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Magnus Hook - One of the best experts on this subject based on the ideXlab platform.
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staphylococcus epidermidis MSCRAMM sesj is encoded in composite islands
Mbio, 2020Co-Authors: Srishtee Arora, Andrew Hillhouse, Kranti Konganti, Sara V. Little, Sara D. Lawhon, David W Threadgill, Samuel A Shelburne, Magnus HookAbstract:ABSTRACT Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates. IMPORTANCES. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.
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Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands
'American Society for Microbiology', 2020Co-Authors: Srishtee Arora, Andrew Hillhouse, Kranti Konganti, Sara V. Little, Sara D. Lawhon, David Threadgill, Samuel Shelburne, Magnus HookAbstract:S. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates
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Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody.
EBioMedicine, 2016Co-Authors: Vannakambadi K Ganesh, Xiaowen Liang, Timothy J Foster, Joan A Geoghegan, Ana Luisa V. Cohen, Nagarajan Venugopalan, Magnus HookAbstract:Abstract The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the "top" of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC 50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.
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a novel MSCRAMM subfamily in coagulase negative staphylococcal species
Frontiers in Microbiology, 2016Co-Authors: Srishtee Arora, Annecatrin Uhlemann, Franklin D Lowy, Magnus HookAbstract:Coagulase negative staphylococci are important opportunistic pathogens. Staphylococcus epidermidis, a coagulase negative staphylococcus, is the third leading cause of nosocomial infections in the US. Surface proteins like Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are major virulence factors of pathogenic gram positive bacteria. Here, we identified a new chimeric protein; SesJ in S. epidermidis, which represents a prototype of a new subfamily of MSCRAMMs. Structural predictions show that SesJ has structural features characteristic of a MSCRAMM along with a N-Terminal repeat region and an Aspartic acid containing repeat region, features that have not been previously observed in staphylococcal MSCRAMMs but have been found in other surface proteins from gram positive bacteria. We identified and analyzed structural homologs of SesJ in three other coagulase negative staphylococci. These homologs of SesJ have an identical structural organization but varying sequence identities within the domains. Using flow cytometry, we also show that SesJ is expressed constitutively on the surface of a representative S. epidermidis strain, from early exponential to stationary growth phase. Thus SesJ is positioned to interact with protein targets in the environment and play a role in S. epidermidis virulence.
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Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs
2016Co-Authors: Xiaowen Liang, Livia Visai, Brandon L. Garcia, Sabitha Prabhakaran, Nicola A. G. Meenan, Jennifer R. Potts, Martin J. Humphries, Magnus HookAbstract:Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5β1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5β1integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5β1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5β1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5β1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5β1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs.
Timothy J Foster - One of the best experts on this subject based on the ideXlab platform.
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the MSCRAMM family of cell wall anchored surface proteins of gram positive cocci
Trends in Microbiology, 2019Co-Authors: Timothy J FosterAbstract:The microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are a family of proteins that are defined by the presence of two adjacent IgG-like folded subdomains. These promote binding to ligands by mechanisms that involve major conformational changes exemplified by the binding to fibrinogen by the ‘dock-lock-latch’ mechanism or to collagen by the ‘collagen hug’. Clumping factors A and B are two such MSCRAMMs that have several important roles in the pathogenesis of Staphylococcus aureus infections. MSCRAMM architecture, ligand binding, and roles in infection and colonization are examined with a focus on recent developments with clumping factors.
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b-Neurexin Is a Ligand for the Staphylococcus aureus
2016Co-Authors: Mscramm Sdrc, Timothy J Foster, Vannakambadi K Ganesh, Magda E Barbu, Chris R Mackenzie, Thomas C SudhofAbstract:Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in b-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the b-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing b-neurexin on their surface. Taken together, our experimental results demonstrate that b-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N2N3 domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possibl
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Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody.
EBioMedicine, 2016Co-Authors: Vannakambadi K Ganesh, Xiaowen Liang, Timothy J Foster, Joan A Geoghegan, Ana Luisa V. Cohen, Nagarajan Venugopalan, Magnus HookAbstract:Abstract The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the "top" of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC 50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.
