INFECTION AND IMMUNITY, Oct. 2005, p. 6736–6741 0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.10.6736–6741.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 10
Isocitrate Lyase Activity Is Required for Virulence of the Intracellular Pathogen Rhodococcus equi Daniel M. Wall, Pamela S. Duffy,1† Chris DuPont,2 John F. Prescott,2 and Wim G. Meijer1* Department of Industrial Microbiology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland,1 and Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada2 Received 30 March 2005/Returned for modification 16 May 2005/Accepted 13 July 2005
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  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 gram-positive bacterium Rhodococcus equi is a major cause of subacute or chronic granulomatous bronchopneumonia in young foals up to 5 months in age and sporadically infects other animals, such as pigs, goats, and cattle. In addition to its animal hosts, R. equi is increasingly responsible for AIDS-associated pneumonia in human immunodeficiency virus-infected individuals (26). The virulence of R. equi is dependent on its ability to infect and proliferate in macrophages, eventually resulting in necrotic death of the infected cell (13, 22). Intramacrophage R. equi appears to be located exclusively within membrane-enclosed vacuoles, and persistence correlates with the absence of phagosome-lysosome fusion (11, 41). All virulent strains isolated from horses contain an 80- to 85-kb plasmid, which is essential for replication and cytotoxicity of the pathogen in macrophages (8, 22, 35, 36). A detailed analysis of the nucleotide sequence of the virulence plasmid of two clinical isolates from foals revealed the presence of a 27-kb pathogenicity island, harboring at least 21 genes (34). One of these genes, encoding the lipid-modified, surface-expressed, virulence-associated protein A (VapA), has been shown to be essential for virulence (18, 33, 37). The pathogenicity island contains an additional seven vapA homologues, two of which are pseudogenes (5, 30, 34). Expression of the VapA gene and other genes in the pathogenicity island is controlled by a range of environmental parameters, including temperature, pH, oxidative stress, and the concentrations of calcium and magnesium (1, 5, 29, 33).
A major area of interest is the determination of what sources of carbon sustain bacterial or fungal pathogens following infection. It has been shown that in contrast to cells grown in liquid medium, Mycobacterium tuberculosis isolated from lungs of infected mice actively oxidizes fatty acids, indicating that these may be an important source of carbon for this pathogen (3). Fatty-acid metabolism proceeds via the dissimilation of these substrates via  oxidation, followed by the assimilation of the resulting acetyl-coenzyme A (CoA) via the glyoxylate shunt. The latter pathway consists of the combined activities of isocitrate lyase and malate synthase, converting isocitrate and acetyl-CoA to succinate and malate. This pathway therefore circumvents the two decarboxylation steps of the citric acid cycle, allowing assimilation of two carbon (C2) substrates, such as acetate, ethanol, and fatty acids (20). A major breakthrough in the analysis of the physiology of intracellular pathogens was the detection of isocitrate lyase mRNA and protein in phagocytosed M. tuberculosis and Mycobacterium avium, followed by the observation that an isocitrate lyase-deficient mutant of M. tuberculosis is impaired in persistence in macrophages and mice (10, 14, 25). Interestingly, the requirement for isocitrate lyase was apparent largely in immunocompetent mice, and the mutant was markedly attenuated for survival in activated but not in resting macrophages. These observations lead to the suggestion that lipids, derived from either macrophage membranes or cell remnants in granulomas, are a major source of carbon for M. tuberculosis (25). Since then, the glyoxylate bypass has been shown to be important for virulence of Rhodococcus fascians and the fungal pathogens Candida albicans, Leptosphaeria maculans, and Magnaporthe grisea (17, 21, 38, 39). Interestingly, isocitrate lyase-deficient mutants of Cryptococcus neoformans and Saccharomyces cerevisiae are not affected in virulence (9). We recently characterized the gene encoding isocitrate lyase of R. equi (19). The aim of the current
