Norio Ohashi

Multiple p44 Genes Encoding Major Outer Membrane Proteins Are Expressed in the Human Granulocytic Ehrlichiosis Agent

Human granulocytic ehrlichiosis (HGE) is caused by infection with an obligatory intracellular bacterium, the HGE agent. We previously cloned a gene encoding HGE agent 44-kDa major outer membrane protein and designated it p44. In this study, we (i) identified five different mRNAs that are transcribed fromp44-homologous genes in the HGE agent cultivated in HL-60 cells; (ii) cloned genes corresponding to the mRNAs from the genomic DNA of the HGE agent; (iii) showed that the genes being expressed were not clustered in the HGE agent genome; (iv) estimated that a minimum copy number of the p44-homologous genes in the genome is 18; (v) detected two different P44-homologous proteins expressed by the HGE agent; and (vi) demonstrated existence of antibodies specific to the two proteins in sera from patients with HGE. These findings showed that p44 multigenes have several active expression sites and the expression is regulated at transcriptional level, suggesting a potentially unique mechanism for generating the diversity in major antigenic outer membrane proteins of the HGE agent. Characterization of p44-homologous genes expressed by the HGE agent in a tissue culture would assist in understanding a role of the p44 multigene family in pathogenesis and immune response in HGE.

Differential expression of VirB9 and VirB6 during the life cycle of Anaplasma phagocytophilum in human leucocytes is associated with differential binding and avoidance of lysosome pathway

Anaplasma phagocytophilum, an obligate intracellular bacterium, is the aetiologic agent of human granulocytic anaplasmosis (HGA). A. phagocytophilum virB/D operons encoding type IV secretion system are expressed in cell culture and in the blood of HGA patients. In the present study, their expression across the A. phagocytophilum intracellular developmental cycle was investigated. We found that mRNA levels of both virB9 and virB6 were upregulated during infection of human neutrophils in vitro. The antibody against the recombinant VirB9 protein was prepared and immunogold and immunofluorescence labelling were used to determine the VirB9 protein expression by individual organisms. Majority of A. phagocytophilum spontaneously released from the infected host cells poorly expressed VirB9. At 1 h post infection, VirB9 was not detectable on most bacteria associated with neutrophils. However, VirB9 was strongly expressed by A. phagocytophilum during proliferation in neutrophils. In contrast, with HL‐60 cells, approximately 80% of A. phagocytophilum organisms associated at 1 h post infection expressed VirB9 protein and were colocalized with lysosome‐associated membrane protein‐1 (LAMP‐1), whereas, VirB9‐undetectable bacteria were not colocalized with LAMP‐1. These results indicate developmental regulation of expression of components of type IV secretion system during A. phagocytophilum intracellular life cycle and suggest that bacterial developmental stages influence the nature of binding to the hosts and early avoidance of late endosome‐lysosome pathway.

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

Immunodominant 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. phagocytophila‐infected 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 σ32‐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.