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.     

Single-nucleotide variations associated with <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> KwaZulu-Natal strains

The occurrence of drug resistance in Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB), is hampering the management and control of TB in the world. Here we present a computational analysis of recently sequenced drug-sensitive (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. Single-nucleotide variations (SNVs) were identified in a pair-wise manner using the anchor-based whole genome comparison (ABWGC) tool and its modified version. For this analysis, four fully sequenced genomes of different strains of M. tuberculosis were taken along with three KwaZulu-Natal (KZN) strains isolated from South Africa including one XDR and one MDR strain. KZN strains were compared with other fully sequenced strains and also among each other. The variations were analysed with respect to their biological influence as a result of either altered structure or synthesis. The results suggest that the DR phenotype may be due to changes in a number of genes. The database on KZN strains can be accessed through the website .     

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.     

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.     

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.     

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.     

Influence of <Emphasis Type="Italic">UPF</Emphasis> genes on severity of <Emphasis Type="Italic">SUP45</Emphasis> mutations

Eukaryotic cells possess a special mechanism for the degradation of mRNAs containing premature termination codons (PTCs), referred to as NMD (nonsense-mediated mRNA decay). The strength of this pathway depends on the recognition of the PTCs by translational machinery and the interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins in Sachromyces cerevisiae yeast. Previously, we have shown that the decrease of eRF1 protein amounts in sup45 nonsense mutants leads to the impairment of NMD. Here we show that the deletion of UPF1 or UPF2 genes leads to an increase in the viability of sup45 mutants, while the effect of UPF3 gene deletion is allele-specific. Two-hybrid data have shown that amino acid residues 1–555 of Upf1 protein interact with eRF1. Any UPF gene deletion leads to allosupression of the adel1-14 mutation without a change in eRF1 content. The Upf1 depletion does not influence the synthetic lethality of sup45 mutations and the [PSI ⁺] prion. It is possible that the absence of Upf1 (or its activator Upf2) leads to a more effective formation of the translation termination complex and consequently to the increased viability of the cells containing mutant termination factors.     

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.     

<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.     

Analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> M145 genes <Emphasis Type="Italic">SCO4164</Emphasis> and <Emphasis Type="Italic">SCO5854</Emphasis> encoding putative rhodaneses

Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.     

Bioinformatics identification of the methylerythritol phosphate pathway associated genes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> with <Emphasis Type="Italic">ceh1</Emphasis> mutant

The methylerythritol phosphate (MEP) pathway for the production of isoprenoids is recently discovered. The current study aimed to identify MEP pathway disorder-related molecular mechanisms and potential genes in Arabidopsis thaliana. Microarray data (GSE61675) obtained from ceh1 mutant plants and corresponding parental lines were retrieved from Gene Expression Omnibus (GEO) database and were applied for differentially expressed genes (DEGs) screening. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs were performed. Protein-protein interaction (PPI) network was then constructed and displayed by Cytoscape software. Total 762 DEGs including 620 up-regulated and 142 down-regulated genes were screened. In addition, a great many of DEGs were mainly involved in biosynthesis and metabolism-related pathways, such as stilbenoid, diarylheptanoid, and gingerol biosynthesis, and biosynthesis of terpenoids and steroids. Moreover, a PPI network contained 90 down-regulated genes and 497 up-regulated genes were obtained. Up-regulated DEGs including glutaredoxin (GRX480, cytochrome BC1 synthase (BCS1, syntaxin of plants 121 (SYP121) and A. thaliana MAP kinase 11 (ATMPK11) with higher degree in this network were hub nodes. Pathways including stilbenoid, diarylheptanoid, and gingerol biosynthesis obtained in our study were consistent with previous studies. Importantly, GRX480, BCS1 and ATMPK11 could have close interactions with the MEP pathway and may play important roles in the biosynthesis of isoprenoids.     

Functional analysis of an intergenic non-coding sequence within <Emphasis Type="Italic">mce1</Emphasis> operon of <Emphasis Type="Italic">M.tuberculosis</Emphasis>

Background  

The mce operons play an important role in the entry of M. tuberculosis into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of mce mutants. Recently, mce1 operon was found to extend over 13 genes, fadD5 (Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to mce1 among the four mce operons of M.tuberculosis. We have examined the function of this region.     

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.