Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis

The glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid (TCA) cycle that allows growth on C2 compounds by bypassing the CO2-generating steps of the TCA cycle. The unique enzymes of this route are isocitrate lyase (ICL) and malate synthase (MS). ICL cleaves isocitrate to glyoxylate and succinate, and MS converts glyoxylate and acetyl-CoA to malate. The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The glyoxylate cycle occurs in Eukarya, Bacteria and Archaea. Recent studies of ICLand MS-deficient strains as well as proteomic and transcriptional analyses show that these enzymes are often important in human, animal and plant pathogenesis. These studies have extended our understanding of the metabolic pathways essential for the survival of pathogens inside the host and provide a more complete picture of the physiology of pathogenic micro-organisms. Hopefully, the recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases.


Introduction
More than half a century ago, Smith & Gunsalus (1954) reported the existence of the enzyme isocitrate lyase (ICL), which cleaves isocitrate to glyoxylate and succinate, in extracts prepared from Pseudomonas aeruginosa. Shortly thereafter Ajl (1956) showed that malate synthase (MS) is able to convert acetyl-CoA and glyoxylate to malate in Escherichia coli. In 1957 Kornberg and co-workers demonstrated that the synthesis of C 4 dicarboxylic acids from acetate occurs by a modified tricarboxylic acid (TCA) cycle that was termed the glyoxylate cycle or glyoxylate bypass (Kornberg & Krebs, 1957;Kornberg & Madsen, 1957;Fig. 1). The pathway consists of the two initial steps of the TCA cycle (catalysed by citrate synthase and aconitase) followed by ICL, MS and malate dehydrogenase (Fig. 1). The glyoxylate cycle serves to bypass the CO 2 -generating steps of the TCA cycle and allow the net assimilation of carbon from C 2 compounds, allowing micro-organisms to replenish the pool of TCA cycle intermediates necessary for gluconeogenesis and other biosynthetic processes. The net result of the glyoxylate cycle is the production of malate and succinate from two molecules of acetyl-CoA derived from acetate or from the degradation of ethanol, fatty acids or poly-b-hydroxybutyrate (Fig. 1). During growth on these compounds, ICL competes with isocitrate dehydrogenase (IDH) for their common substrate, isocitrate. IDH has a much higher affinity for isocitrate and, in bacteria, it is inactivated by phosphorylation by a bifunctional IDH kinase-phosphatase, thus directing isocitrate towards the biosynthetic reactions of the glyoxylate cycle. IDH dephosphorylation (activation) occurs when glycolytic and TCA cycle intermediates are present in the medium, causing isocitrate to be directed towards the energy-yielding TCA cycle (Cozzone, 1998).
The glyoxylate cycle is widespread and well documented in archaea, bacteria, protists, plants, fungi and nematodes [the latter contain an ICL-MS gene fusion (Kondrashov et al., 2006)]. The presence of this metabolic pathway in animals is controversial. ICL and MS activities have been reported in birds and amphibians (Davis et al., 1986(Davis et al., , 1990) and a recent comparative genomic study showed the presence of an ICL gene in nematodes and cnidaria and an MS gene in nematodes, cnidaria, echinoderms, amphibians, fish, and insects. Interestingly, in placental mammals the MS gene is a pseudogene and the ICL gene is absent (Kondrashov et al., 2006). In addition to allowing the growth of bacteria on C 2 compounds, the glyoxylate cycle is important for the growth of higher plant seedlings under most environmental conditions, since it participates in the conversion of stored lipids to carbohydrates that serve as a primary nutrient source prior to the commencement of photosynthesis (Eastmond et al., 2000;Kornberg & Beevers, 1957a, b). In plants, the glyoxylate cycle enzymes are usually localized in peroxisomes, but recent studies with protein-targeting mutants have shown that they can also function effectively in the cytosol (Kunze et al., 2002;McCammon et al., 1990;Piekarska et al., 2008). There is evidence supporting the expression of the glyoxylate cycle during embryogenesis of the nematode Caenorhabditis elegans (Kahn & McFadden, 1980;Liu et al., 1997) and it was suggested that the glyoxylate cycle may allow the growth of halophytic archaea in hypersaline lakes (Oren & Gurevich, 1994;Serrano & Bonete, 2001).
