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Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire

View Article: PubMed Central - PubMed

ABSTRACT

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.

No MeSH data available.


Germline-encoded binding of IGHV4-39 to the NEAT1 domain of IsdB.(a) Crystal structure of an IGHV4-39-derived Fab (D4-10-N1) in complex with NEAT1. The two aromatic resides (Y52 and F53) in CDR-H2 interact with the α-helix1 of NEAT1 which is normally involved in binding haemoglobin. IGHV4-39 CDR-H2 F53 of Fab D4-10-N1 protrudes into a hydrophobic pocket of NEAT1, which is structurally homologous to the heme binding pocket of NEAT2. Crystallization was facilitated by the use of a sandwiching Fab from an antibody (D3–19) that binds NEAT1 at a non-overlapping epitope (bin H). For clarity, the sandwiching Fab is removed from the figure but is included in the Supplementary Data (Supplementary Fig. 10c). (b) CDR-H2 dominates the interaction in terms of BSA. Structural analysis shows that 79% of the BSA is attributed to the heavy chain, and 21% to the light chain. The CDR-H2 contributes about 45% of total BSA. (c) Mutational analysis confirms the structural data and demonstrates that all IGHV derived antibodies in this set bind NEAT1 with a similar mechanism. The KD for all antibodies in this set was determined by SPR-based biosensor binding analysis to recombinant full-length IsdB at 37 °C (KD range, n≥2). The binding of antibody variants at positions 52 and 53 of CDR-H2 to wild type IsdB and the binding of antibodies to NEAT1 variant Y165R (α-helix 1) were evaluated by ELISA (percentage binding relative to binding between original isolated antibodies and wild type IsdB, one representative set of results out of three independent experiments is shown). Every clone was reverted to VH germline sequence and tested for binding to NEAT1 by biosensor analysis and ability to block haemoglobin binding. ND stands for not determined.
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f4: Germline-encoded binding of IGHV4-39 to the NEAT1 domain of IsdB.(a) Crystal structure of an IGHV4-39-derived Fab (D4-10-N1) in complex with NEAT1. The two aromatic resides (Y52 and F53) in CDR-H2 interact with the α-helix1 of NEAT1 which is normally involved in binding haemoglobin. IGHV4-39 CDR-H2 F53 of Fab D4-10-N1 protrudes into a hydrophobic pocket of NEAT1, which is structurally homologous to the heme binding pocket of NEAT2. Crystallization was facilitated by the use of a sandwiching Fab from an antibody (D3–19) that binds NEAT1 at a non-overlapping epitope (bin H). For clarity, the sandwiching Fab is removed from the figure but is included in the Supplementary Data (Supplementary Fig. 10c). (b) CDR-H2 dominates the interaction in terms of BSA. Structural analysis shows that 79% of the BSA is attributed to the heavy chain, and 21% to the light chain. The CDR-H2 contributes about 45% of total BSA. (c) Mutational analysis confirms the structural data and demonstrates that all IGHV derived antibodies in this set bind NEAT1 with a similar mechanism. The KD for all antibodies in this set was determined by SPR-based biosensor binding analysis to recombinant full-length IsdB at 37 °C (KD range, n≥2). The binding of antibody variants at positions 52 and 53 of CDR-H2 to wild type IsdB and the binding of antibodies to NEAT1 variant Y165R (α-helix 1) were evaluated by ELISA (percentage binding relative to binding between original isolated antibodies and wild type IsdB, one representative set of results out of three independent experiments is shown). Every clone was reverted to VH germline sequence and tested for binding to NEAT1 by biosensor analysis and ability to block haemoglobin binding. ND stands for not determined.

Mentions: We also characterized a second class of antibodies that are derived from IGHV4-39 and bind to the NEAT1 domain of IsdB. We determined the crystal structure of the D4-10-N1 Fab in complex with NEAT1 (3.17 Å; Fig. 4a and Table 1; Supplementary Fig. 10c). The structure reveals that binding is again dominated by the heavy chain, particularly by CDR-H2, which contributes 45% of the BSA (Fig. 4b). Specifically, D4-10-N1 utilizes CDR-H2 residues Y52 and F53 to interact with residues Y165 of NEAT1, targeting the same binding region that is responsible for the interaction between haemoglobin and NEAT1 (ref. 6; Supplementary Fig. 13) and therefore providing a mechanistic explanation on how antibodies in this group block haemoglobin binding. Remarkably, CDR-H2 F53 protrudes into a hydrophobic pocket of NEAT1 that is structurally homologous to the heme pocket of NEAT2. Therefore this resembles the IGHV1-69 CDR-H2 interaction with NEAT2 (Supplementary Fig. 14). All antibodies in the NEAT1-binding group have a conserved aromatic residue (Y or F) at positions 52 and 53, and lost binding to NEAT1 when these residues were mutated to A (Fig. 4c). Correspondingly, mutations of NEAT1 at residue Y165 abolished binding for every antibody in this group without disrupting the binding of antibodies that also bind NEAT1 but belong to different epitope bins (Fig. 4c and Supplementary Fig. 11b,d). Collectively, the structural and mutational data strongly suggest that all of the antibodies in this set interact with NEAT1 in a similar fashion. Similar to NEAT2, the sequence of NEAT1 is also highly conserved (Supplementary Fig. 8), therefore we expect these IGHV4-39-derived antibodies to be able to recognize and neutralize IsdB encoded by the vast majority if not all S. aureus strains.


Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
Germline-encoded binding of IGHV4-39 to the NEAT1 domain of IsdB.(a) Crystal structure of an IGHV4-39-derived Fab (D4-10-N1) in complex with NEAT1. The two aromatic resides (Y52 and F53) in CDR-H2 interact with the α-helix1 of NEAT1 which is normally involved in binding haemoglobin. IGHV4-39 CDR-H2 F53 of Fab D4-10-N1 protrudes into a hydrophobic pocket of NEAT1, which is structurally homologous to the heme binding pocket of NEAT2. Crystallization was facilitated by the use of a sandwiching Fab from an antibody (D3–19) that binds NEAT1 at a non-overlapping epitope (bin H). For clarity, the sandwiching Fab is removed from the figure but is included in the Supplementary Data (Supplementary Fig. 10c). (b) CDR-H2 dominates the interaction in terms of BSA. Structural analysis shows that 79% of the BSA is attributed to the heavy chain, and 21% to the light chain. The CDR-H2 contributes about 45% of total BSA. (c) Mutational analysis confirms the structural data and demonstrates that all IGHV derived antibodies in this set bind NEAT1 with a similar mechanism. The KD for all antibodies in this set was determined by SPR-based biosensor binding analysis to recombinant full-length IsdB at 37 °C (KD range, n≥2). The binding of antibody variants at positions 52 and 53 of CDR-H2 to wild type IsdB and the binding of antibodies to NEAT1 variant Y165R (α-helix 1) were evaluated by ELISA (percentage binding relative to binding between original isolated antibodies and wild type IsdB, one representative set of results out of three independent experiments is shown). Every clone was reverted to VH germline sequence and tested for binding to NEAT1 by biosensor analysis and ability to block haemoglobin binding. ND stands for not determined.
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Related In: Results  -  Collection

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f4: Germline-encoded binding of IGHV4-39 to the NEAT1 domain of IsdB.(a) Crystal structure of an IGHV4-39-derived Fab (D4-10-N1) in complex with NEAT1. The two aromatic resides (Y52 and F53) in CDR-H2 interact with the α-helix1 of NEAT1 which is normally involved in binding haemoglobin. IGHV4-39 CDR-H2 F53 of Fab D4-10-N1 protrudes into a hydrophobic pocket of NEAT1, which is structurally homologous to the heme binding pocket of NEAT2. Crystallization was facilitated by the use of a sandwiching Fab from an antibody (D3–19) that binds NEAT1 at a non-overlapping epitope (bin H). For clarity, the sandwiching Fab is removed from the figure but is included in the Supplementary Data (Supplementary Fig. 10c). (b) CDR-H2 dominates the interaction in terms of BSA. Structural analysis shows that 79% of the BSA is attributed to the heavy chain, and 21% to the light chain. The CDR-H2 contributes about 45% of total BSA. (c) Mutational analysis confirms the structural data and demonstrates that all IGHV derived antibodies in this set bind NEAT1 with a similar mechanism. The KD for all antibodies in this set was determined by SPR-based biosensor binding analysis to recombinant full-length IsdB at 37 °C (KD range, n≥2). The binding of antibody variants at positions 52 and 53 of CDR-H2 to wild type IsdB and the binding of antibodies to NEAT1 variant Y165R (α-helix 1) were evaluated by ELISA (percentage binding relative to binding between original isolated antibodies and wild type IsdB, one representative set of results out of three independent experiments is shown). Every clone was reverted to VH germline sequence and tested for binding to NEAT1 by biosensor analysis and ability to block haemoglobin binding. ND stands for not determined.
Mentions: We also characterized a second class of antibodies that are derived from IGHV4-39 and bind to the NEAT1 domain of IsdB. We determined the crystal structure of the D4-10-N1 Fab in complex with NEAT1 (3.17 Å; Fig. 4a and Table 1; Supplementary Fig. 10c). The structure reveals that binding is again dominated by the heavy chain, particularly by CDR-H2, which contributes 45% of the BSA (Fig. 4b). Specifically, D4-10-N1 utilizes CDR-H2 residues Y52 and F53 to interact with residues Y165 of NEAT1, targeting the same binding region that is responsible for the interaction between haemoglobin and NEAT1 (ref. 6; Supplementary Fig. 13) and therefore providing a mechanistic explanation on how antibodies in this group block haemoglobin binding. Remarkably, CDR-H2 F53 protrudes into a hydrophobic pocket of NEAT1 that is structurally homologous to the heme pocket of NEAT2. Therefore this resembles the IGHV1-69 CDR-H2 interaction with NEAT2 (Supplementary Fig. 14). All antibodies in the NEAT1-binding group have a conserved aromatic residue (Y or F) at positions 52 and 53, and lost binding to NEAT1 when these residues were mutated to A (Fig. 4c). Correspondingly, mutations of NEAT1 at residue Y165 abolished binding for every antibody in this group without disrupting the binding of antibodies that also bind NEAT1 but belong to different epitope bins (Fig. 4c and Supplementary Fig. 11b,d). Collectively, the structural and mutational data strongly suggest that all of the antibodies in this set interact with NEAT1 in a similar fashion. Similar to NEAT2, the sequence of NEAT1 is also highly conserved (Supplementary Fig. 8), therefore we expect these IGHV4-39-derived antibodies to be able to recognize and neutralize IsdB encoded by the vast majority if not all S. aureus strains.

View Article: PubMed Central - PubMed

ABSTRACT

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.

No MeSH data available.