Experimental and clinical study test of capreomycin]

Data on the experimental and clinical study of capreomycin in the treatment of tuberculosis are presented. It was shown that capreomycin had low activity with respect to the sensitive strain of Mycobacterium tuberculosis H37 Rv in vitro and the respective infection caused by it in mice. The activity of capreomycin in vitro with respect to streptomycin resistant strains was the same as that with respect to the sensitive strains, while in vivo it increased 3 times. Capreomycin showed a tendency to decreasing its activity with respect to strains highely resistant to canamycin only in vitro. The effect of capreomycin on tuberculosis infection caused by strains resistant to different concentrations of canamycin was the same as that on tuberculosis infection caused by sensitive strains. Cross resistance between florimycin (viomycin) and capreomycin was shown. Clinical trails of capreomycin revealed its moderate therapeutic efficiency, relatively low toxicity and an allergenizing effect on the host. Transient ventibulopatia without pronounced signs of ototoxic action was observed. The nephrotoxic effect was moderate and transient. It was observed predominantly at the peak of the allergic reactions to the antitubercle drugs. The data obtained during the study allow recommendation of capreomycin use in clinics as reserve drug when the causative agent is not resistant to florimycin. The drug should be used under regular control of the blood picture, electrolyte metabolism, state of the kidneys, auditory and vestibular apparatus.     

Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2'-O-methylations in 16S and 23S rRNAs

The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of capreomycin and viomycin at the ribosome subunit interface.     

High Frequency of Resistance,Lack of Clinical Benefit,and Poor Outcomes in Capreomycin Treated South African Patients with Extensively Drug-Resistant Tuberculosis

Background

There are limited data about the epidemiology and treatment-related outcomes associated with capreomycin resistance in patients with XDR-TB. Capreomycin achieves high serum concentrations relative to MIC but whether capreomycin has therapeutic benefit despite microbiological resistance remains unclear.

Methods

We reviewed the susceptibility profiles and outcomes associated with capreomycin usage in patients diagnosed with XDR-TB between August 2002 and October 2012 in two provinces of South Africa. Patients whose isolates were genotypically tested for capreomycin resistance were included in the analysis.

Results

Of 178 XDR-TB patients 41% were HIV-infected. 87% (154/178) isolates contained a capreomycin resistance-conferring mutation [80% (143/178) rrs A1401G and 6% (11/178) were heteroresistant (containing both the rrs A1401G mutation and wild-type sequences)]. Previous MDR-TB treatment, prior usage of kanamycin, or strain type was not associated with capreomycin resistance. 92% (163/178) of XDR-TB patients were empirically treated with capreomycin. Capreomycin resistance decreased the odds of sputum culture conversion. In capreomycin sensitive and resistant persons combined weight at diagnosis was the only independent predictor for survival (p=<0.001). By contrast, HIV status and use of co-amoxicillin/clavulanic acid were independent predictors of mortality (p=<0.05). Capreomycin usage was not associated with survival or culture conversion when the analysis was restricted to those whose isolates were resistant to capreomycin.

Conclusion

In South Africa the frequency of capreomycin conferring mutations was extremely high in XDR-TB isolates. In those with capreomycin resistance there appeared to be no therapeutic benefit of using capreomycin. These data inform susceptibility testing and the design of treatment regimens for XDR-TB in TB endemic settings.     

Cross-resistant relationships among the aminoglucoside antibiotics in Mycobacterium tuberculosis.

Phenotypes of isolates of Mycobacterium tuberculosis H37RV showing resistance to the aminoglucoside antibiotics streptomycin, viomycin, kanamycin, capreomycin, tuberactinomycin N, lividomycin and paromomycin could be grouped into the following types: (I) resistant only to different levels of streptomycins; (2) resistant only to a low level of kanamycin; (3) triply resistant, to low levels of viomycin, tuberactinomycin N and capreomycin; (4) triply resistant, to a low level of kanamycin and high levels of lividomycin and paromomycin; (5) quadruply resistant, to a low level of capreomycin and high levels of kanamycin, lividomycin and paromomycin; (6) hextuply resistant, to high levels of viomycin, tuberactinomycin N, capreomycin, kanamycin, lividomycin, and paromomycin. Three modificatied types of the latter were also observed. Appearance rates of the six types were estimated as 10(-6) to 10(-9), 10(-6), 10(-6) to 10(-7), 10(-8), 10(-8), and 10(-8) to 10(-9), respectively, in a total viable population of the parent strain. Mutations to all phenotypes were considered to be produced by single mutations. According to cross-resistance relationships, aminoglucoside antibiotics were classified into three groups: (I) streptomycin; (II) viomycin, tuberactinomycin N and capreomycin; (III) kanamycin, lividomycin and paromomycin. No cross-resistance relationship between streptomycin and other antibiotics was observed. Resistances to viomycin, tuberactinomycin N and capreomycin occurred by single mutation to type 3. Resistances to kanamycin, lividomycin and paromomycin occurred by single mutations to types 4 and 5. Low resistance to capreomycin was produced by mutation to type 5. Therefore capreomycin was considered to be an intermediate between the second and third groups. These two groups had a close relationship, as resistance to all six agents in these groups could be produced by a single mutation to type 6 (and its modified types).     

