1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) from Mycobacterium tuberculosis: towards understanding mycobacterial resistance to fosmidomycin

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg(2+) ions and exhibits optimal activity between pH 7.5 and 7.9; the K(m) for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 microM, and the K(m) for NADPH was 29.7 microM. The specificity constant of Rv2780c in the forward direction is 1.5 x 10(6) M(-1) min(-1), and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.     

Identification of a Polyprenylphosphomannosyl Synthase Involved in the Synthesis of Mycobacterial Mannosides

We report on the identification of a glycosyltransferase (GT) from Mycobacterium tuberculosis H37Rv, Rv3779, of the membranous GT-C superfamily responsible for the direct synthesis of polyprenyl-phospho-mannopyranose and thus indirectly for lipoarabinomannan, lipomannan, and the higher-order phosphatidyl-myo-inositol mannosides.The mycobacterial cell envelope consists of a multilayered structure of covalently linked peptidoglycan, arabinogalactan, and mycolic acids (the mAGP complex) and, among other important constituents, various noncovalently bound glycosylated lipids, notably the phosphatidyl-myo-inositol mannosides (PIMs) and their more glycosylated end products lipomannan (LM) and lipoarabinomannan (LAM) (6, 8). These glycolipids and lipoglycans exhibit a broad range of immunomodulatory activities implicated in the pathogenesis of tuberculosis and leprosy (for recent reviews, see references 5, 8, and 10).Many steps in the biosynthesis of these phosphoinositides have been defined (for recent reviews, see references 3 and 12). Mannosyltransferases (ManTs) responsible for the polymerization aspects of the synthesis of the higher-order extracytoplasmic PIMs (PIM₄ to PIM₆), LM and LAM, are of the glycosyltransferase C (GT-C) multi-transmembrane domain superfamily, whose members are dependent on polyprenyl-phospho-mannopyranose (polyprenyl-P-Manp), specifically C₃₅-P-Man or C₅₀-P-Man, as the Manp donor, which is in contrast with what occurs in the early steps of the synthesis of the simpler PIMs, which directly utilize GDP-Man (3, 4, 12). However, the biosynthetic origins of C₃₅/C₅₀-P-Man have not been fully explored. In this report, we identify Rv3779, an unassigned GT-C, as a ManT directly responsible for the origins of polyprenyl-P-Man and indirectly for the synthesis of the more polar PIMs, LM and LAM.A survey of the Mycobacterium tuberculosis H37Rv genome for genes with predicted (poly)saccharide-associated functions led to the identification of a cluster of 31 “cell wall biosynthetic genes” (2, 3), including not only arabinogalactan synthetic genes (embCA and embCB, glf, glfT1, and glfT2) but the genes for the putative ABC transporter proteins and mycolyl transferases and Rv3779, among five open reading frames, apparently encoding polyprenyl-P sugar-dependent GT-Cs (3). In particular, the integral membrane protein Rv3779 (666 amino acids) was identified as a putative GT-C, due to a conserved hallmark DXD motif and 12 to 14 predicted membrane-spanning domains (3). However, unlike with other GT-C enzymes, which typically carry the signature DXD motif on extracytoplasmic loops, the DLD motif of Rv3779 (at amino acid position 82) is predicted to map to the second loop and to be on the cytosolic side of the plasma membrane. This observation suggests that unlike other GT-C superfamily enzymes, Rv3779 utilizes a cytosolic sugar donor, presumably a nucleotide sugar. Rv3779 has orthologs in all slow-growing mycobacteria whose genome sequences are available but is not found in Mycobacterium smegmatis or in any fast-growing mycobacteria, with the notable exception of Mycobacterium abscessus. Orthologs of this gene are also not found in any other members of the suborder Corynebacterineae.Since Rv3779 is not naturally present in M. smegmatis, M. smegmatis