Necrotic enteritis locus 1 diguanylate cyclase and phosphodiesterase (cyclic-di-GMP) gene mutation attenuates virulence in an avian necrotic enteritis isolate of Clostridium perfringens.

Necrotic enteritis (NE) caused by netB-positive strains of Clostridium perfringens is an important disease of intensively-reared broiler chickens. It is widely controlled by antibiotic use, but this practice that has come under increasing scrutiny and alternative approaches are required. As part of the search for alternative approaches over the last decade, advances have been made in understanding its pathogenesis but much remains to be understood and applied to the control of NE. The objective of this work was to assess the effect on virulence of mutation of the cyclic-di-GMP signaling genes present on the large pathogenicity locus (NELoc-1) in the tcp-encoding conjugative virulence plasmid, pNetB. For this purpose, the diguanylate cyclase (dgc) and phosphodiesterase (pde) genes were individually insertionally inactivated and the two mutants were subsequently complemented with their respective genes. Southern blotting showed that a single gene insertion was present. Mutation of either gene resulted in almost total attenuation of the mutants to cause NE in experimentally-infected broiler chickens, which was fully restored in each case by complementation of the respective mutated gene. Production of NetB-associated cytotoxicity for Leghorn male hepatoma (LMH) cells was unaffected in mutants. We conclude that the cyclic-di-GMP signaling system is important in controlling virulence in a NE C. perfringens strain and might be a target for control of the disease.

The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry.

Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

Influence of pCP1NetB ancillary genes on the virulence of Clostridium perfringens poultry necrotic enteritis strain CP1.

BACKGROUND: Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. The NetB toxin plays a critical role in the pathogenesis of NE. We previously demonstrated that netB is located within a 42 kb plasmid-encoded pathogenicity locus (NELoc-1), which also encodes 36 additional genes. Although NetB clearly plays a role in pathogenesis, the involvement of the other NELoc-1 genes has not yet been established. The current study was to provide experimental evidence to confirm the involvement of these genes in NE pathogenesis. RESULTS: The present study has characterized a virulent C. perfringens strain (CP1) that has spontaneously lost the NELoc-1-encoding plasmid, pCP1netB. When assessed for cytotoxicity on Leghorn Male Hepatoma (LMH) cells, the culture supernatant of the pCP1netB-deficient CP1 variant (CP1ΔpCP1netB) demonstrated significantly reduced cytotoxicity compared to the wild-type. In addition, CP1ΔpCP1netB was unable to cause intestinal lesions in chickens in a NE disease model. When netB alone was introduced into CP1ΔpCP1netB, in vitro cytotoxicity was restored to the wild-type level; however, it did not completely restore virulence when used to challenge broiler chickens [mean lesion score of 0.71 compared to 3.23 in the wild type control group (n = 14)]. CONCLUSIONS: The results of this study suggest that other genes present in NELoc-1, in addition to netB, are required for full virulence in the chicken challenge model.

A retrospective study on the etiological diagnoses of diarrhea in neonatal piglets in Ontario, Canada, between 2001 and 2010.

Laboratory surveillance data from the Animal Health Laboratory, University of Guelph, on the etiological diagnoses of neonatal diarrhea in piglets were analyzed to determine the relative importance and trends of different enteric pathogens in Ontario. A total of 237 cases, including live and dead 1- to 7-day-old piglets, were submitted for diagnosis of gastrointestinal illness between 2001 and 2010. The combined frequencies for cases of gastrointestinal illness involving Escherichia coli, Clostridium perfringens type A, rotavirus, and Clostridium difficile, either as single pathogens or a complex of pathogens, accounted for 56% of the total cases. In a total of 33% of cases of gastrointestinal illness, an etiological agent was not identified. The frequency of cases diagnosed with enterotoxigenic E. coli was decreased from 2007. Cases submitted in 2010 were more likely to be diagnosed with C. perfringens type A compared to cases submitted in 2002 to 2007 (P < 0.05). There was a significant trend for cases submitted in the winter to be diagnosed with C. perfringens type A, enterotoxigenic E. coli, rotavirus, and Cystoisospora suis (formerly Isospora suis) (P < 0.05). Enterotoxigenic E. coli was less likely diagnosed if C. difficile, C. perfringens, or rotavirus were detected (P < 0.05). Younger piglets were more likely to be diagnosed with C. perfringens type A (P < 0.05) and C. difficile (P < 0.05) than older piglets. This study shows that E. coli, C. perfringens type A, rotavirus, and C. difficile are enteric pathogens of concern for Ontario swine farrowing operations and further research is required to understand the reasons for the cases that are not diagnosed.

