The Arabinosyltransferase EmbC Is Inhibited by Ethambutol in Mycobacterium tuberculosis

Ethambutol (EMB) is an antimycobacterial drug used extensively for the treatment of tuberculosis caused by Mycobacterium tuberculosis. EMB targets the biosynthesis of the cell wall, inhibiting the synthesis of both arabinogalactan and lipoarabinomannan (LAM), and is assumed to act via inhibition of three arabinosyltransferases: EmbA, EmbB, and EmbC. EmbA and EmbB are required for the synthesis of arabinogalactan, and at least one enzyme (M. tuberculosis EmbA [EmbA_Mt]) is essential in M. tuberculosis. EmbC_Mt is also essential for the viability of M. tuberculosis but is involved in the synthesis of LAM. We show that mutations in EmbC_Mt that reduce its arabinosyltransferase activity result in increased sensitivity to EMB and the production of smaller LAM species in M. tuberculosis. Overexpression of EmbC_Mt was not tolerated in M. tuberculosis, but overexpression of Mycobacterium smegmatis EmbC (EmbC_Ms) led to EMB resistance and the production of larger LAM species in M. tuberculosis. Treatment of wild-type M. tuberculosis strains with EMB led to inhibition of LAM synthesis, resulting in the production of smaller species of LAM. In contrast, no change in LAM production was seen in EMB-resistant strains. Overexpression of EmbB_Ms in M. tuberculosis also resulted in EMB resistance, but at a lower level than that caused by EmbC_Ms. Overexpression of EmbA_Mt in M. tuberculosis had no effect on EMB resistance. Thus, there is a direct correlation between EmbC activity and EMB resistance, as well as between EmbC activity and the size of the LAM species produced, confirming that EmbC is one of the cellular targets of EMB action.Tuberculosis (TB), one of the oldest diseases known to humans, remains a major public health threat. It is estimated that one-third of the world''s population are infected with the causative agent, Mycobacterium tuberculosis. The scale of the problem is increasing, and the disease is becoming deadlier as it intersects with the spread of human immunodeficiency virus. The emergence of multidrug-resistant strains establishes the urgent need to fully understand the mechanisms of drug resistance. Ethambutol (EMB) is a bacteriostatic, antimycobacterial drug first described in 1961 (31) and has been prescribed for TB treatment since 1966. EMB is used worldwide for TB therapy in combination with isoniazid, pyrazinamide, and rifampin (rifampicin). It is also effective in the treatment of opportunistic mycobacterial infections of patients with human immunodeficiency virus. Unfortunately, resistance to EMB has been reported in as many as 4% of clinical isolates of M. tuberculosis and is prevalent among multidrug-resistant strains (5).The effects of EMB are highly pleiotropic, and over the past 40 years, many efforts have been made to understand its intracellular target(s). Kilburn and Greenberg (12) were the first to indicate that EMB treatment of Mycobacterium smegmatis caused rapid bacterial declumping, suggesting cell wall changes. It was subsequently demonstrated that EMB inhibited the transfer of mycolic acids to the cell wall (29), resulting in the accumulation of trehalose monomycolate, trehalose dimycolate, and free mycolic acids in the medium (13). This was later related to inhibition of the biosynthesis of the mycobacterial cell wall core polymer, arabinogalactan (AG), leading to a lack of arabinan receptors for the mycolic acids (29). The mycolyl-AG-peptidoglycan complex (in which AG is linked to peptidoglycan and the mycolic acids) is a major structural component of the cell wall, providing a hydrophobic permeability barrier. It was noted subsequently that AG is not the only cell wall arabinan affected by EMB and that EMB also inhibits the synthesis of the arabinan core of lipoarabinomannan (LAM) (6, 17), a key surface molecule in the host-pathogen interaction (16). In M. smegmatis, EMB exposure leads to the accumulation of decaprenol phosphoarabinose (DPA), the arabinosyl donor for arabinan biosynthesis, confirming the effect of EMB on arabinosyltransferases involved in AG and LAM production (32).The genetic basis of resistance to EMB in mycobacteria has been the subject of much scrutiny. Initial work with M. smegmatis led to the identification of a cluster of genes from the related species Mycobacterium avium that conferred EMB resistance when overexpressed (3). The locus has been characterized; it consists of a regulator, EmbR, and two arabinosyltransferases, EmbA and EmbB (3). In M. tuberculosis and M. smegmatis, the emb locus encodes three arabinosyltransferases (EmbC, EmbA, and EmbB); the EmbR regulator is located elsewhere on the chromosome. The identification of point mutations in codons 289 and 292 of M. smegmatis embB that conferred EMB resistance (1, 15) led to a search for similar mutations in codons 303 and 306 of embB in EMB-resistant clinical isolates of M. tuberculosis (15, 30). While mutations have been associated with EMB resistance in several studies (1, 15, 26, 30), there is still some controversy about the role of these mutations, particularly those at codon 306, in mediating EMB resistance, since the most common mutations have been found in fully sensitive isolates (9, 14, 18, 25). Most recently, work using isogenic strains has once again suggested that these mutations are linked to resistance (23). While much attention has been focused on the role of EmbB in mediating resistance, studies have suggested that multiple molecular pathways to an EMB resistance phenotype exist and that these may be associated with mutations elsewhere in the genome (22, 27).The precise mode of action of EMB is still unclear, although given that EmbB has been implicated in EMB resistance, it has been considered to be the primary target of EMB. However, several lines of evidence suggest that EmbB is not the only target and that the target(s) may differ between mycobacterial species. In particular, inhibition of EmbB in M. smegmatis cannot be the sole antibacterial effect, since embB deletion mutants are viable, indicating that the gene product is not essential in culture (7). Thus, in the fast-growing nonpathogenic species, there must be another target of inhibition, and it seems likely that EMB must inhibit two or more of the Emb arabinosyltransferase activities to effect growth inhibition. In contrast, EmbA (2) and EmbC (8) are independently essential in M. tuberculosis, and the available data strongly suggest that EmbB is essential (24). Thus, in theory, inhibition of any one of these could lead to stasis.We investigated the role of EmbC as a target of EMB action. We constructed strains of M. tuberculosis carrying mutated alleles of embC with reduced arabinosyltransferase activity or strains overexpressing EmbC. Phenotypic analyses demonstrated a direct correlation between EmbC activity, the level of resistance to EMB, and the size of the LAM species produced.     

Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope.

Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethambutol, synthesis of the arabinan of lipoarabinomannan was only partially affected, demonstrating a differential effect on arabinan synthesis in the two locales. Further studies of the effect of ethambutol on cell wall biosynthesis revealed that the synthesis of galactan in the cell wall core is strongly inhibited by the drug. In addition, ethambutol treatment resulted in the cleavage of arabinosyl residues present in the mycobacterial cell wall; more than 50% of the arabinan in the cell wall core was removed from the wall 1 h after addition of the drug to growing mycobacterial cultures. In contrast, galactan was not released from the cell wall during ethambutol treatment. The natural function of the arabinosyl-releasing enzyme remains unknown, but its action in combination with inhibition of synthesis during ethambutol treatment results in severe disruption of the mycobacterial cell wall. Accordingly, ethambutol-induced damage to the cell wall provides a ready molecular explanation for the known synergetic effects of ethambutol with other chemotherapeutic agents. Nevertheless, the initial direct effect of ethambutol remains to be elucidated.     

A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis.

Ethambutol [EMB; dextro-2,2'-(ethylenediimino)-di-1-butanol] is an effective drug when used in combination with isoniazid for the treatment of tuberculosis. It inhibits the polymerization of arabinan in the arabinogalactan and lipoarabinomannan of the mycobacterial cell wall. Recent studies have shown that arabinosyltransferases could be targets of EMB. These enzymes are encoded by the emb locus that was identified in Mycobacterium smegmatis, Mycobacterium leprae, Mycobacterium avium, and Mycobacterium tuberculosis. We demonstrate that a missense mutation in the M. smegmatis embB gene, one of the genes of the emb locus, confers resistance to EMB. The level of resistance is not dependent on the number of copies of the mutated embB gene, indicating that this is a true mechanism of resistance. The mutation is located in a region of the EmbB protein that is highly conserved among the different mycobacterial species. We also identified in this region two other independent mutations that confer EMB resistance. Furthermore, mutations have recently been described in the same region of the EmbB protein from clinical EMB-resistant M. tuberculosis isolates. Together, these data strongly suggest that one of the mechanisms of resistance to EMB consists of missense mutations in a particular region of the EmbB protein that could be directly involved in the interaction with the EMB molecule.     

Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis

When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 mug/ml, the cells began to lose their ability to synthesize mycolic acids immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components-alpha-mycolate, methoxymycolate, and beta-mycolate-was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60 min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly, growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence of events which leads to the loss of viability of cells exposed to isoniazid is described.     

