Expression analysis and antibody neutralization of P44 major surface proteins of Anaplasma phagocytophilum during mammalian infection.

摘要

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The immunodominant polymorphic 44-kDa major surface proteins of A. phagocytophilum are encoded by more than 80 p44 paralogs. Although diverse P44 species are shown to be expressed, what drives the P44 antigenic variation in mammals is unknown. Objectives of this study were to (1) characterize the temporal and dynamic expression of p44 genes during the infection of horses, immunocompetent and immunocompromised mice, and in cell culture, (2) examine expression of genes involved in homologous recombination and polycistronic expression of the p44-expression locus, and (3) elucidate P44 antibody neutralization mechanisms in vitro. First, our results showed rapid switch-off of the initial dominant transcript p44-18 occurred in the blood of horses during the logarithmic increase of bacteria growth. Each of the subsequently dominant p44 transcript species was phylogenetically dissimilar from p44-18. When A. phagocytophilum was preincubated with infected horse plasma, the dominance of the p44-18 transcript was rapidly suppressed in vitro and most of the newly emerged p44 transcript species were previously undetected in this horse. Second, we demonstrated two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. These results indicated that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection. Third, immunocompetent mice cleared A. phagocytophilum infection by 3 weeks post inoculation (p.i.), whereas A. phagocytophilum persisted at least two months in C3H/HeJ (TLR4 deficient) and SCID mice. The development of antibodies against P44 N-terminus region was delayed in C3H/HeJ mice. Of seven genes involved in homologous recombination, only recA mRNA level in A. phagocytophilum was significantly greater in mice than in cell culture. Fourth, an electrophoretic migration shift assay showed a specific shift of the promoter region of tr1 in the p44-expression locus upon incubation with A. phagocytophilum lysate. These studies are expected to facilitate the understanding of the mechanisms of P44 antigenic variation: interplay between bacterial recombination and transcriptional regulation and immunoclearance, and a new approach in designing vaccine candidate antigens for the control of HGA.