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a short sequence within subdomain n1 of region a of the staphylococcus aureus MSCRAMM clumping factor a is required for export and surface display
Microbiology, 2014Co-Authors: Niamh Mccormack, Timothy J Foster, Joan A GeogheganAbstract:Clumping factor A (ClfA) is the archetypal fibrinogen-binding surface protein of Staphylococcus aureus and a member of the microbial surface component recognizing adhesive matrix molecules (MSCRAMM) family. An N-terminal signal sequence directs export of the MSCRAMM by the Sec pathway and the C-terminal cell wall-anchoring domain allows covalent attachment of ClfA to peptidoglycan by sortase. Region A of ClfA comprises three independently folded subdomains N1, N2 and N3. Subdomains N2N3 comprise IgG-like folds and promote fibrinogen binding. Nothing is known about the structure or function of subdomain N1. Here we demonstrate an unexpected role for N1 in the export and surface localization of ClfA. Attempted expression of a ClfA variant lacking subdomain N1 resulted in impaired growth of S. aureus and accumulation of ClfA protein in the cytoplasm and cytoplasmic membrane. The presence of residues 211–228 of N1 was required to allow display of ClfA on the bacterial surface. The importance of this region was confirmed when a ClfA variant lacking residues 211–220 was also mislocalized to the cytoplasm and cytoplasmic membrane. However, these residues were not required for export of ClfA lacking the Ser-Asp repeats that link region A to the wall-anchoring domain. Similarly, subdomain N1 of a related MSCRAMM fibronectin-binding protein B was required for export and surface display of the full-length protein, but not a derivative lacking fibronectin-binding repeats. In summary, we demonstrate that residues in the N1 subdomain are required for export and cell wall localization of S. aureus MSCRAMM proteins.
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adhesion invasion and evasion the many functions of the surface proteins of staphylococcus aureus
Nature Reviews Microbiology, 2014Co-Authors: Timothy J Foster, Joan A Geoghegan, Vannakambadi K Ganesh, Magnus HookAbstract:Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is 'decorated' with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections.
Joseph M Patti - One of the best experts on this subject based on the ideXlab platform.
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monoclonal antibodies recognizing the enterococcus faecalis collagen binding MSCRAMM ace conditional expression and binding analysis
Microbial Pathogenesis, 2007Co-Authors: Andrea Hall, Joseph M Patti, Brenda Ames, Elena Gorovits, Peter J. Syribeys, John Vernachio, Paul Domanski, Cathy Y Chang, Jeff T. HutchinsAbstract:Enterococci are opportunistic pathogens known to cause numerous clinical infections and complications in humans. Adhesin-mediated binding to extracellular matrix (ECM) proteins of the host is thought to be a crucial step in the pathogenesis of these bacterial infections. Adhesin of collagen from Enterococcus faecalis (Ace) is a cell-wall anchored protein of E. faecalis that has been shown to be important for bacterial binding to the ECM. In this report, we characterize the conditions for Ace expression and demonstrate Ace binding to mammalian epithelial and endothelial cells as well as to collagens found in the ECM. To further characterize Ace expression and function, we report the generation of a panel of monoclonal antibodies (mAbs) directed against this important E. faecalis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance and flow cytometry, we have characterized this panel of mAbs which may prove to be not only beneficial in studies that address the precise biological role of adhesion of E. faecalis, but may also serve as beneficial therapeutic agents against E. faecalis infections.
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a panel of monoclonal antibodies recognizing the staphylococcus epidermidis fibrinogen binding MSCRAMM sdrg
Hybridoma, 2007Co-Authors: Andrea Hall, Joseph M Patti, Elena Gorovits, Bradley D. Prater, Peter J. Syribeys, John Vernachio, Paul Domanski, Pratisksha Patel, Jeff T. HutchinsAbstract:Staphylococcus epidermidis is an important opportunistic human pathogen that has recently emerged as a major cause of foreign-body infections. The most important stage contributing to the pathogenesis of this bacteria is the initial adherence to host tissue. SdrG is a cell-wall–anchored fibrinogen-binding adhesin of S. epidermidis that has been shown to be necessary for bacterial binding to fibrinogen-coated foreign bodies, such as catheters. Here we report the generation and characterization of a panel of monoclonal antibodies (MAbs) directed against this S. epidermidis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance, and flow cytometry, we have characterized a diverse array of MAbs that may prove to be beneficial in studies that address the precise biologic role of SdrG.
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human immunoglobulin g recognizing fibrinogen binding surface proteins is protective against both staphylococcus aureus and staphylococcus epidermidis infections in vivo
Antimicrobial Agents and Chemotherapy, 2006Co-Authors: John Vernachio, Brenda Ames, Elena Gorovits, Bradley D. Prater, Peter J. Syribeys, Arnold S. Bayer, Dawn Bryant, Joseph M PattiAbstract:A human donor-selected immunoglobulin G for intravenous injection (IGIV) product with elevated titers against the staphylococcal fibrinogen-binding MSCRAMM proteins ClfA and SdrG (INH-A21) was tested in vitro and in vivo. INH-A21 contained a significantly increased ability to inhibit the fibrinogen-binding activity of recombinant forms of both ClfA and SdrG. Evaluation of the opsonizing potential of INH-A21 was evaluated using fluorescently labeled bacteria; this assay indicated an increase in phagocytic activity compared to normal IGIV. The prophylactic efficacy of INH-A21 against an intraperitoneal challenge of methicillin-resistant Staphylococcus epidermidis (MRSE) was evaluated in a neonatal rat model. INH-A21 was also evaluated for prophylactic and therapeutic efficacy in a rabbit model of catheter-induced aortic valve infective endocarditis caused by either MRSE or methicillin-resistant Staphylococcus aureus (MRSA). Results from the in vivo models demonstrated potent prophylactic and therapeutic efficacy against both MRSE and MRSA. These data suggest that INH-A21 may be an important tool for the prevention and treatment of staphylococcal infections, especially in high-risk populations.
