Ning Zhi

Characterization and Transcriptional Analysis of Gene Clusters for a Type IV Secretion Machinery in Human Granulocytic and Monocytic Ehrlichiosis Agents

ABSTRACT 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.

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.

Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks.

ABSTRACT Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of Anaplasma phagocytophila called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal Ixodes scapularis ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different p44 transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a p44-18 gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the p44-18 gene were detected in the HGE agent cultivated in HL-60 cells at 37°C, but their expression levels decreased in the organisms cultivated at 24°C, suggesting that temperature is one of the factors that influence the expression of members of the p44 multigene family. Several additional p44 transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different p44 transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the p44 multigene family in transmission of the HGE agent between mammals and ticks.

Analysis of Sequences and Loci of p44 Homologs Expressed by Anaplasma phagocytophila in Acutely Infected Patients

ABSTRACT 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.

Mechanisms of variable p44 expression by Anaplasma phagocytophilum.

ABSTRACT 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 σ70-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.

Rapid sequential changeover of expressed p44 genes during the acute phase of Anaplasma phagocytophilum infection in horses.

ABSTRACT Anaplasma phagocytophilum immunodominant polymorphic major surface protein P44s have been hypothesized to go through antigenic variation, but the within-host dynamics of p44 expression has not been demonstrated. In the present study we investigated the composition and changes of p44 transcripts in the blood during the acute phase of well-defined laboratory A. phagocytophilum infections in naïve equine hosts. Three traveling waves of sequential population changeovers of the p44 transcript species were observed within a single peak of rickettsemia of less than 1 month. During the logarithmic increase, the rapid switch-off of the initial dominant transcript p44-18 occurred regardless of whether the bacterium was transmitted by ticks or by intravenous inoculation. Each of the subsequently dominant p44 transcript species was phylogenetically dissimilar from p44-18. Development of antibody to the hypervariable region of P44-18 during the rickettsemia suggests the suppression of dominance of immuno-cross-reactive p44 populations. When A. phagocytophilum was preincubated with plasma from the infected horse and then coincubated with HL-60 cells, the dominance of the p44-18 transcript was rapidly suppressed in vitro and most of the newly emerged p44 transcript species were previously undetected in this horse. This work provides experimental evidence of within-host p44 antigenic variation. Results suggest that the rapid and synchronized switch of expression is an intrinsic property of p44s reinitiated after transmission to naïve mammalian hosts and shaped upon exposure to immune plasma.

Western Blot Analysis of Sera Reactive to Human Monocytic Ehrlichiosis and Human Granulocytic Ehrlichiosis Agents

ABSTRACT Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cells as antigen. Concern has been raised that incorrect diagnoses of human monocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between Ehrlichia chaffeensis and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown to be sensitive and specific serodiagnostic antigens for HME and HGE, respectively, could be used to discriminate IFA dually reacting sera. Thirteen dually IFA-reactive sera, three sera that were IFA positive only with E. chaffeensis, and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16 E. chaffeensis IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardless of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positive sera reacted only with rP44, not with rP30. Western immunoblotting using purified E. chaffeensis and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-react between the two organisms. Therefore, Western immunoblot analysis using rP44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactive sera.

Cytokine Gene Expression by Peripheral Blood Leukocytes in Horses Experimentally Infected with Anaplasma phagocytophila

ABSTRACT Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A. phagocytophila or by allowing feeding by infected ticks. The p44 genes encoding the major outer membrane protein P44s of A. phagocytophila were detected by PCR in PBLs of all four horses from 4 to 20 days postexposure. During the 20-day infection period, interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA expression was upregulated in PBLs of all four horses, and IL-8 mRNA expression was upregulated in three horses. Gamma interferon, IL-10, and IL-12 p35 mRNAs were weakly expressed in only one horse each. IL-2, IL-4, IL-6, and IL-12 p40 mRNA expression , however, could not be detected in the PBLs of any of the four horses. These results suggest that IL-1β, TNF-α, and IL-8 generation during A. phagocytophila infection has a primary role in HGE pathogenesis and immunomodulation.

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.

Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, twoBabesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.