Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA.

Aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. Here, we report the structural basis for 30S recognition by the novel plasmid-mediated aminoglycoside-resistance rRNA methyltransferase A (NpmA). These studies are supported by biochemical and functional assays that define the molecular features necessary for NpmA to catalyze m(1)A1408 modification and confer resistance. The requirement for the mature 30S as a substrate for NpmA is clearly explained by its recognition of four disparate 16S rRNA helices brought into proximity by 30S assembly. Our structure captures a "precatalytic state" in which multiple structural reorganizations orient functionally critical residues to flip A1408 from helix 44 and position it precisely in a remodeled active site for methylation. Our findings provide a new molecular framework for the activity of aminoglycoside-resistance rRNA methyltransferases that may serve as a functional paradigm for other modification enzymes acting late in 30S biogenesis.

Epitope specific T-cell responses against influenza A in a healthy population.

Pre-existing human CD4(+) and CD8(+) T-cell-mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross-reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database (www.iedb.org), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super-type coverage. Optimized libraries of CD4(+) and CD8(+) T-cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T-cell responses in healthy donors using interferon-γ ELISPOT assays. Upon stimulation, significant CD4(+) and CD8(+) T-cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4(+) and CD8(+) T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T-cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines.

30S Subunit-dependent activation of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring 16S rRNA methyltransferase Kmr.

Methylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core ß-strands 6 and 7, which forms part of the S-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m(1)A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate.

Respiratory syncytial virus disease burden in adults aged 60 years and older in high-income countries: A systematic literature review and meta-analysis.

BACKGROUND: Respiratory syncytial virus (RSV)-associated acute respiratory infection (ARI) is an underrecognized cause of illness in older adults. We conducted a systematic literature review and meta-analysis to estimate the RSV disease burden in adults ≥60 years in high-income countries. METHODS: Data on RSV-ARI and hospitalization attack rates and in-hospital case fatality rates (hCFR) in adults ≥60 years from the United States, Canada, European countries, Japan, and South Korea were collected based on a systematic literature search (January 1, 2000-November 3, 2021) or via other methods (citation search, unpublished studies cited by a previous meta-analysis, gray literature, and an RSV-specific abstract booklet). A random effects meta-analysis was performed on estimates from the included studies. RESULTS: Twenty-one studies were included in the meta-analysis. The pooled estimates were 1.62% (95% confidence interval [CI]: 0.84-3.08) for RSV-ARI attack rate, 0.15% (95% CI: 0.09-0.22) for hospitalization attack rate, and 7.13% (95% CI: 5.40-9.36) for hCFR. In 2019, this would translate into approximately 5.2 million cases, 470,000 hospitalizations, and 33,000 in-hospital deaths in ≥60-year-old adults in high-income countries. CONCLUSIONS: RSV disease burden in adults aged ≥60 years in high-income countries is higher than previously estimated, highlighting the need for RSV prophylaxis in this age group.

Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria.

X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m(7)G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides.

Expression, purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies.

High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m7G1405) or N1-methyl adenosine 1408 (m¹A1408) modifications at the drug binding site in the bacterial 30S ribosomal subunit decoding center. Two distinct families of 16S ribosomal RNA (rRNA) methyltransferases that incorporate these modifications were first identified in aminoglycoside-producing bacteria but were more recently identified in both human and animal pathogens. These resistance determinants thus pose a new threat to the usefulness of aminoglycosides as antibiotics, demanding urgent characterization of their structures and activities. Here, we describe approaches to cloning, heterologous expression in Escherichia coli, and purification of two A1408 rRNA methyltransferases: KamB from the aminoglycoside-producer Streptoalloteichus tenebrarius and NpmA identified in a clinical isolate of pathogenic E. coli ARS3. Antibiotic minimum inhibitory concentration (MIC) assays and in vitro analysis of KamB and NpmA using circular dichroism (CD) spectroscopy, S-adenosyl-l-methionine (SAM) binding by isothermal titration calorimetry and 30S subunit methylation assays showed both enzymes were soluble, folded and active. Finally, crystals of each enzyme complexed with SAM were obtained, including selenomethionine-derived KamB, that will facilitate high-resolution X-ray crystallographic analyses of these important bacterial antibiotic-resistance determinants.

Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates.

