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PubMed2002

Identification of pulmonary T-lymphocyte and serum antibody isotype responses associated with protection against Rhodococcus equi.

Rhodococcus equi infects and causes pneumonia in foals between 2 and 4 months of age but does not induce disease in immunocompetent adults, which are immune and remain clinically normal upon challenge. Understanding the protective response against R. equi in adult horses is important in the development of vaccine strategies, since those mechanisms likely reflect the protective phenotype that an effective vaccine would generate in the foal. Twelve adult horses were challenged with virulent R. equi and shown to be protected against clinical disease. Stimulation of cells obtained from bronchoalveolar lavage fluid with either R. equi or the vaccine candidate protein VapA resulted in significant proliferation and a significant increase in the level of gamma interferon (IFN-gamma) expression by day 7 postchallenge. The levels of interleukin-4 expression were also increased at day 7 postchallenge; however, this increase was not antigen specific. Anamnestic increases in the levels of binding to R. equi and VapA of all immunoglobulin G (IgG) antibody isotypes [IgGa, IgGb, IgG(T)] examined were detected postchallenge. The levels of R. equi- and VapA-specific IgGa and IgGb antibodies, the IgG isotypes that preferentially opsonize and fix complement in horses, were dramatically enhanced postchallenge. The antigen-specific proliferation of bronchoalveolar lavage fluid cells, the levels of IFN-gamma expression by these cells, and the anamnestic increases in the levels of opsonizing IgG isotypes are consistent with stimulation of a memory response in immune adult horses and represent correlates for vaccine development in foals.

Actinomycetales Infections↗

PubMed1991

Rhodococcus equi: an animal and human pathogen.

Recent isolations of Rhodococcus equi from cavitatory pulmonary disease in patients with AIDS have aroused interest among medical microbiologists in this unusual organism. Earlier isolations from humans had also been in immunosuppressed patients following hemolymphatic tumors or renal transplantation. This organism has been recognized for many years as a cause of a serious pyogranulomatous pneumonia of young foals and is occasionally isolated from granulomatous lesions in several other species, in some cases following immunosuppression. The last decade has seen many advances in understanding of the epidemiology, pathogenesis, diagnosis, treatment, and immunity to infection in foals. The particular susceptibility of the foal is not understood but can be explained in part by a combination of heavy challenge through the respiratory route coinciding with declining maternally derived antibody in the absence of fully competent foal cellular immune mechanisms. R. equi is largely a soil organism but is widespread in the feces of herbivores. Its growth in soil is considerably improved by simple nutrients it obtains from herbivore manure. About one-third of human patients who have developed R. equi infections had contact in some way with herbivores or their manure. Others may have acquired infection from contact with soil or wild bird manure. R. equi is an intracellular parasite, which explains the typical pyogranulomatous nature of R. equi infections, the predisposition to infection in human patients with defective cell-mediated immune mechanisms, and the efficacy of antimicrobial drugs that penetrate phagocytic cells.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Association between a large plasmid and 15- to 17-kilodalton antigens in virulent Rhodococcus equi.

Rhodococcus equi strains showing 15- to 17-kDa antigens in immunoblots were found to be virulent in mice. To study the genes specific to these antigens in virulent R. equi, we compared plasmid profiles, immunoblot profiles, and murine pathogenicity profiles of 10 strains of R. equi. All the strains showing 15- to 17-kDa antigens contained a large plasmid of approximately 85 kbp and were virulent in mice; however, the remaining strains lacked both the antigens and the large plasmid and were avirulent in mice. Mutants of virulent strains ATCC 33701 and L1, which were cured of the large plasmid by repeated passage at 38 degrees C, lacked the 15- to 17-kDa antigens and showed a dramatic decrease in lethality in mice. These results suggested that the presence of an 85-kbp plasmid may be essential for virulence and expression of 15- to 17-kDa antigens of R. equi and offered support for earlier observations that freshly isolated strains of R. equi killed mice, whereas laboratory-adapted strains did not.

Actinomycetales Infections↗

PubMed1992

Role of T-lymphocyte subsets in Rhodococcus equi infection.

Rhodococcus equi, a facultative intracellular gram-positive bacterium, can induce life-threatening infections in immunocompromised patients, especially those with AIDS. We have studied the mechanism of acquired immunity to this pathogen in a murine model. Protective immunity was induced by live but not killed bacteria. Adoptive transfer of resistance was obtained with spleen cells but not immune serum from mice immunized intravenously 30 days earlier with live bacteria. In normal mice, an intravenous challenge of 5 x 10(6) CFU of R. equi was cleared from the spleen, liver, and lungs within 3 weeks, whereas athymic nude mice were unable to clear the bacteria. In vivo depletion with monoclonal antibodies showed that both CD4+ and CD8+ T-cell subsets participate in the clearance of bacteria and that CD8+ T cells play the major role.

