Virulence Of Rhodococcus Equi Research Articles

The N-terminal domain is required for cell surface localisation of VapA, a member of the Vap family of Rhodococcus equi virulence proteins.

Rhodococcus equi pneumonia is an important cause of mortality in foals worldwide. Virulent equine isolates harbour an 80-85kb virulence plasmid encoding six virulence-associated proteins (Vaps). VapA, the main virulence factor of this intracellular pathogen, is known to be a cell surface protein that creates an intracellular niche for R. equi growth. In contrast, VapC, VapD and VapE are secreted into the intracellular milieu. Although these Vaps share very high degree of sequence identity in the C-terminal domain, the N-terminal domain (N-domain) of VapA is distinct. It has been proposed that this domain plays a role in VapA surface localization but no direct experimental data provides support to such hypothesis. In this work, we employed R. equi 103S harbouring an unmarked deletion of vapA (R. equi ΔvapA) as the genetic background to express C-terminal Strep-tagged Vap-derivatives integrated in the chromosome. The surface localization of these proteins was assessed by flow cytometry using the THE2122;-NWSHPQFEK Tag FITC-antibody. We show that VapA is the only cell surface Vap encoded in the virulence plasmid. We present compelling evidence for the role of the N-terminal domain of VapA on cell surface localization using fusion proteins in which the N-domain of VapD was exchanged with the N-terminus of VapA. Lastly, using an N-terminally Strep-tagged VapA, we found that the N-terminus of VapA is exposed to the extracellular environment. Given the lack of a lipobox in VapA and the exposure of the N-terminal Strep-tag, it is possible that VapA localization on the cell surface is mediated by interactions between the N-domain and components of the cell surface. We discuss the implications of this work on the light of the recent discovery that soluble recombinant VapA added to the extracellular medium functionally complement the loss of VapA.

Commonality of Virulence-Promoting Function in Rhodococcus equi Virulence Associated Proteins (Vaps)

Rhodococcus equi is a Gram-positive facultative intracellular pathogen associated with life-threatening bronchopneumonial disease in foals. Key to R. equi’s intracellular survival in host macrophages is the production of virulence associated proteins (Vaps). Numerous vap genes are found on virulence plasmids isolated from different species, and the Vaps share a high degree of sequence identity. VapA has been extensively studied, and although vapK and vapN genes from other R. equi virulence plasmids have been shown to be essential for R. equi intracellular survival, their mode of action is less characterised. We, therefore, examined whether VapK and VapN worked mechanistically in the same way as VapA. Indeed, like VapA, VapK and VapN neutralised lysosomal pH and reduced lysosomal hydrolase activity. A loss of VapA and R. equi virulence could be regained by the presence of either VapK or VapN. The acid-neutralisation activity was also observed to a lesser extent with VapB. There was a differential activity across these virulence-promoting Vaps with the most “acid-neutralising” activity found with VapN, then VapA and K, and finally VapB. These data suggest that VapA production, which is often found in equine infections, can be substituted by VapK and B (produced by plasmids often found in porcine species) or VapN (produced by plasmids often isolated in bovine and human samples). These data imply that the molecular mechanism(s) that VapA uses to neutralise lysosomal acidity should also be seen in VapN and K which will help guide researchers in identifying their precise mode of action and aid the future development of targeted therapeutics.

Antibody activities in hyperimmune plasma against the Rhodococcus equi virulence -associated protein A or poly-N-acetyl glucosamine are associated with protection of foals against rhodococcal pneumonia.