Four new Gram-positive, phenol-degrading strains were isolated from the rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae known to exude high amounts of phenolics in the soil. Isolates were designated Bacillus sp. PS1, Bacillus sp. PS11, Streptomyces sp. PS12, and Streptomyces sp. PN1 based on 16S rDNA sequence and biochemical analysis. In addition to their ability to tolerate and utilize high amounts of phenol of either up to 800 or up to 1,400 mg l(-1) without apparent inhibition in growth, all four strains were also able to degrade a broad range of aromatic substrates including benzene, toluene, ethylbenzene, xylenes, styrene, halogenated benzenes, and naphthalene. Isolates were able to grow in pure culture and in defined mixed culture on phenol and on the mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as a sole source of carbon and energy. Pure culture of Bacillus sp. PS11 yielded 1.5-fold higher biomass amounts in comparison to mixed culture, under all conditions. Strains successfully degraded phenol in the soil model system (2 g kg(-1)) within 6 days. Activities of phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase were detected and analyzed from the crude cell extract of the isolates. While all four strains use ortho degradation pathway, enzyme indicative of meta degradation pathway (catechol 2,3-dioxygenase) was also detected in Bacillus sp. PS11 and Streptomyces sp. PN1. Phenol degradation activities were induced 2 h after supplementation by phenol, but not by catechol. Catechol slightly inhibited activity of catechol 2,3-dioxygenase in strains PS11 and PN1.

Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics.

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles.

Pertussis in high-risk groups: an overview of the past quarter-century.

Infectious diseases can impact chronic medical conditions. However, it is currently not clear how pertussis correlates with preexisting or underlying disorders. We reviewed literature from the last 25 years to describe the burden and impact of pertussis infection in specific risk groups in individuals aged ≥11 years. Our literature search returned 543 hits, of which 18 were eligible for this review. Adolescents and adults with underlying conditions, such as asthma, chronic obstructive pulmonary disease (COPD), or obesity are potentially at increased risk of pertussis infection. Immunodeficiency and smoking have also been associated with worsened pertussis symptoms and an increased pertussis-related hospitalization rate. In patients with pertussis and preexisting asthma or COPD, symptoms were worsened, and health-care costs were consequently increased. Further efforts are needed to close the knowledge gap and to understand the burden of pertussis in at-risk adolescent and adult populations to help inform vaccination strategies and recommendations.

Immune interference (blunting) in the context of maternal immunization with Tdap-containing vaccines: is it a class effect?

INTRODUCTION: Maternal immunization with reduced antigen content tetanus-diphtheria-acellular pertussis (Tdap)-containing vaccines has been recommended to prevent infant pertussis. However, maternal antibodies may interfere with infant responses to routine immunization with diphtheria-tetanus-acellular pertussis (DTaP)-containing vaccines, raising concerns of suboptimal protection after infant vaccination. We performed a narrative literature review to assess whether blunting occurs regardless of the manufacturer of maternal and infant vaccines. Because internationally agreed correlates of protection are lacking, the clinical significance of blunting is not yet fully understood. We have reviewed the evidence available to date. AREAS COVERED: Thirteen studies that evaluated blunting after maternal immunization and infant primary/booster series were identified. Blunting was observed with various combinations of Tdap- and DTaP-containing vaccines for maternal and pediatric immunization. Studies assessing the effectiveness of maternal Tdap immunization beyond the primary infant immunization series in England and in the United States suggested no evidence of a clinically relevant blunting effect so far. EXPERT COMMENTARY: This review indicates that the phenomenon of blunting does not depend on the manufacturer/brand of the pertussis-containing vaccines used for immunizing mothers or children. Currently, there is no epidemiological evidence that children whose mothers received Tdap are at increased risk of pertussis after pediatric vaccinations, although longer follow-up is required.

Detection of anti-NS1 antibodies after pandemic influenza exposure: Evaluation of a serological method for distinguishing H1N1pdm09 infected from vaccinated cases.

BACKGROUND: Reliable exposure information is crucial for assessing health outcomes of influenza infection and vaccination. Current serological methods are unable to distinguish between anti-hemagglutinin (HA) antibodies induced by infection or vaccination. OBJECTIVES: We aimed to explore an alternative method for differentiating influenza infection and vaccination. METHODS: Sera from animals inoculated with influenza viruses or purified H1N1pdm09 HA were obtained. Human samples were selected from a pregnancy cohort established during the 2009 H1N1 pandemic. Unvaccinated, laboratory-confirmed cases (N = 18), vaccinated cases without influenza-like-illness (N = 18) and uninfected, unvaccinated controls (N = 18) were identified based on exposure data from questionnaires, national registries and maternal hemagglutination inhibition (HI) titres at delivery. Animal and human samples were tested for antibodies against the non-structural protein 1 (NS1) and HA from H1N1pdm09, using a Luciferase Immunoprecipitation System (LIPS). RESULTS: Anti-NS1 H1N1pdm09 antibodies were detected in sera from experimentally infected, but not from vaccinated, animals. Anti-HA H1N1pdm09 antibodies were detectable after either of these exposures. In human samples, 28% of individuals with laboratory-confirmed influenza were seropositive for H1N1pdm09 NS1, whereas vaccinated cases and controls were seronegative. There was a trend for H1N1pdm09 NS1 seropositive cases reporting more severe and longer duration of symptomatic illness than seronegative cases. Anti-HA H1N1pdm09 antibodies were detected in all cases and in 61% of controls. CONCLUSIONS: The LIPS method could differentiate between sera from experimentally infected and vaccinated animals. However, in human samples obtained more than 6 months after the pandemic, LIPS was specific, but not sufficiently sensitive for ascertaining cases by exposure.

Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm.

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K(D) (binding constant) of 17.6 microM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.

A Grant Framework as a Push Incentive to Stimulate Research and Development of New Antibiotics.

Antibiotic research and development (R&D) has failed to produce innovative antibiotics in the past two decades, which is due to both scientific and economic factors. We reviewed national and international funding agencies and critically assessed current grant funding mechanisms. Finally, we propose four complementary grant-funding incentives aimed to help developers along the R&D pipeline. Equally important objective of these incentives is to address some of the known R&D risks and bottlenecks.

Antibiotic Pipeline Coordinators.

The World Health Organization (WHO) has published a global priority list of antibiotic-resistant bacteria to guide research and development (R&D) of new antibiotics. Every pathogen on this list requires R&D activity, but some are more attractive for private sector investments, as evidenced by the current antibacterial pipeline. A "pipeline coordinator" is a governmental/non-profit organization that closely tracks the antibacterial pipeline and actively supports R&D across all priority pathogens employing new financing tools.

Should Antibiotics Be Controlled Medicines? Lessons from the Controlled Drug Regimen.

This study aimed to identify the antibiotic-relevant lessons from the controlled drug regimen for narcotics. Whereas several elements of the United Nations Single Convention on Narcotic Drugs (1961) could be advantageous for antibiotics, we doubt that an international legally binding agreement for controlling antibiotic consumption would be any more effective than implementing stewardship measures through national AMR plans.

Distinct T and NK cell populations may serve as immune correlates of protection against symptomatic pandemic influenza A(H1N1) virus infection during pregnancy.

Maternal influenza infection during pregnancy is associated with increased risk of morbidity and mortality. However, the link between the anti-influenza immune responses and health-related risks during infection is not well understood. We have analyzed memory T and NK cell mediated immunity (CMI) responses in pandemic influenza A(H1N1)pdm09 (pdm09) virus infected non-vaccinated pregnant women participating in the Norwegian Influenza Pregnancy Cohort (NorFlu). The cohort includes information on immunization, self-reported health and disease status, and biological samples (plasma and PBMC). Infected cases (N = 75) were defined by having a serum hemagglutination inhibition (HI) titer > = 20 to influenza pdm09 virus at the time of delivery, while controls (N = 75) were randomly selected among non-infected pregnant women (HI titer <10). In ELISpot assays cases had higher frequencies of IFNγ+ CD8+ T cells responding to pdm09 virus or conserved CD8 T cell-restricted influenza A virus epitopes, compared to controls. Within this T cell population, frequencies of CD95+ late effector (CD45RA+CCR7-) and naive (CD45RA+CCR7+) CD8+ memory T cells correlated inversely with self-reported influenza illness (ILI) symptoms. ILI symptoms in infected women were also associated with lower numbers of poly-functional (IFNγ+TNFα+, IL2+IFNγ+, IL2+IFNγ+TNFα+) CD4+ T cells and increased frequencies of IFNγ+CD3-CD7+ NK cells compared to asymptomatic cases, or controls, after stimulation with the pdm09 virus. Taken together, virus specific and functionally distinct T and NK cell populations may serve as cellular immune correlates of clinical outcomes of pandemic influenza disease in pregnant women. Our results may provide information important for future universal influenza vaccine design.

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes.

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.

New polyene macrolide family produced by submerged culture of Streptomyces durmitorensis.

A new polyene macrolide family, closely related to the pentaene macrolide antibiotic roflamycoin, was isolated from the both fermentation broth and biomass of Streptomyces durmitorensis wild-type strain MS405. The main compound was identified by NMR and Fourier transform ion cyclotron resonance mass spectrometry as 32,33-didehydroroflamycoin (1; DDHR). Additional four structurally related compounds were determined solely by MS analysis. DDHR induces cell death by apoptosis in various cancer cell lines as demonstrated by DNA fragmentation. Striking feature of DDHR is its internal fluorescence allowing visualization of labeled plasma membranes and internal membrane structures.

Heterologous Escherichia coli expression, purification and characterization of the GrmA aminoglycoside-resistance methyltransferase.

The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His(6)-EK-GrmA and His(6)-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.