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Letter
Multiple rpoB Mutants
of Mycobacterium tuberculosis and Second-order Selection
Igor Mokrousov*
*St. Petersburg Pasteur Institute, St. Petersburg, Russia
Suggested citation
for this article:
Mokrousov I. Multiple rpoB mutants of Mycobacterium tuberculosis
and second-order selection. [letter]. Emerg Infect Dis [serial on the
Internet]. 2004 Jul [date cited]. Available from: http://www.cdc.gov/ncidod/EID/vol10no7/03-0598.htm
To the Editor: Rad and colleagues recently described variation
in some genes involved in DNA repair (mutT2, mutT4, ogt)
in Mycobacterium tuberculosis strains of different genotypes (1).
This approach can also be used to investigate developing rifampin resistance
in the context of emerging mutator alleles. Resistance to rifampin in
M. tuberculosis strains is usually caused by the point mutations
in the rpoB gene encoding the β-subunit of the DNA-dependent
RNA polymerase, which is a target of the drug. Although a single point
mutation is sufficient for developing rifampin resistance, a number of
articles (2,3) describe multiple rpoB mutants
for M. tuberculosis, i.e., rifampin-resistant strains harboring
mutations in different codons of rpoB. Double, triple, and quadruple
mutations in M. tuberculosis clinical isolates were reported in
studies conducted throughout the world (2,3). Such emergence,
albeit infrequent, of M. tuberculosis rpoB multiple mutants raises
questions about their biologic importance and underlying mechanisms; answers
to both remain elusive.
I propose an explanation of these observations in terms of second-order
selection of hypermutable (mutator) alleles based on alterations in DNA
repair genes. Unlike that of other antituberculosis drugs, resistance
to rifampin is acquired in most M. tuberculosis isolates by altering
a single target molecule and offers the most appropriate and straightforward
model to demonstrate possible hypermutability in this species. In mycobacteria,
hypermutability was demonstrated in vitro for M. smegmatis, a surrogate
model for M. tuberculosis, as an increase in reversion (mutant
to wild-type) rate in rpoB526 or rpsL43 under counterselection
by streptomycin or rifampin, respectively (4). A correlation
between high mutation rate and antimicrobial resistance was reported for
Pseudomonas aeruginosa isolates from lungs of cystic fibrosis patients
(5). The mutator P. aeruginosa strains resulted
from a defective mismatch-repair system (5). In M.
tuberculosis, mismatch-repair genes (mutH, mutL, mutS,
and recJ) were not found in its genome (6). However,
the nucleotide pool in this species is exceptionally clean because of
the presence of several copies of the mutT gene (1,6);
the MutT protein removes oxidized guanines (8-Oxo-dGTP), thus counteracting
replication or transcription errors. Consequently, the MutHLS mismatch-repair
system simply may be not required in M. tuberculosis (6).
Therefore, hypermutability in some strains of this species resulting in
multiple rpoB mutants might develop under certain special (in vivo)
circumstances through inactivation or down-regulation of some mutT
genes. Further, the two most frequently described rpoB mutations
are 531TCG→TTG and 526CAC→TAC. Both are cytosine-to-tymine transitions,
which easily occur by spontaneous cytosine deamination to uracil. Indeed,
M. tuberculosis is a G+C rich organism, therefore, it is naturally
at high risk for cytosine deamination. Furthermore, pathogenic mycobacteria
are at increased risk for deamination because of the production of reactive
oxygen and nitrogen intermediates inside host macrophages. This deamination
process is normally counteracted by uracil-N-glycosylase, the product
of the ung gene, and organisms defective in the removal of uracil
from DNA have an increased spontaneous mutation rate and more G:C→
A:T base-pair transitions (7). Merchant et al., by using
ung+ and ung– Escherichia coli strains, demonstrated
that total nitric oxide exposures in the mmol/L range can lead to C→T
mutations by a mechanism probably involving cytosine deamination (8).
