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. 2004 Jan 8:4:1.
doi: 10.1186/1471-2180-4-1.

Restricted changes in major surface protein-2 (msp2) transcription after prolonged in vitro passage of Anaplasma phagocytophilum

Diana G Scorpio  1 , Karen Caspersen, Hiroyuki Ogata, Jinho Park, J Stephen Dumler
Affiliations

Restricted changes in major surface protein-2 (msp2) transcription after prolonged in vitro passage of Anaplasma phagocytophilum

Diana G Scorpio et al. BMC Microbiol. .

Abstract

Background: Anaplasma phagocytophilum strains often vary in Msp2 expression, a situation assumed to be related to immune evasion. However, Msp2 is also an adhesin, and little is known about the role of endogenous msp2 transcriptional changes in the absence of immune selection. Thus, Msp2 profiles and msp2 transcripts of low passage A. phagocytophilum Webster strain, initially comprised of a single abundant msp2 transcript, were re-examined after > or = 20 in vitro passages without immune selection.

Results: Using an Msp2 monoclonal antibody, immunoblots revealed an unchanged dominant band and several weak bands that appeared with passage. Similarly, msp2 transcript diversity changed, with a decrease in the initially abundant low passage transcript and appearance of a newly abundant and several minor msp2 transcripts with high passage. BLASTN search of the A. phagocytophilum HZ strain genome revealed > or = 52 msp2 paralogs.

Conclusions: Msp2 expression and msp2 transcription modulate even without immune selective pressures. However, the limited diversity of msp2 transcripts in the absence of immune pressure suggests selection for Msp2 by specific functions beyond that of immune evasion, in spite of a large genomic reservoir for Msp2 diversity.

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Figures

Figure 1
Figure 1
Protein immunoblot comparing Msp2 expression at 17 vs. 23 in vitro passages. Note the appearance of a dominant 48 kDa band in both lanes, the presence of a 30 kDa band after 17 passages that is very weakly detected at 23 passages, and the appearance of new bands at 37.5, 40, 43, and 46 kDa
Figure 2
Figure 2
Histogram of 22 msp2 loci and percentage of total RNA transcripts detected at low and high passage for A. phagocytophilum. Frequency of msp2 transcript detection in low passage (7 passages in vitro, represented by shaded bars) [15] and high passage (20 passages in vitro, represented by open bars) A. phagocytophilum Webster strain for each of 22 distinct loci detected.
Figure 3
Figure 3
ClustalX alignment and consensus amino acid sequences of Msp2 hypervariable domains. Domains were translated from genomic and RT-PCR amplicons of A. phagocytophilum Webster strain msp2 after 7 and 20 passages in vitro. The alignment demonstrates the heterogeneity of the hypervariable domains and the lack of significant similarities that would suggest combinatorial gene conversion as a mechanism for ongoing Msp2 antigenic variation. Paralog names are listed on the left, the total number of amino acid residues is shown on the right; the consensus sequence is shown on the bottom – "." indicates a semi-conserved position, ":" indicates a highly conserved position, and "*" indicates a conserved position.
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