title: Mutations in the essential arabinosyltransferase EmbC lead to alterations in Mycobacterium tuberculosis lipoarabinomannan.
abstract: The Mycobacterium tuberculosis cell wall is a complex structure essential for the viability of the organism and its interaction with the host. The glycolipid lipoarabinomannan (LAM) plays an important role in mediating host-bacteria interactions and is involved in modulation of the immune response. The arabinosyltransferase EmbC required for LAM biosynthesis is essential. We constructed recombinant strains of M. tuberculosis expressing a variety of alleles of EmbC. We demonstrated that EmbC has a functional signal peptide in M. tuberculosis. Over- or underexpression of EmbC resulted in reduced or increased sensitivity to ethambutol, respectively. The C-terminal domain of EmbC was essential for activity because truncated alleles were unable to mediate LAM production in Mycobacterium smegmatis and were unable to complement an embC deletion in M. tuberculosis. The C-terminal domain of the closely related arabinosyltransferase EmbB was unable to complement the function of the EmbC C-terminal domain. Two functional motifs were identified. The GT-C motif contains two aspartate residues essential for function in the DDX motif. The proline-rich region contains two highly conserved asparagines (Asn-638 and Asn-652). Mutation of these residues was tolerated, but loss of Asn-638 resulted in the synthesis of truncated LAM, which appeared to lack arabinose branching. All embC alleles that were incapable of complementing LAM production in M. smegmatis were not viable in M. tuberculosis, supporting the hypothesis that LAM itself is essential in M. tuberculosis.
authors.affiliation: From TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington 98102.
authors.forename: Aaron
authors.initials: A
authors.lastname: Korkegian
authors.affiliation: From TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington 98102.
authors.forename: David M
authors.initials: DM
authors.lastname: Roberts
authors.affiliation: From TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington 98102.
authors.forename: Rachel
authors.initials: R
authors.lastname: Blair
authors.affiliation: From TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington 98102 tanya.parish@idri.org.
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
mesh_terms: D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D004977:Ethambutol / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential / Q000235:genetics; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D050296:Microbial Viability / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D021382:Protein Sorting Signals; C050016:lipoarabinomannan; D001224:Aspartic Acid; D001216:Asparagine; D004977:Ethambutol; D010430:Pentosyltransferases; C101967:arabinosyltransferase
keywords: Arabinosyltransferase; Cell Wall; Essential Gene; Ethambutol; Glycolipid; Infectious Disease; Mycobacteria; Mycobacterium tuberculosis