Abstract
BackgroundThe Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppAMTB.Methodology/Principal FindingsOppAMTB was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppAMTB confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1β, IL-6 and TNF-α was also compromised after inactivation of oppD.ConclusionsTaken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.
Highlights
ATP binding cassette (ABC) transporters are found both in eukaryotes and prokaryotes, and play important roles in the transport of ions, amino acids, peptides, proteins, etc. [1]
Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis
We present evidence that MG levels are lower in macrophages infected with an oppD knock out (OppD-KO) of M. tuberculosis compared to the wild type
Summary
ATP binding cassette (ABC) transporters are found both in eukaryotes and prokaryotes, and play important roles in the transport of ions, amino acids, peptides, proteins, etc. [1]. The oligopeptide importers consist of five subunits: two homologous integral membrane proteins OppB and OppC which form the translocation pore, two nucleotide-binding domains OppD and OppF and the substrate-binding lipoprotein (SBP) OppA that determines substrate specificity. In Gram-positive bacteria SBPs are soluble proteins residing in the periplasm. They are either anchored to the membrane through a lipid modification on the N-terminal cysteine or covalently linked to the translocation pore [7]. The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. We report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppAMTB
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