Novel selection for isoniazid (INH) resistance genes supports a role for NAD+-binding proteins in mycobacterial INH resistance.
The genetic basis of isoniazid (INH) resistance remains unknown for a significant proportion of clinical isolates. To identify genes which might confer resistance by detoxifying or sequestering INH, we transformed the Escherichia coli oxyR mutant, which is relatively sensitive to INH, with a Mycobacterium tuberculosis plasmid library and selected for INH-resistant clones. Three genes were identified and called ceo for their ability to complement the Escherichia coli oxyR mutant. ceoA was the previously identified M. tuberculosis glf gene, which encodes a 399-amino-acid NAD+- and flavin adenine dinucleotide-requiring enzyme responsible for catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The proteins encoded by the ceoBC pair were homologous with one another and with the N terminus of the potassium uptake regulatory protein TrkA. Each of the three Ceo proteins contains a motif common to NAD+ binding pockets. Overexpression of the M. tuberculosis glf gene by placing it under the control of the hsp60 promoter on a multicopy plasmid in Mycobacterium bovis BCG produced a strain for which the INH MIC was increased 50% compared to that for the control strains, while similar overexpression of the ceoBC pair had no effect on INH susceptibility in BCG. Mycobacterial extracts containing the overexpressed Glf protein did not bind radiolabeled INH directly, suggesting a more complex mechanism than the binding of unmodified INH. Our results support the hypothesis that upregulated mycobacterial proteins such as Glf may contribute to INH resistance in M. tuberculosis by binding a modified form of INH or by sequestering a factor such as NAD+ required for INH activity.
Department of Molecular Microbiology and Immunology, Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.
P
P
Chen
W R
WR
Bishai
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D002999:Clone Cells; D004268:DNA-Binding Proteins; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000187:drug effects* / Q000235:genetics*; D005816:Genetic Complementation Test; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics; D007538:Isoniazid / Q000494:pharmacology*; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D020917:Receptor, trkA; D012097:Repressor Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; C115388:CeoB protein, Mycobacterium tuberculosis; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C086512:glucose facilitator protein, Zymomonas; C499299:oxyR protein, E coli; D020917:Receptor, trkA; D019751:Intramolecular Transferases; C490824:Glf protein, E coli; C098051:UDP-galactopyranose mutase; D007538:Isoniazid