An Invertron-Like Linear Plasmid Mediates Intracellular Survival andVirulence in Bovine Isolates of Rhodococcus equi

Ana Valero-Rello,a Alexia Hapeshi,a Elisa Anastasi,b Sonsiray Alvarez,b Mariela Scortti,a,b Wim G. Meijer,c Iain MacArthur,b

José A. Vázquez-Bolanda,b,d

Microbial Pathogenesis Unit, School of Biomedical Sciences and Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdoma;Division of Infection and Immunity, Roslin Institute, University of Edinburgh, Edinburgh, United Kingdomb; School of Biomolecular and Biomedical Science, UniversityCollege Dublin, Dublin, Irelandc; Grupo de Patogenómica Bacteriana, Facultad de Veterinaria, Universidad de León, León, Spaind

We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultativeintracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circularvirulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linearplasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathoge-nicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN renderedR. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demon-strated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assignedthe vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmidtype-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted“bovine-type” allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage pro-liferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids.Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in thecommon ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution withspecific animal species.

Rhodococcus equi is a Gram-positive aerobic coccobacillus of theCorynebacteriales associated with chronic or subacute pyo-

genic infections (1, 2). A normal soil inhabitant, the bacteriumuses manure as a growth substrate, multiplies in the herbivore’slarge intestine, and is ubiquitous in the farm environment. Trans-mission occurs via contaminated dust particles, mostly throughairborne exposure (3, 4). R. equi is the causative agent of a majorinfectious disease of horses that affects young foals worldwide.The infection is characterized by multifocal purulent broncho-pneumonia, often accompanied by ulcerative or abcessating le-sions in the intestine (1, 5). While best known as an equine patho-gen, R. equi also infects other animal species (1, 6–8). In abattoirsurveys, R. equi is frequently recovered from porcine submaxillarylymph nodes with granulomatous lesions as well as from appar-ently healthy pigs (9–11). In cattle, it is typically isolated fromcaseating abscesses in respiratory lymph nodes resembling bovinetuberculosis (TB) lesions (12). R. equi is also recognized as anopportunistic pathogen in humans, where it causes severe TB-likepurulent cavitary pneumonia, bacteremia, and localized extrapul-monary infections (8, 13, 14).

R. equi pathogenesis depends on the capacity of the bacteriumto survive and replicate within host macrophages (15–18). Inequine isolates, this ability is conferred by a conjugative circularplasmid of 80 kb (19–21) that promotes intravacuolar survival byinterfering with phagosome maturation (22). These properties aremediated by the vap pathogenicity island (PAI) (23), a horizontalgene transfer (HGT) locus (24). A hallmark of the vap PAI is thepresence of a multigene family encoding homologous virulence-associated proteins (Vaps) (20, 24, 25). One of them, VapA, a19-kDa secreted protein, is essential for virulence. A single vapAgene deletion causes strong attenuation comparable to that caused

by loss of the plasmid, with an inability to both proliferate inmacrophages and survive in vivo in mice (26, 27).

Emerging evidence suggests that the virulence plasmid mayalso play a key role in R. equi host tropism. Early studies showedthat VapA-encoding virulence plasmids were typical of equinestrains (28, 29), while a second plasmid type, encoding VapB, aVapA variant (24), was common among nonequine (pig and hu-man) isolates (10, 30–33). Recently, the existence of a third type ofR. equi virulence plasmid was identified in bovine and humanisolates that were initially deemed to be “plasmidless” (becausevapA and vapB negative) but that tested positive for a traA plasmidconjugal transfer gene marker (34). Molecular epidemiologicalanalysis of a global collection of R. equi isolates established that thevapA�, vapB�, and novel vapAB-negative plasmid types were eachassociated with a specific nonhuman host, i.e., equine, porcine,and bovine, respectively (34). Using a unified nomenclature, these

Received 18 March 2015 Returned for modification 9 April 2015Accepted 16 April 2015

Accepted manuscript posted online 20 April 2015

Citation Valero-Rello A, Hapeshi A, Anastasi E, Alvarez S, Scortti M, Meijer WG,MacArthur I, Vázquez-Boland JA. 2015. An invertron-like linear plasmid mediatesintracellular survival and virulence in bovine isolates of Rhodococcus equi. InfectImmun 83:2725–2737. doi:10.1128/IAI.00376-15.

Editor: A. J. Bäumler

Address correspondence to José A. Vázquez-Boland, v.boland@ed.ac.uk.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00376-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00376-15

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plasmids were designated pVAPA, pVAPB, and pVAPN (for“noA-noB”), respectively (1, 24). In contrast to their unique ani-mal species specificity, the three host-adapted virulence plasmidtypes were commonly detected in human isolates. Besides point-ing to a zoonotic origin of infection, this lack of plasmid typeselectivity was consistent with humans being an opportunistic,nonadapted host for R. equi (34).

Sequencing of the pVAPA and pVAPB virulence plasmids re-vealed that they are essentially the same circular replicon (24). Theanalyzed plasmids, pVAPA1037 and pVAPB1593 (numerical suf-fix indicates the source strain according to proposed harmonizednomenclature for R. equi virulence plasmids) (24), shared a virtu-ally identical backbone encoding replication/partitioning andconjugal transfer functions. In contrast, the vap PAI was moredivergent, differing in both size and vap gene complement, i.e.�21 kb and nine vap genes for pVAPA (vapA, -C, -D, -E, -G, and-H and the pseudogenes vapF, -I, and -X) versus �15 kb and sixvap genes for pVAPB (vapB, -J, -K1, -K2, -L, and -M) (24). Inaddition to major Vap polypeptide sequence diversification, vapmultigene family rearrangements and insertion/deletions affect-ing adjacent genes accounted for the PAI differences. This sug-gested that the vap PAIs were evolving at a higher rate than theconserved housekeeping backbone, consistent with diversifyingselection and a possible role in host-specific adaptation (24).

Here, we report the genomic analysis and characterization ofpVAPN, the bovine-type R. equi virulence plasmid.

