Microbial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defenses. In plants, intracellular nucleotide-binding/leucine-rich repeat receptors (NLRs) detect specific effector interference and trigger immunity by an unknown mechanism. The Arabidopsis-interacting NLR pair, RRS1-R with RPS4, confers resistance to different pathogens, including Ralstonia solanacearum bacteria expressing the acetyltransferase effector PopP2. We show that PopP2 directly acetylates a key lysine within an additional C-terminal WRKY transcription factor domain of RRS1-R that binds DNA. This disrupts RRS1-R DNA association and activates RPS4-dependent immunity. PopP2 uses the same lysine acetylation strategy to target multiple defense-promoting WRKY transcription factors, causing loss of WRKY-DNA binding and transactivating functions needed for defense gene expression and disease resistance. Thus, RRS1-R integrates an effector target with an NLR complex at the DNA to switch a potent bacterial virulence activity into defense gene activation.

The worldwide recrudescence of tuberculosis and widespread antibiotic resistance have strengthened the need for the rapid development of new antituberculous drugs targeting essential functions of its etiologic agent, Mycobacterium tuberculosis. In our search for new targets, we found that the M. tuberculosis pps1 gene, which contains an intein coding sequence, belongs to a conserved locus of seven open reading frames. In silico analyses indicated that the mature Pps1 protein is orthologous to the SufB protein of many organisms, a highly conserved component of the [Fe-S] cluster assembly and repair SUF (mobilization of sulfur) machinery. We showed that the mycobacterial pps1 locus constitutes an operon which encodes Suf-like proteins. Interactions between these proteins were demonstrated, supporting the functionality of the M. tuberculosis SUF system. The noticeable absence of any alternative [Fe-S] cluster assembly systems in mycobacteria is in agreement with the apparent essentiality of the suf operon in Mycobacterium smegmatis. Altogether, these results establish that Pps1, as a central element of the SUF system, could play an essential function for M. tuberculosis survival virtually through its implication in the bacterial resistance to iron limitation and oxidative stress. As such, Pps1 may represent an interesting molecular target for new antituberculous drugs.According to the World Health Organization (http://www .who.int/en/), tuberculosis remains a major public health threat as a first-line infectious disease responsible for 2 to 3 million deaths worldwide annually. Furthermore, the recent recrudescence of infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, is associated with the emergence of multidrug-resistant strains, which has strengthened the need for the rapid development of new antituberculous drugs. The discovery of essential functions of mycobacteria in pathways other than those targeted by present antibiotics appears thus to be necessary to efficiently fight tuberculosis.One singularity of the M. tuberculosis genome is the presence of intein insertion sequences in three genes, namely recA, dnaB, and pps1 (6, 9, 34). While the specificity of the recA and pps1 inteins of M. tuberculosis has been readily applied to the diagnosis of tuberculosis by PCR (46), the presence of an intein in a mycobacterial protein can present additional interest. Effectively, inteins are proteins embedded in-frame in a host protein; they are autocatalytically and posttranscriptionally excised from the peptide precursor to produce the functional host protein (26). Hence, the impediment of the protein splicing of a mycobacterial protein involved in a critical cellular process could represent an unusual way to kill M. tuberculosis (4,8,33).Among the intein-containing proteins, the mycobacterial RecA recombinase, while directly implied in DNA repair and homologous recombination (15, 31, 43), is not essential for the survival of Mycobacterium bovis BCG in a mouse infection model (43). An essenti...

Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by the clinical M. tuberculosis isolate HN878 has been associated with a less efficient Th1 response and increased virulence in mice and rabbits. It has been suggested that the W-Beijing family is the only group of M. tuberculosis strains with an intact pks1-15 gene, required for the synthesis of PGL-tb and capable of producing PGL-tb. We have found that some strains with an intact pks1-15 do not produce PGL-tb while others may produce a variant of PGL-tb. We examined the early host cytokine response to infection with these strains in vitro to better understand the effect of PGL-tb synthesis on immune responses. In addition, we generated a PGL-tb-producing H37Rv in order to determine the effect of PGL-tb production on the host immune response during infection by a strain normally devoid of PGL-tb synthesis. We observed that PGL-tb production by clinical M. tuberculosis isolates affected cytokine production differently depending on the background of the strain. Importantly, while ectopic PGL-tb production by H37Rv suppressed the induction of several pro-and anti-inflammatory cytokines in vitro in human monocytes, it did not lead to increased virulence in infected mice and rabbits. Collectively, our data indicate that, while PGL-tb may play a role in the immunogenicity and/or virulence of M. tuberculosis, it probably acts in concert with other bacterial factors which seem to be dependent on the background of the strain.

THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13

The Mycobacterium tuberculosis Beijing strains are a family highly prevalent in Asia and have recently spread worldwide, causing a number of epidemics, suggesting that they express virulence factors not found in other M. tuberculosis strains. Accordingly, we looked for putative characteristic compounds by comparing the lipid profiles of several Beijing and non-Beijing strains. All the Beijing strains analyzed were found to synthesize structural variants of two well known characteristic lipids of the tubercle bacillus, namely phthiocerol dimycocerosates (DIM) and eventually phenolglycolipids (PGL). These variants were not found in non-Beijing M. tuberculosis isolates. Structural elucidation of these variants showed that they consist of phthiotriol and glycosylated phenolphthiotriol dimycocerosates, eventually acylated with 1 mol of palmitic acid, in addition to the conventional acylation of the ␤-diol by mycocerosic acids. We demonstrated that this unusual lipid profile resulted from a single point mutation in the Rv2952 gene, which encodes the S-adenosylmethionine-dependent methyltransferase participating to the O-methylation of the third hydroxyl of the phthiotriol and phenolphthiotriol in the biosynthetic pathway of DIM and PGL. Consistently, the mutated enzyme exhibited in vitro a much lower O-methyltransferase activity than did the wild-type Rv2952. We finally demonstrated that the structural variants of DIM and PGL fulfill the same function in the cell envelope and virulence than their conventional counterparts.

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...

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.

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