* Corresponding author. Mailing address: Department of Industrial Microbiology, University College Dublin, Dublin 4, Ireland. Phone: 353-1716-1364. Fax: 353-1716-1183. E-mail:
[email protected]. † Present address: Department of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 114 16th St. (114-3503), Charlestown, MA 02129. 6736
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TABLE 1. Bacteria and plasmids used in this study Strain or plasmid
Source or reference
Characteristic(s)
Strains E. coli DH5␣ R. equi 103P⫹ R. equi 103P⫺ R. equi Ace-21
supE44 ⌬lacU169 [80lacZ⌬M15] hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Virulent foal isolate Avirulent foal isolate lacking the virulence plasmid aceA derivative of R. equi 103 P⫹
Stratagene 6 6 This study
Plasmids pBluescript II KS pRE7 pAP1 pICL11 pAIP1 pKICLA
Apr Kmr, R. equi-E. coli shuttle vector Aprr, R. equi suicide vector Apr, pBluescript containing a 1.8-kb HindIII-BamHI DNA fragment harbouring aceA of R. equi Aprr, Suicide vector for R. equi containing an internal 594-bp aceA fragment Kmr, pRE7 containing a 1.8-kb HindIII-BamHI DNA fragment of pICL11 harbouring aceA
Stratagene 40 23 19 This study This study
paper was to determine the role of isocitrate lyase in virulence of R. equi in foals. MATERIALS AND METHODS Bacterial strains and plasmids. Plasmids and strains used in this study are listed in Table 1. Media and growth conditions. Bacterial strains were grown in Luria-Bertani (LB) broth (31) or in R. equi minimal medium containing either succinate (20 mM), acetate (20 mM), or lactate (20 mM) as a source of carbon (19). Where appropriate, the following supplements were added: kanamycin, 50 g ml⫺1 (E. coli) or 200 g ml⫺1 (R. equi); apramycin, 80 g ml⫺1; 5-bromo-4-chloro-3indolyl--D-galactopyranoside, 20 g ml⫺1; isopropyl--D-thiogalactopyranoside, 0.1 mM. For solid media, agar was added to 1.5% (wt/vol). DNA manipulations. Plasmid DNA was isolated with the alkaline lysis method (2) or by using the Wizard Plus SV miniprep (Promega). Chromosomal DNA was isolated as described previously (27). DNA-modifying enzymes were used according to the manufacturer’s recommendations (Roche). Dideoxy sequencing reactions were done with the CEQ DCTS kit as described by the manufacturer (Beckman). The nucleotide sequence was determined using a Beckman CEQ 8000 automatic sequencer; nucleotide sequence data were compiled using the Staden package (32). PCR was carried out using Taq DNA polymerase (Promega) or Deep Vent DNA polymerase (New England Biolabs) as described by the manufacturer. Other DNA manipulations were done in accordance with standard protocol (31). Detection of the virulence plasmid. To verify the presence of the virulence plasmid, two genes specific for this plasmid, virR (ORF4) and vapA (ORF12), were amplified by PCR using Taq DNA polymerase according to the manufacturer’s (Promega) instructions. A 200-bp fragment of the virR gene was amplified using 004F (5⬘-CGGACGAGTTCGACTGGTAT-3) and 004R (5⬘-CAAAGAC GATTTGGGGTACG-3⬘); a 200-bp fragment of vapA was amplified using 012F (5⬘-CAGTACGACGTTCACGGAGA-3⬘) and 012R (5⬘-CACGGCGTTGTAC TGGAAC-3⬘). Construction of disruption vector pAIP1. Plasmid pAP1 contains an origin of replication derived from the vector pBLUESCRIPT and the aacC4 gene specifying resistance to apramycin. This plasmid was digested with HindIII, treated with the Klenow fragment of DNA polymerase, and ligated to a 594-bp fragment of the aceA gene, which was amplified by PCR using the oligonucleotides Icl5 (5⬘-CAACGTCTACGAGCTGCAGA-3⬘) and Icl10 (5⬘-TCGAACATGCCGTA GTTGAG-3⬘). The resulting plasmid, pAIP1, contains an internal fragment of the aceA gene. Electroporation of R. equi. R. equi was made electrocompetent using the method described by Navas et al. (28); plasmids were introduced into R. equi by electroporation as previously described (24). Enzyme assays. Cells were harvested in late-exponential-phase growth (optical density at 600 nm ⫽ 1). Cells were broken by three passages through a French pressure cell (Aminco) at 1,000 lb/in2, followed by centrifugation (10 min, 14,000 ⫻ g, 4°C) to remove cell debris. Isocitrate lyase (EC 4.1.3.1) activity was determined at 37°C by measuring the formation of glyoxylate phenylhydrazone in the presence of phenylhydrazine and isocitrate at 324 nm as described previously (7). Protein was determined according to the method of Bradford using bovine serum albumin as the standard (4).
