Comparison of the Arylamine <Emphasis Type="Italic">N-</Emphasis>Acetyltransferase from <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> and <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Arylamine N-acetyltansferase (NAT) from Mycobacterium tuberculosis (TBNAT) is a potential drug target for anti-tubercular therapy. Recombinant TBNAT is much less soluble and is produced in lower yields than the closely related NAT from Mycobacterium marinum (MMNAT). In order to explore MMNAT as a model for TBNAT in drug discovery, we compare the two mycobacterial NAT enzymes. Two site-directed mutants of MMNAT have been prepared and characterised: MMNAT71, Tyr → Phe and MMNAT209, Met → Thr, in which residues within 6 Å of the active-site cysteine have been replaced with the corresponding residue from TBNAT. Two chimeric proteins have also been produced in which the third domain of MMNAT has been replaced by the third domain of TBNAT and vice versa. The activity profile of the chimeric proteins suggests a role for the third domain in the evolutionary divergence of NAT between these closely related mycobacterial species.     

Novel DNA glycosylases from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Oxidized bases are removed from DNA of Escherichia coli by enzymes formamidopyrimidine DNA glycosylase (Eco-Fpg) and endonuclease VIII (Eco-Nei) of the same structural family Fpg/Nei. New homologs of these enzymes not characterized earlier have been found in genomes of Actinobacteria. We have cloned and expressed two paralogs (Mtu-Nei2 and Mtu-Fpg2) from 36KAZ and KHA94 isolates of Mycobacterium tuberculosis and studied their ability to participate in DNA repair. Under heterologous expression in E. coli, Mtu-Nei2 decreased the rate of spontaneous mutagenesis in the rpoB gene, whereas Mtu-Fpg2 moderately increased it, possibly due to absence of residues crucially important for catalysis in this protein. Mtu-Nei2 was highly active toward double-stranded DNA substrates containing dihydrouracil residues and apurine-apyrimidine sites and was less efficient in cleavage of substrates containing 8-oxoguanine and uracil residues. These lesions, as well as 8-oxoadenine residues, were also recognized and removed by the enzyme from single-stranded DNA. Fpg and Nei homologs from M. tuberculosis can play an important role in protection of bacteria against genotoxic stress caused by oxidative burst in macrophages.     

Genotypic and phenotypic characteristics of tunisian isoniazid-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Forty three isoniazid (INH)-resistant Mycobacterium tuberculosis isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA −15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and sequencing reaction. Mutations at either codon were detected in 67.5% of isolates: katG315 in 37.2, mabA −15C→T in 27.9 and both of them in 2.4%, respectively. katG sequencing showed a G insertion at codon 325 detected in 2 strains and leading to amino acid change T326D which has not been previously reported. Distribution of each mutation, among the investigated strains, showed that katG S315T was associated with multiple-drug profile, high-level INH resistance and loss or decreased catalase activity; whereas the mabA −15C→T was more prevalent in mono-INH resistant isolates, but it was not only associated with a low-level INH resistance. It seems that determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most clinical INH-resistant strains.     

Sulfolipid accumulation in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> disrupted in the <Emphasis Type="Italic">mce2</Emphasis> operon

Mycobacterium tuberculosis, the causative agent of tuberculosis, has a lipid-rich cell wall that serves as an effective barrier against drugs and toxic host cell products, which may contribute to the organism’s persistence in a host. M. tuberculosis contains four homologous operons called nice (mce1–4) that encode putative ABC transporters involved in lipid importation across the cell wall. Here, we analyzed the lipid composition of M. tuberculosis disrupted in the mce2 operon. High resolution mass spectrometric and thin layer chromatographic analyses of the mutant’s cell wall lipid extracts showed accumulation of SL-1 and SL₁₂₇₈ molecules. Radiographic quantitative analysis and densitometry revealed 2.9, 3.9 and 9.8-fold greater amount of [³⁵S] SL-1 in the mce2 operon mutant compared to the wild type M. tuberculosis during the early/mid logarithmic, late logarithmic and stationary phase of growth in liquid broth, respectively. The amount of [³⁵S] SL₁₂₇₈ in the mutant also increased progressively over the same growth phases. The expression of the mce2 operon genes in the wild type strain progressively increased from the logarithmic to the stationary phase of bacterial growth in vitro, which inversely correlated with the proportion of radiolabel incorporation into SL-1 and SL₁₂₇₈ at these phases. Since the mce2 operon is regulated in wild type M. tuberculosis, its cell wall may undergo changes in SL-1 and SL₁₂₇₈ contents during a natural course of infection and this may serve as an important adaptive strategy for M. tuberculosis to maintain persistence in a host.     