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MSCRAMM targeted vaccines and immunotherapy for staphylococcal infection
Current Opinion in Drug Discovery & Development, 2004Co-Authors: Jorge M Rivas, Pietro Speziale, Joseph M Patti, Magnus HookAbstract:Hospital-acquired infections are associated with prolonged hospitalization and an increase in both healthcare costs and resources. Advances in sophisticated medical procedures, an increase in the number of immunocompromised patients, and the continued emergence of resistance to conventional antibiotic therapy has created a need for alternative strategies to prevent and treat infectious bacterial diseases. Immunoprevention and immunotherapy targeting microbial surface components recognizing adhesive matrix molecule (MSCRAMM) proteins are viable approaches to potentially impede bacterial adherence, eliminate colonization, and minimize hematogenous dissemination, thereby halting the inception and progression of infection. This review summarizes several investigative efforts where staphylococcal MSCRAMM proteins are being utilized in the design of subunit vaccines and in the development of innovative therapeutic strategies that could be implemented following the onset of infection to manage severe and life-threatening disease.
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SdrX, a Serine-Aspartate Repeat Protein Expressed by Staphylococcus capitis with Collagen VI Binding Activity
Infection and immunity, 2004Co-Authors: Yule Liu, Brenda Ames, Elena Gorovits, Bradley D. Prater, Peter J. Syribeys, John Vernachio, Joseph M PattiAbstract:Staphylococcus capitis (S. capitis) has been implicated in a large proportion of coagulase-negative staphylococcal infections in very-low-birth-weight infants. To identify potential therapeutic targets, the S. capitis genome was probed for the presence of genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM). By using Southern blot analysis, an S. capitis gene, designated sdrX, that contained sequence motifs consistent with the Sdr family of MSCRAMM proteins was identified. By using monospecific antisera in Western blot and flow cytometry, SdrX was demonstrated to be expressed on the surface of S. capitis. Human collagen type VI was found to bind both the recombinant A domain of SdrX and viable S. capitis expressing SdrX. SdrX is the first collagen-binding Sdr protein described and is the first MSCRAMM protein identified in S. capitis.
Sthanam V L Narayana - One of the best experts on this subject based on the ideXlab platform.
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evidence for the dock lock and latch ligand binding mechanism of the staphylococcal microbial surface component recognizing adhesive matrix molecules MSCRAMM sdrg
Journal of Biological Chemistry, 2008Co-Authors: Gabriela M. Bowden, Sthanam V L Narayana, Karthe Ponnuraj, Sivashankarappa Gurusiddappa, Alejandro P Heuck, Elena Kolosova, Damon Choe, Arthur E Johnson, Magnus HookAbstract:Next Section Abstract Staphylococcus epidermidis is an opportunistic pathogen and a major cause of foreign body infections. The S. epidermidis fibrinogen (Fg)-binding adhesin SdrG is necessary and sufficient for the attachment of this pathogen to Fg-coated materials. Based largely on structural analyses of the ligand binding domain of SdrG as an apo-protein and in complex with a Fg-like peptide, we proposed that SdrG follows a “dock, lock, and latch” mechanism to bind to Fg. This binding mechanism involves the docking of the ligand in a pocket formed between two SdrG subdomains followed by the movement of a C-terminal extension of one subdomain to cover the ligand and to insert and complement a β-sheet in a neighboring subdomain. These proposed events result in a greatly stabilized closed conformation of the MSCRAMM-ligand complex. In this report, we describe a biochemical analysis of the proposed conformational changes that SdrG undergoes upon binding to its ligand. We have introduced disulfide bonds into SdrG to stabilize the open and closed forms of the apo-form of the MSCRAMM. We show that the stabilized closed form does not bind to the ligand and that binding can be restored in the presence of reducing agents such as dithiothreitol. We have also used Forster resonance energy transfer to dynamically show the conformational changes of SdrG upon binding to its ligand. Finally, we have used isothermic calorimetry to determine that hydrophobic interactions between the ligand and the protein are responsible for re-directing the C-terminal extension of the second subdomain required for triggering the β-strand complementation event.
