Structural bases of peptidoglycan recognition by lysostaphin SH3b domain

Mitkowski, Paweł; Jagielska, Elżbieta; Nowak, Elżbieta; Bujnicki, Janusz; Stefaniak, Filip; Niedziałek, Dorota; Bochtler, Matthias; Sabala, I.

doi:10.1038/s41598-019-42435-z

Cited by 63 publications

(68 citation statements)

References 58 publications

(69 reference statements)

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“…a, G5 peptide; b, P4; c, P4-G5; d, P5-G5-P4; f, GM-P5-G5-GM-P4-G5. Titrations confirmed the existence of a narrow cleft previously proposed to bind the PG crossbridges 19 and recently shown to interact with pentaglycine 20 . They also revealed a set of residues interacting with the peptide stem, located on the face of the protein opposite to the G5 binding cleft.…”

Section: Figure 1 Mapping the Interaction Surface Of The Sh3b Domainsupporting

confidence: 76%

“…The specificity of lysostaphin towards staphylococci has been attributed to its binding domain, which recognizes pentaglycine crossbridges 18,19 . Recent crystallographic studies have confirmed early models and showed that the pentaglycine stem is recognized by a shallow groove formed between strands β1-β2 and the RT loop, the binding specificity being essentially conferred by steric hindrance 20 . Despite this exquisite recognition mechanism, the SH3b domain displays a very weak affinity for the pentaglycine stems and binding has been shown to be optimal with multimeric peptidoglycan fragments, suggesting a mechanism more complex than initially anticipated 20,21 .…”

Section: Introductionmentioning

confidence: 68%

“…The B-factors for the high-resolution set are higher than expected, but match that of the Wilson B, and the dataset has a normal intensity distribution. An initial model was solved using the existing apo structure 5LEO 20 as a molecular replacement model in PHASER 41 , and the corresponding structure autobuilt using PHENIX 42 , with the ligand added via visual inspection of the difference map. The structure was updated and refined using COOT 43 , PHENIX 42 and PDB-redo 44 , resulting in a final structure with an R/Rfree of 19.9%/23.0%.…”

Section: Crystallography and Structure Determinationmentioning

confidence: 99%

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Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

et al. 2019

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Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity towards staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA). Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here, we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows a synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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Section: Figure 1 Mapping the Interaction Surface Of The Sh3b Domainsupporting

confidence: 76%

Section: Introductionmentioning

confidence: 68%

Section: Crystallography and Structure Determinationmentioning

confidence: 99%

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Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

et al. 2019

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“…Lysostaphin has an SH3b cell wallbinding domain that recognizes the pentaglycine cross-bridges present exclusively in staphylococcal peptidoglycan, positioning the enzyme to cleave the cross-bridges. Within the SH3b domain resides a shallow groove that appears to sterically select for pentaglycine (112). Just this year, Gonzalez-Delgado et al (110) discovered that the SH3b domain drives not only the specificity, but also high-affinity binding, of lysostaphin toward staphylococcal peptidoglycan.…”

Section: Connecting Cell Wall Biochemistry With Biological Functionmentioning

confidence: 99%

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes

Page

Walker

2020

Journal of Biological Chemistry

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Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall–tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

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“…Other authors also reported a binding spectrum at the genus level for other CBDs from Staphylococcus endolysins 39,52,56 . Some studies suggested that CBDs recognize conserved binding ligands such as the glycine-rich inter-peptide bridge common to most staphylococcal strains [57][58][59] , as it has been reported for the SH3b-like cell wall targeting domain of lysostaphin 60 , which can explain their broad host range. Curiously, the GFP-AMI_SH3 protein demonstrated some binding affinity to two Enterococcus strains and one S. pneumoniae strain.…”

Section: Discussionmentioning

confidence: 86%

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

Costa

Dias

Melo

et al. 2020

Sci Rep

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Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1-5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs. Bloodstream infections (BSIs) are severe diseases caused by the presence of microorganisms, mainly bacteria, in blood and are characterized by high morbidity and mortality 1,2. The BSIs and associated organ dysfunctions (sepsis or septic shock) remain a life-threating disease due, partially, to the inability to rapidly detect and identify the responsible pathogens 3,4. Staphylococcus spp. are Gram-positive facultative anaerobic bacteria that frequently colonize the human body 5,6. These pathogens are becoming increasingly resistant to antibiotics and are well-established in both community and healthcare environments, being commonly isolated in intensive care units (ICU) 6,7. Staphylococcus aureus is a common cause of a variety of infections, from superficial skin infections to life-threatening diseases, including necrotizing pneumonia 8 , infective endocarditis 9 and BSIs 10. Coagulase-negative staphylococci (CoNS) have also been described as harmful to humans, causing several infections, particularly in patients with implanted medical devices 6. The empirical antibiotic therapy remains the standard of BSIs treatments 11 and its correct use within the first hour after the recognition of the BSI is recommended by the Surviving Sepsis Campaign Guidelines 11 and was reported as having a great impact on the patient survival rate 12. Nevertheless, the extensive use of broad-spectrum antibiotics and the large number of patients having negative blood culture samples and thus receiving unnecessary antibiotic treatment, are important contributors to the increase of antimicrobial resistance 13-15. Thus, sensitive, rapid, cost-efficient and specific ...

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Structural bases of peptidoglycan recognition by lysostaphin SH3b domain

Cited by 63 publications

References 58 publications

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

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