Les données de surveillance provenant du Animal Health Laboratory de l'University of Guelph sur les diagnostics étiologiques des diarrhées néonatales des porcelets ont été analysées afin de déterminer l'importance relative et les tendances des différents agents pathogènes entériques en Ontario. Entre 2001 et 2010, 237 cas de porcelets vivants et morts âgés de 1 à 7 jours ont été soumis pour un diagnostic de maladie gastrointestinale. Les fréquences combinées pour les cas de maladies gastrointestinales impliquant Escherichia coli, Clostridium perfringens type A, rotavirus, et Clostridium difficile, soit comme seul agent pathogène ou un complexe d'agents pathogènes, représentaient 56 % du total des cas. Pour 33 % des cas de maladies gastrointestinales, aucun agent étiologique n'a été identifié. La fréquence des cas avec un diagnostic d'E. coli entérotoxigénique a diminué à compter de 2007. Les cas soumis en 2010 étaient plus susceptibles d'avoir un diagnostic d'infection à C. perfringens type A comparativement aux cas soumis de 2002 à 2007 (P < 0,05). Il y avait une tendance significative pour les cas soumis en hiver d'avoir un diagnostic avec C. perfringens type A, E. coli entérotoxigénique, rotavirus, et Cystoisospora suis (anciennement Isospora suis) (P < 0,05). Les E. coli entérotoxigéniques étaient moins souvent diagnostiquées si C. difficile, C. perfringens, ou du rotavirus étaient détectés (P < 0,05). Les porcelets plus jeunes étaient plus susceptibles d'être diagnostiqués avec du C. perfringens type A (P < 0,05) et C. difficile (P < 0,05) que les porcelets plus vieux. Cette étude démontre que E. coli, C. perfringens type A, rotavirus, et C. difficile sont des agents pathogènes d'intérêt pour les opérations de mise-bas en Ontario et des recherches supplémentaires sont requises pour comprendre les raisons pour lesquelles certains cas ne sont pas diagnostiqués.(Traduit par Docteur Serge Messier).

Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

Longitudinal study on the seroprevalence of avian influenza, leptospirosis, and tularemia in an urban population of raccoons (Procyon lotor) in Ontario, Canada.

Raccoons (Procyon lotor) live at high densities, often in close association with people, in urban areas in Ontario and have been implicated as potential reservoirs of numerous zoonotic disease agents. We collected 137 blood samples from 61 apparently healthy raccoons in a small area of Toronto, Ontario, from June to October 2007 as part of a longitudinal study to determine the seasonal patterns of seroprevalence of Francisella tularensis, avian influenza, and Leptospira. In addition, we collected 35 urine samples by cystocentesis from 23 animals to look for evidence of urinary shedding of Leptospira. All samples were serologically negative for F. tularensis and avian influenza. Nineteen of 61 animals (31%) were positive for Leptospira antibodies in one or more trapping periods. The seroprevalence of Leptospira increased from 5% in June to 38% in October. Of the 19 positive animals, 14 were seropositive for serogroup Grippotyphosa, 4 for serogroup Pomona, and 1 for both serogroups Australis and Grippotyphosa. Raccoons were seronegative to serovars representative of serogroups Autumnalis, Canicola, Icterohaemorrhagiae, and Sejroe. Only one urine sample was culture positive for Leptospira (2.9%). Although we found evidence that raccoons in this study were exposed to leptospires belonging to serogroup Grippotyphosa, likely serovar Grippotyphosa, during the summer and able to shed leptospires in urine, further work is required to determine the importance of raccoons as reservoirs of Leptospira in Ontario.

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.

The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

Rhodococcus equi virulence-associated protein A is required for diversion of phagosome biogenesis but not for cytotoxicity.

Rhodococcus equi is a gram-positive facultative intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. Virulence is plasmid regulated and is accompanied by phagosome maturation arrest and host cell necrosis. A replacement mutant in the gene for VapA (virulence-associated protein A), a major virulence factor of R. equi, was tested for its activities during macrophage infection. Early in infection, phagosomes containing the vapA mutant did not fuse with lysosomes and did not stain with the acidotropic fluor LysoTracker similar to those containing virulent wild-type R. equi. However, vapA mutant phagosomes had a lower average pH. Late in infection, phagosomes containing the vapA mutant were as frequently positive for LysoTracker as phagosomes containing plasmid-cured, avirulent bacteria, whereas those with virulent wild-type R. equi were still negative for the fluor. Macrophage necrosis after prolonged infection with virulent bacteria was accompanied by a loss of organelle staining with LysoTracker, suggesting that lysosome proton gradients had collapsed. The vapA mutant still killed the macrophages and yet did not affect the pH of host cell lysosomes. Hence, VapA is not required for host cell necrosis but is required for neutralization of phagosomes and lysosomes or their disruption. This is the first report of an R. equi mutant with altered phagosome biogenesis.