SQ109 targets MmpL3, a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis

SQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core of Mycobacterium tuberculosis, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates. In vitro assays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essential mmpL3 gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.     

Effect of drug concentration on emergence of macrolide resistance in Mycobacterium avium

The emergence of antibiotic resistance in mycobacteria involves the selection of mutant variants within a susceptible bacterial population. However, it is unclear whether antimycobacterial drugs act just as selective agents or can influence the rate of appearance of resistant mutants. The present study was initiated to address this issue by monitoring the effects of antimicrobial agents on the appearance and growth of clarithromycin (CLR)-resistant (CLR(r)) bacilli in broth cultures of Mycobacterium avium. Preexposure of M. avium to CLR had a significant dose effect on the emergence of resistance, with concentrations of 4 to 8 microg/ml resulting in a maximal (approximately 10(4)-fold) increase in the number of CLR(r) bacilli after a 4-day incubation. In addition, a dose effect was found with azithromycin. The use of combinations of CLR with either ethambutol (EMB) or rifabutin (RFB) resulted in fewer resistant bacilli compared to the use of CLR alone. The lowest active concentration of EMB (4 microg/ml) was equivalent to the EMB MIC (4 to 8 microg/ml) for the parental CLR(s) strain and the emergent CLR(r) variants, and thus, the antiresistance effect was probably the result of the bacteriostatic effect of EMB on CLR(r) bacilli. However, RFB was an order of magnitude more active (0.05 microg/ml) at reducing resistance than suggested by the MIC of this agent (0.5 to 1 microg/ml). These results indicate that the emergence of resistance was not simply the selection of a preexisting subpopulation of resistant bacilli. Further analysis suggested that early events in the emergence of resistance involved organisms (progenitors) that acquired a resistance phenotype. In addition, the progenitors appeared to be in a transient state, able to develop into a stable resistant lineage in the presence of CLR, or able to revert to the wild type in nonselective conditions.     

乙型肝炎病毒阿德福韦酯耐药株(RT A181T)感染性克隆的构建及在Huh7细胞中的表达

目的利用重叠延伸聚合酶链反应(SOE-PCR)快速构建乙型肝炎病毒(HBV)阿德福韦酯耐药株(RT A181T)感染性克隆,观察重组质粒在Huh7细胞中的表达,建立HBV阿德福韦酯耐药株(RT A181T)的体外研究细胞模型。方法设计保守引物,从慢性乙型肝炎阿德福韦酯耐药患者血清中扩增全长HBV基因组,利用SOE-PCR技术构建1.3倍基因组长度的感染性克隆质粒pHBV1.3,转染肝癌细胞系Huh7,采Western Blot、Real-time PCR检测感染性克隆复制以及表达情况,同时用抗病毒药物拉米夫定以及阿德福韦酯验证对感染性克隆复制及表达的抑制情况。结果成功构建了HBV阿德福韦酯耐药株感染性克隆质粒pHBV1.3(RT A181T),该质粒在Huh7细胞系中能有效复制、转录和表达。拉米夫定能有效抑制该感染性克隆的复制和表达,阿德福韦酯不能抑制该感染性克隆的复制和表达。结论构建的HBV阿德福韦酯耐药株感染性克隆质粒pHBV1.3(RT A181T)在体外能高效复制及表达蛋白,其转染细胞可用于HBV复制机制及抗病毒研究。     

AG205, a novel agent directed against FabK of Streptococcus pneumoniae

AG205 was identified from high-throughput screening as a potent inhibitor of FabK, the enoyl-ACP reductase in Streptococcus pneumoniae. Specific inhibition of lipid biosynthesis in a macromolecular biosynthesis assay and identification of an Ala141Ser substitution in FabK from spontaneous AG205-resistant mutants indicated that AG205 exerts antibacterial activity against S. pneumoniae through the specific inhibition of FabK.     

耐多药结核分枝杆菌中embB基因突变与乙胺丁醇耐药的相关性研究

目的 研究耐多药结核分枝菌中embB基因突变与乙胺丁醇耐药的相关性. 方法 比例法检测84株耐多药结核分枝杆菌的乙胺丁醇(EMB)耐药性,基因测序检测embB基因的突变,2检验分析二者之间的相关性. 结果 84株耐多药结核分枝杆菌中有43株(51.2%)对EMB耐药,41株(48.8%)对EMB敏感,57株耐多药菌株(67.9%)的embB基因发生突变.在43株EMB耐药菌株中,embB基因突变的菌株为40株(93.0%),而41株EMB敏感菌株中,embB基因突变的菌株为17株(41.5%),embB基因在耐药菌株中的突变频率远高于敏感菌株(2=25.58,P=0.00).embB306是最常见的突变位点,其在耐药菌株的突变率也高于敏感菌株(2=12.37,P=0.00),embB基因和embB306位点检测EMB耐药的敏感度、特异度和准确性分别为93.0%和65.1%,58.5%和73.2%,76.2%和69.0%. 结论 EMB耐药的产生与embB基因和embB306突变有关,二者用于检测EMB耐药有一定的参考意义.     

Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.     

Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 microg/ml to 7, 7, 8.5, and 14 microg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 microg/ml or 28 microg/ml, respectively, to 3 microg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.     

Effect of Ethambutol on the Viable Cell Count in Mycobacterium smegmatis

Soon after a strain of Mycobacterium smegmatis was exposed to ethambutol (EMB), the number of viable cells increased dramatically above the number in a drug-free control. This rapid rise did not occur when the culture was maintained at 4°C instead of 37°C, when an EMB-resistant mutant was used, when auxotrophs were exposed in medium lacking nutrients essential for growth, nor when the levo form of EMB was used. EMB caused no increase in deoxyribonucleic acid synthesis, nor in septum formation of dividing cells. Treated cells changed morphologically, resulting in a lower surface area-to-volume ratio. Whereas EMB did not eliminate cell clusters, the cluster size decreased markedly as detected by filtration and Coulter counter measurements. We concluded that EMB causes a reduced surface-to-volume ratio, leading to reduced cell cohesion and a consequent reduction in cluster size, reflected in an increase in colony-forming units.     

Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis

Ethambutol (EMB) is a central component of drug regimens used worldwide for the treatment of tuberculosis. To gain insight into the molecular genetic basis of EMB resistance, approximately 2 Mb of five chromosomal regions with 12 genes in 75 epidemiologically unassociated EMB-resistant and 33 EMB-susceptible Mycobacterium tuberculosis strains isolated from human patients were sequenced. Seventy-six percent of EMB-resistant organisms had an amino acid replacement or other molecular change not found in EMB-susceptible strains. Thirty-eight (51%) EMB-resistant isolates had a resistance-associated mutation in only 1 of the 12 genes sequenced. Nineteen EMB-resistant isolates had resistance-associated nucleotide changes that conferred amino acid replacements or upstream potential regulatory region mutations in two or more genes. Most isolates (68%) with resistance-associated mutations in a single gene had nucleotide changes in embB, a gene encoding an arabinosyltransferase involved in cell wall biosynthesis. The majority of these mutations resulted in amino acid replacements at position 306 or 406 of EmbB. Resistance-associated mutations were also identified in several genes recently shown to be upregulated in response to exposure of M. tuberculosis to EMB in vitro, including genes in the iniA operon. Approximately one-fourth of the organisms studied lacked mutations inferred to participate in EMB resistance, a result indicating that one or more genes that mediate resistance to this drug remain to be discovered. Taken together, the results indicate that there are multiple molecular pathways to the EMB resistance phenotype.     

Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis

This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa. Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs. As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria. This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin. Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain. Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients. In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs. These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P. aeruginosa.     

Cell wall and membrane changes associated with ethambutol resistance in Mycobacterium tuberculosis H37Ra.

Biochemical variations accompanying the acquisition of ethambutol (EMB) resistance in a single-step mutant of Mycobacterium tuberculosis H37Ra were analyzed. Comparative analysis of phospholipids revealed a reduced content in the EMB-resistant strain, particularly in the cell membrane fraction. Significant alterations were observed in the individual phospholipid content and phospholipid fatty acyl group composition of whole cells and subcellular fractions. Quantitative changes were seen in the chemical constituents of the cell walls of resistant cultures in comparison with those of EMB-susceptible cultures of M. tuberculosis. Alterations in the binding of 1-anilinonaphthalene-8-sulfonate to whole cells of an EMB-resistant strain indicated structural changes on the cell surface. Structural changes in the cell wall may play an important role in the resistance of M. tuberculosis H37Ra to EMB.     

Posaconazole is a potent inhibitor of sterol 14alpha-demethylation in yeasts and molds

Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14alpha-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14alpha-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14alpha-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.     