The genetic regulation of the glyoxylate cycle during bacterial growth on acetate has been reviewed (Cozzone, 1998) and in the last several years it has become evident that the pathway is important in fungal and bacterial pathogenesis (Lorenz & Fink, 2002;Vereecke et al., 2002a). This review focuses on the latter aspect and summarizes the functional role of the glyoxylate cycle in human, animals and plant pathogens as well as in symbionts.

Biochemical characteristics of ICL and MS
As described above, the glyoxylate cycle is a specialized pathway that has been extensively studied in connection with bacterial growth on C 2 compounds, and ICL and MS are the signature enzymes of the pathway (Fig. 1). ICL is a homotetramer requiring Mg 2+ or Mn 2+ and a thiol for activity. During catalysis, isocitrate is deprotonated, forming succinate and glyoxylate. In the ICL of E. coli,  are catalytic, activesite residues, while His-184 is involved in the assembly of the tetrameric enzyme (Diehl & McFadden, 1993Rehman & Mcfadden, 1996. The recent structural determination of the ICLs from E. coli (Britton et al., 2001), Mycobacterium tuberculosis (Sharma et al., 2000) and Aspergillus nidulans (Britton et al., 2000) has revealed key aspects of their functionality. For instance, a remarkable difference between prokaryotic and eukaryotic ICLs is the presence of an additional approximately 100 amino acids located near the centre of the eukaryotic enzyme that have been proposed to function in the localization of ICL in peroxisomes. This also explains the subunit molecular mass difference between prokaryotic (48 kDa) and eukaryotic (67 kDa) enzymes.
The second enzyme of the glyoxylate cycle is MS, which condenses glyoxylate with an acetyl group from acetyl-CoA to produce malate. MS requires Mg 2+ for activity and is competitively inhibited by oxalate, a glyoxylate analogue (Dixon et al., 1960). Two MSs are present in E. coli: MSG and MSA. MSG, with a molecular mass of around 80 kDa, is a monomeric enzyme (encoded by glcB) that functions during growth on glycolate as sole carbon source, and has been found only in bacteria. The active site is composed of the catalytic Asp-631, the residues that bind Mg 2+ (Glu-427, Asp-455), Arg-338, which hydrogen bonds with glyoxylate, and the residues interacting with acetyl-CoA (Tyr-126, Pro-538, Val-18 and Pro-536) (Howard et al., 2000;Tugarinov et al., 2005). Genetic data support these functional assignments, since a D631N mutant had no  detectable activity and an R338K mutant showed only 6 % of wild-type activity (Anstrom et al., 2003).
MSA is a multimeric enzyme with a molecular mass of about 65 kDa (per subunit) that is indispensable for growth on acetate and is found in plants, fungi and bacteria. Lohman et al. (2008) showed recently that the structures of MSA and MSG are very similar and that the difference in size is due to the presence of an additional a/b domain in the G isoform. The active site is formed by Glu-250 and Asp-278, which bind Mg 2+ , while the adenine ring or ribose of acetyl-CoA binds to residues Pro-369, Met-102, Thr-95 Ala-367, Asn-105, Lys-101, Tyr-154 and His-368. MSA contains a highly conserved Cys-438 in the active site, which corresponds to Cys-617 in MSG.

Plant-pathogenic fungi
Several reports indicate an important role for ICL in fungal virulence on plants. Studies with Leptosphaeria maculans, causal agent of blackleg of crucifers (e.g. broccoli, canola and cauliflower) have shown that the icl1 gene is expressed during its infection of Brassica napus cotyledons and inactivation of this locus causes low germination rates of pycnidiospores, reducing the pathogenicity of the fungus on cotyledons as well as limiting its hyphal growth on canola. It was suggested that the reduced pathogenicity of the mutant is due to its inability to utilize carbon sources provided by the plant (Idnurm & Howlett, 2002). Another role for ICL in fungal phytopathogenesis has been reported in Magnaporthe grisea, the rice blast pathogen, which can also infect a number of other agriculturally important cereals including wheat, rye and barley. M. grisea reproduces both sexually and asexually to produce specialized infectious structures known as appressoria. The appressoria attach to the cuticle of the host and from them hyphae emerge and penetrate the plant, the inside of which is rapidly colonized by the fungus, with disease symptoms being observable in a few days. During infection by M. grisea, significant ICL gene expression was found in conidia, appressoria, mycelia and hyphae. Deletion of the M. grisea ICL1 gene caused a reduction in appressorium formation, conidiogenesis and cuticle penetration, and an overall decrease in damage to leaves of rice and barley. Thus ICL is essential for full virulence in this organism (Rauyaree et al., 2001;Wang et al., 2003).
