Comparative genomics of emerging human ehrlichiosis agents

Abstract

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.

Figure 1. Phylogenetic Tree of the α-Proteobacteria

The protein sequences of select conserved genes were concatenated and aligned, and a phylogenetic tree was inferred of all sequenced α-Proteobacteria (see Materials and Methods). The Anaplasmataceae (purple) and the Rickettsiaceae (yellow) are highlighted.

Figure 2. Synteny of the Rickettsiales

Regions of conserved synteny were identified using the ortholog clusters (see Materials and Methods) and visualized with Sybil. The genes along each ordered chromosome were colored on a gradient from yellow to blue. The ortholog clusters for each query genome were then plotted relative to the order of the reference genome. Regions of synteny are then seen as continuous gradients across large regions of the genome. Above the synteny gradient display is the atypical nucleotide composition. Below the gradient display are the predicted coding regions on the plus strand and the minus strand, and the GC-skew. Representatives of all the Rickettsiales (A) and representative Ehrlichia spp. and Anaplasma spp. (B) were compared separately. AMA, A. marginale St. Maries; APH, A. phagocytophilum HZ; ECH, E. chaffeensis Arkansas; ERU, E. ruminantium Welgevonden; NES, N. sennetsu Miyayama; RPR, R. prowazekii Madrid E; WOL, W. pipientis wMel.

Figure 3. Synteny between Anaplasma spp. and Ehrlichia spp.

Anaplasma spp. and Ehrlichia spp. share conserved gene order (synteny) across their chromosomes. E. ruminantium and E. chaffeensis have a single symmetrical inversion near two duplicate Rho termination factors (approximate positions shown in pink). Genomic rearrangements between these Rho termination factors are also apparent in A. marginale (pink). In addition to the synteny breaks near the Rho termination factors, A. marginale has rearrangements located near the msp2- and msp3- expression locus and pseudogenes (approximate positions shown in light blue). Likewise, in A. phagocytophilum, numerous changes in genome arrangement are located near the homologous p44 expression locus and silent genes (approximate positions shown in lavender).

Figure 4. Comparison of the Rickettsiales Gene Sets

The composition of ortholog clusters (see Materials and Methods) of representative Rickettsiales (A), Ehrlichia spp. (B), and Anaplasma spp. (C) were compared. Numbers within the intersections of different ovals indicate ortholog clusters shared by 2, 3, 4, or 5 organisms. Species compared are indicated in diagram intersections as follows. A, R. prowazekii; B, N. sennetsu; C, W. pipientis; D, A. phagocytophilum; E, E. chaffeensis; F, A. marginale; G, E. ruminantium Gardel; and H, E. ruminantium Welgevonden.

Figure 5. Representative Illustrations of p44 Genes

Full-length p44 genes contain conserved start and stop codons, an ORF longer than 1,000 bp, and a central hypervariable region of approximately 280 bp containing a signature of four conserved amino acid regions (C, C, WP, A). These genes can be expressed at their respective current genome location or can recombine into the expression locus (p44ES/APH_1221). A silent/reserve p44 is less than 1,000 bp. It may have either the conserved or alternative start and/or stop codons. A silent/reserve p44 is not likely to be expressed at its current genome location, but can recombine into the expression locus (p44ES/APH_1221). Truncated p44s carry the complete hypervariable region, or a portion thereof, but only one of the two conserved regions. Fragments of p44 have only a conserved region and no hypervariable region. Each annotated p44 is longer than 60 bp. It should be noted that smaller fragments can be identified throughout the genome. These, as well as p44 truncations and fragments, are likely to be nonfunctional remnants of previous recombination events.

Figure 6. Comparative Metabolic Potential of Select Rickettsiales

Metabolic pathways of E. chaffeensis (magenta arrows), A. phagocytophilum (green arrows), N. sennetsu (gold arrows), W. pipientis (lavender arrows), and R. prowazekii (cyan arrows) were reconstructed and compared. The networks of some of the more important pathways are shown with metabolites color coded: red and purple, central and intermediary metabolites; blue, cofactors; green, amino acids; and black, cell structures. Transporters are shown in the membrane and are grouped by predicted substrate specificity: green, inorganic cations; magenta, inorganic anions; red, carbohydrates and carboxylates; blue, amino acids/peptides/amines; yellow, nucleotides/nucleosides; and black, drug/polysaccharide efflux or unknown.