Actinomycetales Infections↗

PubMed1992

Virulence-associated 15- to 17-kilodalton antigens in Rhodococcus equi: temperature-dependent expression and location of the antigens.

Virulent Rhodococcus equi showing 15- to 17-kDa antigens, which is virulent in mice, was found to harbor an 85-kbp plasmid, and the 15- to 17-kDa antigens were found to be associated with possession of the 85-kbp plasmid of R. equi (S. Takai, T. Sekizaki, T. Ozawa, T. Sugawara, Y. Watanabe, and S. Tsubaki, Infect. Immun. 59:4056-4060, 1991). The expression of these antigens was temperature regulated: when cells were grown at a low temperature (25 to 32 degrees C), they did not express them, whereas they expressed them in large amounts when the cells were grown at a higher temperature (34 to 41 degrees C). The antigens were expressed on the cell surface, as evidenced by their susceptibility to proteolysis by a trypsin and by the biotin-avidin protein-blotting technique.

Antigens, Bacterial↗

PubMed1993

Failure of pulmonary clearance of Rhodococcus equi infection in CD4+ T-lymphocyte-deficient transgenic mice.

Pulmonary clearance of Rhodococcus equi requires functional T lymphocytes. In this study, CD8+ T-lymphocyte-deficient transgenic mice cleared virulent R. equi from the lungs while infection in CD4+ T-lymphocyte-deficient transgenic mice persisted. Although both CD4+ and CD8+ T cells function early in pulmonary defense against R. equi, clearance is dependent on CD4+ T lymphocytes.

Actinomycetales Infections↗

PubMed1993

The intracellular bacterium Rhodococcus equi requires Mac-1 to bind to mammalian cells.

Rhodococcus equi is a facultative intracellular bacterium of macrophages that causes disease in immunocompromised individuals, particularly those with AIDS. In this report, we demonstrate that R. equi binding to mammalian cells requires complement and is mediated primarily by the leukocyte complement receptor, Mac-1. Bacteria bind to macrophages poorly unless exogenous complement is added to the incubation medium. The addition of fresh nonimmune serum, which contains no detectable antibodies to R. equi, greatly enhances bacterial binding to macrophages, whereas heat inactivation of this serum or immunological depletion of C3 from the serum reduces binding to levels only slightly higher than those of binding under serum-free conditions. Human serum depleted of C2 or C4 is fully opsonic, indicating that complement activation and fixation occur by the alternative pathway. The serum-dependent binding of rhodococci to macrophages is mediated primarily by the macrophage complement receptor type 3, Mac-1 (CD11b/CD18). Bacteria do not bind to fibroblastoid or epithelial cells that lack this receptor. Most of the bacterial binding to macrophages is inhibited by a monoclonal antibody to Mac-1 but is unaffected by a monoclonal antibody to complement receptor type 1. Furthermore, opsonized, but not unopsonized, bacteria bind to purified Mac-1 immobilized on plastic. In addition, in the presence of opsonic complement, rhodococci bind efficiently to fibroblastoid cells transfected with cloned Mac-1 but relatively poorly to cells transfected with the complement receptor type 1. Hence, R. equi fixes complement by activating the alternative complement pathway, and this fixation is a requirement for bacterial adhesion and invasion. Furthermore, complement fixation defines rhodococcal host cell tropism, since R. equi binds specifically and exclusively to cells expressing Mac-1.

Animals↗

PubMed1995

Cytokine modulation alters pulmonary clearance of Rhodococcus equi and development of granulomatous pneumonia.

Rhodococcus equi, a facultative intracellular bacterium, causes chronic, often fatal granulomatous pneumonia in young horses and in humans with AIDS. The inability of host alveolar macrophages to kill intracellular R. equi results in the development of granulomas and progressive loss of pulmonary parenchyma. Clearance of the organism from the lung requires functional CD4+ T cells. The purpose of this study was to identify the cytokine effector mechanisms that mediate clearance of R. equi from the lung. Mice were treated with monoclonal antibodies (MAbs) to either gamma interferon (IFN-gamma) or interleukin-4 (IL-4) to determine the role of endogenous production of these cytokines in pulmonary clearance of R. equi. Mice treated with an anti-IL-4 or isotype control MAb cleared R. equi by 21 days postinfection and expressed increased levels of IFN-gamma mRNA, as detected by transcriptional analysis of bronchial lymph node CD4+ T cells. In contrast, mice treated with the anti-IFN-gamma MAb failed to express detectable IFN-gamma mRNA, expressed increased levels of IL-4 mRNA, failed to clear pulmonary infection, and developed pulmonary granulomas with large numbers of eosinophils. The enhancement of IL-4 mRNA expression and a predominance of eosinophils in pulmonary lesions of anti-IFN-gamma-treated mice suggest that a nonprotective Th2 response in involved in disease pathogenesis. The association of increased bronchial lymph node CD4+ T-cell IFN-gamma mRNA expression with pulmonary clearance of R. equi suggests that a Th1 response is protective.

