Random insertion mutagenesis of the intracellular pathogen Rhodococcus equi using transposomes

The identification of virulence factors in Rhodococcus equi has been severely hampered by the lack of a method for in vivo random insertion mutagenesis. This study reports the use of transposomes to generate random insertions of a gene conferring kanamycin resistance into the genome of R. equi ATCC 33701. Southern hybridisation using the kanamycin resistance gene as probe showed that insertion of transposome is random. This was confirmed following nucleotide sequence analysis of the junction between the transposome and chromosomal DNA. The presence of a 9 bp duplication of the target sequence showed that random integration of the transposome was due to a bona fide Tn5 transposition event.     

Molecular and infection biology of the horse pathogen Rhodococcus equi

The soil actinomycete Rhodococcus equi is a pulmonary pathogen of young horses and AIDS patients. As a facultative intracellular bacterium, R. equi survives and multiplies in macrophages and establishes its specific niche inside the host cell. Recent research into chromosomal virulence factors and into the role of virulence plasmids in infection and host tropism has presented novel aspects of R. equi infection biology and pathogenicity. This review will focus on new findings in R. equi biology, the trafficking of R. equi -containing vacuoles inside host cells, factors involved in virulence and host resistance and on host–pathogen interaction on organismal and cellular levels.     

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.     

Diversity of isolates of Rhodococcus equi from Australian Thoroughbred horse farms

Pulsed field gel electrophoresis of restriction endonuclease digested genomic DNA from a collection of clinical isolates of Rhodococcus equi was used to compare strain diversity on different Thoroughbred horse farms over time. Restricted diversity was found among the isolates tested, as the same strains were detected on multiple farms and in multiple years. Marked variation occurred in strain prevalence with some strains being represented by single isolates, and the most prevalent by 26 isolates. There were dominant strains on some farms and the prevalence of some strains differed between farms. Infection with multiple strains was noted in some cases where multiple isolates from a single foal were examined.     

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells

Abstract Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent β-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent β-lactam-resistant strains persisted within macrophages for at least 18 min only. A β-lactam-resistant mutant was obtained from a β-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, β-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.     

Serum and mucosal antibodies of infected foals recognized two distinct epitopes of VapA of Rhodococcus equi

Virulence-associated protein A (VapA) of Rhodococcus equi has been proposed for use both as a vaccine and as a target for antibodies in immunotherapy and diagnostic tests. Epitope mapping of VapA allowed the identification of two B cell epitopes associated with R. equi pneumonia. The peptide NLQKDEPGRASDT was confirmed as an immunodominant N-terminal B cell epitope recognized by all sera from infected foals while VSFQYNAVGPYLNINFFDSS (C-terminal B cell epitope) was exclusively recognized by IgA from the tracheal aspirates. Moreover, specific antibodies produced against the VapA-specific peptide reacted with a major protein (approximately 20 kDa) from R. equi antigens separated by two-dimensional gel electrophoresis. The strong reactivity of mucosal IgA from infected foals with the conserved peptides might constitute an attractive target for diagnosis and vaccine.     

Pathogenesis of Rhodococcus equi infection in mice: Roles of virulence plasmids and granulomagenic activity of bacteria

Abstract Virulence of Rhocococcus equi ATCC 33701 and its plasmid-cured derivative ATCC 33701P⁻ was compared in BALB/c and C3H/HeJ mice in terms of bacterial growth kinetics and histological changes in the liver, spleen and lungs, and humoral immune responses. Injection with a sublethal dose of 10⁶ ATCC 33701 in mice resulted in microabscess formation after rapid multiplication in the liver and spleen by day 4, and then the bacteria were gradually eliminated with the formation of granuloma and the production of specific antibodies against 15- to 17-kDa antigens of the virulent bacteria. By contrast, ATCC 33701P⁻ was avirulent as shown by early elimination of viable bacteria and no evidence of net multiplication in the organs. Histopathological changes consisted of only slight, transient infiltration of neutrophils and macrophages in the liver. Although live ATCC 33701P⁻ did not evoke any humoral or histological responses in the mice, a large inoculum (10⁸) of killed ATCC 33701 and ATCC 33701P⁻ resulted in the formation of granuloma in the liver and accelerated extramedullary hemopoiesis in the spleen. These results suggest that the pathogenesis of R. equi infection involves at least two important virulence determinants, both of which play critical roles in the disease: one is the virulence plasmid, which is required for R. equi to resist and grow within host cells; and the other is the granulomagenic activity that is related to the lipids and nature of the cell wall of the species, which induces the characteistic pathological changes.     

Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization

We describe a simple tandem mass spectrometric approach toward structural characterization of mycolic acids, the long-chain α-alkyl-β-hydroxy fatty acids unique to mycobacteria and related taxa. On collisionally activated dissociation in a linear ion trap or tandem quadrupole mass spectrometer, the [M−H]⁻ ions of mycolic acid generated by electrospray ionization undergo dissociation to eliminate the meroaldehyde residue, leading to formation of carboxylate anions containing α-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths of the meromycolate chain and the α-branch. This study revealed that the mycolic acids isolated from pathogenic Rhodococcus equi 103 contained a series of homologous ions having C₃₀ to C₅₀ chain with 0–2 double bonds. The α-branch ranged from C₁₀ to C₁₈ with 0 to 1 double bond, in which 16:0 and 14:0 are the most prominent, whereas the meromycolate chain ranged from C₁₄ to C₃₄ with 0 to 2 double bonds. The major molecular species consisted of more than 3 isomers that differ by the lengths of the α-branch or meromycolate chain, and up to 10 isobaric isomers were identified for some minor ions. We also employed tandem quadrupole mass spectrometry with precursor ion and neutral loss scans for profiling mycolic acid with specific structure in mixtures. The tandem spectra obtained from precursor ion scans of m/z 255 (16:0-carboxylate anion) and m/z 227 (14:0-carboxylate anion) may provide a simple specific means for classification of Rhodococci species, whereas tandem spectra from neutral loss of meroaldehyde residue scans provided a simple approach to reveal the mycolic acid molecules with specific meromycolate chain in mixtures.     

Identification of genes from the obligate intracellular plant pathogen, Plasmodiophora brassicae

Plasmodiophora brassicae is an intracellular pathogen that infects plants in the Brassicaceae family. Although an important pathogen group, information on the genomic makeup of the plasmodiophorids is almost completely lacking. We performed suppression subtractive hybridization (SSH) between RNA from P. brassicae-infected and uninfected Arabidopsis tissue, then screened 232 clones from the resulting SSH library. In addition, we used an oligo-capping procedure to screen 305 full-length cDNA clones from the infected tissue. A total of 76 new P. brassicae gene sequences were identified, the majority of which were extended to full length at the 5' end by the use of RACE amplification. Many of the unisequences were predicted to contain signal peptides for ER translocation. Although we located few sequences in total, these markedly increase available data from the plasmodiophorids, and provide new opportunities to examine plasmodiophorid biology. Our study also points towards the best methods for future plasmodiophorid gene discovery.     

Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi

The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.