strain mc²155 was transformed with a multicopy plasmid (pVV16-Rv3779), allowing the expression of a recombinant C-terminal His₆-tagged Rv3779 protein under the control of the phsp60 promoter (13). A cell-free ManT assay using membranes or whole lysate, similar to the one described by Korduláková et al. (13), was then conducted to determine if Rv3779 is involved in some aspect of PIM/LM/LAM biosynthesis. Thin-layer chromatography (TLC) autoradiography demonstrated an approximately threefold increase in a time-dependent manner in the incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into C₃₅-P-Man and C₅₀-P-Man by mc²155/pVV16-Rv3779 extracts compared with that of the control (Fig. ​(Fig.1A).1A). The two accumulated Man-containing glycolipids were mildly alkali stable and mildly acid labile (data not shown), with TLC mobility properties confirming their identities as precursor glycolipids of the mycobacterial polyisoprenyl-P class (4). An important and substrate concentration-dependent increase in mannosyl transfer following Rv3779 overexpression was also observed when C₅₀-P, the only form of polyprenylphosphate apparently produced by M. tuberculosis (7), was added as an acceptor substrate to the reaction mixture (Fig. 1B and C). This increase in activity over the control was about 27-fold during the first 30 min of the reaction when 0.05 mM C₅₀-P was used in the assay (Fig. ​(Fig.1C).1C). The background polyprenyl-P-Manp syntase activity detected in the control strain most likely resulted from MSMEG_3859/MSMEG_3860 (Ppm1/Ppm2), an M. smegmatis ortholog of the Ppm1 synthase from M. tuberculosis (68% identity, as determined with a 781-amino-acid overlap) (1, 9, 11). Unfortunately but not unexpectedly in light of the reported difficulty of expressing polytopic membrane GTs in Escherichia coli (14), attempts to produce Rv3779 in an active form in E. coli using different expression systems, host strains, growth conditions, and induction protocols were unsuccessful.Open in a separate windowFIG. 1.Effect of Rv3779 overexpression on the incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into mannolipids by cell extracts from M. smegmatis. (A) TLC analysis of an in vitro cell-free assay using GDP-[¹⁴C]Man and crude cell lysates from mc²155/pVV16 and mc²155/pVV16-Rv3779. Lysates were incubated with GDP-[¹⁴C]Man at 37°C for the indicated periods of time. The synthesized mannolipids were extracted with CHCl₃-CH₃OH (2:1, vol/vol), and a 10% aliquot of each sample was analyzed by TLC followed by autoradiography. The TLC plate was developed in CHCl₃-CH₃OH-H₂O-NH₄OH (65:25:4:0.5). Lanes: C, mc²155/pVV16; O, mc²155/pVV16-Rv3779. (B and C) Incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into exogenous decaprenyl-phosphate (C₅₀)-P using membrane extracts from mc²155/pVV16 and mc²155/pVV16-Rv3779. (B) TLC analysis of the first 15 min of the in vitro cell-free assays using 0.5 mM of (C₅₀)-P. (C) Quantification of the Man incorporated by the control (open symbols) and overexpressor (filled symbols) into C₅₀-P-Man over time. The concentrations of (C₅₀)-P used in the assays were 0 (circles), 0.005 (rectangles), and 0.05 (diamonds) mM. The inset shows a closeup of results of the assays using 0 and 0.005 mM (C₅₀)-P.Incubation of whole-cell lysate with GDP-[¹⁴C]Man for more-extended periods (4 to 24 h) and extraction of products with more-polar solvents (e.g., hot phenol) provides a ready estimate of the degree of synthesis of at least the LM metabolic end product. A comparison of the products synthesized by mc²155/pVV16 and mc²155/pVV16-Rv3779 (Fig. ​(Fig.2)2) showed little synthesis of [¹⁴C]LM at 4 h but the production of some low-molecular-weight [¹⁴C]LM at 24 h by the control lysate. In contrast, there was a distinct production of [¹⁴C]LM populations by the lysate from mc²155/pVV16-Rv3779 at both times, indicative of a secondary stimulatory effect of Rv3779 overexpression on LM, and presumably LAM, synthesis.Open in a separate windowFIG. 