Research Update TRENDS in Microbiology 1 Research News Versatile persistence pathways for pathogens of animals and plants Danny Vereecke, Karen Cornelis, Wim Temmerman, Marcelle Holsters and Koen Goethals 66 20 67 68 69 20 20 20 70 Rf 9 10 11 12 B s pg H c se A2 A2 Persistence of pathogens of mammals gc gc v 15 16 17 13 14 ORF5 vicA ORF3 ORF2 ORF1 v 1kb a c gu gl Mt 43 18 c c c 42 18 c 41 18 c 40 18 c 39 18 c 38 18 c 37 18 36 18 c 35 18 34 18 c 33 18 32 18 21 31 18 0 3 18 29 18 8 2 18 7 2 18 26 18 5 2 18 4 2 18 3 2 18 2 2 18 Rv 18 http://tim.trends.com The genus Rhodococcus is closely related to Mycobacterium and includes the species Rhodococcus equi, a facultative intracellular pathogen of macrophages in different animals . The phytopathogenic species R. fascians infects a wide range of plants, provoking the formation of leafy galls consisting of masses of shoot buds that are suppressed for further growth . Pathogenesis relies on a linear plasmid carrying virulence genes involved in synthesizing signal compounds that initiate cell division in plant cortical tissues, leading to the formation of shoot meristems [8,9]. Upon epiphytic colonization, endophytic forms are found in the intercellular spaces of gall tissues and, sometimes, inside plant cells 20 71 72 20 20 73 20 74 20 5 7 20 6 7 20 7 7 2078 2079 20 0 8 20 81 82 20 ML Gene 0 1 2 3 4 5 6 7 8 The glyoxylate shunt is a bypass of the TCA cycle that permits gluconeogenesis starting from acetyl-coenzyme A (acetyl-CoA), which is generated following fatty acid catabolism . The shunt is composed of isocitrate lyase, which cleaves isocitrate to succinate and glyoxylate, and malate synthase, which condenses glyoxylate and acetyl-CoA to malate. As such, it circumvents the CO2-generating steps of the TCA cycle and converts one Persistence of the plant pathogen Rhodococcus fascians MI 20 An important feature of the lifestyle of inter- and intracellular pathogens is the acquisition of nutrients from their host. For intracellular pathogens of mammals, a two-carbon diet through the glyoxylate cycle is crucial for virulence and persistence. The persistence of the bacterium Mycobacterium tuberculosis and the yeast Candida albicans inside macrophages depends on the activity of the glyoxylate shunt of the tricarboxylic acid (TCA) cycle to allow growth in an environment where fatty acids are probably the main carbon source [1–3]. Here, we show that the phytopathogenic bacterium Rhodococcus fascians has similarly adopted the glyoxylate shunt as a persistence pathway in infected plant tissues. macrophage is a glucose-deficient environment. These observations show that for these bacterial and fungal pathogens of mammals persistence and virulence rely on the ability to switch diet within host cells. Furthermore, they suggest that an active glyoxylate cycle has widespread significance in the strategies used by these pathogens. B1 DOI: 10.1016/S0966-842X(02)02457-5 molecule each of acetyl-CoA and isocitrate to two C4 compounds that can be fed into biosynthetic processes. Mycobacterial persistence occurs after the emergence of the adaptive immune response. The intracellular environment of activated macrophages becomes more hostile and bacterial growth is restrained . The pathogen adapts to the nutrient deprivation by shifting its metabolism towards the degradation of fatty acids. Expression of the isocitrate lyase gene is induced after phagocytosis in activated macrophages and its deletion results in reduced virulence, coinciding with a loss of persistence during lung infection of mice . The reliance on a fatty acid diet is further illustrated by the relative abundance of M. tuberculosis genes encoding proteins involved in fatty acid oxidation . Using whole-genome microarray analysis, the genes of the glyoxylate cycle have been shown to be induced in phagocytosed Saccharomyces