Deletion of vapA encoding Virulence Associated Protein A attenuates the intracellular actinomycete Rhodococcus equi

Virulent strains of the facultative intracellular bacterium Rhodococcus equi isolated from young horses (foals) with R. equi pneumonia, carry an 80-90 kb virulence plasmid and express a highly immunogenic 15-17 kDa protein of unknown function called VapA (Virulence Associated Protein A). Recent sequencing of the virulence plasmid identified a putative pathogenicity island encoding a novel family of seven Vap proteins including VapA. These proteins exhibit a significant sequence similarity to each other but have no homologues in other organisms. In this study, we describe the construction of an R. equi mutant lacking a 7.9 kb DNA region spanning five vap genes (vapA, -C, -D, -E and -F ). This vap locus mutant was attenuated for virulence in mice as it was unable to replicate in vivo and was rapidly cleared in comparison to the virulent wild-type strain. Complementation analysis of the vap locus mutant showed that expression of vapA alone could restore full virulence, whereas expression of vapC, -D and -E could not. We subsequently constructed an R. equi strain lacking only the vapA gene and found that it was attenuated for growth in vivo to the same degree as the vap locus mutant. Unlike wild-type R. equi which replicates intracellularly, both of the mutant strains exhibited a growth defect in macrophages although their attachment to the macrophages was unaffected. These studies provide the first proof of a role for vapA in the virulence of R. equi, and demonstrate that its presence is essential for intracellular growth in macrophages.     

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.     

Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.     

A role for bacteriophages in the evolution and transfer of bacterial virulence determinants

A virulence-associated region in the genome of Dichelobacter nodosus has been shown to contain an integrase gene which is highly related to the integrases of Shigella flexneri phage Sf6 and coliphages P4 and φR73, together with open reading frames ( vapB, C and D ) related to genes borne on plasmids in Neisseria gonorrhoeae, Escherichia coli, Actinobacillus actinomycetemcomitans and Treponema denticola . Similar to P4 and φR73, the vap region is bracketed by putative bacteriophage att sites and is adjacent to a tRNA gene, which suggests that the vap region has been derived by the integration of a bacteriophage, or a plasmid carrying a bacteriophage-related integrase gene. Many similarities in genes and genes clusters encoding virulence determinants have been found in distantly related bacteria. These genes are often located on plasmids in one organism but on the chromosome in others, implying that transmission of the genes has been followed by integration. Thus, the events which have generated the vap regions of D. nodosus may represent a common mechanism for transfer of virulence determinants. A number of genes involved in the virulence of bacterial pathogens are found on integrated bacteriophages, and we suggest that others will prove to be associated with tRNA genes and/or integrase genes derived from bacteriophages. The use of tRNA genes as integration sites for many bacteriophages and plasmids may favour intergeneric transmission, as tRNA genes are highly conserved.     

Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4

Rhodococcus erythropolis strain PR4 has been isolated as an alkane-degrading bacterium. The strain harbours one linear plasmid, pREL1 (271 577 bp) and two circular plasmids, pREC1 (104 014 bp) and pREC2 (3637 bp), all with some sequence similarities to other Rhodococcus plasmids. For pREL1, pREC1 and pREC2, 298, 102 and 3 open reading frames, respectively, were predicted. Linear plasmid pREL1 has several regions homologous to plasmid pBD2 found in R. erythropolis BD2. Sequence analysis of pREL1 and pBD2 identified common metal-resistance genes on both, but pREL1 also encodes alkane-degradation genes not found on pBD2, with enzyme constituents some of which are quite different from those of other organisms. The alkane hydroxylase consisted of a cytochrome P450 monooxygenase, a 2Fe-2S ferredoxin, and a ferredoxin reductase. The ferredoxin reductase amino acid sequence resembles the AlkT (rubredoxin reductase) sequence. A zinc-containing alcohol dehydrogenase further oxydizes alkanols, alkane oxidation products catalysed by alkane hydroxylase. Of the circular plasmids, the pREC1 sequence is partially similar to the sequence of pREAT701, the virulence plasmid found in Rhodococcus equi. pREC1 has no pREAT701 virulence genes and encodes genes for beta-oxidation of fatty acids. Thus, joint actions of enzymes encoded by pREL1 and pREC1 may enable efficient mineralization of alkanes.     

