Protein Splicing of SufB Is Crucial for the Functionality of the Mycobacterium tuberculosis SUF Machinery

Abstract

The SufBCD complex is an essential component of the SUF machinery of [Fe-S] cluster biogenesis in many organisms. We show here that in Mycobacterium tuberculosis the formation of this complex is dependent on the protein splicing of SufB, suggesting that this process is a potential new target for antituberculous drugs.The worldwide recrudescence of tuberculosis has been associated with the emergence of multidrug-resistant strains of Mycobacterium tuberculosis, its causative agent. This alarming situation has reinforced the need for the urgent development of new antituberculous drugs targeting novel and specific mycobacterial functions.In a recent work (7), we have identified the M. tuberculosis SUF (mobilization of sulfur) machinery as the unique and essential system of [Fe-S] cluster assembly in mycobacteria. This system is required for the maturation of physiologically important metalloproteins and plays an important role in the resistance to iron limitation and oxidative stress. It is encoded by a mycobacterial operon of seven genes, Rv1460 to Rv1466 (according to the M. tuberculosis genome annotation [5]), among which Rv1461 encoding the highly conserved SufB protein (7) is interrupted by an intein coding sequence (15).In the present study, we show the inability of the unspliced SufB protein to play its role in the SUF machinery owing to its inability to interact with some of its Suf partners. This highlights the prerequisite of protein splicing in SufB maturation and validates the SufB protein splicing as a specific molecular target for the development of novel antituberculous drugs since blocking the protein splicing process of essential proteins was proposed as a singular way to efficiently kill mycobacteria (1,3,6,14).Construction of a sufB mutant and expression of unspliced SufB. The Rv1461 open reading frame (ORF), encoding the M. tuberculosis SufB protein, was cloned in pGADT7 and pGBKT7 vectors (Clontech) for yeast two-hybrid assays (7). In these constructs, the Rv1461 gene was mutated in order to block the protein splicing process of the SufB precursor peptide: the asparagine residue at the C-terminal extremity of the intein sequence (position 611) and the adjacent cysteine (i.e., the first residue of the C-extein at position 612) were replaced by an aspartic acid and a valine, respectively. Site-directed mutagenesis was done using complementary oligonucleotide pairs (5Ј-TTGTAGATCGGTGCGGTGACGTCGTGCACGGCGAA CCCGT-3Ј and 5Ј-ACGGGTTCGCCGTGCACGACGTCAC CGCACCGATCTACAA-3Ј). To verify the protein splicing of the recombinant wild-type mycobacterial SufB protein when expressed in yeast and the blockage effect of the mutation, Saccharomyces cerevisiae strain AH109 (Clontech) was electrotransformed with the wild-type and mutated pGADT7 and pGBKT7 derivatives, plated, and grown at 30°C in minimal DOBA (dropout base with agar; BIO101) medium containing amino acid complement (Complete Supplement Mixture; BIO101) devoid of leucine (Leu) or tryptophan (Trp), to select transformed yeast cells. Two milliliters of a 1...

Identification of a novel α(1→6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan‐ and lipoarabinomannan‐deficient mutant

“…The locus in the bacteria analysed consists of mptB which has in C. glutamicum the locus tag NCgl1505 and in M. tuberculosis Rv1459c. sufR encodes a transcriptional regulator in front of an operon of the SUF machinery of [Fe-S] cluster synthesis ( Huet et al ., 2006 ). The genomic region displayed encompasses 7 kb, and orthologous genes are highlighted accordingly.…”

Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria

“…(Noren et al , 2000) It was shown that unspliced SufB is non-functional because it fails to associate with SufC or SufD by yeast two-hybrid assay. (Huet et al , 2006) These preliminary studies indicate that the Suf pathway and/or intein splicing may be good targets for novel antibiotic therapy to treat drug-resistant TB. Future studies are required to elucidate the biochemical functions of the novel Suf proteins present in Mycobacteria .…”

New Targets and Inhibitors of Mycobacterial Sulfur Metabolism

“…Unspliced SufB cannot interact with other components in the SUF system and is inactive during iron-sulfur cluster protein assembly. Thus, SufB maturation is an interesting target for inhibitors to block iron sulfur cluster biosynthesis in M. tuberculosis [279–281]. …”