A mycolyl transferase mutant of Rhodococcus equi lacking capsule integrity is fully virulent.

Rhodococcus equi is a mucoid Gram-positive facultative intracellular pathogen which can cause severe bronchopneumonia in foals and AIDS patients. A polysaccharide capsule which gives R. equi a mucoid appearance has long been suspected to be a virulence factor. Here, we describe a transposome mutant in the gene fbpA of strain R. equi 103 causing absence of a capsular structure. FbpA is a chromosomal gene homologous to antigen 85 (Ag85) mycolyl chain transferase gene of Mycobacterium tuberculosis. The mutant multiplied normally in isolated macrophages, was able to establish the unusual R. equi-containing vacuole in macrophages, was cytotoxic for macrophages, and was virulent in a mouse model. Colonies had a dry appearance on nutrient agar and defective capsule structure. Surprisingly, fbpA mutants cured of the virulence-associated plasmid were found in a phagosome that was more alkaline than that of the corresponding wild-type bacteria, were more cytotoxic and even multiplied to some extent. This study suggests that the capsule is not an important virulence factor of R. equi and that it may even counteract virulence traits.

Immunization by intrabronchial administration to 1-week-old foals of an unmarked double gene disruption strain of Rhodococcus equi strain 103+.

Rhodococcus equi causes fatal granulomatous pneumonia in foals and immunocompromised animals and humans. However, there is no effective vaccine against this infection. In this study, the chromosomal genes isocitrate lyase (icl) and cholesterol oxidase (choE) were chosen as targets for mutation and assessment of the double mutant as an intrabronchial vaccine in 1-week-old foals. Using a modification of a suicide plasmid previously developed in this laboratory, we developed a choE-icl unmarked deletion mutant of R. equi strain 103+. Five 1-week-old foals were infected intrabronchially with the mutant and challenged intrabronchially with the parent, virulent, strain 2 weeks later. Three of the foals were protected against pneumonia caused by the virulent strain, but the other two foals developed pneumonia caused by the mutant strain during the post-challenge period. Since infection of 3-week-old foals by an icl mutant in an earlier study had shown complete attenuation of the strain, we conclude that a proportion of foals in the 1st week or so of life are predisposed to developing R. equi pneumonia because of an inability to mount an effective immune response. This has been suspected previously but this is the first time that this has been demonstrated experimentally.

In vivo expression of and cell-mediated immune responses to the plasmid-encoded virulence-associated proteins of Rhodococcus equi in foals.

Rhodococcus equi is a facultative intracellular pathogen that causes pneumonia in foals but does not induce disease in adult horses. Virulence of R. equi depends on the presence of a large plasmid, which encodes a family of seven virulence-associated proteins (VapA and VapC to VapH). Eradication of R. equi from the lungs depends on gamma interferon (IFN-gamma) production by T lymphocytes. The objectives of the present study were to determine the relative in vivo expression of the vap genes of R. equi in the lungs of infected foals, to determine the recall response of bronchial lymph node (BLN) lymphocytes from foals and adult horses to each of the Vap proteins, and to compare the cytokine profiles of proliferating lymphocytes between foals and adult horses. vapA, vapD, and vapG were preferentially expressed in the lungs of infected foals, and expression of these genes in the lungs was significantly (P < 0.05) higher than that achieved during in vitro growth. VapA and VapC induced the strongest lymphoproliferative responses for foals and adult horses. There was no significant difference in recall lymphoproliferative responses or IFN-gamma mRNA expression by bronchial lymph node lymphocytes between foals and adults. In contrast, interleukin 4 (IL-4) expression was significantly higher for adults than for foals for each of the Vap proteins. The ratio of IFN-gamma to IL-4 was significantly higher for foals than for adult horses for most Vap proteins. Therefore, foals are immunocompetent and are capable of mounting lymphoproliferative responses of the same magnitude and cytokine phenotype as those of adult horses.

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.

Cervine (Cervus elaphus) cytokine mRNA quantification by real-time polymerase chain reaction.