Kinetic properties of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> bifunctional GlmU

The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg²⁺, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors.     

Association of the Rv0679c protein with lipids and carbohydrates in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>/<Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

The Rv0679c gene in Mycobacterium tuberculosis H37Rv encodes a protein with a predicted molecular mass of 16,586 Da consisting of 165 amino acids which contains a putative N-terminal signal sequence and a consensus lipoprotein-processing motif. Globomycin treatment, Triton X-114 separation and mass spectrometry analyses clarified a property of the Rv0679c protein as a lipoprotein. In addition, trifluoromethanesulphonic acid treatment of the lysate revealed an association of the recombinant Rv0679c protein with carbohydrates. The Rv0679c protein homolog of Mycobacterium bovis BCG was also expressed as the protein associated with lipids and carbohydrates. In Western blot analysis, each of the protein homolog and Lipoarabinomannan (LAM) was detected as a similar pattern by anti-Rv0679c and anti-LAM antibodies, respectively. Interestingly, the Rv0679c protein was detected in commercially available LAM purified from M. tuberculosis. Inhibition assay of LAM synthesis in M. bovis BCG by ethambutol showed an altered migration pattern of the Rv0679c protein to low molecular mass similar to that of LAM. The results suggest that the Rv0679c protein exists as a tight complex with LAM in M. tuberculosis/M. bovis BCG.     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

Few amino acid positions in <Emphasis Type="Italic">rpoB</Emphasis> are associated with most of the rifampin resistance in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mutations in rpoB, the gene encoding the β subunit of DNA-dependent RNA polymerase, are associated with rifampin resistance in Mycobacterium tuberculosis. Several studies have been conducted where minimum inhibitory concentration (MIC, which is defined as the minimum concentration of the antibiotic in a given culture medium below which bacterial growth is not inhibited) of rifampin has been measured and partial DNA sequences have been determined for rpoB in different isolates of M. tuberculosis. However, no model has been constructed to predict rifampin resistance based on sequence information alone. Such a model might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates.     

Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.     

Genotyping of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> using whole genome sequencing

Tuberculosis (TB) is considered one of the most serious infectious diseases worldwide. Effective control of tuberculosis infection involves multiple steps, such as reliable detection, treatment, an epidemiological control as a part of case management, and further surveillance and monitoring of TB spread in the human population. Due to the accelerating advances in molecular biology, especially in DNA sequencing, in the past decade, the application of these methods has become crucial for TB evolution studies, differentiation of Mycobacterium tuberculosis genotypes, and their distribution. Currently, several molecular genetic methods are available. The oldest typing methods (e.g., IS6110-RFLP, spoligotyping, and MIRU-VNTR) can discover the chain of transmission to the patient. Currently, whole genome sequencing facilitates is furthermore able to identify the source of infection, the transmission trays among individuals sharing the same isolate, as well as determination of the TB evolution and its resistance to antituberculotic agents. It is obvious that this technique will become a new gold standard in genotyping methods in tuberculosis molecular epidemiological studies. In this article, molecular genetic typing methods with a special focus on whole genome sequencing and data management are reviewed.     