MATERIALS AND METHODSStrains, culture conditions, and reagents. R. equi PAM1571 is a proto-typic traA� vapAB-negative bovine strain (34) isolated from a heifer’smediastinal lymph node with pyogranulomatous lesions (kindly providedby F. Quigley, Central Veterinary Research Laboratory, Ireland) (12). Itsplasmid-cured derivative, PAM1571�, was obtained by subjecting wild-type bacteria to an electroporation pulse of 12.5 kV/cm at 1,000 � and 25�F (GenePulser Xcell; Bio-Rad) followed by six cycles of plating and sin-gle-colony subculturing in liquid medium at 37°C (27). These two strainsare henceforth designated 1571 and 1571�, respectively. R. equi PAM2012is another traA� vapAB-negative bovine strain, isolated in Germany froma case of lymphadenitis in cattle (kindly provided by C. Lämmler, Veter-inary Faculty, University of Giessen) (35). R. equi 103S is the referencegenome strain, a low-passage clone of equine clinical isolate 103� used indifferent laboratories (24). Its isogenic 103S�vapA and plasmid-cured103S� derivatives were described previously (27). The presence of thevirulence plasmid was routinely checked in all strains by PCR using suit-able oligonucleotide primers (see Table S1 in the supplemental material).R. equi was grown in brain heart infusion (BHI; Difco-BD) or Luria-Bertani (LB; Sigma) medium at 30°C unless stated otherwise. The cloninghost strain Escherichia coli DH5� was grown at 37°C in LB medium. Mediawere supplemented with 1.5% (wt/vol) agar and/or antibiotics as appro-priate. Fluid cultures were incubated with shaking (200 rpm). Chemicalsand primers were purchased from Sigma-Aldrich unless stated otherwise.

DNA techniques. Total DNA extraction and purification from R. equi,PCR techniques, DNA fragment purification and electrophoresis, recom-binant DNA techniques, and plasmid purification and electroporationwere performed as previously described (27, 34, 36). For pulsed-field gelelectrophoresis (PFGE), plugs were formed by embedding R. equi cellsfrom 1-ml aliquots of stationary (24-h) BHI cultures in melted 1% agaroseTris-EDTA (TE). Plugs were incubated in 1 mg/ml lysozyme, 50 mMNaCl, 10 mM Tris-HCl, pH 8.0, at 37°C for 2 h, washed in 50 mM EDTA,20 mM Tris-HCl, pH 8.0, and incubated in 1 mg/ml proteinase K, 100 mMEDTA, 0.2 g/ml sucrose, 10 mM Tris-HCl, pH 8.0, at 50°C overnight.Plugs were then loaded into a 1% Pulsed Field Certified agarose gel (Bio-Rad) prepared with 0.5 Tris-borate-EDTA (TBE) buffer. DNA was sep-

arated in a Chef-DR II pulsed-field electrophoresis system (Bio-Rad) at avoltage of 5 V/cm2, with a switch time ramping from 20 to 30 s and a 23-hrun time at 14°C. Southern blotting was performed by transferring re-solved DNA fragments onto a positively charged nylon membrane aftertreatment of the PFGE gels with 0.25 M HCl for 30 min followed bydenaturing solution (1.5 M NaCl, 0.4 M NaOH) for 20 min (twice) andneutralizing solution (1.5 M NaCl, 0.5 M Tris-Cl2 [pH 7.0]) for 20 min(twice). Membranes were hybridized by using a specific vapN-vapQ PCRfragment (see Table S1 in the supplemental material) labeled with digoxi-genin (DIG High Prime DNA labeling and detection kit; Roche).

pVAPN sequencing and phylogenetic analyses. pVAPN1571 waselectroeluted from preparative PFGE gels by using a model 422 apparatus(Bio-Rad) and paired-end (2 36 bp) sequenced in an Illumina (Solexa) IIgenome analyzer at the Edinburgh Genomics Facility. To complete theplasmid assembly, host strain 1571 DNA was paired-end (2 100 bp)sequenced from a 500-bp PCR-free library using an Illumina HiSeq 2000sequencing system at the Beijing Genomics Institute. pVAPN2012 wasentirely sequenced by using the latter approach. Reads were assessed forquality by using FastQC and then trimmed for adaptors by using Scytheand for low-quality reads by using Sickle. De novo assembly was per-formed by using SPAdes followed by manual verification by PCR map-ping and Sanger resequencing of specific regions. The 5=-end telomericsequence of pVAPN was experimentally confirmed (37) using the suicidevector pSelAct (38) and vector-encoded apramycin resistance for select-ing positive clones. The pVAPN sequence was manually curated and an-notated in Artemis by using the software and databases listed in Table S2in the supplemental material. For phylogenetic analyses, orthologs wereidentified by reciprocal tBlastx analysis with 30% identity over 60% ofthe protein sequence as a minimum similarity score. Paralogous genespredicted based on the topology of neighbor-joining trees and pseudo-genes (except vap pseudogenes) were avoided. Translated products fromeach ortholog cluster were aligned by Muscle (except where otherwisestated) and back-translated in Mega5. The best evolutionary model fornucleotide substitution was selected according to Akaike information cri-terion (AIC) in jModelTest. For multilocus sequence alignment (MLSA),gene alignments were concatenated with Seaview. Maximum likelihood(ML) trees were constructed in PhyML. See Table S2 in the supplementalmaterial for bioinformatics and phylogenetic analysis software references/URLs.

Construction of the vapN deletion mutant. The vapN gene was in-frame deleted from pVAPN1571 by double homologous recombination(36) using 5-fluorocytosine counterselection (38). Briefly, oligonucleo-tide primer pairs Nmutant_a (EcoRI)/Nmutant_b1 (XmaI) and Nmu-tant_c1 (XmaI)/Nmutant_d (SpeI) (see Table S1 in the supplemental ma-terial) were used to PCR amplify two DNA fragments of 908 and 910 bpcarrying the last three 5=-terminal and four 3=-terminal codons of vapNplus adjacent upstream and downstream regions, respectively. The PCRamplicons were joined via the XmaI site introduced by the Nmutant_b1and Nmutant_c1 primers, the ligation product was inserted into the pSe-lAct vector (38) using the external SpeI and EcoRI sites introduced byprimers Nmutant_a and Nmutant_d, and the resulting plasmid was elec-troporated into 1571. Allele exchange was checked by PCR mapping usingsuitable primers (see Table S1 in the supplemental material), and thein-frame deletion was confirmed by DNA sequencing.

