ABSTRACT
Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Cell Wall/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Gene Expression Regulation, Bacterial , Mycobacterium tuberculosis/drug effects , Serine Proteases/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , Cell Wall/genetics , Cell Wall/metabolism , Ethambutol/pharmacology , Galactans/biosynthesis , Gene Expression Profiling , Humans , Ion Pumps/deficiency , Ion Pumps/genetics , Isoniazid/pharmacology , Ligases/genetics , Ligases/metabolism , Mannosyltransferases/genetics , Mannosyltransferases/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Meropenem , Microbial Sensitivity Tests , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Mycolic Acids/metabolism , Peptidoglycan/biosynthesis , Rifampin/pharmacology , Serine Proteases/metabolism , Thienamycins/pharmacology , Vancomycin/pharmacologyABSTRACT
The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1+/-) were generated by homologous recombination. The Asna1+/- mice displayed similar phenotype as the wild-type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E=embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.