2.In vitro LM biosynthesis in M. smegmatis overexpressing Rv3779. Crude cell extracts (4 mg protein) of mc²155/pVV16 and mc²155/pVV16-Rv3779 were incubated with 1.0 μCi of GDP-[¹⁴C]Man (specific activity, 305 mCi/mmol) for 4 and 24 h. The reaction was stopped by adding CHCl₃-CH₃OH (2:1, vol/vol), and the LM/LAM contained in the cell pellet was extracted with hot phenol. LM/LAM separated on 10 to 20% Tricine gel and subsequently blotted to a nitrocellulose membrane was revealed by autoradiography. The leftmost column shows molecular mass markers (in kilodaltons).To confirm these effects of Rv3779, an M. tuberculosis H37Rv Rv3779 knockout mutant (H37RvΔRv3779) was generated by homologous recombination using standard procedures (15) (see Fig. S1 in the supplemental material). H37RvΔRv3779 grew at a much lower rate than WT H37Rv (see Fig. S2 in the supplemental material). Moreover, when examined by scanning electron microscopy, the mutant cells were found to be significantly shorter than their wild-type (WT) parent (Fig. ​(Fig.3).3). Normal growth rate and cell length were restored in the mutant upon complementation with a WT copy of Rv3779 (Fig. ​(Fig.3;3; see Fig. S2 in the supplemental material). TLC profiles of lipid extracts and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses showed a profound decrease in the amounts of acyl-PIM₆ (AcPIM₆) and Ac₂PIM₆ in H37RvΔRv3779 compared to those of the WT strain (Fig. ​(Fig.4A;4A; see Fig. S3 in the supplemental material). Importantly, the synthesis of the simpler PIMs, those arising directly from GDP-Man, was not altered in the mutant (Fig. ​(Fig.4A;4A; see Fig. S3 in the supplemental material). The examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with the monoclonal antibody CS-35 and concanavalin A of the phenol-extracted LM/LAM fraction from WT H37Rv and H37RvΔRv3779 also revealed considerably smaller amounts of both LM and LAM in the mutant (Fig. ​(Fig.4B).4B). Complementation of H37RvΔRv3779 with a WT copy of Rv3779 restored both a normal polar PIM and a normal LM/LAM profile to the mutant.Open in a separate windowFIG. 3.Scanning electron micrographs of M. tuberculosis H37Rv (WT), the mutant H37RvΔRv3779, and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 cultured in 7H9-oleic acid-albumin-dextrose-catalase-Tween 80 broth at 37°C. The length of the H37RvΔRv3779 mutant cells was on average 1.2 ± 0.1 μm, compared to 2.3 ± 0.1 μm for the WT strain and 2.1 ± 0.3 μm for the complemented mutant.Open in a separate windowFIG. 4.Analysis of polar PIMs LM and LAM from WT M. tuberculosis H₃₇Rv, the H37RvΔRv3779 mutant, and H37RvΔRv3779/pVV16-Rv3779. (A) Equal amounts of total cellular lipids from WT H₃₇Rv (lane 1), the Rv3779 mutant H37RvΔRv3779 (lane 2), the mutant carrying an empty plasmid, H37RvΔRv3779/pVV16 (lane 3), and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 (lane 4) were analyzed by TLC developed in CHCl₃-CH₃OH-H₂O-NH₄OH (65:25:4:0.5). (B) LM and LAM extracted from equal-weight cells of WT H₃₇Rv (lane 1), the Rv3779 mutant H37RvΔRv3779 (lane 2), and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 (lane 3) were separated on a 10 to 20% Tricine gel and revealed by periodic acid-Schiff staining. The Western blot analyses were performed on the same samples using concanavalin A (ConA) reacting with the t-Manp residues of LM/LAM, as well as the CS-35 monoclonal antibody known to react with the arabinan segment of LAM.From this evidence, we conclude that Rv3779 is involved in the synthesis of the polar PIMs LM and LAM through its primary role as a polyprenyl-P-Man synthase. The disruption of this gene, which results in important alterations in PIM, LM, and LAM profiles, also has profound effects on cell growth and shape. Rv3779 is the second polyprenyl-P-Man synthase involved in PIM/LM/LAM biosynthesis identified in M. tuberculosis (11).      