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide β-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

Mouse lung infection model to assess Rhodococcus equi virulence and vaccine protection

The pathogenic actinomycete Rhodococcus equi causes severe purulent lung infections in foals and immunocompromised people. Although relatively unsusceptible to R. equi, mice are widely used for in vivo studies with this pathogen. The most commonly employed mouse model is based on systemic (intravenous) infection and determination of R. equi burdens in spleen and liver. Here, we investigated the murine lung for experimental infection studies with R. equi. Using a 107CFU intranasal challenge in BALB/c mice, virulent R. equi consistently survived in quantifiable numbers up to 10 days in the lungs whereas virulence-deficient R. equi bacteria were rapidly cleared. An internally controlled virulence assay was developed in which the test R. equi strains are co-inoculated and monitored in the same mouse. Isogenic R. equi bacteria lacking either the plasmid vapA gene or the entire virulence plasmid were compared using this competitive assay. Both strains showed no significant differences in in vivo fitness in the lung, indicating that the single loss of the virulence factor VapA was sufficient to account for the full attenuation seen in the absence of the virulence plasmid. To test the adequacy of the lung infection model for monitoring R. equi vaccine efficacy, BALB/c mice were immunized with live R. equi and challenged intranasally. Vaccination conferred protection against acute pulmonary challenge with virulent R. equi. Our data indicate that the murine lung infection model provides a useful tool for both R. equi virulence and vaccine studies.

Virulence of Rhodococcus equi Isolated from Lesions of Infected Foals

Virulence of Rhodococcus equi Isolated from Lesions of Infected Foals

The sensor kinase MprB is required for Rhodococcus equi virulence

Rhodococcus equi is a soil bacterium and, like Mycobacterium tuberculosis, a member of the mycolata. Through possession of a virulence plasmid, it has the ability to infect the alveolar macrophages of foals, resulting in pyogranulomatous bronchopneumonia. The virulence plasmid has an orphan two-component system (TCS) regulatory gene, orf8, mutation of which completely attenuates virulence. This study attempted to find the cognate sensor kinase (SK) of orf8. Annotation of the R. equi strain 103 genome identified 23 TCSs encoded on the chromosome, which were used in a DNA microarray to compare TCS gene transcription in murine macrophage-like cells to growth in vitro. This identified six SKs as significantly up-regulated during growth in macrophages. Mutants of these SKs were constructed and their ability to persist in macrophages was determined with one SK, MprB, found to be required for intracellular survival. The attenuation of the mprB− mutant, and its complementation, was confirmed in a mouse virulence assay. In silico analysis of the R. equi genome sequence identified an MprA binding box motif homologous to that of M. tuberculosis, on mprA, pepD, sigB and sigE. The results of this study also show that R. equi responds to the macrophage environment differently from M. tuberculosis. MprB is the first SK identified as required for R. equi virulence and intracellular survival.

Cholesterol oxidase (ChoE) is not important in the virulence of Rhodococcus equi

To analyze further the role in virulence of the prominent cholesterol oxidase (ChoE) of Rhodococcus equi, an allelic exchange choE mutant from strain 103+ was constructed and assessed for virulence in macrophages, in mice, and in foals. There was no difference between the mutant and parent strain in cytotoxic activity for macrophages or in intra-macrophage multiplication. No evidence of attenuation was obtained in macrophages and in mice, but there was slight attenuation apparent in four intra-bronchially infected foals compared to infection of four foals with the virulent parent strain, based on a delayed rise in temperature of the choE-mutant infected foals. However, bacterial colony counts in the lung 2 weeks after infection were not significantly different, although there was a slight but non-significant ( P = 0.12) difference in lung:body weight ratio of the choE mutant versus virulent parent infected foals (mean 2.67 ± 0.25% compared to 4.58 ± 0.96%). We conclude that the cholesterol oxidase is not important for the virulence of R. equi.

The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence

An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.

Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan

This report describes the discovery of two new virulence plasmid types from a crossbred foal with Rhodococcus equi pneumonia in Kumamoto died with severe R. equi pneumonia and ulcerative enteritis. R. equi was isolated in large numbers and isolates from the foal were investigated for the presence of virulence-associated 15–17 kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and by gene coding for VapA by PCR. Plasmid DNAs extracted from the isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII. The digestion patterns that resulted divided the plasmids of these isolates into two closely related types. The digestion patterns were then compared with eight representative virulence plasmid types (85 kb types I, II, III and IV, 87 kb types I and II, 90 kb types I and II), which have already been reported. None of the EcoRI and EcoT22I digestion patterns of the eight representative plasmids matched those of the two plasmid types. We tentatively designated these new plasmid types as 90 kb type III and type IV, since HindIII and BamHI digestion patterns of the two plasmid types were identical with those of a 90 kb type I plasmid. This study, demonstrated that there are at least 10 distinct but closely related plasmids present in isolates from horses in the world.

Evaluation of equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C for use in protecting foals against Rhodococcus equi-induced pneumonia.

To determine whether purified equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C (VapA and VapC) can confer passive protection against R. equi-induced pneumonia in foals. Twenty-eight 3-week-old mixed-breed pony foals. 7 foals received IV injections of equine hyperimmune plasma (HIP) against whole-cell R. equi, and 7 received purified equine immunoglobulin specific for VapA and VapC 1 day prior to intrabronchial infection with R. equi strain 103+. Eleven foals were not treated prior to infection, and 3 control foals were neither treated nor infected. Heart rate, respiratory rate, and rectal temperature were recorded twice daily, and serum fibrinogen concentration and WBC count were determined every other day following infection. Foals were euthanatized 14 days following infection, and lung lesions and concentration of R. equi in lungs were assessed. The onset of clinical signs of pneumonia was significantly delayed in the HIP- and immunoglobulin-treated groups, compared with the untreated infected group. Moreover, pulmonary lesions were less severe in the treated groups, and significantly fewer R. equi organisms were cultured from the lungs of treated foals. Degree of protection against R. equi-induced pneumonia provided by purified immunoglobulin specific for VapA and VapC was similar to that provided by commercially available HIP. Results not only suggest that immunoglobulin is the primary component of HIP that confers protection against R. equi-induced pneumonia in foals but also indicate that antibodies against R. equi VapA and VapC are protective.

Association of disease with isolation and virulence of Rhodococcus equi from farm soil and foals with pneumonia.

To determine whether isolation and virulence of Rhodococcus equi from soil and infected foals are associated with clinical disease. Cross-sectional and case-control study. R equi isolates from 50 foals with pneumonia and soil samples from 33 farms with and 33 farms without a history of R equi infection (affected and control, respectively). R equi was selectively isolated from soil samples. Soil and clinical isolates were evaluated for virulence-associated protein antigen plasmids (VapA-P) and resistance to the beta-lactam antibiotics penicillin G and cephalothin. Microbiologic cultures and VapA-P assays were performed at 2 independent laboratories. VapA-P was detected in 49 of 50 (98%) clinical isolates; there was complete agreement between laboratories. Rhodococcus equi was isolated from soil on 28 of 33 (84.8%) affected farms and 24 of 33 (72.7%) control farms, but there was poor agreement between laboratories. Virulence-associated protein antigen plasmids were detected on 14 of 66 (21.2%) farms by either laboratory, but results agreed for only 1 of the 14 VapA-P-positive farms. We did not detect significant associations between disease status and isolation of R equi from soil, detection of VapA-P in soil isolates, or resistance of soil isolates to beta-lactam antibiotics. No association between beta-lactam antibiotic resistance and presence of VapA-P was detected. On the basis of soil microbiologic culture and VapA-P assay results, it is not possible to determine whether foals on a given farm are at increased risk of developing disease caused by R equi.

Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge

Twelve foals, between 27 and 83 days old, were infected with 2 strains of Rhodococcus equi by intratracheal administration. Ten of the 12 foals were inoculated with 10 4–10 10 colony forming units (cfu) of ATCC 33701 strain. The other 2 foals were inoculated with 10 9 cfu of a plasmid-cured derivative of the ATCC 33701 strain (ATCC 33701P −). All of the 10 foals challenged with the ATCC 33701 strain showed clinical signs of pulmonary disease within 5–13 days, such as gross lesions associated with acute bronchopneumonia and microscopic lesions associated with granulomatous pneumonia. The two foals challenged with the ATCC 33701P − strain showed neither clinical signs of disease nor gross lesions. Apparently, when lacking plasmid, the virulent Rhodococcus equi lost its pathogenicity.

