Research Update
TRENDS in Microbiology
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Research News
Versatile persistence pathways for pathogens of animals
and plants
Danny Vereecke, Karen Cornelis, Wim Temmerman, Marcelle Holsters and Koen Goethals
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Persistence of pathogens of mammals
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The genus Rhodococcus is closely related
to Mycobacterium and includes the
species Rhodococcus equi, a facultative
intracellular pathogen of macrophages
in different animals [6]. 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 [7]. 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
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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 [4]. 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
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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
[3]. 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 [1].
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 [5].
Using whole-genome microarray
analysis, the genes of the glyoxylate cycle
have been shown to be induced in
phagocytosed Saccharomyces cerevisiae
cells [2]. In the related fungal pathogen
C. albicans, both the malate synthase and
isocitrate lyase genes are induced upon
contact with macrophages [2]. 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
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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
[10]. 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
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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 [12].
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 [15] and is required for
chronic persistence of the intracellular
pathogen Brucella abortus [16].
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
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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.
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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.