High avidity drives the interaction between the streptococcal C1 phage endolysin, PlyC, with the cell surface carbohydrates of Group A Streptococcus

Cited by 10 publications

References 69 publications

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

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

Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context

Immunogenicity of Endolysin PlyC

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