Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

Abstract

Background: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain.

Results: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S.

Conclusions: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

Figure 1

Distribution and content of vir gene clusters in eight diverse A. phagocytophilum strains. Top panel. Schematic representation of all vir loci (colored arrows) showing the three conserved gene cluster islands (see text). VirB-7, virB8-2 and virB9-2 are not part of vir gene clusters, but their location relative to surrounding genes is also highly conserved among strains. A small cluster comprising truncated (t) virB6 and virB4 gene fragments is present in all strains, but the Norwegian lamb strains have one additional virB4-t. Bottom panel. Magnification of the virB2 gene cluster. Numbering of paralogs 1–8 is based on the original ApHZ annotated genome (GenBank CP000235). Artificial gaps (stippled lines) were introduced to allow alignment of the more spatially conserved paralogs B2-1, 2–2 and 2–3 at one end, and B2-7 and 2–8 at the other end of the cluster. With the exception of virB2-9, lacking in ApHZ, the number and arrangement (but not necessarily sequence) of virB2 genes is highly conserved in all but the US ruminant ApVar-1 and ApNorLamb-V1, which have several additional virB2 genes. In both strains a sub-cluster of 6 distinct genes was present. Due to the repetitive nature of sequences in this region, combined with the relatively short length of 454 reads (≤550 bp), their placement could not be confidently ascertained (highlighted by arrows and ‘?’). Maps are drawn to scale. Double lines designate interruption in sequences. Genes belonging to the same grouping have the same color. oriC; origin of replication.

Figure 2

Phylogenetic tree to show the relationship of syntenic VirB6 proteins from different strains of A. phagocytophilum. A scale bar is shown underneath representing the number of amino acid substitutions/site.

Figure 3

The 3^′ end of A. phagocytophilum virB6-4 genes is composed of an unusually large tandem repeat region, which exhibits dramatic variability among strains.A. Map of the human HZ strain virB6-3 and virB6-4 genes, highlighting the location and structure of several repeat regions (R1-R4). The most variability occurred in R4; this region is 5.88 kb larger than previously reported for the ApHZ genome (CP000235). The original sequence is diagrammed above the map, with the dashed line representing the segment missing in CP000235. Larger repeated R4 segments of 2.7 kb and 1.15 kb are indicated above. Vertical black bars within each gene designate segments encoding predicted transmembrane domains. BamHI sites, of which there is one in all R4 type 2 repeats (see Figure S2B), are indicated. Also shown are the positions of PCR primers used in C. B. BamHI genomic maps depicting the virB6-4 locus (black arrows).The segment encompassing R4 is highlighted below each respective map. In the regions outside the virB6-4 locus, corresponding BamHI fragments are shown in the same color. Overall, the optical map sizes were in good agreement with the actual sizes, except within R4. This is attributed to the limitation of optical mapping in resolving fragments <2 kb. Despite these discrepancies, the cumulative size of the genomic region encompassing virB6-4 in the optical map is in close agreement with that in the ApDog1 genome sequence. C. The variability in size of PCR products spanning virB6-4 repeat regions R4 and R3/R4 in diverse A. phagocytophilum strains.

Figure 4

Hydrophobicity plots of VirB6-4 proteins from A. phagocytophilum HZ (top) or Dog 1 (bottom) strains.

Figure 5

Phylogenetic trees to show the relationship of syntenic VirB2 proteins from different strains of A. phagocytophilum.

Figure 6

Multiple sequence alignment of VirB2 amino acid sequences from different strains of A. phagocytophilum.

Figure 7

Phylogenetic trees to show the relationship of syntenic VirB4 proteins from different strains of A. phagocytophilum.

Figure 8

Phylogenetic tree to show the relationship of syntenic VirD4 proteins from different strains of A. phagocytophilum.

Figure 9

Phylogenetic tree to show the relationship of other conserved proteins from different strains of A. phagocytophilum. These proteins are: PolA, DNA polymerase I; LeuS, leucyl-tRNA synthetase; AtpA, ATPsynthase F1, alpha subunit; ValS,valyl-tRNA synthetase; RecG, ATP-dependent DNA helicase; LigA, NAD-dependent DNA ligase.