Mating experiments. Transfer of the virulence plasmid between R.equi bacteria was investigated by using a mating protocol essentially aspreviously described (39). Cultures of donor and recipient R. equi strainsgrown overnight in BHI were harvested by centrifugation, resuspended inphosphate-buffered saline (PBS) to a cell density of �107 CFU, mixed�1:1, and spotted in a 5-�l drop onto BHI agar. The recipient 103S�

bacteria carried a chromosomal rifampin resistance (Rmpr) marker.103S�RmpR bacteria were isolated by selection of spontaneously resistantmutants on increasing concentrations of rifampin, from 25 to 100 �g/ml,and stabilization by repeated subculturing in the presence of the highestconcentration of the antibiotic. After incubation of the mating mixture at

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30°C for 72 h, bacteria were collected in 1 ml of PBS, serially diluted, andplated onto BHI agar without and with supplementation with 100 �g/mlrifampin. At this rifampin concentration, no Rmpr colonies were detectedin the donor-only control plates. Transconjugants were identified amongRmpr colonies by simultaneous PCR detection of virulence plasmid-spe-cific markers and of recipient-specific chromosomal gene markers usingad hoc oligonucleotide primers (see Table S1 in the supplemental mate-rial).

Macrophage cultures and infection assay. Low-passage murineJ774A.1 macrophages and human monocyte-like THP-1 cells were ob-tained from the ATCC and cultured at 37°C with 5% CO2 in Dulbecco’sminimal essential medium (DMEM) supplemented with 10% decomple-mented fetal bovine serum, 2 mM glutamine, and 1 mM pyruvate. THP-1cells were initially grown in suspension in RPMI 1640 medium with thesame supplements. Cells were seeded onto 24-well plates at a density of�2 105 cells/well and incubated overnight in DMEM and, for THP-1monocytes, in the presence of 50 ng/ml phorbol 12-myristate 13-acetate(PMA) to allow differentiation into macrophages. Infection assays wereperformed on �80% confluent macrophage monolayers as previouslydescribed (40). Intracellular proliferation data were normalized to theinitial counts at time zero (t � 0) by using an “intracellular growth coef-ficient” (IGC) according to the formula IGC � (IBn � IB0)/IB0, where IBn

and IB0 are the intracellular bacterial numbers at t � n and t � 0, respec-tively (40, 41).

Mouse infections. Experiments were performed at the Animal Facilityof the School of Biological Sciences of the University of Edinburgh usingin-house-bred 6- to 8-week-old BALB/c mice. Mouse intranasal and in-travenous (i.v.) infections and lung competitive virulence assays were per-formed as previously described (27). The relative proportions of compet-ing bacteria were calculated by analyzing at least 40 random colonies fromthe plated organ homogenate by PCR using suitable oligonucleotideprimers (see Table S1 in the supplemental material). Competitive index(CI) values were calculated by using the formula CI � (test/reference logCFU ratio at t � n)/(input test/reference log CFU ratio in the inoculum)(27). Mouse experiments were approved by the University of EdinburghEthical Review Committee and were covered by a project license grantedby the United Kingdom Home Office under the Animals (Scientific Pro-cedures) Act of 1986.

Statistics. Intracellular proliferation and uptake data were comparedby using two-way analysis of variance (ANOVA) and one-way ANOVA,respectively, followed by Šidák post hoc multiple-comparison tests. One-sample Student’s t tests were used to determine if CI values differed sig-nificantly from 1 (i.e., the expected CI value if the ratio of the competingstrains remains the same with respect to the ratio at t � 0). Statisticalanalyses were performed by using Prism 6.0 software (GraphPad, SanDiego, CA).

Nucleotide sequence accession numbers. The pVAPN1571 andpVAPN2012 genome sequences have been deposited in GenBank underaccession no. KF439868 and KP851975, respectively.

RESULTS AND DISCUSSIONIdentification and sequencing of pVAPN. Attempts to isolate thenovel traA� vapAB-negative plasmid type (34) from 1571 andother bovine isolates by using the procedure for R. equi circularvirulence plasmid extraction (34) were unsuccessful. However,PFGE analysis of undigested genomic DNA revealed a distinctband in the range of �100 kb in all bovine strains tested from ourcollection (n � 22). This band was not detected in R. equi strainscarrying a circular virulence plasmid, e.g., 103S harboring pVAPA(Fig. 1). Similarity searches of exploratory low-coverage whole-genome 454 pyrosequencing assemblies from 1571 with thepVAPA reference sequence from R. equi 103S (40) identified con-tigs harboring vap PAI-homologous genes. Southern blotting us-ing a probe from this novel vap PAI identified the �100 kb PFGEband as the putative pVAPN virulence plasmid (Fig. 1). Most(95%) tested traA� vapAB-negative bovine isolates (34) were pos-itive for pVAPN by PCR using a plasmid-specific vap PAI marker(vapN, the counterpart of the equine vapA and porcine vapB genes[see below]). The �100-kb band from strain 1571 was isolatedfrom PFGE gels and shotgun sequenced. The pVAPN1571 ge-nome sequence was completed as described in Materials andMethods.

pVAPN1571 is 119,931 bp long and contains 148 open readingframes (ORFs), 10 of which are pseudogenes (Fig. 2). The averageG�C content is 66.2%, similar to that of R. equi genomic DNA(68.7%) (40). pVAPN1571 is predicted to be a linear repliconbased on its PFGE migration pattern (42), the presence of a traBconjugal translocase determinant (see below), phylogenetic relat-edness to other Rhodococcus linear plasmids (Fig. 3A), and thepresence of telomeric invertron-like terminal inverted repeats(TIRs) with multiple palindromic secondary structures (Fig. 4)(43–45). The TIR sequences of pVAPN are 569 bp long and 99%identical. The nucleotide sequence of a second example of thepVAPN plasmid, from a bovine isolate from Germany(PAM2012), was virtually identical to that of pVAPN1571 exceptfor the presence of two additional ORFs at the left end (see Fig. S1in the supplemental material).

Comparative analysis and functional overview. Rhodococcusspecies characteristically possess large plasmids, circular or linearif 100 kb in size (46). These plasmids consist of a vertically evolv-ing backbone, encoding plasmid maintenance and conjugal trans-fer functions, and a horizontally acquired variable region (VR)providing specific niche-adaptive properties to the host bacterium(24, 37, 44, 45, 47). The housekeeping backbone of pVAPN isunrelated to that of the circular pVAPA/B (equine/porcine-type)R. equi virulence plasmids. pVAPN is instead closely related to thelinear plasmid pNSL1 from Rhodococcus sp. NS1 (48) in terms ofgenetic structure and synteny (Fig. 2). pNSL1 has a size (117,252bp) similar to that of pVAPN, and its backbone is perfectly colin-

FIG 1 Detection of pVAPN by PFGE. (A) Genomic DNA of bovine isolate1571 and equine isolate 103S; three and two independent lysates per strain areshown. Relevant positions of the lambda PFGE marker (New England Bio-Labs) are indicated. pVAPN is observable as a distinct band of �100 kb in thebovine isolate. (B) Southern blot analysis of bovine isolates PAM1571,PAM1533, and PAM1554 (strain 103S was used as a negative control). (Left)Relevant sections of the PFGE gel; (right) membrane hybridized with apVAPN-specific DNA probe (600-bp fragment encompassing the 3= region ofvapN and the 5= region of vapQ). The arrow indicates the pVAPN band.