The copper (II) ion as a carrier for the antibiotic capreomycin against Mycobacterium tuberculosis

In recent years, the bacterium responsible for tuberculosis has been increasing its resistance to antibiotics resulting in new multidrug resistant Mycobacterium tuberculosis (MR-TB) and extensively drug-resistant tuberculosis (XDR-TB). In this study we use several analytical techniques including NMR, FT-ICR, TOF-MS, LC–MS and UV/Vis to study the copper–capreomycin complex. The copper (II) cation is used as a carrier for the antibiotic capreomycin. Once this structure was studied using NMR, FT-ICR, and MALDI-TOF-MS, the NIH-NIAID tuberculosis cell line for several Tb strains (including antibiotic resistant strains) were tested against up to seven variations of the copper–capreomycin complex. Different variations of copper improved the efficacy of capreomycin against Tb up to 250 fold against drug resistant strains of Tb.     

Isolation of somatic cell mutants defective in the biosynthesis of phosphatidylethanolamine

An in situ autoradiographic assay for CDP-ethanolamine:1,2-sn-diacylglycerol ethanolamine phosphotransferase (EC 2.7.8.1) activity in Chinese hamster ovary cells was developed and used to screen approximately 10,000 individual mutagen-treated colonies attached to filter paper (Esko, J. D., and Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1190-1193). A variant (strain 40.11) was isolated in which the ethanolamine phosphotransferase specific activity in vitro was 6-10-fold less than in the parent, but the level of CDP-choline:1,2-sn-diacylglycerol choline phosphotransferase (EC 2.7.8.2) activity was normal. In extracts, the mutant was also defective in the synthesis of ethanolamine plasmalogen. In vivo, the short term kinetics of labeling with [32P]phosphate or [14C]ethanolamine was correspondingly altered. However, the long tem growth rate and steady state phospholipid compositions of the mutant and parent were quite similar. These results show that the ethanolamine and choline phosphotransferases of Chinese hamster ovary cells are distinct as judged by genetic criteria, while the biosynthesis of phosphatidylethanolamine and its plasmalogen share common enzymatic component(s).     

Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium.

Inhibition of cellular adenylate cyclase activity by sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system was reliant on the activities of the protein components of this enzyme system and on a gene designated crrA. In bacterial strains containing very low enzyme I activity, inhibition could be elicited by nanomolar concentrations of sugar. An antagonistic effect between methyl alpha-glucoside and phosphoenolpyruvate was observed in permeabilized Escherichia coli cells containing normal activities of the phosphotransferase system enzymes. In contrast, phosphoenolpyruvate could not overcome the inhibitory effect of this sugar in strains deficient for enzyme I or HPr. Although the in vivo sensitivity of adenylate cyclase to inhibition correlated with sensitivity of carbohydrate permease function to inhibition in most strains studied, a few mutant strains were isolated in which sensitivity of carbohydrate uptake to inhibition was lost and sensitivity of adenylate cyclase to regulation was retained. These results are consistent with the conclusions that adenylate cyclase and the carbohydrate permeases were regulated by a common mechanism involving phosphorylation of a cellular constituent by the phosphotransferase system, but that bacterial cells possess mechanisms for selectively uncoupling carbohydrate transport from regulation.     

Capreomycin susceptibility is increased by TlyA-directed 2'-O-methylation on both ribosomal subunits

The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2'-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers resistance to capreomycin and viomycin. We report here that TlyA orthologues occur in diverse bacteria and fall into two distinct groups. One group, now termed TlyA(I) , has shorter N- and C-termini and methylates only C1920; the second group (now TlyA(II) ) includes the mycobacterial enzyme, and these longer orthologues methylate at both C1409 and C1920. Ribosomal subunits are the preferred substrates for both groups of orthologues. Amino acid substitutions at the N-terminus of TlyA(II) reduce its ability to methylate these substrates. Growing pairs of recombinant TlyA(II) Escherichia coli strains in competition shows that even subtle changes in the level of rRNA methylation lead to significant differences in susceptibility to sub-inhibitory concentrations of capreomycin. The findings reveal that 2'-O-methyls at both C1409 and C1920 play a role in facilitating the inhibitory effects of capreomycin and viomycin on the bacterial ribosome.     

Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA.

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.     

Insulin action on Escherichia coli. Regulation of the adenylate cyclase and phosphotransferase enzymes.

Insulin on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotransferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, the rate of alpha-methylglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. In vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin on E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of alpha-methylglucoside.     

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.     

氧化还原对NDPK-A及突变体磷酸转移酶活性的影响

对核苷二磷酸激酶A(NDPK-A)及其4种半胱氨酸突变体进行诱导表达及纯化,测定它们在氧化还原条件及正常条件下的磷酸转移酶活性,研究氧化还原及二硫键异构对NDPK-A及突变体活性的影响。将实验室之前构建成功的野生型NDPK-A(PBV-NDPK-A)及4种突变型NDPK-A基因(PBV-NDPK-A C4S,PBV-NDPK-A C109S,PBV-NDPK-A C145S,PBV-NDPK-A C4/109/145S)在大肠杆菌中高效表达;以DEAE-sepharose Fast Flow离子交换层析与Cibacron Blue 3GA Sepharose CL-4B亲和层析技术纯化目的蛋白;HPLC法测定比较野生型NDPK-A及突变体在氧化还原和正常环境下磷酸转移酶活性。结果显示,NDPK-A及突变体在大肠杆菌中高效表达;经纯化分别获得了均一的NDPK-A蛋白及突变体蛋白,纯度均达到98%;在还原环境下NDPK-A及突变体的磷酸转移酶活性均高于正常环境下的活性,但是在氧化环境下的磷酸转移酶活性明显低于正常环境下。氧化还原环境对NDPK-A结构异构及磷酸转移酶活性有一定的影响,提示氧化还原环境可能调控NDPK-A二硫键的形成,影响蛋白的聚集状态,从而影响蛋白的磷酸转移酶活性,并且NDPK-A结构中可能有更为复杂的氧化还原调控酶活性机制。     

Pyrophosphate: fructose 6-phosphate phosphotransferase in germinating castor bean seedlings

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP_i:fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP_i:fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.     

The effect of dietary carbohydrate and fat on the activities of some enzymes responsible for glycerolipid synthesis in rat liver.

1. Male rats were fed for 14 days on diets containing (by wt.) 53% of starch, or on diets in which 20% of the starch was replaced by sucrose, corn oil or lard. 2. The hepatic activities of the microsomal glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase and choline phosphotransferase, and of the soluble phosphatidate phosphohydrolase, were measured. 3. The soluble phosphatidate phosphohydrolase activity was higher in those rats fed on lard than in those fed on the starch diet. Choline phosphotransferase activity was higher in the rats fed on corn oil than in those fed on the starch diet. 4. The rate of hepatic glycerolipid synthesis was measured in vivo 1 min after injection of [1,3-3H]glycerol and [1-14C]palmitate into the portal veins. 5. The relative rate of phosphatidylcholine synthesis in vivo was increased after feeding with corn oil and the higher specific activity of choline phosphotransferase may contribute to this result. The equivalent rate of triacylglycerol synthesis was increased by feeding with lard rather than corn oil, and the increased activity of phosphatidate phosphohydrolase may partly explain this. The latter changes probably contribute to the increased concentration of triacylglycerol which other authors have observed in the livers and sera of animals fed on saturated and monounsaturated fats.     

Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis

Glycerol and glycerol 3-phosphate uptake in Bacillus subtilis does not involve the phosphotransferase system. In spite of this, B. subtilis mutants defective in the general components of the phosphotransferase system, EnzymeI or Hpr, are unable to grow with glycerol as sole carbon and energy source. Here we show that a Hpr mutant can grow on glycerol 3-phosphate and that glycerol 3-phosphate, but not glycerol, can induce glpD encoding glycerol-3-phosphate dehydrogenase. Induction of glpD also requires the glpP gene product which is a regulator of all known glp genes. Thus the phosphotransferase system general components do not interfere with the overall regulation of the glp regulon. Revertants of a Hpr mutant which can grown on glycerol carry mutations closely linked to the glp region at 75 degrees on the B. subtilis chromosomal map. This region contains the glpP, the glpFK and the glpD operons. The glpFK operon encodes the glycerol uptake facilitator (glpF) and glycerol kinase (glpK). The present results demonstrate that one of these genes, or their gene products, is the target for phosphotransferase system control of glycerol utilisation. Furthermore we conclude that utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in B. subtilis.     

Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization.

Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.     