Stagonospora nodorum is a necrotropic fungal pathogen producing leaf and glume blotch disease on wheat and other cereals of economic importance. Infection begins by germination of pycnidiospores on the leaf followed by hyphal penetration of the host and sporulation at the end of the infection cycle. In the initial steps of infection the pathogen depends on internal, stored carbon sources and it is thought that lipids are metabolized via the glyoxylate pathway to produce glucose and support fungal development. Expression analysis of the MS gene (mls1) in this pathogen during its interaction with wheat showed an increased expression in ungerminated spores followed by a dramatic decrease in transcription after germination. Paradoxically, the opposite pattern was seen using MS activity measurements, where activity was undetectable in ungerminated spores but increased to a significant level after germination. Spores of an mls1 null mutant inoculated onto wheat seedlings and leaves were unable to induce necrotic lesions on either tissue, indicating that this gene is essential for virulence on wheat. The biological reason for this phenotype is that the mls1-deficient strain has dramatically decreased spore germination and a reduction in the length of hyphae (Solomon et al., 2004). Asakura et al. (2006) demonstrated the functional role of ICL in Colletotrichum lagenarium, which causes anthracnose on a considerable number of plants of agricultural interest such as cucumber, watermelon, muskmelon, cantaloupe, winter squash and bittermelon. An icl1 mutant of C. lagenarium failed to grow on acetate or fatty acids, similar to other ICL mutants of fungi and prokaryotes. For the cellular localization of ICL, the encoding gene was fused to that of the green fluorescent protein (GFP) and icl1-gfp expression was detected in peroxisomes, conidia, appressoria and hyphae of the fungus. The icl1 mutant was able to germinate and develop appressoria and was capable of degrading lipid bodies as well as the wild-type strain. However, conidia from the icl1-deficient mutant inoculated onto cucumber leaves and cotyledons formed a reduced number of lesions on leaves, and especially on cotyledons, but nevertheless remained pathogenic. In invasive experiments such as the inoculation of conidia into wound sites, no defect was observed in the icl1 mutant, while in penetration assays on cucumber cotyledons the mutant was unable to develop penetrating hyphae, indicating a requirement for ICL at this early stage of C. lagenarium infection.

Candida albicans
Support for the direct involvement of ICL in virulence has also come from studies of Candida albicans, a commensal of the mammalian microbiota inhabiting the skin, mouth, gastrointestinal tract, gut and vagina. In immunocompromised patients, this diploid fungus is responsible for mucosal surface infections as well as life-threatening systemic infections. C. albicans is able to survive and grow inside macrophages. Transcriptional profiles of phagocytosed populations of C. albicans showed that all the steps of the glyoxylate cycle are induced (Lorenz et al., 2004). Northern blot and differential display experiments with C. albicans in the presence of macrophages revealed that both ICL and MS are induced (Lorenz & Fink, 2001;Prigneau et al., 2003). In addition, both enzymes are induced in C. albicans exposed to human neutrophils (Fradin et al., 2005). High enzymic activities of ICL and MS were detected in C. albicans strains isolated from diabetic patients suffering from vulvovaginal candidiasis (Lattif, et al., 2006). Furthermore, evaluation of ICL mutants in a mouse model demonstrated that activity of this enzyme is essential for fungal virulence (Lorenz & Fink, 2001). In a model that mimics C. albicans bloodstream infection, ICL and MS were downregulated in the initial stages of infection (10 min), upregulated beginning about 20 min after infection and reached a 20-fold increase after 60 min, suggesting blood-specific expression and an important biological role for the glyoxylate cycle genes in bloodstream infections (Fradin et al., 2003). In an interesting study Barelle et al. (2006) showed a specific activation of the C. albicans ICL1 when the pathogen was exposed to neutrophils or macrophages, but because icl was not expressed in infected kidney cells it was concluded that ICL1 contributes to virulence but is not essential for systemic infection.