It has been difficult to perform cytokine studies for many wildlife and nontraditional species because of a lack of immunologic reagents at the protein level. Recently, simple and rapid assays for quantifying mRNA expression by real-time reverse transcription-polymerase chain reaction (RT-PCR) have been used for analysis of cytokine profiles in humans and other mammalian species. This report describes the development and application of real time RT-PCR to measure the expression of several important elk (Cervus elaphus) cytokine mRNAs, including interleukin (IL)-2, IL-4, IL-10, IL-12p40, interferon-gamma, tumor necrosis factor (TNF)-alpha, and the enzyme-inducible nitric oxide synthase, all of which are involved in immune responses and regulation. For the broadest potential application of the assay, primers and probes were designed using consensus sequences from several species of interest. To obtain standardized quantitative results, external controls consisting of a DNA template for each target gene were used to generate linear standard curves over a 6 to 8 log range with detection of as few as 10 copies of amplicon per reaction. Sample-to-sample variation in the efficiency of the RT, as well as in the quantity and quality of the starting RNA, was compensated for by normalizing the results to the endogenous housekeeping gene beta(2)-microglobulin. The assay was evaluated by monitoring the kinetics of cytokine mRNA synthesis induced by mitogenic and antigenic stimulation of peripheral blood mononuclear cells (PBMCs) from Mycobacterium bovis-infected elk. Concanavalin A-stimulated PBMCs demonstrated a rapid but transient increase in cytokine mRNA expression following in vitro mitogenic activation with optimal mRNA induction observed after 4 to 16 hr. The PBMCs stimulated with the mycobacterial recall antigen, bovine-purified protein derivative (PPD-bovis), demonstrated variable mRNA induction kinetics for each cytokine. Whereas PPD-bovis optimally induced IL-2 mRNA after 8 hr of in vitro stimulation, longer in vitro stimulation times were necessary for the optimal induction of IL-4 and TNF-alpha mRNA (up to 48 hr). We demonstrate real-time RT-PCR to be a rapid, sensitive, and reproducible technique, which will make it a valuable tool in the study of immunologic responses and cytokine profiles of cervids and other nontraditional livestock and wildlife species.

In vitro and intra-macrophage gene expression by Rhodococcus equi strain 103.

Rhodococcus equi is a facultative intracellular respiratory pathogen of foals that persists and multiplies within macrophages. In foals, virulence is associated with 80-90 kb plasmids, which include a pathogenicity island (PI) containing the virulence-associated protein (vap) gene family, but detailed understanding of the basis of virulence is still poor. A 60 spot-based DNA microarray was developed containing eight PI genes and 42 chromosomal putative virulence or virulence-associated genes selected from a recent partial genome sequence in order to study transcription of these genes by R. equi grown inside macrophages and under in vitro conditions thought to simulate those of macrophages. In addition to seven PI genes, nine chromosomal genes involved in fatty acid and lipid metabolism (choD, fadD13, fbpB), heme biosynthesis (hemE), iron utilization (mbtF), heat shock resistance and genes encoding chaperones (clpB, groEL), a sigma factor (sigK), and a transcriptional regulator (moxR) were significantly induced in R. equi growing inside macrophages. The pattern of R. equi chromosomal genes significantly transcribed inside macrophages largely differed from those transcribed under in vitro conditions (37 degrees C, pH 5.0 or 50mM H2O2 for 30 min). This study has identified genes, other than those of the virulence plasmid, the transcription of which is enhanced within equine macrophages. These genes should be investigated further to improve understanding of how this organism survives intracellularly.

Isocitrate lyase activity is required for virulence of the intracellular pathogen Rhodococcus equi.

Rhodococcus equi is an important pathogen of foals, causing severe pyogranulomatous pneumonia. Virulent R. equi strains grow within macrophages, a process which remains poorly characterized. A potential source of carbon for intramacrophage R. equi is membrane lipid-derived fatty acids, which following beta oxidation are assimilated via the glyoxylate bypass. To assess the importance of isocitrate lyase, the first enzyme of the glyoxylate bypass, in virulence of a foal isolate of R. equi, a mutant was constructed by a strategy of single homologous recombination using a suicide plasmid containing an internal fragment of the R. equi aceA gene encoding isocitrate lyase. Complementation of the resulting mutant with aceA showed that the mutant was specific for this gene. Assessment of virulence in a mouse macrophage cell line showed that the mutant was killed, in contrast to the parent strain. Studies in the liver of intravenously infected mice showed enhanced clearance of the mutant. When four 3-week-old foals were infected intrabronchially, the aceA mutant was completely attenuated, in contrast to the parent strain. In conclusion, the aceA gene was shown to be essential for virulence of R. equi, suggesting that membrane lipids may be an important source of carbon for phagocytosed R. equi.

