Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever

Abstract

Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. sennetsu showed that 758 (88.2%) of protein-coding genes are conserved between N. risticii and N. sennetsu. Four-way comparison of genes among N. risticii and other Anaplasmataceae showed that most genes are either shared among Anaplasmataceae (525 orthologs that generally associated with housekeeping functions), or specific to each genome (>200 genes that are mostly hypothetical proteins). Genes potentially involved in the pathogenesis of N. risticii were identified, including those encoding putative outer membrane proteins, two-component systems and a type IV secretion system (T4SS). The bipolar localization of T4SS pilus protein VirB2 on the bacterial surface was demonstrated for the first time in obligate intracellular bacteria. These data provide insights toward genomic potential of N. risticii and intracellular parasitism, and facilitate our understanding of PHF pathogenesis.

Figure 1.

Phylogenetic tree of the family Anaplasmataceae. 16S rRNA sequences of members of the family Anaplasmataceae were aligned using the Clustal W method, and a phylogenetic tree was built. Gray box highlights Neorickettsia species.

Figure 2.

Circular representation of the genome of N. risticii. From outside to inside, the first two circles represent predicted protein-coding sequences (ORFs) on the plus and minus strands, respectively. Colors indicate the role categories of ORFs: dark gray: hypothetical proteins or proteins with unknown functions; gold: amino-acid and protein biosynthesis; sky blue: purines, pyrimidines, nucleosides and nucleotides; cyan: fatty acid and phospholipid metabolism; light blue: biosynthesis of cofactors, prosthetic groups and carriers; aquamarine: central intermediary metabolism; royal blue: energy metabolism; pink: transport and binding proteins; dark orange: DNA metabolism and transcription; pale green: protein fate; tomato: regulatory functions and signal transduction; peach puff: cell envelope; pink: cellular processes; maroon: mobile and extrachromosomal element functions. The third and fourth circles show unique ORFs compared to N. sennetsu. The fifth and sixth circles represent RNA genes, including tRNAs (blue), rRNAs (orange), and sRNAs (red). The seventh circle represents G–C skew values [(G – C)/(G + C)] with a windows size of 1 kb.

Figure 3.

Synteny plots between N. risticii Illinois (horizontal axis) and N. sennetsu Miyayama, A. phagocytophilum HZ, E. chaffeensis Arkansas, and the Wolbachia endosymbiont of Brugia malayi. Numbers represent base pairs. Each dot represents a pair of probable sequence fragments defined as reciprocal BLAST best hits with E-value <0.001 (red: sequences match at the forward strand; blue: sequences match at the reverse complemented strand).

Figure 4.

Comparison of the gene sets in members of the family Anaplasmataceae. Venn diagram showing the comparison of conserved and unique genes between Neorickettsia spp. (A), or among selected members of the family Anaplasmataceae (B). Numbers within the intersections of different circles indicate ortholog clusters shared by 2, 3, or 4 organisms. (C) Comparison of gene sets by functional role category breakdown. Species indicated in the diagram are as follows: N. sennetsu (NSE), N. risticii (NRI, A), E. chaffeensis (ECH, C), A. phagocytophilum (APH, C), and the Wolbachia endosymbiont of Brugia malayi (WBM, D).

Figure 5.

Genomic organization of T4SS virB/D clusters and phylogenic analysis of virB2 genes in the family Anaplasmataceae. (A) Circular representation of T4SS virB/D genes in genomes of the family Anaplasmataceae. From outside to inside circles: first (black)—N. risticii (879 977 bp); second (red)—N. sennetsu (859 006 bp); third (blue)—A. phagocytophilum (1 471 282 bp); fourth (green)—E. chaffeensis (1 176 248 bp). The individual virB/D genes were color-coded in the clusters for better visualization. Note that all T4SS genes of N. risticii and N. sennetsu, and most T4SS genes of A. phagocytophilum and E. chaffeensis are encoded in the ‘−’ strand, therefore, the location of most genes appear close to the inner circles. Genes encoded in the ‘+’ strand are followed by red arrows. (B) Phylogenetic tree of virB2 genes in the family Anaplasmataceae. Nucleotide sequences of virB2 from members of the family Anaplasmataceae were aligned using the Clustal W method, and a phylogenetic tree was built. APHxxxx: locus ids for virB2 genes of A. phagocytophilum HZ; ECHxxxx: locus ids of E. chaffeensis Arkansas; NSExxxx: locus ids of N. sennetsu Miyayama; NRIxxxx: locus ids of N. risticii Illinois; ATU6168: Agrobacterium tumefaciens C58 pilin subunit, TrbC/VirB2 family protein (Accession No. NP_396488); RP192: Rickettsia prowazekii Madrid E TrbC/VirB2 family protein (Accession No. NP_359878); RC241: Rickettsia conorii Malish 7 TrbC/VirB2 family protein (Accession No. NP_359878); CC2417: Caulobacter crescentus CB15 TrbC/VirB2 family protein (Accession No. NP_421220).

Figure 6.

Expression and localization of N. risticii VirB2 (NRI_0738/NRI_0740). (A) RNAs were prepared from N. risticii-infected P388D₁ cells at 3 days p.i. DNase-treated total RNA was reverse-transcribed (RT+) and subsequently PCR-amplified using primers specific to virB2 genes: NRI_0738 and NRI_0740. RT–: negative control without reverse transcriptase; 738: PCR with primers targeting NRI_0738 only; 740: PCR with primers targeting NRI_0740 only; 738 + 740: PCR with primers spanning NRI_0738 and NRI_0740. The relative sizes of molecular mass standards are shown (in base pairs) on the left. (B) Whole-cell lysates from N. risticii-infected P388D₁ cells (3 days p.i.) were prepared and subjected to western blotting using antibody against N. risticii VirB2 (NRI_0738), or host α-tubulin as a loading control. The molecular mass standards are shown (in kDa) on the right. (C) N. risticii-infected P388D₁ cells at 3 days p.i. were fixed, permeabilized with 0.1% saponin and dual labeled with horse anti-N. risticii (NRI) and rabbit anti-VirB2-1 (NRI_0738) antibodies. Results are representative of three independent experiments. Note bright dots of VirB2 on most of intracellular bacteria. Bar: 5 μm. (D) Host cell-free N. risticii purified from infected P388D₁ cells at 3 days p.i. were fixed and dual labeled with horse anti-N. risticii (NRI) and rabbit anti-VirB2 antibodies. The right panel is a 5× amplification of the inlet from the merged image. Results are representative of three independent experiments. Note average bipolar localization of VirB2 per bacterium on the surface of N. risticii and variable sizes of bacteria.

Figure 7.

Genomic organization and alignment of N. risticii putative membrane proteins NSP and SSA. (A and D) Genomic organization of N. risticii genes encoding putative membrane proteins NSP (A) and SSA (D). Color bars inside SSA genes indicate repeat sequences. NCR: non-coding region. (B and E) Phylogenetic tree of nsp (B) and ssa (E) genes in N. risticii and N. sennetsu. Nucleotide sequences of nsp or ssa from N. risticii and N. sennetsu were aligned using the Clustal W method, and a phylogenetic tree was built. NRI25-D: N. risticii strain 25-D; NRI90-12: N. risticii strain 90-12. (C and F) Alignment of N. risticii NSP (C) and SSA (F) protein sequences using blast2seq.