A clear role for ICL in the pathogenesis of C. albicans emerges from the data summarized above. However, the function of this enzyme in C. albicans is peculiar because it is probably involved in processes other than lipid utilization or gluconeogenesis, since an ICL-deficient mutant is unable to utilize acetate, ethanol, citrate, glycerol, oleate, lactate, pyruvate, peptone, glutamate or alanine for growth, unlike the parental strain (Ramírez & Lorenz, 2007;Piekarska et al., 2006Piekarska et al., , 2008Brock, 2009). ICL expression is also detected during growth on Casamino acids, glutamate or peptone, and under starvation conditions (Barelle et al., 2006;Brock, 2009). Interestingly a fox2 mutant lacking the second enzyme of the b-oxidation pathway is also unable to utilize acetate, ethanol, lactate and oleic acid and is significantly attenuated in virulence. Therefore both the fox2 mutant and the ICL-deficient mutant are unable to utilize nonfermentable carbon sources and have reduced virulence in mice, indicating a role for the b-oxidation pathway in virulence. This contention is not supported by the finding that a mutant in peroxisome biogenesis (pex5), which is strongly reduced in b-oxidation activity and is unable to utilize oleic acid, is able to use acetate, ethanol or lactate and is not affected in virulence (Piekarska et al., 2006). This shows that acetyl-CoA derived from b-oxidation is not the carbon source inducing the glyoxylate cycle in the pex5 mutant. Thus, ICL1 expression in virulence may result not from lipid metabolism (Piekarska et al., 2006;Ramírez & Lorenz, 2007;Brock, 2009) but from the conversion of carbon sources such as lactate into C 2 units for metabolism by the glyoxylate cycle (Piekarska et al., 2006). ICL in C. albicans, unlike other organisms, is necessary for the utilization of a large variety of carbon sources. The fact that ICL is probably interconnected with multiple metabolic networks important in virulence encourages the development of specific inhibitors against this enzyme.

Other fungal pathogens
Upregulation of the glyoxylate cycle genes has also been detected in other intracellular fungal pathogens such as Paracoccidioides brasiliensis, which causes paracoccidioido-mycosis in humans. RT-PCR analysis showed that transcript levels of the ICL and MS genes in this fungus increased following phagocytosis by murine macrophages (Derengowski et al., 2008). Penicillium marneffei is a dimorphic fungus that can cause systemic mycosis in humans. The incidence of this fungal infection has increased substantially during the past few years, occurring most often in patients infected with HIV. To evaluate if the glyoxylate cycle was involved in the virulence of P. marneffei, Northern blot experiments were performed; these showed that after macrophage internalization of conidia the ICL-encoding gene (acuD) was highly expressed, suggesting a potential role for the cycle in the pathogen's adaptation inside macrophages (Thirach et al., 2008). Together, these data directly or indirectly support the relevance of the glyoxylate pathway in fungal virulence in plants, animals and humans.
While a functional role for the glyoxylate cycle in fungal virulence is widespread, it is not universal. In the animal pathogen Aspergillus fumigatus, ICL expression was detected in hyphae and in conidia (Ebel et al., 2006) but tissues of patients infected with the fungus were negative for the enzyme after immunostaining, and a mutant deleted of the ICL gene (acuD) was fully virulent in a murine model (Schöbel et al., 2007). Similar results were obtained with the human-pathogenic fungus Cryptococcus neoformans, where ICL in a rabbit meningitis model was upregulated after 7 days in the subarachnoid space but an icl1 mutant showed the same number of subarachnoidal yeast cells as the wild-type after 10 days in immunosuppressed rabbits. In addition, in an inhalation model of murine cryptococcosis, no differences in survival were observed between an icl1 mutant and the wild-type, and similar growth was observed for both C. neoformans strains inside macrophages (Rude et al., 2002). These findings show a lack of correlation between ICL gene expression and biological function in these systems.