Novel mutations in <Emphasis Type="Italic">katG</Emphasis> gene of a clinical isolate of isoniazid-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Most of isoniazid-resistant Mycobacterium tuberculosis evolved due to mutation in the katG gene encoding catalase-peroxidase. A set of new mutations, namely T1310C, G1388T, G1481A, T1553C, and A1660G, which correspond to amino acid substitutions of L437P, R463L, G494D, I518T, and K554E, in the katG gene of the L10 clinical isolate M. tuberculosis was identified. The wild-type and mutant KatG proteins were expressed in Escherichia coli BL21(DE3) as a protein of 80 kDa based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. The mutant KatG protein exhibited catalase and peroxidase activities of 4.6% and 24.8% toward its wild type, respectively, and retained 19.4% isoniazid oxidation activity. The structure modelling study revealed that these C-terminal mutations might have induced formation of a new turn, perturbing the active site environment and also generated new intramolecular interactions, which could be unfavourable for the enzyme activities.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Identification of wild-type <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates and point mutations associated with isoniazid resistance

Isoniazid resistance in Mycobacterium tuberculosis (MBT) is associated with point mutations in codon 315 of the katG gene. Two PCR technique were developed for detection of point mutations in codon 315. Most frequent point mutations (AGC → ACC and AGC → AGA) were identified in codon 315 by using two sets of primers, either of which included an additional competitive blocking primer with a 3′-terminal phosphate group in order to prevent nonspecific amplification. PCR with a set of two primers, one of which contained five locked nucleic acid monomers (LNA), permits one to detect any of six known mutations in codon 315 of katG and, thereby, discriminate between isoniazid-sensitive and resistant MBT isolates. The structure and purity of the 17-nt long LNA-containing oligonucleotides were characterized by MALDI-TOF mass spectrometry; and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation.     

Modeling metabolic adjustment in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> upon treatment with isoniazid

Complex biological systems exhibit a property of robustness at all levels of organization. Through different mechanisms, the system tries to sustain stress such as due to starvation or drug exposure. To explore whether reconfiguration of the metabolic networks is used as a means to achieve robustness, we have studied possible metabolic adjustments in Mtb upon exposure to isoniazid (INH), a front-line clinical drug. The redundancy in the genome of M. tuberculosis (Mtb) makes it an attractive system to explore if alternate routes of metabolism exist in the bacterium. While the mechanism of action of INH is well studied, its effect on the overall metabolism is not well characterized. Using flux balance analysis, inhibiting the fluxes flowing through the reactions catalyzed by Rv1484, the target of INH, significantly changes the overall flux profiles. At the pathway level, activation or inactivation of certain pathways distant from the target pathway, are seen. Metabolites such as NADPH are shown to reduce drastically, while fatty acids tend to accumulate. The overall biomass also decreases with increasing inhibition levels. Inhibition studies, pathway level clustering and comparison of the flux profiles with the gene expression data indicate the activation of folate metabolism, ubiquinone metabolism, and metabolism of certain amino acids. This analysis provides insights useful for target identification and designing strategies for combination therapy. Insights gained about the role of individual components of a system and their interactions will also provide a basis for reconstruction of whole systems through synthetic biology approaches.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> strains: <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Triacylglycerol: nourishing molecule in endurance of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The ability of Mycobacterium tuberculosis (M. tuberculosis) to accumulate lipid-rich molecules as an energy source obtained from host cell debris remains interesting. Additionally, the potential of M. tuberculosis to survive under different stress conditions leading to its dormant state in pathogenesis remains elusive. The exact mechanism by which these lipid bodies generated in M. tuberculosis infection and utilized by bacilli inside infected macrophage for its survival is still not understood. In this, during bacillary infection, many metabolic pathways are involved that influence the survival of M. tuberculosis for their own support. However, the exact energy source derived from infecting host cells remain elusive. Therefore, this study highlights several alternative energy sources in the form of triacylglycerol (TAG) and fatty acids, i.e. oleic acids accumulation, which are essential in dormancy-like state under M. tuberculosis infection. The prominent stage in tuberculosis (TB) infection is re-establishment of M. tuberculosis under stress conditions and deployment of a confined strategy to utilize these biomolecules for its persistence survival. So, growing in our understanding of these pathways will help us in accelerating therapies, which could reduce TB prevalence world widely.