Anaplasma phagocytophilum p44 mRNA Expression Is Differentially Regulated in Mammalian and Tick Host Cells: Involvement of the DNA Binding Protein ApxR

Abstract

The natural life cycle of Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum-infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum-infected HL-60 cells cultured at 37°C than in A. phagocytophilum-infected HL-60 cells cultured at 28°C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus (p44E) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR. ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E.The obligatory intracellular bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (5, 11), is well adapted to a natural cycle of alternately infecting vertebrates and blood-sucking ticks. A. phagocytophilum surface proteins at the host-pathogen interface are expected to be differentially regulated in the two divergent host environments so that the bacterium can infect and acquire nutrients from the hosts and evade killing by the host immune systems.The polymorphic 44-kDa major outer membrane proteins (P44s, Msp2s) of A. phagocytophilum are the primary bacterial surface-exposed antigens recognized by immune systems of human granulocytic anaplasmosis patients, experimentally infected mice, and horses (8,14,20,30,(33)(34)(35). P44 has been demonstrated to function as a porin, facilitating transport of some sugars and amino acids through the bacterial membrane (13). Notably, 113 p44 genes are present in the A. phagocytophilum genome, including 22 full-length p44 genes, 64 shorter p44 genes (without a start codon), 21 fragmented p44 genes (containing only the 5Ј or 3Ј conserved region), and 6 truncated p44 genes (containing only the hypervariable region) ...

Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein

“…The expression of ecxR was autoregulated, like many other transcription factors (20,29,30), in response to an unknown signal. The mechanism of the downregulation of the virBD genes has not been determined yet.…”

“…Therefore, the regulation of the virBD genes in these bacteria may be similar to the EcxR regulation of the virBD genes in E. chaffeensis. In A. phagocytophilum, the EcxR homolog ApxR regulates the expression of a putative transcription factor, tr1 (31), and the downstream p44E locus (30). p44E encodes the immunodominant pleomorphic 44-kDa major surface protein (31), suggesting that in E. chaffeensis the expression of tr1 and the downstream omp-1/p28 locus, which encodes the major outer membrane proteins (22), might be also regulated by EcxR.…”

Mechanisms of Obligatory Intracellular Infection with Anaplasma phagocytophilum

“…The differential expression of A. phagocytophilum genes in HL-60 cells and ISE6 tick cells (164,228) and the temporal transcriptional and protein expression profiles during A. phagocytophilum infection in HL-60 cells and neutrophils (120,128,166,167) imply that the host environment and bacterial concentration regulate the transcription of A. phagocytophilum. However, genes encoding the nutritional stress response protein RelA/SpoT and proteins required for the biosynthesis of a quorum-sensing pheromone have not been found in A. phagocytophilum.…”

Cloning of the Major Outer Membrane Protein Expression Locus in Anaplasma platys and Seroreactivity of a Species-Specific Antigen

“…Tr1, a putative transcription factor, is more highly expressed in tick cells infected with A. phagocytophilum than in human leukemic HL-60 cells infected with A. phagocytophilum, suggesting that Tr1 may regulate genes involved in the bacterial infection cycle in ticks (44,64). In contrast, Tr is expressed similarly in bovine red blood cells and IDE8 tick cell cultures infected with A. marginale (5).…”

“…It is not known whether A. platys p44ES undergoes nonsegmental gene conversion (as in A. phagocytophilum to generate identical P44s from a large number of donor loci) or segmental gene conversion (as in A. marginale to generate mosaic Msp2ES from a small number of donor loci) (39,47). P44 has a role in the interaction between A. phagocytophilum and host cells (36,49,64). P44 also has porin activity that allows for passive diffusion of hydrophilic solutes (30).…”