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the enterococcus faecalis MSCRAMM ace binds its ligand by the collagen hug model
Journal of Biological Chemistry, 2007Co-Authors: Qing Liu, Vannakambadi K Ganesh, Sthanam V L Narayana, Karthe Ponnuraj, Barbara E Murray, Jouko Sillanpaa, Magnus HookAbstract:We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). This segment is composed of two subdomains, N1 and N2, each adopting an IgG-like fold and forming a putative collagen binding surface at the interface between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed. We suggest that ACE binds collagen by a similar mechanism and present the first biochemical evidence for this binding model. Replacing residues in the putative collagen binding trench of ACE N2 with Ala residues affected collagen binding. A closed conformation of ACE stabilized by an engineered disulfide bond is unable to bind collagen. Finally, the importance of the residues in the N2 extension in stabilizing the MSCRAMM-ligand complex is demonstrated by selected point and truncation mutations.
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a novel variant of the immunoglobulin fold in surface adhesins of staphylococcus aureus crystal structure of the fibrinogen binding MSCRAMM clumping factor a
The EMBO Journal, 2002Co-Authors: Champion Deivanayagam, Magnus Hook, Elisabeth R Wann, Wei Chen, Mike Carson, Kanagalaghatta R Rajashankar, Sthanam V L NarayanaAbstract:We report here the crystal structure of the minimal ligand‐binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This fibrinogen‐binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE‐variant or the DEv‐IgG fold. This subgroup includes the ligand‐binding domain of the collagen‐binding S.aureus MSCRAMM CNA, and many other structures previously classified as jelly rolls. Structure predictions suggest that the four fibrinogen‐binding S.aureus MSCRAMMs identified so far would also contain the same DEv‐IgG fold. A systematic docking search using the C‐terminal region of the fibrinogen γ‐chain as a probe suggested that a hydrophobic pocket formed between the two DEv‐IgG domains of the clumping factor as the ligand‐binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues 408 AGDV 411 of the γ‐chain, resulted in proteins with no or markedly reduced affinity for fibrinogen.
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crystallization and preliminary x ray crystallographic analysis of ace a collagen binding MSCRAMM from enterococcus faecalis
Biochimica et Biophysica Acta, 2002Co-Authors: Karthe Ponnuraj, Magnus Hook, Champion Deivanayagam, D Moore, Lluis Boque, Sthanam V L NarayanaAbstract:Abstract Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3121 or P3221 with unit cell parameters a=b=80.24, c=105.91 A; α=β=90 and γ=120°. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.
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structural organization of the fibrinogen binding region of the clumping factor b MSCRAMM of staphylococcus aureus
Journal of Biological Chemistry, 2001Co-Authors: Samuel E Perkins, Evelyn J Walsh, Timothy J Foster, Champion Deivanayagam, Sthanam V L Narayana, Magnus HookAbstract:The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the approximately 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta-sheets with little or no discernible alpha-helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain N1 appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.
Vannakambadi K Ganesh - One of the best experts on this subject based on the ideXlab platform.
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b-Neurexin Is a Ligand for the Staphylococcus aureus
2016Co-Authors: Mscramm Sdrc, Timothy J Foster, Vannakambadi K Ganesh, Magda E Barbu, Chris R Mackenzie, Thomas C SudhofAbstract:Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in b-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the b-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing b-neurexin on their surface. Taken together, our experimental results demonstrate that b-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N2N3 domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possibl
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Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody.
EBioMedicine, 2016Co-Authors: Vannakambadi K Ganesh, Xiaowen Liang, Timothy J Foster, Joan A Geoghegan, Ana Luisa V. Cohen, Nagarajan Venugopalan, Magnus HookAbstract:Abstract The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the "top" of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC 50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.
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adhesion invasion and evasion the many functions of the surface proteins of staphylococcus aureus
Nature Reviews Microbiology, 2014Co-Authors: Timothy J Foster, Joan A Geoghegan, Vannakambadi K Ganesh, Magnus HookAbstract:Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is 'decorated' with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections.
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A Structural Model of the Staphylococcus aureus ClfA– Fibrinogen Interaction Opens New Avenues for the Design of Anti-Staphylococcal Therapeutics
2013Co-Authors: Vannakambadi K Ganesh, Elisabeth R Wann, Emanuel Smeds, Jose Rivera J. ¤a, Gabriela M. Bowden, Ross J. Fitzgerald, Magnus HookAbstract:The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) c-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin aIIbb3 and ClfA bind to the same segment in the Fg c-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the ‘‘Dock, Lock and Latch’ ’ mechanism previously described for the Staphylococcus epidermidis SdrG–Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not aIIbb3 may allow the design of novel antistaphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may hav
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targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins
Journal of Biological Chemistry, 2012Co-Authors: Cana L Ross, Xiaowen Liang, Magnus Hook, Qing Liu, Barbara E Murray, Vannakambadi K GaneshAbstract:The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N1 and N2 subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.