Identification of catalytic residues in the beta-glucoside permease of Escherichia coli by site-specific mutagenesis and demonstration of interdomain cross-reactivity between the beta-glucoside and glucose systems

beta-Glucoside Enzyme II (IIBgl) of the Escherichia coli phosphotransferase system transports and phosphorylates beta-glucosides, whereas the glucose Enzyme II-III pair (IIGlc-IIIGlc) transports and phosphorylates glucose as well as certain aliphatic alpha- and beta-glucosides. Comparisons of their respective amino acid sequences previously revealed that both systems are homologous and must be evolutionarily related. To gain more insight into the details of the transport mechanism, we made use of the observed homologies among phosphotransferase system permeases to design a suitable set of site-specific mutants within the gene encoding IIBgl. This set was used to study in vivo fermentation and to analyze in vitro P-enolpyruvate-dependent sugar phosphorylation as well as sugar phosphate-dependent sugar transphosphorylation. The following results were obtained. (i) IIBgl transports and phosphorylates glucose as well as aryl- and alkyl-beta-glucosides; (ii) histidyl 547 is essential for the phosphorylation of IIBgl by the histidine-containing phosphoryl carrier protein of the phosphotransferase system (HPr) (first phosphorylation site); (iii) both cysteyl 24 and histidyl 306 are essential for the transfer of the phosphoryl group to the sugar; (iv) replacement of Cys-24 by serine leads to uncoupling of sugar transport from phosphorylation; and (v) histidyl 183 is important for substrate specificity. Our studies also revealed heterologous phosphoryl transfer between the beta-glucoside and glucose permease components which probably occurs as follows: 1) HPr-P----IIBgl (His-547)----IIGlc----alkyl-alpha- or -beta-glucosides or glucose (but not aryl-beta-glucosides) and 2) HPr-P----IIIGlc----IIBgl (Cys-24 or His-306)----alkyl- or aryl-beta-glucosides or glucose (but not methyl-alpha-glucoside). In addition to the essential residues noted above, several residues in IIBgl were identified which when mutated reduced the in vitro catalytic efficiency of the enzyme more than 10-fold. Thus, aspartyl 551 and arginyl 625 appeared to function together with histidyl 547 in phosphoryl transfer involving the first phosphorylation site in the permease, whereas histidyl 183 appeared to function together with cysteyl 24 and histidyl 306 in phosphoryl transfer involving the second phosphorylation site in the permease.     

Biochemical basis of resistance to hygromycin B in Streptomyces hygroscopicus--the producing organism

Hygromycin B, an aminocyclitol antibiotic that strongly inhibits both 70S and 80S ribosomes, is synthesized by Streptomyces hygroscopicus. Ribosomes from this Gram-positive mycelial bacterium are inhibited in vitro by the antibiotic. In contrast, the streptomycete is highly resistant to the drug in vivo since it possesses hygromycin B phosphotransferase activity. This enzyme has been shown by gel filtration to have a molecular weight of 42000, and to modify its antibiotic substrate to produce 7"-O-phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in vitro.     

Galactose transport systems in Streptococcus lactis

Galactose-grown cells of Streptococcus lactis ML3 have the capacity to transport the growth sugar by two separate systems: (i) the phosphoenolpyruvate-dependent phosphotransferase system and (ii) an adenosine 5'-triphosphate-energized permease system. Proton-conducting uncouplers (tetrachlorosalicylanilide and carbonyl cyanide-m-chlorophenyl hydrazone) inhibited galactose uptake by the permease system, but had no effect on phosphotransferase activity. Inhibition and efflux experiments conducted using beta-galactoside analogs showed that the galactose permease had a high affinity for galactose, methyl-beta-D-thiogalactopyranoside, and methyl-beta-D-galactopyranoside, but possessed little or no affinity for glucose and lactose. The spatial configurations of hydroxyl groups at C-2, C-4, and C-6 were structurally important in facilitating interaction between the carrier and the sugar analog. Iodoacetate had no inhibitory effect on accumulation of galactose, methyl-beta-D-thiogalactopyranoside, or lactose via the phosphotransferase system. However, after exposure of the cells to p-chloromercuribenzoate, phosphoenolpyruvate-dependent uptake of lactose and methyl-beta-D-thiogalactopyranoside were reduced by 75 and 100%, respectively, whereas galactose phosphotransferase activity remained unchanged. The independent kinetic analysis of each transport system was achieved by the selective generation of the appropriate energy source (adenosine 5'-triphosphate or phosphoenolpyruvate) in vivo. The maximum rates of galactose transport by the two systems were similar, but the permease system exhibited a 10-fold greater affinity for sugar than did the phosphotransferase system.