Affiliation:
1. Laboratory Research Branch, National Hansen's Disease Programs at Louisiana State University, Baton Rouge, Louisiana,1 and
2. Anandaban Leprosy Hospital, Kathmandu, Nepal2
Abstract
ABSTRACT
Two
Mycobacterium leprae
genes,
folP1
and
folP2
, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a
folP
knockout mutant of
Escherichia coli
(C600Δ
folP
::Km
r
). Expression of
M. leprae folP1
in C600Δ
folP
::Km
r
conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type
E. coli
C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC
50
]) the DHPS activity of this recombinant. When the
folP1
genes of several dapsone-resistant
M. leprae
clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600Δ
folP
::Km
r
knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type
M. leprae folP1
. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC
50
68-fold. These results prove that the
folP1
of
M. leprae
encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of
M. leprae
. Transformants created with
M. leprae folP2
did not confer growth on the C600Δ
folP
::Km
r
knockout strain, and DNA sequences of
folP2
from dapsone-susceptible and -resistant
M. leprae
strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in
M. leprae
.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Reference38 articles.
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2. Identification of a class of sulfonamides highly active against dihydropteroate synthase form Toxoplasma gondii, Pneumocystis carinii, and Mycobacterium avium
3. Rapid identification of a point mutation of the Mycobacterium tuberculosis catalase-peroxidase (katG) gene associated with isoniazid resistance.;Cockrill F. R.;J. Infect. Dis.,1995
4. The dihydropteroate synthase gene, folP, is near the leucine tRNA gene, leuU, on the Escherichia coli chromosome
5. Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100
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