Unraveling the mode of action of the antimalarial choline analog G25 in Plasmodium falciparum and Saccharomyces cerevisiae

Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor of Plasmodium falciparum growth in vitro and in vivo. Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known. Here we show that, similar to its effects on P. falciparum, G25 prevents choline entry into Saccharomyces cerevisiae cells and inhibits S. cerevisiae growth. However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1. An hnm1Delta yeast mutant, which lacks the only choline transporter gene HNM1, was not altered in the transport of a labeled analog of this compound. Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25. Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding gene PSD1 (but not PSD2) were found to be highly resistant to this compound. Based on these data for S. cerevisiae, labeling studies in P. falciparum were performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Labeling studies in P. falciparum and in vitro studies with recombinant P. falciparum phosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine. Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity.     

Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol

In performing radiometric susceptibility testing on over 2,000 patient isolates of Mycobacterium tuberculosis during the past 6 years, we found that resistance to 7.5 microg/ml ethambutol (EMB) occurred only in isolates that are also resistant to 0.4 microg/ml isoniazid (INH). Using 157 selected isolates in the present study, we performed radiometric and agar proportion susceptibility tests and DNA sequencing of genetic regions associated with resistance to these two drugs. The goal was to study the occurrence of the common mutations associated with resistance to each drug and also to determine whether any particular INH-resistance-associated mutation occurred more often in combination with any particular EMB-resistance-associated mutation. In an analysis of 128 isolates resistant to 0.4 microg/ml INH, we found that a mutation at katG Ser315 was more common in isolates also resistant to 7.5 microg/ml EMB (61 of 67=91.0%) than in isolates either susceptible to EMB or resistant to 2.5 microg/ml EMB (39 of 60=65.0%). These observations suggest that INH-resistant strains with a mutation at katG Ser315 are more likely to acquire resistance to 7.5 microg/ml EMB than are isolates with INH-resistance-associated mutations at other sites. In addition, we found that 64 of 67 (95.5%) isolates resistant to 7.5 microg/ml EMB contained a mutation in either codon 306 or codon 406 of embB. Met306Val was the most common embB mutation, present in 52 (77.6%) of the 67 isolates. Most occurrences of this mutation (49 of 52=94.2%) were found in isolates that also contained the katG Ser315Thr mutation. Finally, sequencing this region of embB appears to be sufficiently sensitive for use as a rapid screening tool for detection of high-level resistance to EMB.     

Relationship between the uptake of isoniazid and its action on in vivo mycolic acid synthesis in Mycobacterium tuberculosis

A direct relationship was established between the rate of uptake of isoniazid and the action of this drug on in vivo mycolic acid synthesis in Mycobacterium tuberculosis H37Ra. The rate of uptake of isoniazid increased linearly with its external concentration and appeared to reach a maximal value of 52 pmoles per hr per 10(9) cells at an external concentration of about 13 mum. Correspondingly, the rate of inhibition of mycolic acid synthesis increased with the rise in the rate of uptake of the drug. A 50% inhibition of mycolic acid synthesis occurred when the uptake of isoniazid reached 5.2 pmoles per 10(9) cells. Calculations showed that this level of drug uptake represents an internal cellular concentration of 9 mum. These results show clearly that the action of isoniazid on the mycolate synthetase system of M. tuberculosis is rapid and that this enzyme system is highly sensitive to the drug.     

5-Fluorocytosine antagonizes the action of sterol biosynthesis inhibitors in Candida glabrata.

The concentration-dependent antagonistic interaction between 5-fluorocytosine and a sterol biosynthesis inhibitor (SBI) was studied using intact cells and cell-free extracts of Candida glabrata. 5-Fluorocytosine promoted incorporation of radioactivity into 4-desmethylsterols (P < 0.01), and enhanced the relative and absolute increases of ergosterol (P < 0.05) in C. glabrata incubated aerobically with an SBI (miconazole or amorolfine). Further aerobic incubation of C. glabrata with combinations of a nucleic acid or protein synthesis inhibitor (rifampicin or chlortetracycline) and an SBI (miconazole) promoted a similar increase in ergosterol biosynthesis. In contrast, 5-fluorocytosine reduced the incorporation of radioactivity into 4,4-dimethylsterols (P < 0.01), but had no obvious effect on the absolute ergosterol level in C. glabrata incubated statically with miconazole. In cell-free extracts of cultures previously incubated with 5-fluorocytosine, ergosterol synthesis was less sensitive to the action of miconazole. Antagonism between 5-fluorocytosine and the SBI is thus mediated by a reversal of inhibition of intracellular ergosterol synthesis. The possible mechanisms underlying antagonism between 5-fluorocytosine and SBIs that inhibit different sites of the sterol biosynthesis pathway, as well as its clinical relevance to combination therapy, are discussed.