Simultaneous variation of the immunodominant outer membrane proteins, MSP2 and MSP3, during anaplasma marginale persistence in vivo

Abstract

Vector-borne bacterial pathogens persist in the mammalian host by varying surface antigens to evade the existing immune response. To test whether the model of surface coat switching and immune evasion can be extended to a vector-borne bacterial pathogen with multiple immunodominant surface proteins, we examined Anaplasma marginale, a rickettsia with two highly immunogenic outer membrane proteins, major surface protein 2 (MSP2) and MSP3. The simultaneous clearance of variants of the two most immunodominant surface proteins of A. marginale followed by emergence of unique variants indicates that the switch rates and immune selection for MSP2 and MSP3 are sufficiently similar to explain the cyclic bacteremia observed during infection in the immunocompetent host.

FIG. 1.

Bacteremic peaks 1 and 2. The plot illustrates two peaks of A. marginale bacteremia with clearance between the two peaks. Percent parasitized erythrocytes (PPE) is shown on the y axis.

FIG. 2.

Schematic representations of the msp2 and msp3 expression sites. (A) The msp2 expression site contains four open reading frames with msp2 at the 3′ end (3). Primers used to clone msp2 are shown: msp2 forward (msp2 for) sits in opag1 and is specific for the single-copy operon; msp2 reverse (msp2 rev) sits in the 3′ conserved region of msp2. (B) The msp3 expression site contains three open reading frames. Primers used to clone msp3 are shown: AB973 is a unique sequence tag 5′ to the open reading frames; msp3 3′ con and msp3 5′ con sit in the msp3-specific conserved regions flanking the hypervariable region. Msp2 sequences (black bars), msp3 sequences (white bars), hypervariable regions (HV) (dark gray bars), and operon-associated genes (light grey bars) are shown.

FIG. 3.

Expression site sequences for msp2 and msp3. (A) Amino acid sequences deduced from the msp2 genes found at bacteremic peaks 1 and 2. (B) Amino acid sequences deduced from the msp3 genes found at bacteremic peaks 1 (first sequence) and 2. All sequences are truncated to show the HVRs only. Regions corresponding to oligonucleotide probes are underlined (msp2 peak 1 and 2; msp3 peak 2) or overlined (msp3 peak 1). Numbers above the sequence correspond to the amino acid positions relative to those of pCKR11.2 (18) for msp2 and msp3E-SM16D3 for msp3 (16). Clone designations begin with 1 or 2 to indicate that the clone was obtained from bacteremic peak 1 or 2, respectively. The numbers in parentheses indicate the number of sequences obtained for each variant. Sequence conservation in the different clones is indicated by white type on black background. Gaps introduced to maximize alignment are indicated by dashes.

FIG. 3.

Expression site sequences for msp2 and msp3. (A) Amino acid sequences deduced from the msp2 genes found at bacteremic peaks 1 and 2. (B) Amino acid sequences deduced from the msp3 genes found at bacteremic peaks 1 (first sequence) and 2. All sequences are truncated to show the HVRs only. Regions corresponding to oligonucleotide probes are underlined (msp2 peak 1 and 2; msp3 peak 2) or overlined (msp3 peak 1). Numbers above the sequence correspond to the amino acid positions relative to those of pCKR11.2 (18) for msp2 and msp3E-SM16D3 for msp3 (16). Clone designations begin with 1 or 2 to indicate that the clone was obtained from bacteremic peak 1 or 2, respectively. The numbers in parentheses indicate the number of sequences obtained for each variant. Sequence conservation in the different clones is indicated by white type on black background. Gaps introduced to maximize alignment are indicated by dashes.