Macrophage infection. Murine J774A.1 macrophages were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum, 2 mM glutamine, and 10 g/ml gentamicin. Plates were seeded at a concentration of 1 ⫻ 106 macrophages per well (final volume, 1 ml) and left for 48 h, feeding with growth medium after 24 h. Overnight LB broth cultures of R. equi 103P⫹, 103P⫺, and Ace-21 were grown to a density of 108 CFU per ml. R. equi was washed twice with cation-free phosphate-buffered saline and resuspended in phagocytosis buffer (12). Monolayers were washed with warm DMEM, and phagocytosis buffer and normal mouse serum (5% [vol/vol]) were added. Macrophages were subsequently infected with wild-type and mutant strains at a multiplicity of infection of 15. Plates were incubated for 30 min at 37°C in 5% CO2. Monolayers were subsequently washed with phagocytosis buffer to remove unbound bacteria and incubated for a further 30 min to allow internalization of the bacteria. Phagocytosis buffer was replaced with DMEM supplemented with 10% (vol/vol) fetal calf serum, 2 mM glutamine, and 50 g/ml gentamicin for 15 min. The medium was subsequently replaced with same containing 10 g/ml gentamicin. Medium was removed and monolayers washed with phosphatebuffered saline. Macrophages were then lysed with 0.5% (wt/vol) sodium dodecyl sulfate (SDS). Exposure to SDS was brief (20 s), allowing lysis of the macrophage but not affecting subsequent growth of R. equi. R. equi was subsequently enumerated by plate counts on LB agar plates. Mouse infection. Six groups of eight 6- to 8-week-old female CD1 mice were injected intravenously with 100 l of 5 ⫻ 106 CFU/ml of R. equi 103P⫹, 103P⫺, or Ace-21. The mice were euthanized 2 or 4 days after infection, and their livers were aseptically removed and ground in phosphate-buffered saline, pH 7.2. The suspended ground tissue was diluted in a 10-fold series and 50-l aliquots plated on Trypticase soy agar (Difco). Bacterial colonies were counted after 48 h incubation at 37°C. Significant differences in bacterial numbers between mice infected with R. equi 103P⫹ and those infected with R. equi Ace-21 were determined by the two-tailed Student t test. Foal infection. Infection and subsequent clinical assessment of foals was carried out as described previously (8). Briefly, four mixed-breed pony foals at 21 days of age were challenged with R. equi Ace-21 and four similarly aged foals were challenged with R. equi P⫹ by introducing 25 ml of cell suspension (5 ⫻ 108 cells/ml) into both main bronchi (total, 50 ml). Foals were clinically assessed based on daily complete physical examinations and twice-daily heart rate, respiratory rate, and temperature recordings. Foals were euthanized 14 days postinfection, and post-mortem examinations were performed to determine the lungto-body-weight ratio, and to take lung samples to determine R. equi counts per gram of lung tissue from six preselected sites. Lesions, if any, in the lungs were examined for consistency with R. equi infection. The proportion of Ace-21 reverting to the wild type in foals infected with this mutant was determined by enumeration of R. equi on Trypticase soy agar plates with and without apramycin. Nucleotide sequence accession number. The GenBank accession number of the sequence reported in this paper is AY738741.
