Limits...
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 and allelic specificity of IGHV4-39-derived NEAT1 binders.(a) Clone D4-10-N1 was reverted to all allelic variants with amino acid differences relative to IGHV4-39*01. No differences in IsdB binding were observed. Results shown are an average of three independent experiments. Error bars are defined as s.d. (b) IGHV4-39*01 has high sequence homology to IGHV4-30*04 and IGHV4-61*01 (they only differ by six amino acids in the variable region), and they all have the critical Y52 and Y53 residues in the CDR-H2. (c) The VH of four clones, one from each donor, were reverted to both IGHV4-30*04 and IGHV4-61*01, and their binding to IsdB was tested by ELISA. All IGHV4-30*01-derived variants were unable to bind IsdB, while most of the IGHV4-61*01-derived variants exhibited significantly loss of binding. ELISA data is an average of three independent experiments. Error bars are defined as s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120205&req=5

f5: Germline and allelic specificity of IGHV4-39-derived NEAT1 binders.(a) Clone D4-10-N1 was reverted to all allelic variants with amino acid differences relative to IGHV4-39*01. No differences in IsdB binding were observed. Results shown are an average of three independent experiments. Error bars are defined as s.d. (b) IGHV4-39*01 has high sequence homology to IGHV4-30*04 and IGHV4-61*01 (they only differ by six amino acids in the variable region), and they all have the critical Y52 and Y53 residues in the CDR-H2. (c) The VH of four clones, one from each donor, were reverted to both IGHV4-30*04 and IGHV4-61*01, and their binding to IsdB was tested by ELISA. All IGHV4-30*01-derived variants were unable to bind IsdB, while most of the IGHV4-61*01-derived variants exhibited significantly loss of binding. ELISA data is an average of three independent experiments. Error bars are defined as s.d.

Mentions: Given the prominent role of the IGHV4-39 germline-encoded CDR-H2 in binding IsdB NEAT1, we next measured the affinity of heavy chain V-gene germline-reverted (framework 1–framework 3) antibodies for all IGHV4-39 antibodies. Remarkably, all IGHV4-39 germline-reverted antibodies exhibited very high affinities (with KD values at 37 °C in the single- to triple-digit nanomolar range; Fig. 4c), supporting the idea that the IGHV4-39 germline has intrinsic potential for recognizing IsdB NEAT1. These germline-reverted antibodies can also block haemoglobin binding (Fig. 4c; Supplementary Fig. 7). This feature appeared to be specific for IGHV4-39, as reverting selected antibodies to two other highly homologous germlines23, IGHV4-30*04 and IGHV4-61*01, resulted in significant loss of binding for the antibodies evaluated (Fig. 5). Unlike the IGHV1-69 NEAT2 binders, allelic variation did not appear to affect the ability of IGHV4-39-derived clones to bind NEAT1 (ref. 23; Fig. 5).


Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
Germline and allelic specificity of IGHV4-39-derived NEAT1 binders.(a) Clone D4-10-N1 was reverted to all allelic variants with amino acid differences relative to IGHV4-39*01. No differences in IsdB binding were observed. Results shown are an average of three independent experiments. Error bars are defined as s.d. (b) IGHV4-39*01 has high sequence homology to IGHV4-30*04 and IGHV4-61*01 (they only differ by six amino acids in the variable region), and they all have the critical Y52 and Y53 residues in the CDR-H2. (c) The VH of four clones, one from each donor, were reverted to both IGHV4-30*04 and IGHV4-61*01, and their binding to IsdB was tested by ELISA. All IGHV4-30*01-derived variants were unable to bind IsdB, while most of the IGHV4-61*01-derived variants exhibited significantly loss of binding. ELISA data is an average of three independent experiments. Error bars are defined as s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120205&req=5

f5: Germline and allelic specificity of IGHV4-39-derived NEAT1 binders.(a) Clone D4-10-N1 was reverted to all allelic variants with amino acid differences relative to IGHV4-39*01. No differences in IsdB binding were observed. Results shown are an average of three independent experiments. Error bars are defined as s.d. (b) IGHV4-39*01 has high sequence homology to IGHV4-30*04 and IGHV4-61*01 (they only differ by six amino acids in the variable region), and they all have the critical Y52 and Y53 residues in the CDR-H2. (c) The VH of four clones, one from each donor, were reverted to both IGHV4-30*04 and IGHV4-61*01, and their binding to IsdB was tested by ELISA. All IGHV4-30*01-derived variants were unable to bind IsdB, while most of the IGHV4-61*01-derived variants exhibited significantly loss of binding. ELISA data is an average of three independent experiments. Error bars are defined as s.d.
Mentions: Given the prominent role of the IGHV4-39 germline-encoded CDR-H2 in binding IsdB NEAT1, we next measured the affinity of heavy chain V-gene germline-reverted (framework 1–framework 3) antibodies for all IGHV4-39 antibodies. Remarkably, all IGHV4-39 germline-reverted antibodies exhibited very high affinities (with KD values at 37 °C in the single- to triple-digit nanomolar range; Fig. 4c), supporting the idea that the IGHV4-39 germline has intrinsic potential for recognizing IsdB NEAT1. These germline-reverted antibodies can also block haemoglobin binding (Fig. 4c; Supplementary Fig. 7). This feature appeared to be specific for IGHV4-39, as reverting selected antibodies to two other highly homologous germlines23, IGHV4-30*04 and IGHV4-61*01, resulted in significant loss of binding for the antibodies evaluated (Fig. 5). Unlike the IGHV1-69 NEAT2 binders, allelic variation did not appear to affect the ability of IGHV4-39-derived clones to bind NEAT1 (ref. 23; Fig. 5).

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.