Primary Structural Variation in Anaplasma marginale Msp2 Efficiently Generates Immune Escape Variants

Abstract

Antigenic variation allows microbial pathogens to evade immune clearance and establish persistent infection. Anaplasma marginale utilizes gene conversion of a repertoire of silent msp2 alleles into a single active expression site to encode unique Msp2 variants. As the genomic complement of msp2 alleles alone is insufficient to generate the number of variants required for persistence, A. marginale uses segmental gene conversion, in which oligonucleotide segments from multiple alleles are recombined into the expression site to generate a novel msp2 mosaic not represented elsewhere in the genome. Whether these segmental changes are sufficient to evade a broad antibody response is unknown. We addressed this question by identifying Msp2 variants that differed in primary structure within the immunogenic hypervariable region microdomains and tested whether they represented true antigenic variants. The minimal primary structural difference between variants was a single amino acid resulting from a codon insertion, and overall, the amino acid identity among paired microdomains ranged from 18 to 92%. Collectively, 89% of the expressed structural variants were also antigenic variants across all biological replicates, independent of a specific host major histocompatibility complex haplotype. Biological relevance is supported by the following: (i) all structural variants were expressed during infection of a natural host, (ii) the structural variation observed in the microdomains corresponded to the mean length of variants generated by segmental gene conversion, and (iii) antigenic variants were identified using a broad antibody response that developed during infection of a natural host. The findings demonstrate that segmental gene conversion efficiently generates Msp2 antigenic variants.

FIG 1

A. marginale Msp2 mechanism of antigenic variation. The mosaics generated through genomic recombination result in expression of new antigenically distinct Msp2 variants, which compose sequential bacteremic waves and allow persistence within the host. (A) Chromosomal msp2 loci, 7 silent alleles, and a single active expression site (ES) with a central hypervariable domain (HVR). P1, E6/F7, G11, 1, 2, 9H1, and 3H1 are the 7 conserved donor alleles for the Msp2 HVR, with 5 of them being unique (allele P1 is a duplication of allele G11, and allele 2 is a duplication of allele 3H1). (B) Segmental gene conversion from an oligonucleotide segment of a donor allele into the expression site, generating an expression site mosaic. (C) Bacteremic peaks characterized by expression of unique Msp2 variants, variants generated by recombination of a “whole” donor allele, predominate in acute bacteremia, followed by a persistent phase in which the mosaics generated by segmental gene conversion predominate. The modeled protein structure with the peptide sequences generated by a donor allele or segmental gene conversion is indicated above the bacteremic peak.

FIG 2

Graphical representation of the A. marginale St. Maries HVR microdomains and corresponding flanking tethers. T1, N-terminal flanking tether; T2, spacing tether between microdomain 1 and microdomain 2; T3, spacing tether between microdomain 2 and microdomain 3.

FIG 3

Primary sequences of the Nayarit microdomain 1 variants compared to the homologous St. Maries strain. The microdomain 1 sequences encoded by the Nayarit strains are indicated below the corresponding sequence encoded by the St. Maries strain alleles 2, E6F7, and G11 (boldface). The flanking N-terminal (L/VSK/QKVC) and C-terminal (CG) tethers and conserved amino acids within the hypervariable microdomain are indicated in purple. Insertions relative to the St. Maries sequence are indicated in green, deletions in yellow, and substitutions in red. Changes in charge and hydrophobicity relative to the St. Maries sequence are indicated as follows: a, positive to neutral; b, negative to neutral; c, neutral to positive or insertion of a positive charge; d, neutral to negative or insertion of a negative charge.

FIG 4

Primary sequences of the Nayarit microdomain 2 variants compared to the homologous St. Maries strain. The microdomain 2 sequences encoded by the Nayarit strains are indicated below the corresponding sequence encoded by St. Maries strain alleles 9H1, 1, and E6F7 (boldface). The flanking N-terminal (CG) and C-terminal (KL/I) tethers and conserved amino acids within the hypervariable microdomain are indicated in purple. Insertions relative to the St. Maries sequence are indicated in green, deletions in yellow, and substitutions in red. Changes in charge and hydrophobicity relative to the St. Maries sequence are indicated as follows: a, positive to neutral; b, negative to neutral; c, neutral to positive or insertion of a positive charge; d, neutral to negative or insertion of a negative charge.