Reinvestigation of the virulence of Rhodococcus equi isolates from patients with and without AIDS

Rhodococcus equi emerged as a zoonotic pathogen of human immunodeficiency virus-infected patients over the last three decades. Two virulence plasmid types of R. equi, pVAPA and pVAPB associated with equine and porcine isolates, have been recognized, and more recently, pVAPN, a novel host-associated virulence plasmid in R. equi, was found in bovine and caprine isolates. We reinvestigated 39 previously reported isolates of R. equi from patients with and without acquired immunodeficiency syndrome (AIDS) by detecting vapA, vapB and vapN using PCR and plasmid profiling. After excluding one isolate that could not be cultured from frozen storage, eight isolates carried a virulence plasmid encoding vapA (pVAPA), 10 carried a virulence plasmid encoding vapB (pVAPB), seven carried a virulence plasmid encoding vapN (pVAPN) and 13 were negative for those genes. Of the 29 isolates from patients with AIDS, 7, 10 and 5 harboured pVAPA, pVAPB and pVAPN respectively. Among nine isolates from patients without AIDS, one and two harboured pVAPA and pVAPN respectively. This study demonstrated that pVAPN-positive R. equi existed in human isolates before 1994 and reaffirmed that equine-associated pVAPA-positive, porcine-associated pVAPB-positive and bovine- or caprine-associated pVAPN-positive R. equi are widely spread globally. Because domestic animals might be major sources of human infection, further research is needed to reveal the prevalence of pVAPN-positive R. equi infection in cattle and goats.     

The completely sequenced plasmid pEST4011 contains a novel IncP1 backbone and a catabolic transposon harboring tfd genes for 2,4-dichlorophenoxyacetic acid degradation

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D(+) phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D(+) phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazine-catabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 beta subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the alpha, beta, and gamma subgroups) that it belongs to a new IncP1subgroup, the delta subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids.     

pFiD188, the linear virulence plasmid of Rhodococcus fascians D188

Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.     

Interaction of Shigella sonnei phase-I strains carrying F'- or R386 plasmids with continuous HeLa- and L-line cells

The region of S. sonnei chromosome, located to the left of the gene lac I, has been found to be linked with the capacity of these bacteria for penetrating epithelial cells: this capacity is sharply suppressed in transconjugates carrying plasmids F' which cover the above-mentioned chromosomal region in recipients. The loss of virulence by transconjugates with transferred plasmids F'lac is not linked with the transfer of F factor proper, as those transconjugates which have acquired plasmids F' from E. coli donor strains K = 12 X 363 or F'his 131, not covering the lactose region to the left of the gene lac I, retain their virulence. The transfer of plasmid R 386 having no analogs of bacterial chromosomal genes leads to the loss of virulence due to the oss of the invasive capacity of bacteria.     

Genetic organization of the duplicated vap region of the Dichelobacter nodosus genome.

