Evolutionary origin of aminoglycoside phosphotransferase resistance genes

Summary The protein sequences of seven 3-aminoglycoside phosphotransferases falling into the six identified types and three 6-aminoglycoside phosphotransferases were analyzed to give a rooted phylogenetic tree. This tree supports the origin of these groups of enzymes in an ancestor closely related to the actinomycetes, and that horizontal transfer of the resistance genes occurred, possibly via transposons. The implications for genetic engineering of a novel antibiotic are discussed.     

The prevalence of gentamicin 2'-N-acetyltransferase in the Proteeae and its role in the O-acetylation of peptidoglycan

Abstract The prevalence of aac(2')-Ia , a gene coding for gentamicin 2'-JV-acetyltransferase in Providenda stuartii , among species of the Proteeae was investigated to determine if it is a common resistance factor and whether the correlation observed in P. stuartii between its expression and the levels of peptidoglycan O -acetylation represents a general feature of bacteria producing this form of modified peptidoglycan. An evaluation of the MICs of gentamicin for each of the species of the Proteeae did not reveal any apparent relationship between resistance and the degree of O-acetylation of peptidoglycan. The entire aac(2')-Ia gene was used as a probe in Southern hybridization experiments against genomic DNA from each species of the Proteeae. A sequence with strong homology to aac(2')-Ia was present only in Proteus penneri while weak hybridization was also observed to the restriction digested DNA from Providenda rettgeri . Other bacteria that O -acetylate peptidoglycan were also screened with this probe and a homologous DNA sequence was only found in Neisseria subflava . These data suggest that AAC(2')-Ia may contribute to the rO -acetylation of peptidoglycan in P. stuartii , but a more specific enzyme must also be produced for this function.     

顺铂和噪声联合作用对听觉器官的影响

研究了顺铂与噪声联合作用对耳蜗的毒性,发现联合作用后听阈偏移大于单独顺铂作用后阈移和单独噪声作用后阈移的总和。此外,联合作用后听觉脑干诱发电位振幅下降非常明显。结果表明,顺铂与噪声联合作用对内耳的毒性有协同作用。     

大肠埃希菌和肺炎克雷伯菌对三种氨基糖苷类抗生素的耐药性分析

【摘 要】 目的 评价庆大霉素、妥布霉素及阿米卡星三种氨基糖苷类抗生素（AGs）对大肠埃希菌和肺炎克雷伯菌的体外抗菌活性。方法 对902株大肠埃希菌和404株肺炎克雷伯菌,采用VITEK-2全自动微生物分析仪配套的AST-GN13药敏卡进行庆大霉素、妥布霉素及阿米卡星的体外药敏试验。结果 大肠埃希菌和肺炎克雷伯菌的产超广谱β-内酰胺酶（ESBLs）菌株检出率分别为40.8％和36.6％;产ESBLs的大肠埃希菌对庆大霉素、妥布霉素及阿米卡星的敏感率分别为42.4%、39.1%和96.5%,与非产ESBLs菌株比较,敏感率差异均有统计学意义（P<0.01）;产ESBLs的肺炎克雷伯菌对庆大霉素、妥布霉素及阿米卡星的敏感率分别为64.2%、62.8%和91.9%,与非产ESBLs菌株比较,敏感率差异均有统计学意义（P<0.01）;阿米卡星对产与非产ESBLs的大肠埃希菌和肺炎克雷伯菌均高度敏感,敏感率均在91%以上。结论 本地区大肠埃希菌和肺炎克雷伯菌产ESBLs菌株流行严重,ESBLs的产生可使大肠埃希菌和肺炎克雷伯菌对AGs的耐药情况加重,提示ESBLs和AGs引起的耐药可能存在一定的相关性。     

Aminoglycoside antibiotics reduce glucose reabsorption in kidney through down-regulation of SGLT1

Nephrotoxicity is known to be a major clinical side effect of aminoglycoside antibiotics. Aminoglycosides cause damage to proximal tubular cells in kidney, however the mechanism of toxicity is still unclear. In order to elucidate the mechanism of nephrotoxicity, we studied the effect of aminoglycoside antibiotics on glucose transport systems in vitro and in vivo. As a result, we found that the aminoglycosides significantly reduced Na(+)/glucose cotransporter (SGLT1)-dependent glucose transport and also down-regulated mRNA and protein levels of the SGLT1 in pig proximal tubular LLC-PK(1) cells. To obtain evidence about SGLT1 down-regulation in vivo, we studied the mRNA expression of SGLT1 using gentamicin C-treated murine kidney and found that gentamicin C down-regulated SGLT1 in vivo as well as in vitro. Furthermore, the gentamicin C-treated mice showed significant rise in urinary glucose excretion. These results indicate that one of the mechanisms of aminoglycoside nephrotoxicity is the down-regulation of SGLT1, which causes reduction in glucose reabsorption in kidney.     

Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl₂ for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl₂. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.     

Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and non-syndromic hearing loss

We report here the clinical, genetic, and molecular characterization of two Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects. Penetrances of hearing loss in BJ105 and BJ106 pedigrees are 67% and 33%, respectively. In particular, three of 10 affected matrilineal relatives of BJ105 pedigree had aminoglycoside-induced hearing loss, while seven affected matrilineal relatives in BJ105 pedigree and six affected matrilineal relatives in BJ106 pedigree did not have a history of exposure to aminoglycosides. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mtDNA variants belonging to haplogroups F3 and M7b. These variants showed no evolutionary conservation, implying that mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycosides and nuclear backgrounds appear to be major modifier factors for the phenotypic manifestation of the A1555G mutation in these Chinese families.     

A Novel, Drug-based, Cellular Assay for the Activity of Neurotoxic Mutants of the Prion Protein

In prion diseases, the infectious isoform of the prion protein (PrP^Sc) may subvert a normal, physiological activity of the cellular isoform (PrP^C). A deletion mutant of the prion protein (Δ105–125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP^C and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Δ105–125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Δ105–125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Δ105–125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.     

Extremely low penetrance of deafness associated with the mitochondrial 12S rRNA mutation in 16 Chinese families: implication for early detection and prevention of deafness

Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic, and molecular characterization of 16 Chinese pedigrees (a total of 246 matrilineal relatives) with aminoglycoside-induced impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects, although these subjects share some common features: being bilateral and sensorineural hearing impairment. Strikingly, these Chinese pedigrees exhibited extremely low penetrance of hearing loss, ranging from 4% to 18%, with an average of 8%. In particular, nineteen of 246 matrilineal relatives in these pedigrees had aminoglycoside-induced hearing loss. Mutational analysis of the mtDNA in these pedigrees showed the presence of homoplasmic 12S rRNA A1555G mutation, which has been associated with hearing impairment in many families worldwide. The extremely low penetrance of hearing loss in these Chinese families carrying the A1555G mutation strongly supports the notion that the A1555G mutation itself is not sufficient to produce the clinical phenotype. Children carrying the A1555G mutation are susceptible to the exposure of aminoglycosides, thereby inducing or worsening hearing impairment, as in the case of these Chinese families. Using those genetic and molecular approaches, we are able to diagnose whether children carry the ototoxic mtDNA mutation. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside therapy, and eventually to decrease the incidence of deafness.     

Aminoglycoside 2'-phosphotransferase IIIa (APH(2')-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2' kinases

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2'-phosphotransferase IIIa (APH(2')) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2')-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.