HUG Domain Is Responsible for Active Dimer Stabilization in an NrdJd Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.

NUDT6 and NUDT9, two mitochondrial members of the NUDIX family, have distinct hydrolysis activities.

The 22 members of the NUDIX (NUcleoside DIphosphate linked to another moiety, X) hydrolase superfamily can hydrolyze a variety of phosphorylated molecules including (d)NTPs and their oxidized forms, nucleotide sugars, capped mRNAs and dinucleotide coenzymes such as NADH and FADH. Beside this broad range of enzymatic substrates, the NUDIX proteins can also be found in different cellular compartments, mainly in the nucleus and in the cytosol, but also in the peroxisome and in the mitochondria. Here we studied two members of the family, NUDT6 and NUDT9. We showed that NUDT6 is expressed in human cells and localizes exclusively to mitochondria and we confirmed that NUDT9 has a mitochondrial localization. To elucidate their potential role within this organelle, we investigated the functional consequences at the mitochondrial level of NUDT6- and NUDT9-deficiency and found that the depletion of either of the two proteins results in an increased activity of the respiratory chain and an alteration of the mitochondrial respiratory chain complexes expression. We demonstrated that NUDT6 and NUDT9 have distinct substrate specificity in vitro, which is dependent on the cofactor used. They can both hydrolyze a large range of low molecular weight compounds such as NAD+(H), FAD and ADPR, but NUDT6 is mainly active towards NADH, while NUDT9 displays a higher activity towards ADPR.

Investigating the composition and recruitment of the mycobacterial ImuA'-ImuB-DnaE2 mutasome.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III ß subunit (ß clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted ß clamp-binding motif abolishes ImuB-ß clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this ß clamp-binding antibiotic collapses pre-formed ImuB-ß clamp complexes. These observations establish the essentiality of the ImuB-ß clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

14-3-3 Activated Bacterial Exotoxins AexT and ExoT Share Actin and the SH2 Domains of CRK Proteins as Targets for ADP-Ribosylation.

Bacterial exotoxins with ADP-ribosyltransferase activity can be divided into distinct clades based on their domain organization. Exotoxins from several clades are known to modify actin at Arg177; but of the 14-3-3 dependent exotoxins only Aeromonas salmonicida exoenzyme T (AexT) has been reported to ADP-ribosylate actin. Given the extensive similarity among the 14-3-3 dependent exotoxins, we initiated a structural and biochemical comparison of these proteins. Structural modeling of AexT indicated a target binding site that shared homology with Pseudomonas aeruginosa Exoenzyme T (ExoT) but not with Exoenzyme S (ExoS). Biochemical analyses confirmed that the catalytic activities of both exotoxins were stimulated by agmatine, indicating that they ADP-ribosylate arginine residues in their targets. Side-by-side comparison of target protein modification showed that AexT had activity toward the SH2 domain of the Crk-like protein (CRKL), a known target for ExoT. We found that both AexT and ExoT ADP-ribosylated actin and in both cases, the modification compromised actin polymerization. Our results indicate that AexT and ExoT are functional homologs that affect cytoskeletal integrity via actin and signaling pathways to the cytoskeleton.

Mycobacterium tuberculosis Rv3160c is a TetR-like transcriptional repressor that regulates expression of the putative oxygenase Rv3161c.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a major health threat listed among the top 10 causes of death worldwide. Treatment of multidrug-resistant Mtb requires use of additional second-line drugs that prolong the treatment process and result in higher death rates. Our team previously identified a 2-pyridone molecule (C10) that blocks tolerance to the first-line drug isoniazid at C10 concentrations that do not inhibit bacterial growth. Here, we discovered that the genes rv3160c and rv3161c are highly induced by C10, which led us to investigate them as potential targets. We show that Rv3160c acts as a TetR-like transcriptional repressor binding to a palindromic sequence located in the rv3161c promoter. We also demonstrate that C10 interacts with Rv3160c, inhibiting its binding to DNA. We deleted the rv3161c gene, coding for a putative oxygenase, to investigate its role in drug and stress sensitivity as well as C10 activity. This Δrv3161c strain was more tolerant to isoniazid and lysozyme than wild type Mtb. However, this tolerance could still be blocked by C10, suggesting that C10 functions independently of Rv3161c to influence isoniazid and lysozyme sensitivity.

Evaluating the role of vitamin D receptor polymorphisms on susceptibility to tuberculosis among iranian patients: a case-control study.

Many genetic studies recently have focused on HLA, VDR, NRAMP1, MBL, TNF-a, and their relationships with susceptibility to diseases such as pulmonary tuberculosis. Some studies showed predisposing and protective roles for VDR polymorphisms in pulmonary tuberculosis. Through a case-control study, blood samples were taken from tuberculosis case (n= 164) and control (n= 50) groups. DNA was extracted from white blood cells by PCR-RFLP technique using special primers and enzymes for each polymorphism. VDR polymorphisms are known as ApaI, BsmI, FokI, and Taq I which were evaluated within the two mentioned groups. Combined genotypes AbfT and AabbFfTT were the only statistically significant factors which protected people against pulmonary TB in this study. Two mentioned genotypes were protective factors against TB and this study could not find any predisposing genotype to TB. More study is requested on this matter.

Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system.

Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo. One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants.

Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE.

σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor.

Publisher Correction: Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The NRAMP1, VDR, TNF-α, ICAM1, TLR2 and TLR4 gene polymorphisms in Iranian patients with pulmonary tuberculosis: A case-control study.

The innate immune response drives early events in Mycobacterium tuberculosis infection. Since human genetic variation is an important determinant in the outcome of infection with M. tuberculosis, we typed polymorphisms in the innate immune molecules, such as natural-resistance-associated macrophage protein 1 (NRAMP1), Vitamin D receptor (VDR), Tumor necrosis factor alpha (TNF-α), intercellular adhesion molecule1 (ICAM-1), Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in a case-control study of pulmonary tuberculosis in Iranian population. We conducted an association study and included 96 patients and 122 matched healthy individuals. We used single ARMS-PCR technique to simultaneously genotype fourteen polymorphisms in this survey. Among all fourteen polymorphisms that were examined, three polymorphisms were significantly different between case and control groups. The TNF -308A polymorphism showed significant increase in allele and genotype frequencies among patients compared to control individuals [-308A allele: 19.3 vs. 9.4%, GA genotype: 28.1 vs. 17.2%, AA genotype: 5.2 vs. 0.8%; Corrected P (Pc)<0.05], and the TLR4 variant allele and genotypes prevalence (D299G and T399I) were significantly higher among patients compared to controls [DG genotype: 14.6 vs. 5.7%, Pc<0.05 and I399 allele: 4.2 vs. 0.8%, TI genotype: 8.3 vs. 1.6%; Pc<0.05], respectively. In conclusion, our data suggest that TLR4 (D299G and T399I) and TNF (-308G/A) genetic polymorphisms may influence the risk of developing tuberculosis after exposure to Mycobacterium.

PE_PGRS33 Contributes to Mycobacterium tuberculosis Entry in Macrophages through Interaction with TLR2.

PE_PGRS represent a large family of proteins typical of pathogenic mycobacteria whose members are characterized by an N-terminal PE domain followed by a large Gly-Ala repeat-rich C-terminal domain. Despite the abundance of PE_PGRS-coding genes in the Mycobacterium tuberculosis (Mtb) genome their role and function in the biology and pathogenesis still remains elusive. In this study, we generated and characterized an Mtb H37Rv mutant (MtbΔ33) in which the structural gene of PE_PGRS33, a prototypical member of the protein family, was inactivated. We showed that this mutant entered macrophages with an efficiency up to ten times lower than parental or complemented strains, while its efficiency in infecting pneumocytes remained unaffected. Interestingly, the lack of PE_PGRS33 did not affect the intracellular growth of this mutant in macrophages. Using a series of functional deletion mutants of the PE_PGRS33 gene to complement the MtbΔ33 strain, we demonstrated that the PGRS domain is required to mediate cell entry into macrophages, with the key domain encompassing position 140-260 amino acids of PE_PGRS33. PE_PGRS33-mediated entry into macrophages was abolished in TLR2-deficient mice, as well as following treatment with wortmannin or an antibody against the complement receptor 3 (CR3), indicating that PE_PGRS33-mediated entry of Mtb in macrophages occurs through interaction with TLR2.

Rapid and simultaneous detection of vitamin D receptor gene polymorphisms by a single ARMS-PCR assay.

BACKGROUND: Vitamin D has various roles in many biological actions such as calcium homeostasis, cell proliferation, and cell differentiation to many target tissues. These effects are mediated by the active form of vitamin D, 1,25(OH)2D3, which binds to a cytoplasmic protein called vitamin D receptor (VDR). VDR gene has four common single nucleotide polymorphisms (SNPs) that are defined by the presence of restriction sites for FokI (F/f), TaqI (T/t), BsmI (B/b), and ApaI (A/a). The association of VDR gene polymorphisms with several diseases has been investigated. In most studies, VDR genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays, which are cumbersome and time consuming, and their results are sometimes difficult to interpret. OBJECTIVE: We modified previously reported primers for VDR genotyping and set up a single amplification-refractory mutation system (ARMS)-PCR method for simultaneous genotyping of four common VDR polymorphisms. METHODS: In this study, 218 DNA samples were analyzed for VDR genetic variants by this ARMS-PCR technique; 136 of them were re-genotyped by PCR-RFLP assays to compare genotyping results. RESULT: We obtained allelic frequencies of 69 vs. 31 % for F/f, 34 vs. 66 % for B/b, 70 vs. 30 % for T/t, and 52 vs. 48 % for A/a in this sample of the Iranian population. In addition, comparisons of the results of these two methods showed good uniformity in VDR genotypes; although, in some samples, ambiguity in restriction patterns was present. CONCLUSION: As ARMS-PCR is more rapid, economic, and user friendly than PCR-RFLP, its substitution would be welcomed in disease association and pharmacogenetic studies of VDR variants.

Impact of protein domains on PE_PGRS30 polar localization in Mycobacteria.

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.