R. equi Bovine-Associated Virulence Plasmid pVAPN

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ear with pVAPN. No significant overall similarity with other se-quenced linear plasmids from the genus Rhodococcus was detectedin pairwise alignments (see Fig. S3 in the supplemental material).However, MLSA analysis of gene orthologs from the housekeep-ing backbones of a representation of rhodococcal linear and cir-cular plasmids placed pVAPN within a monophyletic clade to-gether with the linear replicons, indicating that they all share acommon origin (Fig. 3A).

(i) Conjugation genes. pVAPN encodes a MOBf (TrwC) fam-ily conjugal relaxase (49) homologous to TraA from pVAPA/B(24). Detection of its coding sequence (pVAPN_0650) by PCRusing conserved traA target sequences from pVAPA/B allowed thediscovery of the traA� vapAB-negative bovine pVAPN plasmid inthe first instance (34). Relaxases play a key role in the conjugationof circular plasmids, nicking the supercoiled double-strandedDNA (dsDNA) and leading the nascent DNA strand into the re-cipient cell in conjunction with a type IV secretion system (T4SS),which forms the transport channel (50, 51). Indeed, deletion oftraA has been shown to prevent the transfer of the equine pVAPAcircular virulence plasmid (39). Interestingly, however, thepVAPN traA relaxase gene is corrupted (5=-terminal deletion af-fecting the first 75 codons, including the gene start and part of theTrwC relaxase domain, and frameshifts in the 3=-terminal region)and probably nonfunctional. This traA pseudogene is located out-side the pVAPN conjugation module at the left boundary of the

vap PAI. It is immediately contiguous to three ORFs, encodingphage excisionase, Rep, and CopG (regulator of plasmid copynumber) homologs, also present in the pVAPA/B backbone (24)but absent in pNSL1. These three ORFs and the adjacent pVAPNvap PAI were identified as HGT acquired based on local DNAcompositional bias, as determined by the Alien_hunter program(52) (Fig. 2). This suggests that the traA pseudogene-phage exci-sionase-rep-copG genes are remnants of a lateral gene exchange,probably the same one that mobilized the vap PAI from the circu-lar virulence plasmid.

Despite traA being a pseudogene and a relaxase-associatedT4SS apparatus being absent, pVAPN is transferable by mating(see below). This is probably mediated by pVAPN_0320, encod-ing a TraB-like plasmid translocase, also present in pNSL1. TraBtranslocases are evolutionarily related to the septal FtsK/SpoIIEfamily proteins involved in chromosome segregation (51) andmediate a novel relaxase/T4SS-independent mechanism of conju-gation in Streptomyces linear replicons (53). They all share a sim-ilar structural arrangement, with an AAA� motor ATPase domainwith characteristic Walker A and B boxes, a transmembrane do-main, and a C-terminal DNA-binding winged helix-turn-helixmotif (53). In translocase-mediated conjugation, TraB binds toplasmid dsDNA and forms a transmembrane DNA-conductinghexameric channel through which the plasmid is transferred tothe recipient bacterium in an ATP-dependent manner. The

FIG 2 Genome alignments of the linear virulence plasmid pVAPN, circular virulence plasmids pVAPA and pVAPB, and the respective closest homologs fromnonpathogenic rhodococcal species (pNSL1 from Rhodococcus sp. NS1 [48] and pREC1 from R. erythropolis [44]). The alignments were built with EasyFig(http://easyfig.sourceforge.net/). The circular plasmids (pVAPA, pVAPB, and pREC1) were linearized starting from the first conserved gene of the housekeepingbackbone. Regions with significant similarity between plasmids are connected by gray stripes (tblastx E value threshold of 0.1); grayscale indicates percentsimilarity. ORFs are color coded as follows: hypothetical proteins (gray), conjugation or DNA replication/recombination/metabolism (red), DNA mobility genes(magenta), transcriptional regulators (blue), secreted proteins (dark green), membrane proteins (pale green), metabolic functions (yellow), vap family genes(black), and pseudogenes (brown). Green and pale red bars below the genes indicate conjugation and replication/partitioning functional modules, respectively;dashed underline indicates HGT regions identified by Alien_hunter (52); and the triangle indicates a putative origin of replication. Abbreviations: 3oxoACPr,3-oxoacyl-ACP reductase; 3oxoACPs, 3-oxoacyl-ACP synthase; Endo, endonuclease; Exo, exonuclease; Lig, ligase; LT, lytic transglycosylase; MT, methyltrans-ferase; Mtase, methylase; PK, protein kinase; PR, pentapeptide repeat protein; ssRec, site-specific recombinase; TPR, TPR repeat protein; FA met, fatty acidmetabolism.

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pVAPN (and pNSL1) conjugation module also comprises (i) anadditional AAA� ATPase with sequence similarity to the conjuga-tive coupling factor TraD (pVAPN_0360); (ii) a homolog of aSoj/ParA family ATPase (pVAPN_0300), involved in chromo-somal and plasmid DNA segregation (54) and recruitment of con-jugative DNA to the transfer channel (55); (iii) a putative M23endopeptidase family/lysozyme-like lytic murein transglycosy-lase/cell wall hydrolase (pVAPN_0390), probably involved in con-jugation channel formation (a homolog of which is also present inthe circular pVAPA/B virulence plasmids and the related Rhodo-coccus erythropolis plasmid pREC1) (Fig. 2); and (iv) a number ofputative membrane-associated proteins. In addition, pVAPN_0550, at the other side of an interposed plasmid replication/parti-tioning (rep-parA) module, encodes a putative cutinase. A cuti-nase gene is also present at the boundary of this replication/partitioning module and the conjugation module in the circularpVAPA/B and pREC1 replicons (Fig. 2). Bacterial cutinase-likeproteins, common among mycolic acid-containing actinomy-cetes (40, 56), have esterase/lipolytic activity (56–58). In phagesthat infect mycolata, they form part of the LysB lipolytic enzymecomplement, thought to aid in the breakdown of the lipid-rich

envelope during phage penetration or lytic egress (59). Recently, acutinase from the R. fascians pFiD188 linear virulence plasmid wasshown to be required for efficient conjugation, probably by facil-itating the penetration of the DNA translocation complex in therhodococcal cell envelope (45).