The presence and the content of Monacolins in Red Yeast rice prepared from Thai glutinous rice

Red yeast rice which is a product of solid fermentation was prepared from several kinds of Thai glutinous rice (Oryza sativa L.) cv. Korkor 6 (RD6), Kam (Kam), and Sanpatong1 (SPT1). Monascus purpureus CMU001 isolated from available Chinese red yeast rice was used as the fermentation starter. The analysis for the presence and the content of monacolins, the cholesterol-lowering compounds, were carried out using high performance liquid chromatography (HPLC). The presence of the monacolins was confirmed by the retention time of the reference compounds and LC-MS. The results were compared to those obtained from the Chinese red yeast rice and Thai non-glutinous rice (O. sativa L. cv. Mali105). The chromatograms show the presence of monacolin K acid form (MKA), compactin (P1), monacolin M acid form (MMA), monacolin K (MK), monacolin M (MM), and dehydromonacolin K (DMK). A large peak of a compound with the molecular weight of 358 was also detected but could not be identified. The amount of two important monacolins, compactin, and monacolin K, were determined. It was found that the highest amount of compactin and monacolin K were 21.98 and 33.79 mg/g, respectively, when using Thai rice varity O. sativa L. cv. RD6 which was fermented without adding soybean milk.     

N Glycolylation of the nucleotide precursors of peptidoglycan biosynthesis of Mycobacterium spp. is altered by drug treatment

The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N-glycolylmuramic rather than N-acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis. The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N-acylmuramyl-pentapeptide precursor molecules also contain a mixture of N-acetyl and N-glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with d-cycloserine consisted entirely of N-glycolyl muropeptides. However, nucleotide-linked peptidoglycan precursors isolated from M. tuberculosis treated with d-cycloserine contained only N-glycolylmuramyl-tripeptide precursors, whereas those from similarly treated M. smegmatis consisted of a mixture of N-glycolylated and N-acetylated residues. The full pentapeptide intermediate, isolated following vancomycin treatment of M. smegmatis, consisted of the N-glycolyl derivative only, whereas the corresponding M. tuberculosis intermediate was a mixture of both the N-glycolyl and N-acetyl products. Thus, treatment with vancomycin and d-cylcoserine not only caused an accumulation of nucleotide-linked intermediate compounds but also altered their glycolylation status, possibly by altering the normal equilibrium maintained by de novo biosynthesis and peptidoglycan recycling.     

Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium tuberculosis

The thiourea isoxyl (thiocarlide; 4,4'-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 microg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Delta9-stearoyl desaturase as the drug target. Development of a cell-free assay for Delta9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Delta9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Delta9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Delta9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.     

Seasonal effects on the sleep–wake cycle,the rest–activity rhythm and quality of life for Japanese and Thai older people

Background: The sleep-wake cycle and the rest–activity rhythm are known to change with aging, and such changes have been implicated in higher levels of depression as well as an increased incidence of dementia. However, information supporting seasonal changes in the sleep–wake cycle, the rest–activity rhythm and quality of life in older community-dwelling people remains insufficient. The aim of the present study was to prospectively investigate seasonal effects on the sleep–wake cycle, the rest–activity rhythm and quality of life among older people living in areas of Japan or Thailand with different climate classifications.