cerevisiae cells . In the related fungal pathogen C. albicans, both the malate synthase and isocitrate lyase genes are induced upon contact with macrophages . When the isocitrate lyase gene is deleted, the virulence of C. albicans is reduced, suggesting that the interior of a B The glyoxylate cycle and the glycine cleavage system are part of conserved metabolic pathways involved in the chronic persistence of microorganisms in animal hosts. In the chromosome of the plant pathogen Rhodococcus fascians, the vic locus has been identified as a region containing genes essential for persistence inside induced leafy galls. Sequence analysis showed that this 18-kb locus is syntenic with chromosomal regions of Mycobacterium species that encompass the ‘persistence’ loci of these mammalian pathogens. Hence, the ability to switch diet inside the host appears to be governed by ‘persistence’ enzymes that are conserved between pathogens of animals and plants. TRENDS in Microbiology Fig. 1. Overview of the genes located in the syntenic regions of the chromosomes of Mycobacterium tuberculosis (Mt), Mycobacterium leprae (Ml) and Rhodococcus fascians (Rf). ORFs that occur in all three bacteria are in red, those found only in the mycobacteria in yellow, only in R. fascians in blue, those only in M. tuberculosis in green, and those that are absent in M. leprae in brown; the pale colored ORFs (2080, 2079, 2078, 2071, 2068 and 2067) in M. leprae and gene 10 in R. fascians represent pseudogenes; triangles on the R. fascians sequence indicate the location of mutations. 0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(02)02457-5 2 Research Update TRENDS in Microbiology Table 1. Syntenic genes in Mycobacterium tuberculosis, M. leprae and Rhodococcus fascians M. tuberculosis (bp, aa) M. leprae (bp, aa) Rv1821 (2424, 808) b R. fascians (bp, aa) ML2082 (2337, 778) Rv1822 (627, 209) ML2081 (621, 206) Gene 1 (606, 202) Rv1823 (921, 307) Rv1824 (363, 121) Rv1825 (876, 292) Rv1826 (402, 134) ML2080 (927, PG) ML2079 (325, PG) ML2078 (692, PG) ML2077 (399, 132) Gene 2 (855, 285) Gene 3 (441, 147) Gene 4 (732, 244) Gene 5 (423, 141) Rv1827 (486, 162) Rv1828 (741, 247) Rv1829 (492, 164) Rv1830 (675, 225) Rv1831 (255, 85) Rv1832 (2823, 941) ML2076 (489, 162) ML2075 (756, 251) ML2074 (495, 164) ML2073 (696, 231) Gene 6 (498, 166) Gene 7 (738, 245) Gene 8 (474, 158) Gene 9 (678, 226) Gene 10 (315, PG) Gene 11 (2853, 951) Gene 12 (1137, 379) Rv1833c (858, 286) Rv1834 (864, 288) Rv1835c (1884, 628) Rv1836c (2031, 677) Rv1837c (2223, 741) Rv1838c (393, 131) Rv1839c (261, 87) Rv1840c (1545, 515) c Function secA2 Gene 0 (627, 209) ML2072 (2859, 952) Gene a ML2071 (803, PG) ML2070 (2202, 733) ML2069 (2196, 731) ORF5 (1715, 571) vicA (2174, 724) Rv1841c (1035, 345) ML2068 (948, PG) ORF3 (915, 305) ORF2 (1144, 381) Rv1842c (1365, 455) ML2067 (1303, PG) ORF1 (1350, 450) Rv1843c (1437, 479) ML2066 (1437, 478) Preprotein translocase subunit (Class III.D: protein and peptide secretion) mutT2 Homologue of MutT/NudiX-family protein of Brucella melitensis (Q8YII2) pgsA2 CDP-diacylglycerol-glycerol-3-P-3-phosphatidyltransferase (Class I.H.3: lipid biosynthesis) Conserved hypothetical protein (Class V) Membrane protein (Class II.C.5: cell envelope) Unknown hypothetical protein (Class VI) gcvH Glycine cleavage system protein H (Class I.C.1: general central intermediary metabolism) Conserved hypothetical protein (Class V) Conserved hypothetical protein (Class V) Conserved hypothetical protein (Class V) Conserved hypothetical protein (Class V) Unknown hypothetical protein (Class VI) gcvB Glycine cleavage system protein P (Class I.C.1:general central intermediary metabolism) Homologue of putative integral membrane protein of Streptomyces coelicolor (AL353872) Class IV.I: miscellaneous phosphatases, lyases and hydrolases Conserved hypothetical protein (Class V) Conserved hypothetical protein (Class V) Conserved hypothetical protein (Class V) glcB Malate synthase (Class I.B.4: glyoxylate bypass) Conserved hypothetical protein (Class V) Conserved hypothetical protein (Class V) PE_PGRS Class IV.C.1.b: PE family, PGRS subfamily Unknown hypothetical protein Putative integral membrane protein (Class II.C.5: cell envelope) Putative integral membrane protein (Class II.C.5: cell envelope) guaB1 Inosine-5'-monophosphate dehydrogenase (Class I.F.1: purine ribonucleotide biosynthesis) a Abbreviations: aa, amino acid(s); bp, base pair(s); PG, pseudogene. The sequence of the region of the R. fascians chromosome is submitted under accession number AJ301559. The protein classes are according to the functional classification of the gene products of the Mycobacterium genomes (http://www.sanger.ac.uk/Projects/M_tuberculosis/ and http://www.sanger.ac.uk/Projects/M_leprae/). b c . The persistent presence and activity of this bacterial subpopulation is essential for the growth and maintenance of the leafy gall [11,12]. We have characterized a chromosomal locus (vic) that contributes to virulence: an insertion mutant was strongly affected in gall formation and the few buds formed were weakly suppressed for further growth. The insertion mutation maps to a gene homologous to malate synthase and although the vic mutant grew equally well in rich medium and defined glycerol medium, it failed to grow in a medium with acetate as sole carbon source. The mutant also lacked traceable malate synthase activity, and accumulated glyoxylate in acetate medium. The expression of the malate synthase gene was induced by extracts of plant http://tim.trends.com and gall tissues. Growth of the mutant ceased rapidly when confronted with gall extracts, but not with extracts from uninfected plants. In symptomatic plant tissues the number of vic mutant bacteria was significantly lower than that of wild-type bacteria. These results suggest that there is a shift in the diet of R. fascians during plant infection that requires specific metabolic reactions involving vic-encoded biochemistry. The absence of an active malate synthase in the vic mutant results in the accumulation of glyoxylate when bacteria are grown on gall extracts. Glyoxylate accumulation interferes with the metabolism of the bacteria and, ultimately, their viability, with a reduced virulence phenotype as a consequence . Syntenic regions comprise ‘persistence’ loci Interestingly, the malate synthase gene is part of a locus carrying 14 genes that are highly conserved both in sequence and organization with the corresponding genomic regions of M. tuberculosis and Mycobacterium leprae (Fig. 1). Also comparable to M. tuberculosis, the malate synthase gene of R. fascians is not linked to the previously cloned isocitrate lyase gene; its gene product has high sequence identity (55%) with malate synthase G, which is involved in glycolate utilization, and a lower identity (20%) with malate synthase A of Escherichia coli [5,12–14]. In addition to genes encoding hypothetical and membrane-targeted proteins, the syntenic region also carries the genes comprising the glycine cleavage system (Table 1), which is responsible Research Update TRENDS in Microbiology for the oxidative decarboxylation of glycine, generating CO2 and NADH. This pathway is thought to be involved (as glycine synthase in the reverse reaction) in the maintenance of the NAD pool under O2-limiting conditions in M. tuberculosis  and is required for chronic persistence of the intracellular pathogen Brucella abortus . Mutagenesis of the R. fascians gene encoding the glycine dehydrogenase P homologue results in a marked decrease in virulence. This gene is not transcribed in a medium containing glycine as the sole nitrogen source, as has been found for the genes of E. coli and