Actinomycetales Infections↗

PubMed1996

Transfer of a CD4+ Th1 cell line to nude mice effects clearance of Rhodococcus equi from the lung.

Rhodococcus equi, and intracellular respiratory pathogen, causes sever e granulomatous pneumonia in humans with AIDS and in young horses. Pulmonary clearance of R. equi requires functional CD4+ T cells and gamma interferon (IFN-gamma) expression from bronchial lymph node cells. The purpose of this study was to investigate whether R. equi-specific CD4+ Th1 cells could effect clearance of R. equi from the lung. Adoptive transfer of a clearance of R. equi from the lungs. In contrast, mice transfused with a R. equi-specific CD4+ Th2 cell line expressed interleukin-4 but not IFN-gamma mRNA, failed to clear pulmonary infection, and developed granulomas in the lung. Control mice, which did not receive cells, did not produce IFN-gamma or interleukin-4 and developed small pulmonary granulomas. These results clearly show that a Th1 response is sufficient to effect pulmonary clearance of R. equi.

Animals↗

PubMed1998

Cytokine induction in murine macrophages infected with virulent and avirulent Rhodococcus equi.

To look for a possible correlation between the virulence of Rhodococcus equi and its cytokine-inducing capacity, we evaluated intracellular survival and measured cytokine induction by mouse macrophages infected with a virulent strain containing an 85-kb plasmid and expressing VapA (103+), its avirulent plasmid-cured derivative (103-), and heat-killed 103+ (HK). After incubation with similar numbers of bacteria, macrophages infected with 103- contained significantly more organisms than those infected with 103+ or HK. The number of bacteria in the macrophages infected with 103- and HK decreased progressively, whereas the 103+ numbers remained constant over 48 h. Interleukin 1beta (IL-1beta), IL-6, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA induction peaked at 4 h and returned to baseline between 12 and 48 h postinfection. IL-1beta, IL-6, IL-10, and TNF-alpha concentrations assessed by enzyme-linked immunosorbent assay generally agreed well with mRNA expression; IL-12 could, however, not be detected. For all the cytokines detected, mean concentrations in the supernatants were consistently higher in the 103(-)-infected monolayers than in those infected with 103+, although, with the exception of IL-1beta, the differences were not statistically significant. R. equi HK was a poor inducer of cytokine production. In conclusion, virulent and avirulent R. equi strains induced similar levels of cytokine synthesis. The slightly greater induction of most cytokines observed following infection with 103- is likely secondary to greater uptake by macrophages rather than to a direct role of VapA or another plasmid-encoded product in downregulating cytokine induction.

Animals↗

PubMed1999

Modulation of cytokine response of pneumonic foals by virulent Rhodococcus equi.

The ability of Rhodococcus equi to induce pneumonia in foals depends on the presence of an 85- to 90-kb plasmid. In this study, we evaluated whether plasmid-encoded products mediate virulence by modulating the cytokine response of foals. Foals infected intrabronchially with a virulence plasmid-containing strain of R. equi had similar gamma interferon (IFN-gamma) and interleukin-12 (IL-12) p35 but significantly higher IL-1beta, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA expression in lung tissue compared to foals infected with the plasmid-cured derivative. IFN-gamma mRNA expression levels in CD4+ T lymphocytes isolated from bronchial lymph nodes (BLN) were similar for the two groups of R. equi-infected foals on day 3 postinfection. However, on day 14, in association with pneumonia and marked multiplication of virulent R. equi but with complete clearance of the plasmid-cured derivative, IFN-gamma mRNA expression in BLN CD4+ T lymphocytes was significantly (P < 0.001) higher in foals infected with the plasmid-cured derivative. These results suggests an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-gamma mRNA expression by CD4+ T lymphocytes.

Actinomycetales Infections↗

PubMed1999

Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.

Actinomycetales Infections↗

PubMed2000

DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103.

The virulence plasmids of the equine virulent strains Rhodococcus equi ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (vap) genes, including vapA. These vap genes have extensive homology to vapA, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the vap genes. The vap genes were present as a cluster of three (vapA, vapC, and vapD), as a pair (vapE and vapF), or individually (vapG; vapH). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual vap genes. There was extensive homology among the C-terminal halves of all vap genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.

Amino Acid Sequence↗

PubMed2000

Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcus equi by activated macrophages.

Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-gamma), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO(-)), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS(-/-)) or the oxidative burst (gp91(phox-/-)) are more susceptible to lethal R. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O(2)(-)) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO(-) efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO(-) in situ. Inhibition of nitric oxide production by activated macrophages by using N(G)-monomethyl-L-arginine blocks their production of ONOO(-) and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equi by IFN-gamma-activated macrophages.

Actinomycetales Infections↗

PubMed2001

Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins.

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37 degrees C but not at 30 degrees C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins.

Amino Acid Sequence↗

PubMed2002

H(2)O(2), which causes macrophage-related stress, triggers induction of expression of virulence-associated plasmid determinants in Rhodococcus equi.

The response of the intracellular pathogen Rhodococcus equi to H(2)O(2) treatment, a situation potentially encountered after the oxidative burst of alveolar macrophages, was analyzed. Compared to other bacteria, including Deinococcus radiodurans, R. equi showed exceptionally high resistance to this stress. A proteomic approach showed that four polypeptides present in the wild-type strain (85F) are missing in the plasmid-cured strain 85F(P-), and by using a DNA macroarray, we identified two plasmid-encoded vap genes, vapA and vapG, whose expression was highly induced by H(2)O(2) treatment. Whereas the transcript size of vapA was compatible with a monocistronic mRNA, the transcript of vapG was considerably longer. Rapid amplification of cDNA ends PCRs showed that the transcriptional start sites of the two operons were 69 and 269 nucleotides (nt) upstream of the start codon, respectively. Analysis of these leader sequences revealed the presence of a small open reading frame named podG, which encodes a sequence of 55 amino acids preceded by a putative ribosome binding site sequence in the vapG transcript. Taking this result into account, the untranslated leader of the podG/vapG operon is 87 nt. Alignment of this sequence with the leader sequences of vapA and vapD, genes previously shown to be induced by acid, revealed significant homologies. Since our results showed that vapA, vapD, and vapG are genes highly induced by macrophage-related stresses, their gene products may, within the Vap protein family, play a dominant role inside these phagocytic cells and may be the most promising candidates for vaccination strategies.

5' Untranslated Regions↗

PubMed2003

Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection.

Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth system was developed for obtaining R. equi CFS antigens. Potential immunogens for prevention of equine rhodococcal pneumonia were identified by using immunoblots. The 48-h CFS contained five virulence-associated protein bands that migrated between 12 and 24 kDa and were recognized by sera from R. equi-infected foals and immune adult horses. Notably, the CFS contained the previously characterized proteins VapC, VapD, and VapE, which are encoded by genes on the R. equi virulence plasmid. R. equi CFS was also examined for the ability to stimulate a type 1-like memory response in immune horses. Three adult horses were challenged with virulent R. equi, and cells from the bronchoalveolar lavage fluid were recovered before and 1 week after challenge. In vitro stimulation of pulmonary T-lymphocytes with R. equi CFS resulted in significant proliferation and a significant increase in gamma interferon mRNA expression 1 week after challenge. These results were consistent with a memory effector response in immune adult horses and provide evidence that R. equi CFS proteins are antigen targets in the immunoprotective response against R. equi infection.

Actinomycetales Infections↗

PubMed2004

Rhodococcus equi-infected macrophages are recognized and killed by CD8+ T lymphocytes in a major histocompatibility complex class I-unrestricted fashion.

The goal of this research was to examine the role of cytotoxic T lymphocytes (CTL) in the control of Rhodococcus equi and specifically to determine if R. equi-specific CD8+ CTL occurred in the blood of immune horses. Equine peripheral blood mononuclear cells stimulated with antigen-presenting cells either infected with R. equi or exposed to soluble R. equi antigen lysed R. equi-infected target cells. Lysis was decreased to background by depletion of either CD2+ or CD3+ cells, indicating that the effector cell had a T-lymphocyte, but not NK cell, phenotype. Stimulation induced an increased percentage of CD8+ T cells in the effector population, and depletion of CD8+ T cells resulted in significantly decreased lysis of infected targets. Killing of R. equi-infected macrophages by effector cells was equally effective against autologous and equine leukocyte antigen A (classical major histocompatibility complex [MHC] class I) mismatched targets. To evaluate potential target antigens, target cells were infected with either virulent (80.6-kb plasmid-containing) or avirulent (plasmid-cured) R. equi. The degree of lysis was not altered by the presence of the plasmid, providing evidence that the virulence plasmid, which is required for survival within macrophages, was not necessary for recognition and killing of R. equi-infected cells. These data indicate that immunocompetent adult horses develop R. equi-specific CD8+ CTL, which may play a role in immunity to R. equi. The apparent lack of restriction via classical MHC class I molecules suggests a novel or nonclassical method of antigen processing and presentation, such as presentation by CD1 or other nonclassical MHC molecules.

Animals↗