RESULTS Isocitrate lyase gene of R. equi 103. We previously characterized the aceA gene of R. equi ATCC 33701. Although this is a well-characterized strain, it has been noted by us and others
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FIG. 1. Strategy for disruption of the aceA gene of Rhodococcus equi. The suicide plasmid pAIP1 harboring the aacC4 gene conferring apramycin resistance and an internal fragment of aceA was introduced into R. equi by homologous recombination. Insert A shows the results of a PCR amplification of the virR and vapA genes of two apramycin-resistant R. equi strains (Ace-20 and Ace-21) and the wild-type strain (103). The molecular weight marker is a 100-bp marker. Insert B shows the result of a Southern blot of chromosomal DNA of two apramycin-resistant R. equi strains (Ace-20 and Ace-21) and the wild-type strain (103) digested with BglII using the aceA gene as probe.
that R. equi 103 is more amenable to genetic manipulation than strain ATCC 33701 due to an approximately 100-fold-higher transformation frequency and reduced frequency of illegitimate recombination. We therefore chose strain 103 with which to construct an aceA mutant. The virulence plasmids of both strains have been sequenced and shown to be virtually identical, suggesting that the genomes of both strains are equally conserved (34). Prior to constructing an aceA mutant strain, we amplified a 1,126-bp DNA fragment containing an internal fragment of the aceA gene of R. equi 103. Nucleotide sequence analysis showed it to be identical to the gene of strain 33701, except for a neutral substitution in codon 47 encoding threonine in which ACC is replaced by ACG. Construction of an isocitrate lyase-deficient R. equi mutant. To construct an R. equi mutant lacking isocitrate lyase activity, the 2.9-kb plasmid pAIP1 was created, which contains the aacC4 gene conferring apramycin resistance as a selectable marker and a 594-bp internal fragment of aceA. This plasmid was introduced into R. equi by electroporation, and the transformation mixture was subsequently plated onto LB agar plates containing apramycin. Because pAIP1 is unable to replicate in R. equi, apramycin-resistant colonies will contain pAIP1 integrated into the chromosome. To determine whether this was indeed the case, chromosomal DNA was isolated from two of the apramycin-resistant mutants, digested with BglII, and analyzed by Southern hybridization using the 594-bp internal aceA fragment as a probe. Since a BglII restriction site does not occur within either the aceA gene or pAIP1, insertion of pAIP1 will increase the size of the BglII fragment harboring aceA by 2.9 kb. Following hybridization of the aceA probe to BglII-digested chromosomal DNA of two apramycin-resistant mutants (R. equi Ace-20 and Ace-21) and the wild-type strain, a single 8-kb hybridizing band was observed in the two mu-
tants, whereas a 5-kb BglII fragment was observed in the-wild type strain (Fig. 1). To further confirm that pAIP1 had inserted correctly into the aceA gene, chromosomal DNA of one of the mutants (R. equi Ace-21) was digested with BglII and subsequently ligated. The ligation mixture was used to transform E. coli, followed by selection for apramycin-resistant colonies, which contained an 8-kb plasmid harboring the disrupted aceA gene. Subsequent determination of the nucleotide sequence of the junction between the aceA gene and the point of insertion of pAIP1 showed that insertion had occurred via homologous recombination between aceA and the 594-bp internal aceA fragment (data not shown). Using virulence plasmid-specific primers amplifying the virR and vapA genes, it was shown that the aceA disruption mutant had retained the virulence plasmid (Fig. 1). Characterization of R. equi Ace-21. We previously determined that R. equi contains high activities of isocitrate lyase following growth on acetate- or lactate-containing minimal medium (19). Isocitrate lyase activity is essential for growth on acetate, since it allows assimilation of this C2 compound via the glyoxylate bypass. However, unless lactate is metabolized using lactate oxidase, which converts lactate to CO2 and acetate, isocitrate lyase is not required for lactate metabolism. As expected, R. equi Ace-21 failed to grow on acetate, whereas growth on lactate- and succinate-containing minimal media was comparable to that of the wild type. The activity of isocitrate lyase in cell extracts of wild-type and mutant strains following growth on lactate was 586 nmol per min per mg of protein for the wild type, whereas this enzyme activity was absent in R. equi Ace-21. These results demonstrate that although aceA is expressed during growth on lactate, it is dispensable for the metabolism of this substrate. In order to rule out that the failure of R. equi Ace-21 to grow on acetate
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FIG. 2. Survival and proliferation of isogenic R. equi strains in a murine macrophage-like cell line (J774A.1). Monolayers were infected with R. equi P⫹ (F), R. equi P⫺ (■), isocitrate lyase-deficient strain R. equi Ace-21 (‚), and R. equi Ace-21 containing pKICLA harboring an intact aceA gene (Œ). Following a 30-min incubation to allow phagocytosis, monolayers were washed and treated with gentamicin to kill remaining extracellular bacteria. Intracellular bacteria were enumerated by plate counts following macrophage lysis. The data represent the averages for two independent experiments. Plate counts were carried out in duplicate. Values are expressed as means ⫾ standard deviations (error bars).
