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.
Highlights
The arabinosyltransferase EmbC plays an essential role in the synthesis of the cell wall component lipoarabinomannan
To investigate the critical function and role EmbC plays in the physiology of M. tuberculosis, we looked for phenotypic changes resulting from modulating the activity of the enzyme via site-directed mutagenesis, truncations, allele hybrids, and alterations to expression level
EmbC plays an essential role in the biosynthesis of LAM in M. tuberculosis
Summary
The arabinosyltransferase EmbC plays an essential role in the synthesis of the cell wall component lipoarabinomannan. Results: We identified a key motif for EmbC catalytic activity and determined that its structural features are unique over other arabinosyltransferases. The Mycobacterium tuberculosis cell wall is a complex structure essential for the viability of the organism and its interaction with the host. 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 prolinerich 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
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