(ii) Replication/partitioning. The pVAPN self-replication de-terminant includes a module encoding a Rep protein (pVAPN_0480), which probably directs the bidirectional replication of theplasmid toward the telomeres, and the plasmid-partitioning pro-tein/ATPase ParA (pVAPN_0500). A 26-bp semipalindromic se-quence (5=-AAAACCCCCAGGTGGGGGTGGG-TTTT) similarto that determined to be the origin of replication of the pNSL1plasmid (48) was identified at the same position upstream of therep gene in pVAPN (Fig. 2). The rep-parA module is detected asHGT genetic material in pNSL1 and is conserved in the circularpVAPA/B and R. erythropolis pREC1 plasmids (also identified asHGT acquired). In pVAPN, it is flanked on the right by a phageexcisionase gene (pVAPN_0520), which is conserved in pNSL1and, interestingly, also in the circular replicons despite these beingderived from a different ancestor (Fig. 2). This lends additionalsupport to the hypothesis that the rep-parA determinant formspart of an “exchangeable” gene cassette subjected to HGT betweendifferent rhodococcal plasmids (24). This replication/partitioningregion appears to serve as an insertion platform for HGT-acquiredDNA (24), as suggested by the fact that the VR either is immedi-ately adjacent to (pVAPA/B circular plasmids) or interrupts(pVAPN, pNSL1, and the larger circular plasmid pREC1) it (Fig.2). Interestingly, in contrast to the circular pVAPA/B plasmids(and pREC1), pVAPN (and pNSL1) does not encode the ParBcomponent of the ParAB replicon segregation system (60). Thelack of a parB gene appears to be a hallmark of the �400-kb rho-dococcal linear extrachromosomal replicons, as exemplified bypREL1 or pBD2 from R. erythropolis (44), pRHL2 and pRHL3from R. jostii (37), or pFiD188 from R. fascians (45).

(iii) Plasticity region (VR). The colinearity with pNSL1 isabruptly interrupted at the level of the traA pseudogene, markingthe start of the VR. The VR of pVAPN is interrupted by an islandof homology with ORFs from the right end of the pNSL1 back-bone, suggesting that it was formed by two independent DNAacquisition events (Fig. 2). The left VR section comprises thepVAPA/B-homologous phage excisionase-rep-copG sequencemodule (see above) plus the vap PAI; the right section encodesrhodococcal/actinobacterial conserved hypothetical proteins anda number of products with various predicted functions (Fig. 2).The complete left VR section with the vap PAI was identified asHGT material (Fig. 2), suggesting that it is a more recent acquisi-tion.

vap PAI. The genetic structure of the vap PAI of pVAPN issimilar to that of pVAPA/B (Fig. 5). It is 15.1 kb in length andcontains 21 ORFs, including (i) a complement of six vap genes(vapN, vapO, vapP, vapQ pseudogene, vapR, and vapS) encodingpolypeptides differing in amino acid sequence identity withpVAPA/B Vaps by 20 to 81% (see Table S3 in the supplementalmaterial); (ii) a vir locus encoding the two key vap PAI transcrip-tional regulators, VirR (LysR type) and VirS (orphan two-compo-nent response regulator) (61, 62), the major facilitator superfam-ily (MFS) transporter IcgA (63), VapP, and a conserved protein ofunknown function; and (iii) several additional non-vap genes(Fig. 5). Four of the latter are conserved as functional genes in thethree virulence plasmids, indicating that they are core compo-

FIG 3 ML trees of Rhodococcus plasmid backbones (A) and the R. equi vapmultigene family (B). The Hasegawa-Kishino-Yano with gamma distribution(HKY�G) evolutionary model was used. (A) ML tree based on a concatenatedalignment of orthologs from a selection of rhodococcal extrachromosomalreplicons (total of 7,802 nucleotides). The genes used are indicated by dots inFig. 2. Values 50 for 100 bootstrap replicates are indicated. Symbols: trian-gles, linear plasmids; circles, circular plasmids. (B) vap family members de-rived from each of the predicted seven precursor vap genes in the MRCA of theextant pVAPA, pVAPB, and pVAPN PAIs are in gray balloons.

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nents of the PAI: pVAPN_0700 (pVAPA/B_0420), encoding a hy-pothetical protein with similarity to a CopG family transcriptionalregulator (here designated cgf), which is probably the first gene ofthe vap PAI instead of the downstream lsr2 gene considered ini-tially (24); pVAPN_0720 (pVAPA/B_0440), encoding a putativenucleoid-associated protein similar to Lsr2, which in mycobacte-ria is involved in a number of virulence-related functions (64–66);pVAPN_0760 (pVAPA/B_0470), encoding an S-adenosylmethio-nine (SAM)-dependent methyltransferase with a potential regu-latory role via protein, nucleic acid, or lipid methylation; andpVAPN_870 (pVAPA/B_0570), also known as the vap-coregu-lated vcgB gene in pVAPA, encoding a hypothetical protein con-served in pathogenic mycobacteria (67) (Fig. 5). At the right end,the putative transposon invertase/resolvase gene invA, found inpVAPA/B and in the VR of the related rhodococcal circularpREC1 plasmid (24), is replaced in pVAPN by tniA-like trans-posase/integrase and tniQ-like transposase helper protein pseudo-genes (Fig. 5).

vap PAI evolution. Maximum likelihood (ML) trees groupedthe vap multigene family from pVAPN, pVAPA, and pVAPB into

several well-supported clades (Fig. 3B; see also Fig. S4A in thesupplemental material). vap family members were not clus-tered by plasmid; instead, vap sequences from different viru-lence plasmids were grouped under each of the nodes, suggestingthat they are allelic variants of a vertically evolving vap precursorgene. Three of the clades contained vap sequences from only oneor two of the plasmid types, suggesting a loss of vap alleles. Inaddition, in two cases, the clades included more than one vapsequence from the same PAI, consistent with instances of vap geneduplication (Fig. 3B; see also Fig. S4A in the supplemental mate-rial). To help pinpoint the gene duplication and loss events un-derlying the evolution of the R. equi vap family, the vap gene treeand a “species” tree of the three PAIs based on their conservednon-vap genes (see Fig. S4B in the supplemental material) werecompared by using Notung phylogenetic reconciliation software(68) (see Fig. S5 in the supplemental material). The phylogeneticdata were then interpreted in combination with a detailed com-parative analysis of the genetic structure of the PAIs (Fig. 5). Fromthese analyses, we inferred that the most recent common ancestor(MRCA) of the three vap PAIs probably comprised seven precur-

FIG 4 pVAPN telomeric sequences. (A) Clustal� alignment of the left- and right-end 200 terminal nucleotides. Identical nucleotides are shaded (dark and lightblue, purines and pyrimidines, respectively). Inverted repeats are indicated above the sequence. In red are four conserved palindromic sequences with the centralmotif GCTNCGC identified in the binding site of telomere-associated proteins involved in Streptomyces linear plasmid replication (72). Several “GCTNCGC”palindromic sequences are normally present in the telomeres of rhodococcal linear plasmids (43–45) (see Fig. S2 in the supplemental material). (B) Secondarystructures potentially formed by the palindromic sequences in pVAPN telomeres, as numbered in panel A. Structures were determined with mFold. Free energy(�G) values: �33.84 kcal/mol (left), �37.95 kcal/mol (right).