The recombinant plasmid pJIR318 contains a fragment of the Dichelobacter nodosus genome which is associated with virulence. Sequence analysis of the pJIR318 insert has shown that it contains four vap (virulence-associated protein) genes which are homologous to open reading frames found on the Escherichia coli F plasmid and the Neisseria gonorrhoeae cryptic plasmid (M. E. Katz, R. A. Strugnell, and J. I. Rood, Infect. and Immun. 60:4586-4592, 1992). The plasmid pJIR318 hybridizes to three regions of the D. nodosus genome, each of which has now been isolated. Regions 1 and 3 were found to be adjacent in the genome of D. nodosus A198, and the order of the vap genes in vap regions 1 and 2 were shown to be identical. Partial sequence analysis and Southern blot analysis of the vap regions showed that the three regions probably arose by a duplication event(s) followed by insertions and/or deletions. A recombinant plasmid, pJIR749, was isolated from a library of a benign D. nodosus strain, 305. This plasmid contained sequences from both ends of vap region 2. Analysis of pJIR749 showed that the sequences on either side of vap region 2 were separated by 324 bp in the genome of benign strain 305 and that the orientations of the sequences were different. It is clear that a simple insertion or deletion event did not generate the benign and virulent strains studied. A model which describes the evolution of the duplicated vap regions in D. nodosus A198 is presented.     

Identification of the origin of replications and partial characterization of plasmid pRK100

In search for the evolutionary origin of the conjugative F-like plasmid pRK100, the plasmid's functional replication regions were identified. Additionally targeted genetic analysis was used to investigate origins of other regions of the plasmid. Construction of minireplicons via ligation of Tn1725 with plasmid fragments and targeted cloning of putative replication regions, followed by sequence analysis indicated two functional replication regions, a F plasmid related RepFIB and a R1 plasmid related RepFIIA replication region. Partial nucleotide sequencing of regions of the plasmid revealed genes that encode a putative enterochelin iron uptake system previously associated with an Escherichia coli pathogenicity island, PAI III536, and the pColV-like aerobactin genes. In addition, a homologue of the R100 plasmid related rmoA gene was found that exhibits strong similarity to hha/ymoA encoding the Hha/YmoA class of modulators of gene expression. PCR and hybridization experiments further demonstrated that pRK100 harbors multiple IS2 and IS3 insertion sequences that may have facilitated in the acquisition of elements from other DNA molecules. These data together with the previous identification of a F-like tra region and a pColIa-like colicin Ia, indicate that pRK100 has a highly mosaic structure with elements derived from many different known large natural plasmids.     

VapI, a new member of the Rhodococcus equi Vap family

Rhodococcus equi is a facultative intracellular bacterium which can cause bronchopneumonia in foals and AIDS patients. In this report we show that the ORF13-protein coded by the virulence associated plasmid of R. equi is clearly homologous to VapE. Nucleotide sequence analysis revealed frame shift mutations that shorten the sequence of the ORF13-protein. A theoretical extension of the sequence of ORF13 by the introduction of a single nucleotide yields a translated amino acid sequence that is highly homologous to VapE and other members of the␣Vap family. The data provided in this study indicate that the ORF13-protein is a novel member of the Vap family and is therefore designated VapI.     

Nucleotide sequence of mkaD, a virulence-associated gene of Salmonella typhimurium containing variable and constant regions

We have identified the nucleotide (nt) sequence of mkaD, a virulence-associated gene of the Salmonella typhimurium virulence plasmid, pEX102. The gene shows 98% homology on nt sequence level to mkfA, a corresponding gene of the S. typhimurium virulence plasmid pIP1350. The few nt changes, however, caused more extensive changes on the amino-acid level. The differences between mkaD and mkfA were clustered in distinct variable regions rather than being randomly scattered along the sequence. A third salmonellar virulence plasmid, pLT2, contained an mkaD gene identical to that of pEX102. Our observation suggests that the conserved virulence determinant on the plasmids of Salmonellae may contain different alleles of the same gene.     

Characterization of the α-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding α-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire α- hly CABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited α- hly determinants. The α- hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli α-hly plasmid pHly152, in particular, the structural α- hly CABD genes (99.2% identity) and the regulatory hly R regions (98.8% identity). pEO5 and α-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural α- hly CABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS 2 element upstream of the hly C gene in pHly152. The presence of transposon-like structures at both ends of the α- hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.     

Comparative analysis of the structure of incompatibility plasmids N, P and W

Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.