minimal medium was due to a mutation other than disruption of the aceA gene, plasmid pKICLA harboring an intact aceA gene and its promoter was introduced in R. equi Ace-21. In contrast to the mutant strain, a strain harboring this plasmid was able to use acetate as the sole source of carbon. R. equi Ace-21 is unable to proliferate in macrophages. Intracellular survival and proliferation of R. equi Ace-21 in the murine macrophage-like cell line (J774A.1) was compared to those of the wild-type strain and its avirulent plasmid-free derivative. To this end, macrophages were incubated with R. equi and allowed to internalize, after which gentamicin was added to kill extracellular bacteria. Intracellular bacteria were subsequently enumerated by plate counts following SDS-induced macrophage lysis. The number of intracellular wild-type R. equi bacteria increased 11-fold over a 48-h period, whereas the avirulent plasmid-free strain failed to proliferate, as observed previously (8). In contrast, the number of intracellular R. equi Ace-21 bacteria increased after 12 h of infection but then declined by three orders of magnitude, indicating that isocitrate lyase and a functional glyoxylate bypass are essential for long-term survival and proliferation of R. equi in macrophages. The ability of R. equi Ace-21 to proliferate in macrophages was restored following the introduction of pKICLA, which contains an intact aceA gene, showing that the attenuated phenotype of this strain was due to disruption of the aceA gene (Fig. 2). R. equi Ace-21 is attenuated in mice. Although immunocompetent mice eventually clear R. equi, the increase in R. equi numbers in the liver and spleen 2 to 4 days after intravenous injection provides a good indication of R. equi virulence in vivo
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FIG. 3. Virulence of isogenic R. equi strains in mice. Mice were intravenously infected with R. equi P⫹ (black bar) and Ace-21 (white bar). Two and four days following infection, the numbers of R. equi bacteria present in the liver were determined. The avirulent strain R. equi P⫺ was completely cleared after 2 days (data not shown). Values are expressed as means ⫾ standard deviations (error bars).
(8). To assess the virulence of R. equi Ace-21, mice were intravenously injected with this strain, and the number of R. equi Ace-21 bacteria in the liver was compared to those in mice infected with isogenic virulent (103P⫹) or avirulent (103P⫺) R. equi. The avirulent plasmid-free strain was completely cleared 2 days after infection. In contrast, both the virulent and isocitrate lyase-deficient strain could be detected in the livers of mice 2 and 4 days following infection. However, there was a significant difference (day 2, P ⫽ 9.5 ⫻ 10⫺5; day 4, P ⫽ 9.6 ⫻10⫺3) in bacterial load between the two strains (Fig. 3), showing that R. equi Ace-21 is partially attenuated in mice. In vivo infection of foals. The previous experiments using macrophages and mice to assess virulence of R. equi Ace-21 indicated that R. equi Ace-21 is attenuated. However, unlike foals, mice are naturally resistant to R. equi infections, and the route of infection in mice by intravenous injection is different from that in foals, which are challenged via the respiratory route. The outcome of the macrophage and mouse studies may therefore not necessarily reflect virulence of R. equi Ace-21 in foals. To evaluate virulence of R. equi Ace-21 in its native host, 3-week-old pony foals were intrabronchially infected with R. equi Ace-21, and results were compared to those for foals infected with R. equi 103 P⫹. The temperature and heart and respiratory rates of foals infected with R. equi P⫹ started to increase 9 days postinfection, whereas these parameters remained unaltered in foals infected with R. equi Ace-21 (Fig. 4). The foals were subjected to a post-mortem analysis immediately following euthanasia. The lungs from foals infected with virulent R. equi displayed severe lesions of suppurative to pyogranulomatous bronchopneumonia. In contrast, of the four foals infected with R. equi Ace-21, none showed any signs of granulomatous or other pneumonia in the lung tissue upon post-mortem examination. The lungs were completely normal both on gross visual inspection and on palpation. The lung weight/body weight ratio of control foals infected with R. equi 103P⫹ was 3.83% ⫾ 1.18%, whereas this ratio was 1.15% ⫾ 0.06% in foals infected with the isocitrate lyase-deficient strain. These values compare to 4.58% ⫾ 0.96% for infected historical
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FIG. 4. Heart rates (A), temperatures (B), and respiratory rates (C) of foals infected with R. equi P⫹ (F) or R. equi Ace-21 (‚). Values are shown as the means ⫾ standard deviations (error bars) within each group.