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sor vap genes. These genes gave rise to the contemporary plasmidtype-specific allelic variants as schematized in Fig. 3B (see also Fig.S4A and S5 in the supplemental material for additional details).The seven MRCA vap genes likely originated by successive dupli-cation events from a primordial vap gene (Fig. 6; see also Fig. S5 inthe supplemental material), probably acquired by HGT from an-other organism. Indeed, while being R. equi specific among the

actinomycetes, Vap homologs are found in other bacteria fromdifferent phyla or even fungi (see Fig. S4A in the supplementalmaterial).

The presence in pVAPN, adjacent to the PAI, of an orphan,corrupted copy of traA plus other sequences from the pVAPA/Bhousekeeping backbone (phage excisionase-rep-copG HGT se-quence module) (Fig. 2 and 5) suggests that the vapN PAI was

FIG 5 Genetic structure of the vap PAIs from pVAPN (15.1 kb), pVAPA (21.5 kb), and pVAPB (15.9 kb). Color codes of genes: vap family (black), DNAconjugation/partitioning (red), DNA mobility/recombination (magenta), transcriptional regulators (blue), other regulators (cyan), membrane proteins (green),metabolic reactions (yellow). Orthologs are in the same color shade and linked by gray bands. ORFs encoding hypothetical proteins are represented in lightblue-gray and in white if they are outside the PAI. White arrowheads point to the first and last genes of the consensus PAI. The traA pseudogene/phageexcisionase-rep-copG HGT cluster presumably acquired by pVAPN from the pVAPA backbone is boxed. The figure also schematizes the probable evolutionaryrelationships of the vap multigene family as inferred from phylogenetic analyses (Fig. 3B; see also Fig. S4 and S5 in the supplemental material) and PAI geneticstructure; the model minimizes the number of vap gene loss events. Solid lines/arrows connect vap genes belonging to the same monophyletic group (thus likelyrepresenting allelic variants of a common vap gene ancestor). Curved lines/arrows indicate vap gene duplications within a PAI. Crosses denote vap genes that werelost, and asterisks indicate pseudogenes. Two alternative evolutionary paths are shown for vapA-vapB-vapK1-vapK2-vapN (see the legend of Fig. S5 in thesupplemental material for additional details). The black dots indicate the non-vap genes used for the MLSA shown in Fig. S4B in the supplemental material.

FIG 6 Hypothetical reconstruction of vap PAI evolution. (A) Model of vap multigene family evolution. Lines indicate the evolutionary path of the vap genesbetween ancestral PAIs, the most recent common ancestor (MRCA), and extant PAIs. Pre-pVAPA designates the hypothetical direct precursor of the currentpVAPA PAI. Gene duplication events are indicated by red squares, gene loss events by crosses, and pseudogenes by asterisks and white borders. (B) Fate of thevap PAI and R. equi virulence plasmid evolution. (a) Acquisition by rhodococcal circular replicon of vap PAI ancestor conferring the ability to colonizemacrophages; (b) mobilization of vap PAI from pre-pVAPA plasmid to rhodococcal linear replicon; (c) evolution of species specificity.

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mobilized to the linear replicon from an ancestor of the circularplasmid pVAPA/B and not vice versa. A gene translocation iden-tical to that observed for the allelic variants vapG (pVAPA) andvapO (pVAPN) is unlikely to have occurred twice independently,indicating that the vapN PAI probably originated from a directprecursor of pVAPA after diversification from pVAPB. This inter-pretation is supported by a phylogenetic analysis performed withthe non-vap genes of the PAI (see Fig. S4B in the supplemental

material). It also accounts for the presence in the vapN PAI ofpVAPA’s vapI-vapE and vapC-vapF putative allelic variants vapRand vapS, which are absent in pVAPB (Fig. 5; see also Fig. S5 in thesupplemental material). The possibility that the vapA PAI is de-rived from pVAPN is less plausible because it implies the occur-rence, after the mobilization of the PAI, of a second, independenthorizontal transfer/recombination event to convey the traA-phage excisionase-rep-copG module from pVAPA/B to pVAPN.The probable evolutionary history of the vap PAI in the threehost-adapted R. equi virulence plasmids is schematized in Fig. 6.

pVAPN and its vapN gene are essential for intracellular pro-liferation in macrophages. To determine the role of pVAPN invirulence, we obtained an isogenic plasmid-cured derivative of1571 (1571�) and examined its behavior in in vitro infection as-says in mouse J774A.1 and human THP-1 macrophages. Studieswith the equine plasmid previously showed that VapA is essentialfor R. equi virulence (26, 27), in contrast to other pVAPA-encodedVap products (i.e., VapC, VapD, VapE, VapF [26], VapG [23], orVapH [our unpublished data]), which are dispensable or acces-sory. Since, according to our data, vapN is pVAPN=s ortholog/allelic variant of vapA, an unmarked, in-frame vapN deletion mu-tant was also constructed and tested. A plasmidless derivative anda vapA deletion mutant of equine isolate 103S (strains 103S� and103S�vapA, respectively) (27) were used as controls.

Figure 7A shows that both 1571� and 1571�vapN had lost theability to proliferate in J774A.1 and THP-1 cells. The effects wereessentially identical to those observed for 103S� and 103S�vapA,respectively (Fig. 7B). These results demonstrate that the bovineplasmid pVAPN is, like the equine plasmid pVAPA, necessary forfacilitating R. equi parasitization of host macrophages. These re-sults also show that VapN appears to perform an essential func-tion in pathogenesis, similarly to VapA in the equine plasmid (26).The uptake of 1571� and 1571�vapN remained unaffected, as alsoobserved for 103S� and 103S�vapA (see Fig. S6 in the supplemen-tal material), indicating that the effect of the two plasmids andtheir cognate VapN and VapA products is specifically related tointracellular survival and/or replication.

