Activation of a p44 pseudogene in Anaplasma phagocytophila by bacterial RNA splicing: a novel mechanism for post‐transcriptional regulation of a multigene family encoding immunodominant major outer membrane proteins

Abstract

SummaryImmunodominant 44 kDa major outer membrane proteins of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) are encoded by the p44 multigene family. One of the paralogues, p44-18 is predominantly expressed by A. phagocytophila in mammalian hosts, but is downregulated in the arthropod vector. The expression of p44-18 was upregulated in A. phagocytophila cultivated in HL-60 cells at 37∞ ∞ ∞ ∞ C compared with 24∞ ∞ ∞ ∞ C. However, the molecular mechanism of such gene expression was unclear, as p44-18 has a pseudogene-like structure, i.e. it lacks an AUG start codon and is out of frame with an upstream overlapping paralogue, p44-1 . In the present study, we found that an amplicon detected by reverse transciption-polymerase chain reaction (RT-PCR) [808 basepair (bp)] for the p44-1 / p44-18 gene locus was smaller than that detected by PCR with the genomic DNA (1652 bp) in the A. phagocytophilainfected HL-60 cells cultured at 37∞ ∞ ∞ ∞ C. A circularized RNA molecule corresponding to the 844 bp region missing from the locus in the RT-PCR product was detected by inverse RT-PCR, indicating that this is an intron (designated p44-1 intervening sequence, p44-1 IVS). The splicing event of p44-1 IVS was also observed when the p44-1 IVS-carrying plasmid was introduced into Escherichia coli , suggesting that the splicing is sequence-dependent. Structural analysis and in vitro splicing experiments of p44-1 IVS suggested that this is likely to represent a new class of introns in eubacteria. The primer extension analysis showed the presence of a putative s s s s 32 -type promoter in region upstream from p44-1 . Collectively, the novel RNA splicing and the temperature-dependent transcription may account for the dominant p44-18 expression in mammals.

Groupings of highly similar major surface protein (p44)-encoding paralogues: a potential index of genetic diversity amongst isolates of Anaplasma phagocytophilum

“…We did find one 'similarity group' that comprised paralogues from ruminant UK and human USA isolates, which was the 'p44-18 group'. It has been hypothesized that the p44-18 paralogue may have a somewhat specific role in the initial phase of infection of previously naïve animals and humans (Zhi et al, 2002b).…”

“…Plasmids containing appropriately sized inserts (400-600 bp) were identified using standard protocols, purified (Wizard Plus SV Miniprep DNA Purification System; Promega) and their inserts were sequenced using primers T4 and SP7. The DNA sequences obtained were aligned in GCG with all (65 in number) of the available, published p44 paralogue gene sequences of A. phagocytophilum (Caspersen et al, 2002;Lin et al, 2002;Zhi et al, 1999Zhi et al, , 2002b, including those of the Webster and BDS 'strains', and the NY31, NY36, NY37, HZ and LL isolates from the USA (GenBank accession nos AF443396 to AF443413 and AF443415 to AF443419, AF059181, AF135263, AF135254 to AF135257, AF412818 to AF412831, AF414591 and AY064513 to AY064530).…”

“…Much of this difference was due to a difference in the likelihood that different isolates from the same country contained highly similar paralogues: 35/65 (54 %) paralogues from USA isolates had >90 % similarity with a paralogue in a different USA isolate, while 4/68 (6 %) paralogues from UK isolates had >90 % similarity with a paralogue in a different UK isolate. The one group containing highly similar paralogues from UK and USA isolates comprised the p44-18 paralogue from the HZ 'strain' isolated from a human in the USA (Zhi et al, 2002b), and the paralogues FGB6 and Cairn7 from the UK Feral Goat and Cairn 'strains', respectively (Fig. 4).…”

Introns in the Cytolethal Distending Toxin Gene ofActinobacillus actinomycetemcomitans

“…Based on our results, it is unlikely that AacdtIVS-1 and AacdtIVS-2 belong to group I or II introns. Recently, the p44-1 IVS of Anaplasma phagocytophilum (the causative agent of the tick-borne disease human granulocytic ehrlichiosis) was reported to self-splice in vitro but to be structurally and mechanistically dissimilar to group I and II introns (45). Based on these findings, the authors suggested that p44-1 IVS could belong to a new group of bacterial introns.…”

“…Intervening sequences found in the tcdA and recA genes of C. difficile and B. anthracis, respectively, are removed from their precursor mRNAs, giving rise to proteins that are functionally indistinguishable from their intronless counterparts (2,15). More recently, bacterial RNA splicing was shown to function in a temperature-dependent fashion, as a novel means of regulating the expression of the major outer membrane protein gene (p44-18) of A. phagocytophilum (45). In this light, splicing of the cdt transcripts could serve as an avenue for posttranscriptional control, regulating the expression of Cdt proteins.…”