control foals and to 1.13% ⫾ 0.07% observed previously for noninfected historical control foals (8). The mean number of R. equi bacteria per gram of tissue in Ace-21 infected foals was 103.85 ⫾ 0.59 per gram of lung tissue, compared to 109.61 ⫾ 0.30 for 103P⫹-infected foals. DISCUSSION This paper focused on the role of the glyoxylate bypass in virulence of R. equi by analyzing virulence of an isocitrate lyase-deficient mutant. The glyoxylate bypass, which consists of the combined activities of isocitrate lyase and malate synthase, circumvents two decarboxylation reactions of the citric acid
cycle (20). Without this pathway, assimilation of carbon substrates, which enter the central metabolic pathways at the level of acetyl-CoA, would not be possible, since they would exclusively be converted to CO2. Examples of two carbon compounds are ethanol and acetate, but also fatty acids, which are metabolized to acetyl-CoA by -oxidation. Virulence of the isocitrate lyase mutant was assessed in three ways: macrophage survival, clearance in mice after intravenous infection, and infection in foals. The data show that the three different methods of assessing virulence gave similar results. A comparison of the three approaches has not previously been done, although there appears to be an assumption that these are correlated (8, 18). Survival in macrophages (Fig. 2) and in mice (Fig. 3) measure the ability of the organism to survive the innate immunity provided by macrophages. The observed attenuation of R. equi Ace-21 in these models was supported by infection studies with 3-week-old foals, in which the aceA mutant was found to be completely attenuated, since clinical and pathological findings were similar to those associated with infection of foals with a virulence plasmid-negative and therefore avirulent isolate (8, 15). The observed attenuation of isocitrate lyase-deficient R. equi is in agreement with the results from previous experiments, which showed that isocitrate lyase-negative M. tuberculosis and C. albicans strains are attenuated (21, 25). The dependence on the glyoxylate cycle for virulence strongly indicates that these intracellular pathogens utilize fatty acids derived from macrophage membrane lipids as a source of carbon. However, R. equi appears to be more dependent on the glyoxylate bypass than M. tuberculosis. In the mouse model, isocitrate lyase-deficient M. tuberculosis initially behaved like the wild-type strain, leading to comparable bacterial burdens in the lungs of infected mice 2 weeks following infection. Only in the subsequent weeks did the difference in virulence between the isocitrate lyasedeficient and wild strains become apparent, with the former being progressively cleared whereas the latter persisted. The lack of a functional isocitrate lyase did not seriously affect M. tuberculosis survival in resting macrophages, since only in activated macrophages was there a dramatic difference in survival between wild-type and mutant strains (21, 25). In contrast, the isocitrate lyase-deficient R. equi strain failed to induce disease in foals, and although R. equi could be recovered from lungs of foals infected with R. equi Ace-21, the number of bacteria was about six orders of magnitude lower than usually observed with the wild-type strain (9, 14). Furthermore, the isocitrate lyase-deficient strain failed to proliferate in resting macrophages, whereas the wild-type strain grew with a doubling time of approximately 8 h. It thus appears that R. equi is less versatile than M. tuberculosis with regard to the number of host-derived carbon sources it can metabolize. R. equi is the latest addition to a growing number of pathogenic microorganisms that require the glyoxylate bypass for virulence (17, 21, 25, 38, 39). The dependence on this pathway for virulence appears to be a unifying factor; however, the underlying reasons for this requirement differ. M. tuberculosis, C. albicans, and R. equi are intracellular pathogens of mammalian phagocytic cells, which most likely require the glyoxylate cycle for assimilation of macrophage lipids (21, 25). Virulence of the plant pathogen R. fascians is dependent on malate synthase, which may be required for the assimilation of