Mycobacterium tuberculosis
The actinobacterium Mycobacterium tuberculosis kills 3 000 000 people worldwide every year and is assumed to utilize fatty acid degradation products when growing in the host (Segal & Bloch, 1956;Srivastava et al., 2008). The genome sequence of this bacterium (Cole et al., 1998) contains more than 250 genes encoding proteins annotated as being involved in fatty acid metabolism, providing indirect support for the role of fatty acid degradation and C 2 metabolism in the pathogenesis of M. tuberculosis. ICL activity increases in pellicles in synthetic media as a consequence of fatty acid degradation (Murthy et al., 1973) as well as under microaerophilic growth conditions (Wayne & Lin, 1982). Increased aceA (icl) mRNA expression in response to human macrophages is also documented (Dubnau et al., 2002;Graham & Clark-Curtiss, 1999). M. tuberculosis expresses a 50 kDa protein during intracellular infection (Sturgill-Koszycki et al., 1997) and this was shown to be encoded by a second copy of an ICL gene that is present in several Mycobacterium species. Expression analysis and biochemical characterization of ICL activity clearly show that M. tuberculosis and Mycobacterium avium have two functional ICLs, ICL and AceA (Höner Zu Bentrup et al., 1999). ICL seems to be the principal enzyme in the processing of isocitrate and McKinney et al. (2000) reported that single ICL mutations had no dramatic effect on the growth of M. tuberculosis in mouse lung during the first 2 weeks of infection. However, lungs infected with the mutant showed few changes between 2 and 16 weeks, suggesting that the ICL mutant had a reduced ability to sustain the infection. In contrast, at 16 weeks the lungs of mice infected with the virulent Erdman strain showed inflammatory lesions, enlargement and multiple expanding and coalescing tubercles. icl mRNA levels markedly increase in lungs of mice (Timm et al., 2003) and in human lung granulomas, as well as in the lymphocyte region of necrotic granulomas. In contrast, icl expression was not detected in the transition zone and in the central region of necrotic granulomas, supporting the notion that icl has a pivotal role in bacterial persistence in the host (Fenhalls et al., 2002). Muñoz-Elías & McKinney (2005) reported that single mutations in icl or aceA had no dramatic effect on bacterial growth on fatty acids, while an icl aceA double mutant was unable to grow on this carbon source. The double mutant inoculated into mice was eliminated from lungs and spleen and was unable to induce splenomegaly or alterations in lungs. ICL activity is thus essential for M. tuberculosis survival in the host. Additionally, ICL and to a lesser extent AceA were required for the growth of M. tuberculosis on propionate and on odd-chain fatty acids as a carbon source (Muñoz-Elías & McKinney, 2005;Muñoz-Elías et al., 2006). The propionate or propionyl-CoA derived from b-oxidation of odd-chain fatty acids can be catabolized by the methylcitrate cycle, consisting of the enzymes 2-methylisocitrate lyase (MICL), methylcitrate synthase and methylcitrate dehydratase, encoded by the prpB, prpC and prpD genes, respectively. The M. tuberculosis genome contains homologues of prpC and prpD, but not prpB. However, structural and biochemical studies have demonstrated that unlike other ICLs and MICLs, the M. tuberculosis ICL possesses dual ICL/MICL activity and can support growth on acetate and propionate (Gould et al., 2006). The prpC and prpD genes are upregulated during infection of macrophages (Schnappinger et al., 2003), suggesting that the methylcitrate cycle could be important in M. tuberculosis pathogenesis. However, studies with a prpC prpD double mutant show that the methylcitrate cycle is required for M. tuberculosis replication in non-activated murine bonemarrow-derived macrophages, but that in IFN-c-activated macrophages or in the lungs and spleen of inoculated mice the double mutant shows no alteration of in vivo growth, persistence or virulence. Thus, the functional role of ICL in M. tuberculosis virulence is in the glyoxylate cycle rather than the methylcitrate cycle (Muñoz-Elías et al., 2006). Recent reports show that M. tuberculosis phoP mutants are attenuated but persist in macrophages and mouse organs apparently because the mutant expresses higher levels of icl. The PhoP-deficient strain would be a good candidate for vaccine production, since prolonged exposure of the immune system to the persistent, attenuated strain could result in long-term immunogenicity (Gonzalo-Asensio et al., 2008).
The single MS present in M. tuberculosis may also contribute to pathogenicity. Studies in vitro show that MS is secreted and enhances the adherence of the pathogen to lung epithelial cells, supporting the notion that it may function as an adhesin as well as an enzyme (Kinhikar et al., 2006). MS can also be used as a biomarker because it is recognized in the humoral response of tuberculosis patients, making possible its use in serodiagnostic assays for identification of this pulmonary disease (Achkar et al., 2006;Melo Cardoso Almeida et al., 2008;Samanich et al., 2001;Wanchu et al., 2008).

