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. 2004 Dec;72(12):6852-9.
doi: 10.1128/IAI.72.12.6852-6859.2004.

Rapid sequential changeover of expressed p44 genes during the acute phase of Anaplasma phagocytophilum infection in horses

Xueqi Wang  1 , Yasuko Rikihisa, Tzung-Hui Lai, Yumi Kumagai, Ning Zhi, Stephen M Reed
Affiliations

Rapid sequential changeover of expressed p44 genes during the acute phase of Anaplasma phagocytophilum infection in horses

Xueqi Wang et al. Infect Immun. 2004 Dec.

Abstract

Anaplasma phagocytophilum immunodominant polymorphic major surface protein P44s have been hypothesized to go through antigenic variation, but the within-host dynamics of p44 expression has not been demonstrated. In the present study we investigated the composition and changes of p44 transcripts in the blood during the acute phase of well-defined laboratory A. phagocytophilum infections in naive equine hosts. Three traveling waves of sequential population changeovers of the p44 transcript species were observed within a single peak of rickettsemia of less than 1 month. During the logarithmic increase, the rapid switch-off of the initial dominant transcript p44-18 occurred regardless of whether the bacterium was transmitted by ticks or by intravenous inoculation. Each of the subsequently dominant p44 transcript species was phylogenetically dissimilar from p44-18. Development of antibody to the hypervariable region of P44-18 during the rickettsemia suggests the suppression of dominance of immuno-cross-reactive p44 populations. When A. phagocytophilum was preincubated with plasma from the infected horse and then coincubated with HL-60 cells, 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. This work provides experimental evidence of within-host p44 antigenic variation. Results suggest that the rapid and synchronized switch of expression is an intrinsic property of p44s reinitiated after transmission to naive mammalian hosts and shaped upon exposure to immune plasma.

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Figures

FIG. 1.
FIG. 1.
Levels of A. phagocytophilum organisms in the blood of two horses during the course of infection, as determined by p44 C-PCR. EQ005 was infected by tick attachment, and EQ006 was inoculated i.v.
FIG. 2.
FIG. 2.
Relative proportion of each of the p44 transcript species in the blood of horses EQ005 (A) and EQ006 (B). The vertical axes show the percentage of cDNA clones of each p44 species at each time point. The horizontal axes show the various p44 cDNA clone species detected.
FIG. 3.
FIG. 3.
Development of anti-hvP44-18 antibody in the plasma of A. phagocytophilum-infected horses. (A) Three micrograms of affinity-purified rhvP44-18 (AP-rhvP44-18) was subjected to SDS-PAGE followed by Coomassie blue staining. M, molecular size marker. Therecombinant hvP44-18 is indicated by the arrow. (B) Purified rhvP44-18 (2.5 μg/lane) was separated by SDS-PAGE and blotted to the membrane. The membrane was incubated with the hvP44-18-specific MAb (3E65) or pan-P44-specific MAb (5C11). (C) Purified rhvP44-18 (2.5 μg/lane) and A. phagocytophilum (3 μg/lane) were separated by SDS-PAGE and blotted to the membrane. The membrane was incubated with horse sera collected at the indicated days p.t. or p.i. Numbers on the right indicate molecular masses (in kilodaltons) based on the broad-range prestained standards (Bio-Rad).
FIG. 4.
FIG. 4.
Infected horse plasma reduced dominance of the p44-18 transcript in cell culture. (A) Colony hybridization of p44 cDNA clones from A. phagocytophilum HZ incubated with horse (EQ005) day zero plasma (pre-tick attachment) and day 22 plasma at 6 days p.c. hvp44-18 and pan-p44 probes each were hybridized to one of the duplicated membranes each with 100 p44 cDNA clones. Two cDNA clones with p44-18 and p44-40 inserts were used as a positive and a negative control, respectively. A pCRII plasmid with a non-p44 insert was also used as negative control. Approximately 14% of p44 cDNA clones had the p44-18 insert with A. phagocytophilum incubated with day 22 p.t. plasma, and approximately 80% of p44 cDNA clones had the p44-18 insert with those incubated with day zero plasma. (B) Temporal changes in the p44-18 transcript population in A. phagocytophilum incubated with plasma obtained on day zero (preinfection) and day 31 p.t. from EQ005, FBS, or RPMI 1640 medium. Colony hybridization analysis was performed on 100 p44 cDNA clones in each specimen at each day p.c. to determine the percentage of cDNA clones with p44-18 inserts.
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