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products of photorespiration, such as glycolate (38). The fungus Magnaporthe grisea appears to employ the glyoxylate bypass to metabolize fungal lipids for turgor generation and appressorium formation prior to infection (39). The aceA gene is the second gene after vapA (18) to be shown to be essential for virulence of R. equi. In addition to being essential for assimilation of fatty acids, isocitrate lyase is also required for assimilation of acetate, which is present in significant amounts in the large intestine of horses. R. equi grows well on this substrate, and it has been shown previously that acetate stimulates growth of this pathogen in dung (16). It therefore is clear that although isocitrate lyase is not a true virulence factor like VapA, it plays a critical role in growth and survival of R. equi both within and outside the host environment. ACKNOWLEDGMENTS This work was supported by Research Project Grant 133/99 from the Health Research Board, the Natural Research and Engineering Research Council of Canada, and the Ontario Horseracing Industry Association. We thank Vivian Nicholson for expert technical assistance and Laura Palumbo for care of the foals. REFERENCES 1. Benoit, S., A. Benachour, S. Taouji, Y. Auffray, and A. Hartke. 2002. H2O2, which causes macrophage-related stress, triggers induction of expression of virulence-associated plasmid determinants in Rhodococcus equi. Infect. Immun. 70:3768–3776. 2. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening of recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523. 3. Bloch, H., and W. Segal. 1956. Biochemical differentiation of Mycobacterium tuberculosis grown in vivo and in vitro. J. Bacteriol. 72:132–141. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 73:248–254. 5. Byrne, B. A., J. F. Prescott, G. H. Palmer, S. Takai, V. M. Nicholson, D. C. Alperin, and S. A. Hines. 2001. Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins. Infect. Immun. 69:650–656. 6. de la Pena-Moctezuma, A., and J. F. Prescott. 1995. Association with HeLa cells by Rhodococcus equi with and without the virulence plasmid. Vet. Microbiol. 46:383–392. 7. Dixon, G. H., and H. L. Kornberg. 1959. Assay methods for key enzymes of the glyoxylate cycle. Biochem. J. 72:3p. 8. Gigue`re, S., M. K. Hondalus, J. A. Yager, P. Darrah, D. M. Mosser, and J. F. Prescott. 1999. Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi. Infect. Immun. 67:3548–3557. 9. Goldstein, A. L., and J. H. McCusker. 2001. Development of Saccharomyces cerevisiae as a model pathogen. A system for the genetic identification of gene products required for survival in the mammalian host environment. Genetics 159:499–513. 10. Graham, J. E., and J. E. Clark-Curtiss. 1999. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc. Natl. Acad. Sci. USA 96:11554–11559. 11. Hietala, S. K., and A. A. Ardans. 1987. Interaction of Rhodococcus equi with phagocytic cells from R. equi-exposed and non-exposed foals. Vet. Microbiol. 14:307–320. 12. Hondalus, M. K., M. S. Diamond, L. A. Rosenthal, T. A. Springer, and D. M. Mosser. 1993. The intracellular bacterium Rhodococcus equi requires Mac-1 to bind to mammalian cells. Infect. Immun. 61:2919–2929. 13. Hondalus, M. K., and D. M. Mosser. 1994. Survival and replication of Rhodococcus equi in macrophages. Infect. Immun. 62:4167–4175. 14. Ho ¨ner zu Bentrup, K., A. Miczak, D. L. Swenson, and D. G. Russell. 1999. Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis. J. Bacteriol. 181:7161–7167. 15. Hooper-McGrevy, K. E., S. Gigue`re, B. N. Wilkie, and J. F. Prescott. 2001. Evaluation of equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C for use in protecting foals against Rhodococcus equi-induced pneumonia. Am. J. Vet. Res. 62:1307–1313.
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