Other bacterial pathogens
The importance of a functional glyoxylate cycle in some intracellular human pathogens such as Salmonella has been evaluated, and the results obtained with an ICL-deficient strain of Salmonella enterica serovar Typhimurium show that ICL is required for persistence during chronic infection, but not for acute lethal infection in mice (Fang et al., 2005;Kim et al., 2006;Tchawa Yimga et al., 2006). In Brucella suis, the glyoxylate cycle has been shown to be unnecessary for virulence (Kohler et al., 2002). The pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis induce ICL and MS during growth on acetate but not on xylose, while Yersinia pestis synthesizes both enzymes on both carbon sources (Hillier & Charnetzky, 1981b). A natural mutation in iclR, encoding a repressor protein, explains why Y. pestis constitutively produces ICL (Sebbane et al., 2004), and this constitutive enzymic activity has been used to identify Y. pestis in humans, animals, water, soil and food (Hillier & Charnetzky, 1981a;Quan et al., 1982). Sebbane et al. (2004) showed that mutations in the sole ICL gene (aceA) of Y. pestis prevented growth on acetate but did not affect pathogenesis in a mouse model.
Rhodococcus equi is a Gram-positive intracellular bacillus causing pneumonia and enteritis in foals and is also able to infect cats, dogs and pigs, producing submandibular adenitis. R. equi causes infection in immunocompromised humans and is easily isolated from soil contaminated with faeces. The fact that R. equi uses acetate (Kelly et al., 2002) and probably lipids as carbon sources in soil and within macrophages suggests that it may utilize the glyoxylate cycle in its interaction with foals. To validate this hypothesis an ICL-deficient (aceA mutant) strain of R. equi was evaluated in macrophages (Wall et al., 2005). The population of the ICL-deficient strain increased in macro-phages after 12 h but then declined significantly, indicating that ICL is essential for long-term survival and proliferation in macrophages, consistent with the finding that the aceA mutant was partially attenuated in a mouse model. In foals, the wild-type R. equi strain induced severe lesions of suppurative to pyogranulomatous bronchopneumonia in lung, while the aceA mutant was unable to produce any alteration, probably because the mutant population was six orders of magnitude lower than that of the wild-type strain (Wall et al., 2005).
Another pathogen infecting lungs is Pseudomonas aeruginosa, which produces lung dysfunction and mortality in humans with cystic fibrosis. P. aeruginosa upregulates genes in vivo that are needed for replication in the lung environment, including those encoding enzymes for fatty acid and lipid metabolism (alcohol dehydrogenase), choline metabolism (betaine aldehyde dehydrogenase), amino acid degradation (arginine deiminase), nitrogen metabolism (respiratory nitrate reductase) and the glyoxylate cycle (ICL). These metabolic genes almost certainly contribute to carbon and nitrogen nutrition, allowing the replication and persistence of P. aeruginosas inside the host (Son et al., 2007). Support for this hypothesis was obtained by a study in which several metabolic mutants of P. aeruginosa were isolated and evaluated on alfalfa seedlings and in a mouse model (Lindsey et al., 2008). An ICL (aceA) mutant derived from strain PAO1 displayed reduced virulence on alfalfa seedlings and a reduction in histopathology in rat lungs. Thus the glyoxylate cycle has a pivotal role in the interaction of P. aeruginosa with both plant and mammalian hosts.

Inhibitors of ICL and MS
The development of specific inhibitors against ICL and MS is an attractive prospect, since in a variety of humanpathogenic bacteria and fungi the expression of the glyoxylate cycle genes is detected in specific stages of the interaction (Table 1). Several inhibitors of ICL have been identified, including itaconate, itaconic anhydride, bromopyruvate, nitropropionate, oxalate and malate (Höner Zu Bentrup et al., 1999;McFadden & Purohit, 1977). However, these are not pharmacologically suitable for use in vivo since they are toxic and non-specific. For instance, nitropropionate inhibits ICL but also inhibits succinate dehydrogenase, a pivotal enzyme of the TCA cycle (Alston et al., 1977;Fig. 1). Efforts to isolate natural ICL inhibitors from plants revealed that extracts of Illicium verum and Zingiber officinale inhibit the ICL of Mycobacterium tuberculosis (Bai et al., 2007).
Because the glyoxylate cycle is important in many types of fungal pathogenesis, natural inhibitors of fungal ICLs have been sought. Those isolated from the tropical sponge Hippospongia sp. are halisulfates that are able to inhibit ICL activity, appressorium formation and C 2 utilization in the rice blast fungus Magnaporthe grisea (Lee et al., 2007). Natural glyoxylate cycle inhibitors such as 5-hydroxyin-dole-type alkaloids are potent inhibitors of the Candida albicans ICL (Lee et al., 2009).
