Activation of foal neutrophils at different ages by CpG oligodeoxynucleotides and Rhodococcus equi

Toll-like receptor 9 (TLR9) activation stimulates protective immune responses against intracellular pathogens by phagocytes, including neutrophils. This study examined TLR9-mediated neutrophil activation in neonatal foals. Unmethylated CpGs, ligands for TLR9, were used to stimulate equine neutrophils, either purified or in contact with other peripheral blood leukocytes. Rhodococcus equi was used as another stimulus in parallel. TLR9 mRNA was constitutively expressed at a similar level in purified equine neutrophils across different ages from birth to adulthood, and expression was not affected by either CpG or R. equi. Purified foal neutrophils were directly sensitive to CpG stimulation, reflected by enhanced reactive oxygen species generation following fMLP stimulation, and by expressing significantly (P < 0.05) greater mRNA of IFN-γ, IL-8, IL-12p35, and significantly (P < 0.05) decreased TNF-α mRNA. In comparison, purified foal neutrophils stimulated by R. equi showed significantly (P < 0.05) increased mRNA production of IL-6, IL-8, IL-23p19, and TNF-α. Neutrophils co-cultured with other leukocytes expressed a distinct profile of cytokine mRNA than purified neutrophils in response to CpG stimulation, whereas the profile was very similar following R. equi stimulation irrespective of neutrophil purity. When co-cultured with other leukocytes, foal neutrophils were significantly (P < 0.05) activated at birth by B-class CpGs and produced IL-6, IL-8, IL-12p40, and IL-23p19 at similar magnitudes to those at 2 months of age. In foal neutrophils at birth, R. equi significantly (P < 0.05) induced all cytokines stimulated by CpGs (except IL-12p40), as well as TNF-α. Our results indicate that foal neutrophils were sensitive to CpG or R. equi activation as early as at birth, and that B-class CpGs enhanced foal neutrophil functions in vitro.     

Serogrouping of Rhodococcus equi

The serological relationships among 27 isolates of Rhodococcus equi selected from a total of 1,195 isolates were investigated by cross-agglutination and absorption tests. The presence of capsular material was demonstrated in all the 27 isolates by electron microscopic observation. Antisera were prepared by employing formalized antigen of each isolate. In the cross-agglutination test with formalized antigen, 13 antisera reacted with homologous antigens alone, but the remaining 14 antisera reacted not only with homologous antigens but also with one to four heterologous antigens. When these 14 antisera possessing heterologous agglutinins were absorbed with each of the cross-reacting antigens, 14 specific antisera were obtained. The cross-agglutination test with these 27 antisera proved the 27 strains examined to be serologically distinct from one another. These strains were designated serogroups 1 to 27. Thus the same number of grouping antisera were prepared. The distribution of each serogroup among the 1,195 isolates and 15 reference strains was investigated by the slide agglutination test. All the strains were found to be groupable. Most of them belonged to serogroups 1 to 4, 7 to 9, 11, 14, and 15. Of the serogroups designated, 4, 16, 2, 12, 21, 1, and 9 were identical with Prescott's serovars 1, 2, 3, 4, 5, 6, and 7, respectively.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

P-chirality-dependent immune activation by phosphorothioate CpG oligodeoxynucleotides

Many of the biologic activities of phosphorothioate oligodeoxynucleotides (PS-oligos) are affected by the sense of chirality of the phosphorus atoms of the internucleotide linkages. Some of the activities are increased by the Rp stereoisomer, and others are increased by the Sp stereoisomer. In previous studies, we showed that PS-oligos containing unmethylated CpG dinucleotides in particular sequence contexts can stimulate B cells and other immune cells. These CpG PS-oligos trigger mitogenactivated protein kinase (MAPK) signaling pathways, causing the induction of B cell proliferation and cytokine and immunoglobulin secretion. We investigated whether the immune stimulation by CpG PS-oligos depends on the sense of their P-chirality. CpG PS-oligos synthesized with internucleotide phosphorothioates of Rp configuration at P-atom showed much stronger MAPK activation and induction of I kappa B degradation after 40 minutes of stimulation compared with PS-oligos synthesized with Sp linkages. In order to determine if the enhanced stimulatory effects of the Rp stereoisomer may result from differential cellular uptake, we examined the rates at which fluorescently labeled Rp or Sp CpG PS-oligos were taken up by B cells, but these were found to be identical to each other and to stereorandom PS-oligos. The stronger stimulatory effect of the R stereoisomer did not last for 48 hours, and (3)H-thymidine incorporation assays at this point showed that only the S stereoisomer was active--to approximately the same level as induced by PS-oligos with stereorandom phosphorothioate linkages. This loss of activity of the R stereoisomer most likely resulted from rapid degradation of the oligonucleotides rather than from reduced interaction with the CpG receptor because PS-oligos in which only the CpG dinucleotide was stereodefined were most stimulatory when the CpG was Rp but not when the CpG was Sp. These studies demonstrate that the sense of Pchirality of PS-oligos plays a major role in determining the biologic activities of CpG motifs. Rp-chirality at the CpG is preferred for best stimulation at early time points, but Sp-chirality of the PS-oligo appears to improve stability and may provide more durable effects in prolonged tissue culture systems.     

