The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions

Abstract

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

Figure 1. Comparative genomics and phylogenomics of R. equi 103S.

(A) Pairwise chromosome alignments of R. equi 103S, R. jostii RHA1, N. farcinica IFM10152, M. tuberculosis (Mtb) H37Rv and S. coelicolor A3(2) genomes. Performed with Artemis Comparison Tool (ACT), see Table S12. Red and blue lines connect homologous regions (tBLASTx) in direct and reverse orientation, respectively. Mean identity of shared core orthologs between R. equi and: R. jostii RHA1, 75.08%; N. farcinica, 72.1%; Mtb, 64.6% (see also Figures S1, S2, and S5). (B) Phylogenomic analysis of Rhodococcus spp. and four other representative actinobacterial species. Unrooted neighbor-joining tree based on percent amino-acid identity of a sample of 665 shared core orthologs. The scale shows similarity distance in percentage.

Figure 2. Role of gene duplication and horizontal gene transfer (HGT) in rhodococcal genome evolution.

Scatter plots of (A) duplicated (paralogous) genes and (B) HGT genes versus the total number of genes in rhodococcal and actinobacterial genomes (curve fits of rhodococcal data in red, general trendline in black). HGT genes were excluded from the paralogy analyses.

Figure 3. Schematic overview of relevant metabolic and virulence-related features of R. equi 103S.

Complete glycolytic, PPP, and TCA cycle pathways, and all components for aerobic respiration, are present. The TCA cycle incorporates the glyoxylate shunt, which diverts two-carbon metabolites for biosynthesis. The methylcitrate pathway enzymes (pprCBD, REQ09040-60) are also present. The lutABC operon may take over the function of the D-lactate dehydrogenase (cytochrome) REQ00650, which is a pseudogene in 103S. REQ15040 (L-lactate 2-monoxygenase) and REQ27530 (pyruvate dehydrogenase [cytochrome]) can directly convert lactate and pyruvate into acetate. Unlike Mtb and other actinomycete pathogens, R. equi 103S has no secreted phospholipase C (Plc), only a cytosolic phospholipase D (Pld, REQ09260); a secreted Plc is however encoded in the genomes of environmental Rhodococcus spp. Rbt1/IupS (REQ08140-60) is a dimodular BhbF-like siderophore synthase . Rbt1 rhequibactins are synthesized from (iso)chorismate via 2,3-dihydroxybenzoate (DHB) as for enterobactin or bacillibactin (REQ08130-100 encode homologs of Ent/DhbCAEB) , . Two MFS transporters and a siderophore binding protein (REQ08180-200) encoded downstream from iupS may be involved in rhequibactin export/uptake. There is also a putative Ftr1-family iron permease (REQ12610). R. equi may store intracellular iron via two bacterioferritins (REQ01640-50) and the Dps/ferritin-like protein (REQ14900). IdeR- (REQ20130), DtxR- (REQ19260) and Fur- (REQ04740-furA, REQ29130-furB)-like regulators may contribute to iron/metal ion regulation. Homologs of the Mtb DosR (dormancy) regulon are also present in the R. equi genome (Table S6).

Figure 4. R. equi pilus locus (rpl).

(A) The 9 Kb rpl HGT island (REQ18350-430) is absent from nonpathogenic Rhodococcus spp. rpl genes have been detected in all R. equi clinical isolates (P. Gonzalez et al., manuscript in preparation). Putative rpl gene products: A, prepilin peptidase; B, pilin subunit; C, TadE minor pilin; D, putative lipoprotein; E, CpaB pilus assembly protein; F, CpaE pilus assembly protein; GHI, Tad transport machinery . (B) Electron micrograph of R. equi 103S pili (indicated by arrowheads; generally 2–4 per bacterial cell). Bar = 0.5 µm. (C) R. equi 103S pili visualized by immunofluorescence microscopy (×1,000 magnification).

Figure 5. Network analysis of virulence plasmid–chromosome regulatory crosstalk.

(A) Integration of the virulence plasmid vap PAI in the R. equi regulatory network. 3D graph of the R. equi 103S transcriptome (see text for experimental conditions) constructed with BioLayout Express^3D, an application for the visualization and cluster analysis of coregulated gene networks , . Settings used: Pearson correlation threshold, 0.85; Markov clustering (MCL) algorithm inflation, 2.2.; smallest cluster allowed, 3; edges/node filter, 10; rest of settings, default. Network graph viewable in Dataset S1. Each gene is represented by a node (sphere) and the edges (lines) represent gene expression interrelationships above the selected correlation threshold; the closer the nodes sit in the network the stronger the correlation in their expression profile. Note that the plasmid vap PAI genes (red spheres) are embedded within, and establish multiple functional connections with, chromosomal nodes (see also Figure S12A) whereas those of the plasmid housekeeping backbone lie outside the main network, reflecting an independent regulatory pattern. (B) Isolated subgraph of the R. equi transcription network obtained with r = 0.95 Pearson correlation threshold, showing the coregulation of the chromosomal genes REQ23860 (putative AroQ chorismate mutase) and REQ23850 (putative TrpEG-like bifunctional anthranilate synthase) (see Figure 7) with the virulence plasmid vap PAI genes. Color codes for nodes as indicated in (A) (spheres, vap PAI-coregulated cluster; cubes, plasmid housekeeping backbone cluster). MCL inflation, 2.2, smallest cluster allowed, 3; rest of settings, default. See Dataset S3.

Figure 6. Intracellular growth kinetics of ΔREQ23860 and ΔREQ23850 mutants in J774 macrophages.

Data were normalized to the initial bacterial counts at t = 0 using an intracellular growth coefficient (IGC); see Materials and Methods. Positive IGC indicates proliferation, negative values reflect decrease in the intracellular bacterial population. Bacterial counts per well at t = 0: 103S (wild type), 9.84±0.55×10⁴; 103S^P−, 4.67±0.62×10⁴; ΔREQ23860 (putative CM), 11.26±2.78×10⁴; complemented ΔREQ23860, 4.24±0.10×10⁴; ΔREQ23850 (putative AS), 9.67±0.12×10⁴; complemented ΔREQ23850, 8.29±0.22×10⁴. Means of at least three independent duplicate experiments ±SE. Asterisks denote significant differences from wild type with P≤0.001 (two-tailed Student's t test). Except for the intracellular proliferation defect, the two mutants were phenotypically indistinguishable from the wild-type parental strain 103S, including growth kinetics in broth medium.

Figure 7. Structure of the chromosomal locus of the putative chorismate mutase (CM) and anthranilate synthase (AS) genes REQ23860 and REQ23850.

The locus contains two additional genes, REQ23840 and REQ23830, encoding a putative prephenate dehydrogenase (PD) and a hypothetical protein (HP), respectively. The four genes are conserved at the same chromosomal location in the environmental Rhodococcus spp (CDS numbers indicated), including R. opacus B4.