S. cerevisiae [17, 18], but, similar to the malate synthase gene, expression is strongly induced by extracts of infected plant tissues (D. Vereecke et al., unpublished). sustain growth of R. fascians? Is the photorespiration level higher in leafy galls? Are the photorespiration intermediates used by R. fascians? If so, how are they obtained by the intercellular population? What is the role of isocitrate lyase in these processes, and hence in virulence? In conclusion, it is apparent that R. fascians and its close relative M. tuberculosis have adopted similar metabolic pathways that are linked to persistence in animal hosts or particular plant tissues. The integration of the corresponding biochemistry into the host’s metabolism might differ between pathogens of plants and animals, but relies on common genetic grounds, thus illustrating the versatility of these persistence pathways in adaptation to different host environments. What are the nutrient sources for R. fascians in leafy galls? Acknowledgements In congruence with Mycobacterium, the vic locus together with isocitrate lyase can be involved in the specific metabolism of fatty acids in leafy gall tissues. However, the physiology of the niche that is occupied by R. fascians – arial plant parts that are active in photosynthesis – could suggest other gall-derived nutrients. From our data, it is conceivable that the vic locus functions in the metabolism of glycine, glycolate and/or glyoxylate, in a pathway that does not require isocitrate lyase. Interestingly, these compounds are shuttled between several cellular organelles of C3 plants during a process known as photorespiration [19,20]. Assuming that photorespiration is high in leafy galls, these intermediates could be produced in sufficiently large amounts to be scavenged by R. fascians and metabolized by the vic-derived enzymes. Future research Several questions remain to be answered to demonstrate further the analogy between the strategies used by R. fascians and M. tuberculosis and their respective hosts. What is the relationship between the metabolic adaptation and the intra- and intercellular forms of R. fascians? Are there leafy gall-abundant fatty acids that can http://tim.trends.com We thank Wilson Ardiles for sequencing the vic locus, Martine De Cock for help with the manuscript, and Rebecca Verbanck for artwork. W.T. and K.C. are indebted to the Vlaams Instituut voor de Bevordering van het WetenschappelijkTechnologisch Onderzoek in de Industrie and the ‘von Humbolt Gesellschaft’ for a postdoctoral fellowship, respectively. References 1 McKinney, J.D. et al. (2000) Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735–738 2 Lorenz, M.C. and Fink, G.R. (2001) The glyoxylate cycle is required for fungal virulence. Nature 412, 83–86 3 Höner zu Bentrup, K. and Russell, D.G. (2001) Mycobacterial persistence: adaptation to a changing environment. Trends Microbiol. 9, 597–605 4 Kornberg, H.L. (1966) The role and control of the glyoxylate cycle in Escherichia coli. Biochem. J. 99, 1–11 5 Cole, S.T. et al. 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(1996) Specific induction by glycine of the gene for the P-subunit of glycine decarboxylase from Saccharomyces cerevisiae. Mol. Microbiol. 19, 611–623 19 Husic, D.W. et al. (1987) The oxidative photosynthetic carbon cycle or C2 cycle. CRC Crit. Rev. Plant Sci. 5, 45–100 20 Kozaki, A. and Takeba, G. (1996) Photorespiration protects C3 plants from photooxidation. Nature 384, 557–560 Danny Vereecke Wim Temmerman Marcelle Holsters* Koen Goethals Dept of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. *e-mail: [email protected] Karen Cornelis Present address: Entwicklungsgenetik, Zentrum für Molekularbiologie der Pflanzen, Universität Tübingen, Auf der Morgenstelle 3, D-72076 Tübingen, Germany.
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