Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells

Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues.     

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.     

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.     

Bisphosphonate-related osteonecrosis of jaw (BRONJ): an anti-angiogenic side-effect?

Malakoplakia is a disease especially of the urinary tract with typical plaques most frequently observed in the urinary bladder's mucosa. In the context of immunosuppression malakoplakia can also occur in other organs. Some of these extravesical malakoplakias are associated with an infection by Rhodococcus equi, a rare human pathogen well known from veterinary medicine. Here we present the first case of a pleural malakoplakia without lung involvement caused by a proved Rhodococcus equi infection.     

Defective production of interferon-gamma and tumour necrosis factor-alpha by AIDS mononuclear cells after in vitro exposure to Rhodococcus equi

The production of interferon-gamma and tumour necrosis factor-alpha was evaluated in the peripheral blood mononuclear cells (PBMCs) from healthy donors and AIDS patients after Rhodococcus equi infection in vitro. PBMCs from healthy donors secreted elevated levels of IFN-gamma and TNF-alpha when challenged in vitro with killed R. equi, whereas the release of both cytokines was impaired in supernatant cultures from AIDS patients. We conclude that the failure of IFN-gamma generation in AIDS patients in response to R. equi is not antigen-specific but it may reflect the global impairment of T-cell function. In such patients, however, the infection with R. equi, a facultative intracellular pathogen which survives and replicates within macrophages, may be responsible for the impairment in the TNF-alpha release, possibly enhancing the HIV-induced macrophage dysftmction.     

The steroid catabolic pathway of the intracellular pathogen Rhodococcus equi is important for pathogenesis and a target for vaccine development

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3'-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.     

Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis

Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.     

Immune response to the Rhodococcus equi infection in high and low antibody-producing mice (Selection IV-A)

Rhodococcus equi is a gram-positive, facultative intracellular bacterium which infects macrophages and causes rhodococcal pneumonia and enteritis in foals. Recently, this agent has been recognized as an opportunistic pathogen for immunocompromised humans. Several murine experimental models have been used to study R. equi infection. High (H(IV-A)) and Low (L(IV-A)) antibody (Ab)-producers mice were obtained by bi-directional genetic selections for their ability to produce antibodies against sheep and human erythrocytes (Selection IV-A). These lines maintain their phenotypes of high and low responders also for other antigens than those of selection (multispecific effect). A higher macrophage activity in L(IV-A) mice has been described for several intracellular infectious agents, which could be responsible for their intense macrophage antigens (Ag)-handling and low Ab production. Due to these differences, L(IV-A) mice were found to exhibit a better performance to trigger an effective immune response towards intracellular pathogens. The objective of this work was to characterize the immune response of Selection IV-A against R. equi. H(IV-A) and L(IV-A) mice were infected with 2.0x10(6) CFU of ATCC 33701+R. equi by intravenous route. With regards to bacterial clearance and survival assays, L(IV-A) mice were more resistant than H(IV-A) mice to virulent R. equi. L(IV-A) mice presented a higher hydrogen peroxide (H2O2) and nitric oxide (NO) endogenous production by splenic macrophages than H(IV-A) mice. L(IV-A) expressed the most intense cellular response, available by the Delayed-Type Hypersensitivity (DTH) reaction, which activated macrophages and produced more H2O2 and NO. The three times higher specific antibodies titres in H(IV-A) indicated that Selection IV-A maintained the multispecific effect and the polygenic control of humoral and cellular responses also to R. equi.     

Site-specific integration of Streptomyces PhiC31 integrase-based vectors in the chromosome of Rhodococcus equi

StreptomycesPhiC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the PhiC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.     

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.     

Associations between the ecology of virulent Rhodococcus equi and the epidemiology of R. equi pneumonia on Australian thoroughbred farms

The ecology of virulent strains of Rhodococcus equi on horse farms is likely to influence the prevalence and severity of R. equi pneumonia in foals. This study examined the association between the ecology of virulent R. equi and the epidemiology of R. equi pneumonia by collecting air and soil samples over two breeding seasons (28 farm-year combinations) on Thoroughbred breeding farms with different reported prevalences of R. equi pneumonia. Colony blotting and DNA hybridization were used to detect and measure concentrations of virulent R. equi. The prevalence of R. equi pneumonia was associated with the airborne burden of virulent R. equi (both the concentration and the proportion of R. equi bacteria that were virulent) but was not associated with the burden of virulent R. equi in the soil. Univariable screening and multivariable model building were used to evaluate the effect of environmental and management factors on virulent R. equi burdens. Lower soil moisture concentrations and lower pasture heights were significantly associated with elevated airborne concentrations of virulent R. equi, as were the holding pens and lanes, which typically were sandy, dry, and devoid of pasture cover. Few variables appeared to influence concentrations of virulent R. equi in soil. Acidic soil conditions may have contributed to an elevated proportion of virulent strains within the R. equi population. Environmental management strategies that aim to reduce the level of exposure of susceptible foals to airborne virulent R. equi are most likely to reduce the impact of R. equi pneumonia on endemically affected farms.     

Innate immune responses to Rhodococcus equi

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.     

Quantitative culture of Rhodococcus equi from the feces of horse

The selective isolation of R. equi in NANC medium was made by quantitative culture of the organism in the feces. R. equi was observed in the feces of all the mares and foals investigated. The mean viable count of R. equi organisms in 1 gram of feces was 8.42 X 10(2) in the mares and 7.57 X 10(2) in the foals, and its 95% confidence limits were in a range of 6.48 X 10(2) to 1.09 X 10(3) and 4.19 X 10(2) to 1.37 X 10(3), respectively. This result indicates that R. equi is a member of the normal intestinal flora in the horse.     

The cell wall of the pathogenic bacterium Rhodococcus equi contains two channel-forming proteins with different properties

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.     

Diagnosis of lung cancer

Lung cancer is the most common malignant tumor among male and second among female. The most effective diagnostic tool would be the early detection of the lung cancer. The diagnosis of tumors has to be based on patho-morphological findings. The invasive diagnostic tools can be used if the proved lung process has any therapeutic consequence. Only an experienced team has the chance to examine quickly and effectively the patients.In Hungary there are pulmonological centers where these teams consisting of pneumonologist, radiologists and pathologists are working effectively.     

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.     

Identification of methionine-processed HPr in the equine pathogen Streptococcus equi

Using preparative electrophoresis, a low molecular weight protein has been partially purified from a cell extract of the equine pathogen Streptococcus equi susp. equi. N-terminal sequence analysis and Western blotting revealed the protein to be HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Interestingly, the only form of the HPr protein detected in S. equi was one with the amino-terminal methionine removed, a modification that has previously been associated with surface localization of streptococcal HPr proteins.