Role of pVAPN and vapN in virulence in vivo. 1571 and plas-midless 1571� were also tested in mice by using a competitive lunginfection model (27). Immunocompetent BALB/c mice were in-fected via the intranasal route with a �1:1 mix of both bacteria,and R. equi burdens were determined by plate counting over a4-day period, where R. equi numbers remain stable in the lung(27). The relative proportions of the two strains at each time pointwere then determined by PCR, and the corresponding CIs werecalculated (see Materials and Methods).

The plasmidless 1571� strain was cleared from the lungs at amuch higher rate than the 1571 parent strain (Fig. 8A). Except fort � 0, when similar numbers of 1571 and 1571� were recovered,the CI was significantly lower than 1 at all time points (Table 1). Byday 3, most (96.3%) of the bacteria were plasmid positive, and atday 4, the 1571� strain was not detected despite total CFU num-bers remaining stable in the lungs, indicating that the plasmid-cured bacteria were strongly outcompeted (Fig. 8A). This patternmirrored the results observed when the same experiment was per-formed with R. equi 103S and 103S� (27). These data demonstratethat the bovine-type pVAPN plasmid, like equine pVAPA, confersto R. equi the ability to survive in vivo in an animal host.

We next analyzed the role of VapN in pVAPN-promoted vir-ulence in mice. Since, according to the macrophage data, the lack

FIG 7 Intracellular proliferation in murine (J774A.1) and human (THP-1)macrophages. Data are expressed as normalized IGC values (see Materials andMethods). Means of data from three duplicate experiments � standard errorsare shown. Statistical significance was analyzed by 2-way ANOVA; P valuesdetermined by Šidák post hoc multiple-comparison tests at each time point areshown if �0.05. (A) Plasmidless derivative and in-frame �vapN mutant ofbovine isolate 1571. Two-way ANOVA P values � 0.0007 for J774A.1 and0.0160 for THP-1. (B) Plasmidless derivative and in-frame �vapA mutant ofequine isolate 103S. Two-way ANOVA P values � 0.0112 for J774A.1 cells and 0.0001 for THP-1 cells.

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of VapN was likely to cause strong attenuation in vivo, we com-pared the competitive ability of 1571�vapN against the nonviru-lent derivative 1571� (27). This approach takes advantage of thegreater sensitivity of competitive tests in assessing small differ-ences in virulence (69, 70). To ascertain the relative importance ofVapN and other pVAPN products in R. equi virulence, it is moreinformative than a comparison with the fully virulent parentstrain.

While 1571� was readily outcompeted by the plasmid-positive1571, sizable numbers of both �vapN and 1571� bacteria wererecovered at all time points (Fig. 8B). This is similar to the behav-ior of 103S�vapA and 103S� under the same experimental condi-tions (27), demonstrating that the loss of VapN is sufficient to

cause a reduction in virulence comparable to that in the absence ofits coding pVAPN plasmid. Nevertheless, the CI data showed par-tial outcompetition of plasmid-cured strain 1571� by the �vapNstrain, particularly at the two first time points (Table 1). No suchdifferences were previously observed in competition assays be-tween 103S�vapA and 103S� (27). This suggests that pVAPNproducts other than VapN are also potentially important for R.equi survival in mice and that there may be some differences in thecontributions of VapN and VapA to virulence in their respectiveplasmid backgrounds.

Collectively, our data support the notion that vapN and vapAare allelic variants of a precursor vap gene essential for R. equivirulence because required for supporting intramacrophage pro-liferation.

pVAPN transferability by conjugation. We finally testedwhether pVAPN is transferable by mating, as predicted from thesequence data. Experiments were carried out with 1571 as thedonor and a rifampin-resistant (Rmpr) plasmidless 103S�

(103S�RmpR) as the recipient. The two strains belong to differentR. equi chromosomal genogroups (our unpublished data). As acontrol, conjugation tests with pVAPA from 103S, for whichtransfer frequencies in the range of 10�2 were previously reported(39), were performed using the same recipient. Transconjugantswere determined by screening a total of 900 random Rmpr (100�g/ml) colonies using suitable recipient- and virulence plasmid-specific PCR markers. Transfer of pVAPA to 103S� was observedat a frequency of 1.25 10�2, but transfer of pVAPN could not bedetected. We reasoned that the linear plasmid pVAPN could betransferable at a low frequency, unworkable for the PCR-basedscreening method used. To circumvent this, a transconjugant se-lection strategy was devised based on the ability of the virulenceplasmid to promote R. equi survival in vivo. BALB/c mice wereinfected i.v. with �4 108 CFU of a mating mix of 1571 and103S�RmpR, followed by plating of spleen and liver homogenatesonto rifampin-containing plates at days 0, 3, and 5 after infection.Based on previously reported data on i.v. infection in mice (27),this time course was expected to lead to a progressive eliminationof plasmid-negative R. equi and a concomitant increase of theplasmid-positive population. The recovered pVAPN-positive/Rmpr bacteria were confirmed to be transconjugants by PCR us-ing suitable strain-specific gene markers and determination ofstrain-specific DNA sequences (see Materials and Methods; seealso Table S1 in the supplemental material).

Figure 9A demonstrates a steady enrichment of pVAPN-posi-tive transconjugants, from 0.5% at day 0 to 34.5% at day 3 and80.3% at day 5. These data show that pVAPN is transferable be-tween different R. equi strains. The positive selection of 103S�

pVAPN transconjugants in mice indicates that the bovine plasmidpromotes R. equi virulence irrespective of the strain hosting it.Experiments in J774A.1 cells demonstrated that the acquisition of

FIG 8 Competitive virulence assay in mouse lung. BALB/c mice (n � 4 pertime point) were infected intranasally with a �1:1 mixture of the test bacteria,and the competing populations were monitored 60 min after infection (t � 0)and then on four consecutive days. Bar height denotes total lung CFU, and thelight and dark gray areas within bars indicate the proportions of the competingbacteria. Corresponding CI values are shown in Table 1. (A) Competitionbetween wild-type bovine isolate 1571 and the isogenic plasmidless derivative1571� at an infection dose of 3.7 107 CFU/mouse (2.3 107 and 1.4 107

CFU, respectively). (B) Competition between the avirulent 1571� strain andan in-frame 1571�vapN deletion mutant at an infection dose of 7.8 107

CFU/mouse (3.2 107 and 4.6 107 CFU, respectively).