The iron-regulated iupABC operon is required for saprophytic growth of the intracellular pathogen Rhodococcus equi at low iron concentrations.

Rhodococcus equi is a facultative intracellular pathogen which proliferates rapidly in both manure-enriched soil and alveolar macrophages. Although both environments are characterized by extremely low concentrations of free iron, very little is known regarding the strategies employed by R. equi to thrive under these conditions. This paper reports the characterization of an R. equi transposome mutant that fails to grow at low iron concentrations. The transposome was shown to be inserted into iupA, the first gene of the iupABC operon encoding an ABC transport system highly similar to siderophore uptake systems. Disruption of the iupA gene also resulted in a failure of R. equi to utilize heme and hemoglobin as a source of iron. Introduction of the iupABC operon in trans restored the wild-type phenotype of the mutant strain. iupABC transcripts were 180-fold more abundant in R. equi grown in iron-depleted medium than in organisms grown in iron-replete medium. Proliferation of the iupABC mutant strain in macrophages was comparable to that of the wild-type strain. Furthermore, the iupABC mutant was not attenuated in mice, showing that the iupABC operon is not required for virulence.

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.

Analysis of virulence plasmid gene expression of intra-macrophage and in vitro grown Rhodococcus equi ATCC 33701.

Rhodococcus equi is a soil organism that infects macrophages of foals and immunocompromised humans. Virulence in foal isolates is tightly associated with an 80kb plasmid, which includes a pathogenicity island (PI) with a virulence-associated gene family, vap. A DNA microarray containing 66 of 69 putative open reading frames (ORFs) of the virulence plasmid was developed. Virulence plasmid gene expression of R. equi grown in macrophages or under different conditions in vitro was compared against in vitro growth at 30 degrees C, pH=7. When grown in macrophages, all seven vap family genes as well as six ORFs within, but not outside, the PI were induced. Cluster analysis of the gene expression matrix assembled from different growth conditions suggested that those genes that actively responded to environmental changes divided broadly into two groups. One group, orf1, 2, 5, 6-8, 12-15, 19, and 20 (which includes all the vap genes), was induced at 37 degrees C, mostly by low iron, and to a lesser extent by the synergy of low calcium and pH=5. The second group, orf3, 9, and 10, was induced at 37 degrees C by magnesium depletion (produced by EDTA treatment of growth medium). Temperature (37 degrees C) was the most important factor inducing gene expression for the both groups. Iron restriction led to down-regulation of Group II genes and magnesium restriction led to down-regulation of Group I genes. A putative consensus IdeR binding site was identified upstream of vapA, suggesting that vapA is a member of an IdeR regulon in R. equi. Expression of genes inside macrophages was most closely but not completely mimicked by growth of bacteria at 37 degrees C, pH=5, under conditions of restricted iron, calcium and magnesium; that is, similar to environmental factors found inside macrophages.

A novel lipoarabinomannan from the equine pathogen Rhodococcus equi. Structure and effect on macrophage cytokine production.

Rhodococcus equi is a major cause of foal morbidity and mortality. We have investigated the presence of lipoglycan in this organism as closely related bacteria, notably Mycobacterium tuberculosis, produce lipoarabinomannans (LAM) that may play multiple roles as virulence determinants. The lipoglycan was structurally characterized by gas chromatography-mass spectrometry following permethylation, capillary electrophoresis after chemical degradation, and (1)H and (31)P and two-dimensional heteronuclear nuclear magnetic resonance studies. Key structural features of the lipoglycan are a linear alpha-1,6-mannan with side chains containing one 2-linked alpha-d-Manp residue. This polysaccharidic backbone is linked to a phosphatidylinositol mannosyl anchor. In contrast to mycobacterial LAM, there are no extensive arabinan domains but single terminal alpha-d-Araf residue capping the 2-linked alpha-d-Manp. The lipoglycan binds concanavalin A and mannose-binding protein consistent with the presence of t-alpha-d-Manp residues. We studied the ability of the lipoglycans to induce cytokines from equine macrophages, in comparison to whole cells of R. equi. These data revealed patterns of cytokine mRNA induction that suggest that the lipoglycan is involved in much of the early macrophage cytokine response to R. equi infection. These studies identify a novel LAM variant that may contribute to the pathogenesis of disease caused by R. equi.