Method: The survey was conducted from March 2016 to May 2017, and 109 participants were recruited from Japan and Thailand: 47 older people living in Akita prefecture, Japan, and 62 older people living in Chiang Mai or Nakhon Ratchasima, Thailand. According to the Köppen–Geiger classification of Asian climates comprising tropical, desert, steppe, temperate and subarctic climates, Akita prefecture, which is located in northern Japan, is classified as a humid subarctic climate, while the Thai study areas are classified as tropical savanna. To monitor parameters of the sleep–wake cycle during nighttime (e.g. total sleep time, sleep latency, sleep efficiency, awaking time and frequency of sleep interruptions) and to calculate parameters of the rest–activity rhythm over the 24 h profile (e.g., interdaily stability, intradaily variability, relative amplitude, mean of least active 5 h period and mean of most active 10 h period), all the participants from both countries wore an Actiwatch 2 device on their nondominant wrist continuously for 7 days during each local season. The World Health Organization Quality of Life Questionnaire-BREF (WHOQOL-BREF) was also assessed during each local season.

Results: The final sample size was 37 older people living in Akita prefecture, Japan, and 44 older people living in Thailand; these subjects completed the data collections during each local season. The dropout rates were 21% in Japan and 29% in Thailand. The results for the Japanese subjects showed a significantly shorter sleep time with higher levels of activity during the nighttime on summer (p < 0.001) and a fragmented rest–activity rhythm over the 24 h profile on winter (p < 0.001). The older Thai participants exhibited a poor state of night sleeping year-round, and a significant relationship was observed between seasonal variations in motor activity and the social domain of WHOQOL-BREF for each Thai season (|r| = 0.4, p < 0.01).

Conclusion: These findings provide new and important information regarding seasonal effects on the sleep–wake cycle, the rest–activity rhythm and quality of life in older community-dwelling people living in two different Asian climates. Consequently, clinical preventions targeting such seasonal variations might be useful for improving the quality of life of older Japanese and Thai individuals.     

A Large Cohort of Hemoglobin Variants in Thailand: Molecular Epidemiological Study and Diagnostic Consideration

Background

Hemoglobin (Hb) variants are structurally inherited changes of globin chains. Accurate diagnoses of these variants are important for planning of appropriate management and genetic counseling. Since no epidemiological study has been conducted before, we have investigated frequencies, molecular and hematological features of Hb variants found in a large cohort of Thai subjects.

Materials and Methods

Study was conducted on 26,013 unrelated subjects, inhabiting in all geographical parts of Thailand over a period of 11 years from January 2002-December 2012. Hb analysis was done on high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Mutations causing Hb variants were identified using PCR and related techniques.

Results

Among 26,013 subjects investigated, 636 (2.4%) were found to carry Hb variants. Of these 636 subjects, 142 (22.4%) carried α-chain variants with 13 different mutations. The remaining included 451 (70.9%) cases with 16 β-chain variants, 37 (5.8%) cases with Hb Lepore (δβ-hybrid Hb) and 6 (0.9%) cases with a single δ-chain variant. The most common α-globin chain variant was the Hb Q-Thailand (α^{74GAC-CAC, Asp-His}) which was found in 101 cases (15.8%). For β-globin chain variants, Hb Hope (β^{136GGT-GAT, Gly-Asp}) and Hb Tak (β^{146+AC, Ter-Thr}) are the two most common ones, found in 121 (19.0%) and 90 (14.2%) cases, respectively. Seven Hb variants have never been found in Thai population. Hb analysis profiles on HPLC or CE of these variants were illustrated to guide presumptive diagnostics.

Conclusions

Hb variants are common and heterogeneous in Thai population. With varieties of thalassemias and hemoglobinopathies in the population, interactions between them leading to complex syndromes are common and render their diagnoses difficult in routine practices. Knowledge of the spectrum, molecular basis, genotype-phenotype correlation and diagnostic features should prove useful for prevention and control of the diseases in the region.