Genetic Reconstruction of Protozoan rRNA Decoding Sites Provides a Rationale for Paromomycin Activity against Leishmania and Trypanosoma

Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics

Understanding the Origins of Bacterial Resistance to Aminoglycosides through Molecular Dynamics Mutational Study of the Ribosomal A-Site

Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations

Gentamicin Rapidly Inhibits Mitochondrial Metabolism in High-Frequency Cochlear Outer Hair Cells

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies

A rapid method for detection of five known mutations associated with aminoglycoside-induced deafness

<p>Abstract</p> <p>Background</p> <p>South Africa has one of the highest incidences of multidrug-resistant tuberculosis (MDR-TB) in the world. Concomitantly, aminoglycosides are commonly used in this country as a treatment against MDR-TB. To date, at least five mutations are known to confer susceptibility to aminoglycoside-induced hearing loss. The aim of the present study was to develop a rapid screening method to determine whether these mutations are present in the South African population.</p> <p>Methods</p> <p>A multiplex method using the SNaPshot technique was used to screen for five mutations in the <it>MT-RNR1 </it>gene: A1555G, C1494T, T1095C, 961delT+C(n) and A827G. A total of 204 South African control samples, comprising 98 Mixed ancestry and 106 Black individuals were screened for the presence of the five mutations.</p> <p>Results</p> <p>A robust, cost-effective method was developed that detected the presence of all five sequence variants simultaneously. In this pilot study, the A1555G mutation was identified at a frequency of 0.9% in the Black control samples. The 961delT+C(n) variant was present in 6.6% of the Black controls and 2% of the Mixed ancestry controls. The T1095C, C1494T and A827G variants were not identified in any of the study participants.</p> <p>Conclusion</p> <p>The frequency of 0.9% for the A1555G mutation in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. Future larger studies are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. The high frequencies of the 961delT+C(n) variant observed in the controls suggest that this change is a common non-pathogenic polymorphism. This genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is important in a low-resource country like South Africa where, despite their adverse side-effects, aminoglycosides will continue to be used routinely and are accompanied with very limited or no audiological monitoring.</p

Environmental and vegetation controls on the spatial variability of CH4 emission from wet-sedge and tussock tundra ecosystems in the Arctic

Aims Despite multiple studies investigating the environmental controls on CH4 fluxes from arctic tundra ecosystems, the high spatial variability of CH4 emissions is not fully understood. This makes the upscaling of CH4 fluxes from plot to regional scale, particularly challenging. The goal of this study is to refine our knowledge of the spatial variability and controls on CH4 emission from tundra ecosystems. Methods CH4 fluxes were measured in four sites across a variety of wet-sedge and tussock tundra ecosystems in Alaska using chambers and a Los Gatos CO2 and CH4 gas analyser. Results All sites were found to be sources of CH4, with northern sites (in Barrow) showing similar CH4 emission rates to the southernmost site (ca. 300 km south, Ivotuk). Gross primary productivity (GPP), water level and soil temperature were the most important environmental controls on CH4 emission. Greater vascular plant cover was linked with higher CH4 emission, but this increased emission with increased vascular plant cover was much higher (86 %) in the drier sites, than the wettest sites (30 %), suggesting that transport and/or substrate availability were crucial limiting factors for CH4 emission in these tundra ecosystems. Conclusions Overall, this study provides an increased understanding of the fine scale spatial controls on CH4 flux, in particular the key role that plant cover and GPP play in enhancing CH4 emissions from tundra soils

Lichen response to ammonia deposition defines the footprint of a penguin rookery

Ammonia volatilized from penguin rookeries is a major nitrogen source in Antarctic coastal terrestrial ecosystems. However, the spatial extent of ammonia dispersion from rookeries and its impacts have not been quantified previously. We measured ammonia concentration in air and lichen ecophysiological response variables proximate to an Adèlie penguin rookery at Cape Hallett, northern Victoria Land. Ammonia emitted from the rookery was 15N-enriched (δ15N value +6.9) and concentrations in air ranged from 36–75 µg m−3 at the rookery centre to 0.05 µg m−3 at a distance of 15.3 km. δ15N values and rates of phosphomonoesterase (PME) activity in the lichens Usnea sphacelata and Umbilicaria decussata were strongly negatively related to distance from the rookery and PME activity was positively related to thallus N:P mass ratio. In contrast, the lichen Xanthomendoza borealis, which is largely restricted to within an area 0.5 km from the rookery perimeter, had high N, P and 15N concentrations but low PME activity suggesting that nutrient scavenging capacity is suppressed in highly eutrophicated sites. An ammonia dispersion model indicates that ammonia concentrations sufficient to significantly elevate PME activity and δ15N values (≥0.1 µg NH3 m−3) occurred over c. 40–300 km2 surrounding the rookery suggesting that penguin rookeries potentially can generate large spatial impact zones. In a general linear model NH3 concentration and lichen species identity were found to account for 72 % of variation in the putative proportion of lichen thallus N originating from penguin derived NH3. The results provide evidence of large scale impact of N transfer from a marine to an N-limited terrestrial ecosystem

Plant growth enhancement by elevated CO2 eliminated by joint water and nitrogen limitation

http://www.nature.com/ngeo/Rising atmospheric CO2 concentrations can fertilize plant growth. The resulting increased plant uptake of CO2 could, in turn, slow increases in atmospheric CO2 levels and associated climate warming. CO2 fertilization e ects may be enhanced when water availability is low, because elevated CO2 also leads to improved plant water-use e ciency. However, CO2 fertilization e ects may be weaker when plant growth is limited by nutrient availability. How variation in soil nutrients and water may act together to influence CO2 fertilization is unresolved. Here we report plant biomass levels from a five-year, open-air experiment in a perennial grassland under two contrasting levels of atmospheric CO2, soil nitrogen and summer rainfall, respectively. We find that the presence of a CO2 fertilization e ect depends on the amount of available nitrogen and water. Specifically, elevated CO2 levels led to an increase in plant biomass of more than 33% when summer rainfall, nitrogen supply, or both were at the higher levels (ambient for rainfall and elevated for soil nitrogen). But elevated CO2 concentrations did not increase plant biomass when both rainfall and nitrogen were at their lower level. We conclude that given widespread, simultaneous limitation by water and nutrients, large stimulation of biomass by rising atmospheric CO2 concentrations may not be ubiquitous

Mutation K42R in Ribosomal Protein S12 Does Not Affect Susceptibility of Mycobacterium smegmatis 16S rRNA A-Site Mutants to 2-Deoxystreptamines

Recent studies have suggested that ribosomal protein S12 modulates 16S rRNA function and susceptibility to 2-deoxystreptamine aminoglycosides. To study whether the non-restrictive K42R mutation in RpsL affects 2-deoxystreptamine susceptibility in Mycobacterium smegmatis, we studied the drug susceptibility pattern of various mutants with genetic alterations in the 16S rRNA decoding A-site in the context of wild-type and mutant protein S12. RpsL K42R substitution was found not to affect the drug resistance pattern associated with mutational alterations in 16S rRNA H44.European Commission (PAR, FP7-HEALTH-2009-241476