Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens.

Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.

Anaplasma (Ehrlichia) phagocytophila and Ehrlichia chaffeensis, the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight virB and virD genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight virB and virD genes were clustered at two separate loci in each genome. Five of the genes (virB8, virB9, virB10, virB11, and virD4) were located downstream from a ribA gene. These five genes in both A. phagocytophila and E. chaffeensis were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the virB8 gene in human leukemia cell lines. The virB9 gene of A. phagocytophila was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes (virB3, virB4, and virB6) of both A. phagocytophila and E. chaffeensis were arranged downstream from a sodB gene and cotranscribed with the sodB gene through one or more sodB promoters in human leukocytes. This suggests that transcription of the three virB genes in these two Anaplasma and Ehrlichia spp. is regulated by factors that influence the sodB gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of Anaplasma and Ehrlichia spp. in granulocytes or monocytes.Human granulocytic ehrlichiosis (HGE) and human monocytic ehrlichioses (HME) are newly discovered tick-borne febrile illnesses of increasing importance in the United States (1, 7) and have been recognized in several countries in Europe and Africa and in Mexico. The clinical signs of HGE and HME are similar. The patients frequently require hospitalization, and they may die when the treatment is delayed or they are immunocompromised (15,30). HGE and HME are caused by obligatory intracellular bacteria, Anaplasma (Ehrlichia) phagocytophila (7, 13) and Ehrlichia chaffeensis (1), which have tropism for granulocytes and monocytes, respectively. These agents reside and replicate within membrane-bound inclusions (parasitophorous vacuoles) in the cytoplasm of phagocytes. The 16S rRNA gene sequences of A. phagocytophila and E. chaffeensis are 7.5% divergent (7), and their ultrastructures, antigenic compositions, inclusion compartments, sensitivities to intracellular iron depletion, and properties of the major outer membrane protein gene family are distinct (5,6,20,24,25,28,29,32,(42)(43)(44).Type IV secretion systems are ancestrally related to the bacterial conjugal system and are thought to function to deliver effector macromolecules produced by parasitic or symbiotic bacteria into ...

The human intragranulocytic bacterium Anaplasma phagocytophilum promotes variation of P44s, which are surface-exposed proteins encoded by a p44 multigene family. In the present study, the specific p44 gene expression loci in four strains of A. phagocytophilum were identified and it was determined that each consisted of four tandem genes, tr1, omp-1X, omp-1N, and p44. A putative 70 -type promoter was found upstream of tr1. The p44 genes include a central hypervariable region flanked by conserved regions. The hypervariable region sequence in the p44 expression locus was duplicated and, regardless of the expression status, conserved at another locus in both low-and high-passage cell cultures of strain NY-37. No significant differences in the hypervariable region were found when we compared p44 sequences, at the level of cDNA, within the expression locus and within other loci in the genomes of strains NY-37 and HZ. Similarly, in cDNA isolated from patients and from assorted cultures of strains NY-31, NY-36, and NY-37, hypervariable regions of 450 deduced amino acid sequences of various p44s within each strain were found to be identical, as were those of p44 sequences in the genome of strain HZ. These data suggest that variations in p44 sequences at the level of the p44 expression locus occur through unidirectional conversion of the entire (nonsegmental) p44 hypervariable region including flanking regions with a corresponding sequence copied from one of the conserved donor p44 genomic loci. The data suggest that the P44 antigenic repertoire within the hypervariable region is restricted.

Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.