It was recently proposed that instead of ICL or MS, other enzymes could make better targets for bringing about inhibition of the glyoxylate cycle. For instance, inactivation of the kinase-phosphatase that phosphorylates and inactivates isocitrate dehydrogenase would be a good candidate, since its absence would promote carbon flow through the full TCA cycle and avoid its assimilation by ICL (Singh & Ghosh, 2006). Other proposed targets for indirect inhibition of the glyoxylate cycle are the enzymes of the PHB cycle, since this metabolic route can provide acetyl-CoA in a manner that circumvents the link between glycolysis and the TCA cycle (Purohit et al., 2007).

Roles of ICL and MS in other plant-bacteria interactions
The functionality of the glyoxylate cycle was evaluated in the plant pathogen Rhodococcus fascians, which causes leafy gall disease on a variety of monocots and dicots, including Nicotiana tabacum (tobacco). A malate synthase mutant of R. fascians gave a diminished number of bacteria inside symptomatic tobacco tissues in comparison to tissues infected by the wild-type (Vereecke et al., 2002a, b). In the plant pathogen Xanthomonas campestris, MS was induced during infection of tomato plants and a MS-deficient strain induced fewer and smaller lesions on 75 % of inoculated leaves as compared to the wild-type strain (Tamir-Ariel et al., 2007). Thus, in these two pathogens the glyoxylate cycle has an important role in the plant-microbe interaction.
Carbon metabolism has long been studied in Rhizobium spp., bacteria that form a nitrogen-fixing symbiosis with leguminous plants. Large quantities of acetate and fatty acids were reported in soybean nodules formed by Bradyrhizobium japonicum (Johnson et al., 1966), and radiorespirometric studies of B. japonicum bacteroids indicated that up to 50 % of the acetyl-CoA entering the TCA cycle was metabolized via MS (Stovall & Cole, 1978). It was also shown that acetate can be used by isolated B. japonicum bacteroids to support ex planta nitrogen fixation (Peterson & LaRue, 1981. The existence of a complete glyoxylate cycle in nitrogen-fixing bacteroids is in doubt because ICL activity is not detected in the microsymbionts isolated from soybean, pea, alfalfa and clover nodules (Green et al., 1998;Johnson et al., 1966). However, ICL activity has been detected in bacteroids from senesced nodules formed by B. japonicum (Wong & Evans, 1971). In contrast, MS activity was found in bacteroids isolated from pea, alfalfa and clover nodules, and substantially higher activities were detected in bacteroids isolated from bean, cowpea and soybean nodules (Green et al., 1998;Johnson et al., 1966). Based on these data we decided to genetically evaluate the role of the glyoxylate cycle in the Rhizobium-Leguminosae symbiosis. We showed that neither MS nor or ICL is required for symbiosis, since MS (glcB) mutants of Rhizobium legumi-IP: 54.70.40.11 On: Sat, 01 Jun 2019 12:48:34 nosarum evaluated during their interaction with Pisum sativum have the same levels of nitrogen fixation and nodulation as the wild-type strain (García-de los Santos et al., 2002). Similarly, a glcB mutant of Sinorhizobium meliloti is able to normally nodulate and fix nitrogen in symbiosis with Medicago sativa. To evaluate the role of ICL, null mutants were constructed in S. meliloti and Rhizobium tropici and their symbiotic performance was evaluated on M. sativa and Phaseolus vulgaris, respectively. The results demonstrated that in both symbiotic models ICL is not involved in nodulation or nitrogen fixation (Ramírez-Trujillo et al., 2007).

Conclusions
The study of the metabolic pathways involved in the pathogenesis of bacterial and fungal infections is critical for public health, crop productivity and animal welfare. The glyoxylate cycle is an important metabolic pathway in this regard, since substantial evidence supports its importance in many host-pathogen systems. The knowledge generated about the role of this pathway in pathogenesis is important, since it provides the opportunity to develop specific inhibitors of ICL and MS that could be used to combat fungal and bacterial diseases. from DGAPA/UNAM (IN206802, IN206705 IN214808) and Conacyt (89337) J. A. R.-T. was a PhD student in the Programa de Doctorado en Ciencias Bioló gicas-UNAM and acknowledges the fellowships from the Consejo Nacional de Ciencia y Tecnología (México).