On the other hand, in M. smegmatis, the abrogation of the Ung activity
leads not only to increased mutator phenotype but also to growth inhibition
by reactive nitrogen intermediates (7). In summary, I
speculate that mutations in ung that do not completely impair function,
but do decrease synthesis of its product, might tolerably increase the
spontaneous C→T mutations, including those in the respective positions
in the rpoB codons 531 and 526. This assumption seems likely because
both of the aforementioned particular mutations were described in spontaneous
mutants of H37Rv obtained in vitro and had a Darwinian fitness slightly
less than or equal to that of the rpoB wild-type-susceptible parental
strain (9). In contrast, the translesion synthesis-based
pathways appear less likely to contribute to emergence of such mutants,
although at least one of the translesion synthesis genes (dinP)
is present in the genome of M. tuberculosis. In the E. coli
in vitro model, a translesion synthesis enzyme (dinB encoded DNA
polymerase IV) activity clearly promoted more important frameshift mutations
(single-base deletions) in two thirds of the spontaneous mutants (10).
From an evolutionary point of view, the multiple rpoB mutations
in M. tuberculosis have been hypothesized to arise as a compensatory
mechanism to ameliorate the fitness costs of the original resistance mutation
by a secondary mutation (11). The process of adaptation
to the fitness costs of chromosomally encoded resistance has been studied
in E. coli and Salmonella enterica serovar Typhi for mutations
that affect translation in the rpsL and fusR genes (11)
and for rpoB mutations in E. coli K12 strain (11).
In the last instance, the rpoB multiple mutants were selected in
vitro in a stepwise fashion, and one double mutant, L511Q+D516G (also
described in M. tuberculosis strain [3]), exhibited
a relative fitness either greater than or equal to either single mutant
or the wild type. Reynolds (11) suggested that this
allele is favored not merely as a combination of two low-level resistance
mutations but also because these mutations together boost resistance and
preserve fitness. Whether the same is true for other multiple mutant alleles
in M. tuberculosis rpoB remains to be seen. Studying the costs
of resistance of multiple rpoB mutations in a more realistic environment
of animal models of TB infection seems promising.
References
- Rad ME, Bifani P, Martin C, Kremer K, Samper S, Rauzier
J, et al. Mutations
in putative mutator genes of Mycobacterium tuberculosis strains
of the W-Beijing family. Emerg Infect Dis. 2003;9:838–45.
- Mani C, Selvakumar N, Narayanan S, Narayanan PR. Mutations
in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis
clinical isolates from India. J Clin Microbiol. 2001;39:2987–90.
- Pozzi G, Meloni M, Iona E, Orru G, Thoresen OF, Ricci ML, et al. rpoB
mutations in multi-drug resistant strains of Mycobacterium tuberculosis
isolated in Italy. J Clin Microbiol. 1999;37:1197–9.
- Karunkaran P, Davies J. Genetic
antagonism and hypermutability in Mycobacterium smegmatis.
J Bacteriol. 2000;182:3331–5.
- Oliver A, Canton R, Campo P, Baquero F, Blazquez J. High
frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis
lung infection. Science. 2000;288:1251–3.
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mycobacterial genomics. Curr Opin Microbiol. 1998;1:567–71.
- Venkatesh J, Kumar P, Krishna PSM, Manjunath R, Varshnay U. Importance
of uracil DNA glycosylase in Pseudomonas aeruginosa and Mycobacterium
smegmatis, G+C rich bacteria, in mutation prevention, tolerance
to acidified nitrite, and endurance in mouse macrophages. J Biol
Chem. 2003;278:24350–8.
- Merchant K, Chen H, Gonzalez TC, Keefer LK, Shaw BR. Deamination
of single-stranded DNA cytosine residues in aerobic nitric oxide solution
at micromolar total NO exposures. Chem Res Toxicol. 1996;9:891–6.
- Billington OJ, McHugh TD, Gillespie SH. Physiological
cost of rifampin resistance induced in vitro in Mycobacterium tuberculosis.
Antimicrob Agents Chemother. 1999;43:1866–9.
- Wagner J, Nohmi T.
Escherichia coli DNA Polymerase IV mutator activity: genetic
requirements and mutational specificity. J Bacteriol. 2000;182:4587–95.
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