Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

Defining RNA motif–aminoglycoside interactions via two-dimensional combinatorial screening and structure–activity relationships through sequencing

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3 × 3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure–activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif–aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site.     

Aminoglycoside-RNA interactions

The structural and physico-chemical parameters promoting the binding of aminoglycosides to RNAs are becoming clear. The strength of the interaction is dominated by electrostatics, with the positively charged aminoglycosides displacing metal ions. Although aminoglycosides inhibit most known ribozymes, aminoglycosides or polyamines are able to catalyze specific RNA cleavage in the absence of metal ions.     

Mitochondrial 12S rRNA A827G mutation is involved in the genetic susceptibility to aminoglycoside ototoxicity

We have analyzed the clinical and molecular characterization of a Chinese family with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluations revealed that only those family members who had a history of exposure to aminoglycoside antibiotics subsequently developed hearing loss, suggesting mitochondrial genome involvement. Sequence analysis of the mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, a mutation that was identified previously in a few sporadic patients and in another Chinese family with non-syndromic deafness. The pathogenicity of the A827G mutation is strongly supported by the occurrence of the same mutation in two independent families and several genetically unrelated subjects. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly conserved in mammals. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. However, incomplete penetrance of hearing impairment indicates that the A827G mutation itself is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Indeed, aminoglycosides may contribute to the phenotypic manifestation of the A827G mutation in this family. In contrast with the congenital or early-onset hearing impairment in another Chinese family carrying the A827G mutation, three patients in this pedigree developed hearing loss only after use of aminoglycosides. This discrepancy likely reflects the difference of genetic backgrounds, either mitochondrial haplotypes or nuclear modifier genes, between two families.     

Sensitive high-performance liquid chromatographic assay for aminoglycosides in biological matrices enables the direct estimation of bacterial drug uptake

Following the development of a sensitive high-performance liquid chromatographic (HPLC) assay for gentamicin in biological matrices, the utility of this assay for the determination of other clinically important aminoglycosides (neomycin, netilmicin and sisomicin) in bacterial culture media or plasma is demonstrated. The high sensitivity of the assay enables direct measurement of the aminoglycoside content of bacterial cells cultured in the presence of unlabelled drug.     

Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment

Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.     

细菌对氨基糖苷类抗生素产生抗性的机理及其控制策略

氨基糖苷类抗生素在治疗感染性疾病尤其是革兰氏阴性菌引起的严重感染方面起着重要作用 ,但是耐药菌株的出现较大地限制了此类抗生素的发展 ,因此 ,如何控制耐药性已经成为一项迫切需要解决的任务。细菌对氨基糖苷类抗生素产生抗性的机制很多 ,目前普遍接受的主要有三种 :1. 通过减少对氨基糖苷类抗生素的摄取或减少药物在体内的累积而产生抗性。 2. 通过改变核糖体结合位点而产生抗性。 3. 通过表达氨基糖苷类抗生素修饰酶而产生抗性。目前细菌耐药性的控制主要集中在对原有氨基糖苷类抗生素进行改造或合成新的抗生素 ,开发氨基糖苷类抗生素修饰酶抑制剂。     

Inhibition of Hepatitis Delta Virus Genomic Ribozyme Self-Cleavage by Aminoglycosides

Subgenomic regions of hepatitis delta virus (HDV) RNA contains ribozyme whose activities are important to viral life cycles and depend on a unique pseudoknot structure. To explore the characters of HDV ribozyme, antibiotics of the aminoglycoside, which has been shown inhibiting self-splicing of group I intron and useful in elucidating its structure, were tested for their effect on HDV genomic ribozyme. Aminoglycosides, including tobramycin, netromycin, neomycin and gentamicin effectively inhibited HDV genomic ribozyme self-cleavage in vitro at a concentration comparable to that inhibiting group I intron self-splicing. The extent of inhibition depended upon the concentration of magnesium ion. Chemical modification mapping of HDV ribozyme RNA indicated that the susceptibility of nucleotide 703 to the modifying agent was enhanced in the presence of tobramycin, suggesting a conformational shift of HDV ribozyme, probably due to an interaction with the aminoglycoside. Finally, we examined the effect of aminoglycoside on HDV cleavage and replication in cell lines, however, none of the aminoglycoside effective in vitro exerted suppressive effects in vivo. Our results represented as an initial effort in utilizing aminoglycoside to probe the structure of HDV ribozyme and to compare its reaction mechanism with those of other related ribozymes.     

多重耐药黏液型铜绿假单胞菌 氨基糖苷类修饰酶基因检测

摘要：目的 研究多重耐药黏液型铜绿假单胞菌（MDR-mPA）氨基糖苷类修饰酶基因的分布,为合理应用抗生素提供依据。方法 采用纸片扩散（K-B）法对临床分离的MDR-mPA进行药敏试验,用聚合酶链反应（PCR）法检测氨基糖苷类修饰酶。结果 61株MDR-mPA中共有23株检出氨基糖苷类修饰酶,其中aac(3)-Ⅱ阳性12株（48%）,aac(6′)-Ⅱ阳性9株（36%）,aac(6′)-Ⅰ阳性3株（12%）,ant(2″)-Ⅰ阳性1株（4%）。结论 黏液型铜绿假单胞菌对氨基糖苷类抗菌药物的耐药与氨基糖苷类修饰酶基因表达有关。     

A-site model RNAs

Oligonucleotide model systems have played a key role for the exploration of structure, dynamics and ligand binding of the ribosomal decoding-site (A site). An overview is given on the merits and limitations of various A-site models that were used for solution and crystallographic work.