Chemoselective biotransformation of nitriles by Rhodococcus equi A4

Resting cells of Rhodococcus equi A4 (free or immobilized in hydrogels) produced monomethyl isophtalate 1c and monomethyl terephtalate 2c from methyl-3-cyanobenzoate 1a and methyl-4-cyanobenzoate 2a, respectively, via the intermediate carboxamides 1b and 2b. The use of dried immobilized biocatalysts resulted in an increased formation of the unwanted cyano acids 1d and 2d. © Rapid Science Ltd. 1998     

Numerical classification of Rhodococcus equi and related actinomycetes

Eighty-three strains received as Corynebacterium or Rhodococcus equi and marker cultures of Corynebacterium and Rhodococcus species were subjected to numerical phenetic analyses using 112 unit characters. The data were examined using the simple matching ( S _SM) and Jaccard ( S _J) coefficients and clustering was achieved using the average linkage algorithm. Cluster composition was not markedly affected by the coefficient used or by test error, estimated at 2.9%. Most of the strains received as R. (C.) equi formed a distinct and homogeneous cluster in the aggregate Rhodococcus taxon, the strains of which were sharply separated from marker cultures of C. pseudotuberculosis and C. renale. The taxon R. equi was redescribed and Nocardia calcarea Metcalf & Brown reduced to a subjective synonym of R. erythropolis (Gray & Thornton) Goodfellow & Alderson.     

Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.     

VapA of Rhodococcus equi binds phosphatidic acid

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi‐containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA^32‐189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host‐derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA^32‐189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.     

Study of Lysozyme Resistance in Rhodococcus equi

Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.     

Binding and uptake of immunostimulatory CpG oligodeoxynucleotides by human neuroblastoma cells

Oligodeoxynucleotides (ODNs) that contain unmethylated CpG dinucleotides (CpG-ODN) trigger a strong innate immune response in vertebrates. They have been used to eradicate experimental neuroblastoma, but a direct interaction of CpG-ODN with neuroblastoma cells has not been investigated. We have analyzed uptake, binding, and intracellular distribution of CpG-ODN in the neuroblastoma cells line SKNSH. Our results indicate that cellular uptake of CpG-ODN is dose, time, temperature, and energy dependent but independent of the CpG motif. After internalization, CpGODN localized to the cytoplasm and showed a typical speckled distribution pattern. The intracellular distribution pattern and binding proteins are CpG motif independent as well. Thus, CpG-ODNs are taken up by neuroblastoma cells by a nonspecific transfer mechanism for oligonucleotides and interact with intracellular proteins. These mechanisms might help us to understand the biodistribution of oligo within tumors and might be helpful in evaluating the therapeutic effects of oligonucleotides and rational drug design.     

Degradation of Estrogens by Rhodococcus zopfii and Rhodococcus equi Isolates from Activated Sludge in Wastewater Treatment Plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17β-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17β-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17β-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17β-estradiol into substances without estrogenic activity.     

Microbial transformation of sterols to C19-steroids by Rhodococcus equi

A wild-type strain of Rhodococcus equi, isolated from soil, degraded cholesterol, -sitosterol, stigmasterol and mixed sterois to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). A definite preference for a relatively simply structured cholesterol side chain was observed. Highest specific cholesterol side-chain cleavage was associated with active growth of the culture. Maximum yield of ADD was obtained when sodium acetate and cholesterol were incorporated together in the medium. Specific side-chain cleavage required the presence of 2,2-dipyridyl, an inhibitor of ring cleavage.S. Ahmad and B.N. Johri are with the Department of Microbiology, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantriagar 263 145, Nainital, UP, India. P.K. Roy, A.W. Khan and S.K. Basu are at Fermentation Technology Division, Central Drug Research Institute, Lucknow, India.     

Hydrolysis of lysergamide to lysergic acid by Rhodococcus equi A4

From a mixture of lysergamide and its epimer isolysergamide, Rhodococcus equi A4 containing amidase preferentially hydrolyzed lysergamide into lysergic acid.     

Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides

Immune stimulation has been widely recognized as an undesirable side effect of certain antisense oligodeoxynucleotides (ODN) which can interfere with their therapeutic application. It is now clear that these dose-dependent immune stimulatory effects primarily result from the presence of an unmethylated CpG dinucleotide in particular base contexts ('CpG motif). The sequence-specific immune activation is not just an experimental artifact, but is actually a highly evolved immune defense mechanism whose actual 'goal' is the detection of microbial nucleic acids. In contrast to vertebrate DNA, in which CpG dinucleotides are 'suppressed' and are highly methylated, microbial genomes do not generally feature CpG suppression or methylation [1]. Immune effector cells such as B cells, macrophages, dendritic cells, and natural killer cells appear to have evolved pattern recognition receptors (PRR) that by binding the microbe-restricted structure of CpG motifs, trigger protective immune responses. Although the specific immune activation appears to have a variety of potential therapeutic applications, it is generally undesirable in antisense ODN. Immune stimulation may be avoided in antisense oligos by the selection of CpG-free target sequences, by the use of ODN backbones that do not support immune stimulation, or by selective modifications of the cytosine in any CpG dinucleotides.     

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10⁶ cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10⁷ cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.     

Safety of CpG oligodeoxynucleotides in veterinary species

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.     

Cooperative stimulation of dendritic cells by Cryptococcus neoformans mannoproteins and CpG oligodeoxynucleotides

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4(+) T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.     

Biotransformations of aromatic diniitriles using Rhodococcus equi cells

Short incubation (1 – 5 h) of aromatic dinitriles with resting cells of Rhodococcus equi A4 produces the corresponding cyano amides in 48 – 81% yield. The substrate reactivity decreases in order 1,4- 1,3- > 1,2-disubstitution. Prolonged treatment (20 – 50 h) is needed to achieve the conversion to acid or for transformation of the second cyano group.