TABLE 1 Competitive indexesa

Competing strains

Mean CI � SEM (P value) on day:

0 1 2 3 4

1571� and 1571 1.54 � 0.20 (0.0763) 0.42 � 0.13 (0.0225) 0.40 � 0.12 (0.0407) 0.06 � 0.06 (0.0051) 0.0 (0.0001)�vapN and 1571� 0.88 � 0.13 (0.4761) 2.24 � 0.16 (0.0049) 3.36 � 0.41 (0.0107) 1.47 � 0.09 (0.0363) 2.68 � 0.64 (0.1197)a Shown are CI values for the mouse infection experiments depicted in Fig. 8. A CI equal to 1 is the theoretical value for two strains with the same competitive ability. Significance ofthe data was calculated by comparing the experimental CI value at each time point to a theoretical value of 1 (one-sample Student’s t test).

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pVAPN is sufficient to confer to R. equi the capacity for intracel-lular proliferation in macrophages (Fig. 9B).

Conclusions. Our previous work established that the equine-type pVAPA and porcine-type pVAPB virulence plasmids are thesame circular replicon in which the HGT-acquired vap PAIevolved divergently, presumably by host-driven selection (24).Our new data show that the bovine pVAPN plasmid originated byhorizontal mobilization of the vap PAI to a linear replicon. ThepVAPN vap locus, like that of pVAPA/B, is an HGT island and isflanked by DNA mobility genes, consistent with a recent lateralacquisition, probably involving a phage or an integrative element.

Both the circular pVAPA/B (24) and linear pVAPN backbonesshare common ancestry with other extrachromosomal conjuga-tive replicons found in environmental rhodococci. Rhodococcalplasmids play a key role in facilitating adaptation to different hab-itats via plasticity regions rich in HGT material. In environmentalbiodegradative rhodococci, these plasticity regions typically en-code catabolic, detoxification, or secondary metabolic determi-nants, while in pathogenic species (R. equi and R. fascians) they arevirulence related (1, 46, 71). In the phytopathogen R. fascians,

virulence is conferred by a linear conjugative plasmid (45) withoutobvious similarity to pVAPN, illustrating that multiple extrachro-mosomal elements serve as platforms for the expression and dy-namic exchange of niche-adaptive traits in the genus Rhodococcus.

Our findings suggest the following hypothetical scenario forthe evolution of pathogenicity in R. equi (Fig. 6). First, the acqui-sition of an ancestral vap PAI by a circular conjugative plasmidendowed a “pre-R. equi” obligate saprotroph with intracellularsurvival capability in macrophages, promoting its conversion intoa facultative parasite by a process known as “co-optive” virulenceevolution (40). During coevolution with animal hosts, porcine-and equine-specific tropism evolved as a secondary trait of the PAIin the circular plasmid (Fig. 6B), involving gene duplication andsequence diversification within the vap multigene family (Fig.6A). Finally, acquisition of the vap PAI by a linear plasmid, pre-sumably from a direct precursor of the equine pVAPA plasmid(Fig. 6B), gave rise to the bovine-adapted pVAPN plasmid, inwhich another set of specific vap genes evolved (Fig. 6A).

Our analyses with pVAPN confirm the notion that the primaryfunction of the host-adapted R. equi virulence plasmids is to sup-port intracellular proliferation in macrophages. This primordialfunction is clearly dissociable from host tropism, since epidemio-logical or experimental evidence indicates that R. equi plasmidspromote virulence in accidental (nonadapted) animal hosts, suchas humans or mice, regardless of their species-specific type. Ourdata indicate that a specific vap gene, which was already present inthe nearest common ancestor of the contemporary PAIs andevolved into the allelic variants vapA in pVAPA and vapN inpVAPN (and possibly vapB in porcine pVAPB), is critical for in-tracellular survival in macrophages.

Why bovine host tropism evolved in a linear replicon andnot by further host-driven diversification of the PAI in thecircular pVAP replicon remains unclear. Since pVAPA andpVAPB share a virtually identical circular backbone, equine-and porcine-specific infectivity most likely resides in their di-vergent vap PAI. Whether determinants outside the vap PAI inthe unrelated pVAPN backbone contribute adaptive featureswhich optimize the interaction of R. equi with the bovine hostrequires further investigation.

The findings in this study establish R. equi as a novel paradigmof a multihost-adapted pathogen. The pVAPN plasmid reportedhere, together with the previously characterized equine- and por-cine-associated plasmids, provides a unique model system to gaina better understanding of the bacterial mechanisms of intramac-rophage survival and host tropism.

ACKNOWLEDGMENTS

We thank F. Quigley and C. Lämmler for providing the bovine isolatesfrom which the pVAPN plasmid was sequenced, M. Letek for his earlycontributions to the R. equi genome program, J. Navas for initial helpwith mating experiments, P. Iglesias for technical assistance, and U. Fog-arty and colleagues at the Irish Equine Centre for encouragement andsupport. We also thank Edinburgh Genomics (http://genomics.ed.ac.uk/)for pVAP1571 plasmid DNA sequencing and preliminary read assembly.

This work was supported by the Horserace Betting Levy Board(grants vet/prj/712 and 753 to J.A.V.-B.), partially by the Research Stim-ulus Fund (grant RSF 06 379 to J.A.V.-B. and W.M.), and by core BBSRCfunding from the Roslin Institute. A.V.-R. was supported by a EuropeanUnion Marie Curie postdoctoral fellowship, A.H. and E.A., respectively,by BBSRC-funded Ph.D. and (Zoetis-sponsored) CASE studentships

FIG 9 Transfer of pVAPN by mating confers virulence to a plasmid-negativeR. equi recipient strain. (A) In vivo selection of pVAPN transconjugants inmice. Note the progressive enrichment of the recipient 103S�RmpR strain uponacquisition of the pVAPN plasmid. Time zero is 60 min after infection. (B)Intracellular proliferation in J774A.1 macrophages. Acquisition of pVAPN(and control pVAPA) promotes intracellular proliferation in the recipient103S�RmpR strain. Data are expressed as normalized IGC values (see Materialsand Methods). Means of data from three duplicate experiments � standarderrors; P values (determined by 2-way ANOVA and Šidák post hoc multiple-comparison tests) are indicated. ns, not significant.

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from the Centre for Infectious Diseases of the University of Edinburgh(UoE), and I.M. by a Charles Darwin international scholarship from UoE.

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