Pathogenesis and virulence of Rhodococcus equi

Inhalation of the soil-borne organism, Rhodococcus equi, can lead to a chronic and severe pyogranulomatous pneumonia in young horses and immunocompromised people. In addition, ulcerative colitis is a common sequela to infection in foals, and dissemination from the lung to other body sites is not uncommon in either the horse or man. Although the facultative intracellular bacterium is susceptible to neutrophil-mediated killing, it is able to resist innate macrophage defenses and establish residence within the intracellular environment of that phagocyte. Definitive virulence factors of R. equi have not yet been determined, but potential candidates include capsular polysaccharide, the exoenzyme cholesterol oxidase, cell wall mycolic acids, and the products encoded by a virulence-associated plasmid. The ability to replicate within the macrophage is associated with virulence, and correlates in animals with the possession of a large plasmid and expression of the plasmid-encoded, surface-expressed lipoprotein, VapA. All strains of R. equi isolated from horses with clinical disease possess a large plasmid and express VapA antigens. In addition, bacterial clearance and granuloma development in mice is linked to plasmid possession and VapA expression. Plasmid containing strains replicate within the tissues of the mouse whereas plasmid-cured strains are rapidly cleared. At present, the function of the VapA protein is unknown. In contrast to what is observed in the foal, only a small percentage of R. equi strains isolated from humans with rhodococcal disease express VapA antigens, although a high proportion of others express a related protein which is associated with reduced virulence and is also plasmid-encoded. In a limited number of plasmid-negative human isolates, virulence has been linked to β-lactam resistance, and preliminary evidence suggests that the phenotype may be phage encoded. It is likely that the immune status of the patient can influence whether a particular strain of R. equi is able to produce clinical disease, and certainly experimental infection in mice has confirmed that an intact cellular immune response is necessary for clearance of the organism.

Assessment of the immunogenic potential of Rhodococcus equi virulence associated protein (VapA) in mice

The development of immunity to Rhodococcus equi, particularly to a virulence-associated protein (VapA) based antigen preparation, was examined in CD1 and BALB c mice after intraperitoneal vaccination. Immunization with VapA based antigen without adjuvant markedly enhanced organ clearance in CD1 mice but not in BALB c mice. Delayed type hypersensitivity response and antibody titres in VapA based antigen immunized BALB c mice were less than in CD1 mice. By constrast also to CD1 mice, sera from immunized BALB c mice did not react as strongly with VapA in western blots. Use of adjuvants (aluminium hydroxide, iscoms) interfered markedly with the immunogenic properties of the VapA based antigen, in the case of aluminium hydroxide by apparently driving a Th2 type of response. Unexpectedly, isocom adjuvants also impaired immunity and, despite the highest DTH response, produced a low IgG2a response, suggesting that iscomization of the antigen produced a low interferon gamma and high interleukin 2 response. Passive immunization of BALB c mice with serum from mice immunized with live virulent strain 103+ resulted in only temporary and slight enhancement of organ clearance, supporting the central importance of cellular immunity to R. equi. Immunization with live virulence plasmid- and VapA-positive R. equi strain 103 resulted in marked liver clearance, in marked DTH response and high antibody titres. By contrast, immunization with live virulence plasmid- and VapA-negative strain 103 resulted in slight but variable enhancement of clearance, but insignificant DTH and antibody. The virulence plasmid, and by implication VapA, was thus shown to be critical in determining a highly effective protection to live organisms.

Virulence of Rhodococcus equi for mice.

Virulence of Rhodococcus equi for mice.