Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death.

Aminoglycoside antibiotics, such as gentamicin and kanamycin, directly target the ribosome, yet the mechanisms by which these bactericidal drugs induce cell death are not fully understood. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here, we use systems-level approaches and phenotypic analyses to provide insight into the pathway whereby aminoglycosides ultimately trigger hydroxyl radical formation. We show, by disabling systems that facilitate membrane protein traffic, that mistranslation and misfolding of membrane proteins are central to aminoglycoside-induced oxidative stress and cell death. Signaling through the envelope stress-response two-component system is found to be a key player in this process, and the redox-responsive two-component system is shown to have an associated role. Additionally, we show that these two-component systems play a general role in bactericidal antibiotic-mediated oxidative stress and cell death, expanding our understanding of the common mechanism of killing induced by bactericidal antibiotics.

A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis.

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

Large-scale mapping and validation of Escherichia coli transcriptional regulation from a compendium of expression profiles.

Machine learning approaches offer the potential to systematically identify transcriptional regulatory interactions from a compendium of microarray expression profiles. However, experimental validation of the performance of these methods at the genome scale has remained elusive. Here we assess the global performance of four existing classes of inference algorithms using 445 Escherichia coli Affymetrix arrays and 3,216 known E. coli regulatory interactions from RegulonDB. We also developed and applied the context likelihood of relatedness (CLR) algorithm, a novel extension of the relevance networks class of algorithms. CLR demonstrates an average precision gain of 36% relative to the next-best performing algorithm. At a 60% true positive rate, CLR identifies 1,079 regulatory interactions, of which 338 were in the previously known network and 741 were novel predictions. We tested the predicted interactions for three transcription factors with chromatin immunoprecipitation, confirming 21 novel interactions and verifying our RegulonDB-based performance estimates. CLR also identified a regulatory link providing central metabolic control of iron transport, which we confirmed with real-time quantitative PCR. The compendium of expression data compiled in this study, coupled with RegulonDB, provides a valuable model system for further improvement of network inference algorithms using experimental data.

Combination drugs, an emerging option for antibacterial therapy.

The emerging and sustained resistance to antibiotics and the poor pipeline of new antibacterials is creating a major health issue worldwide. Bacterial pathogens are increasingly becoming resistant even to the most recently approved antibiotics. Few antibiotics are being approved by regulatory organizations, which reflects both the difficulty of developing such agents and the fact that antibiotic discovery programs have been terminated at several major pharmaceutical companies in the past decade. As a result, the output of the drug pipelines is simply not well positioned to control the growing army of resistant pathogens, although academic institutions and smaller companies are trying to fill that gap. An emerging option to fight such pathogens is combination therapy. Combinations of two antibiotics or antibiotics with adjuvants are emerging as a promising therapeutic approach. This article provides and discusses clinical and scientific challenges to support the development of combination therapy to treat bacterial infections.

Continuous movement behavior of humpback whales during the breeding season in the southwest Indian Ocean: on the road again!

BACKGROUND: Humpback whales are known to undertake long-distance migration between feeding and breeding sites, but their movement behavior within their breeding range is still poorly known. Satellite telemetry was used to investigate movement of humpback whales during the breeding season and provide further understanding of the breeding ecology and sub-population connectivity within the southwest Indian Ocean (SWIO). Implantable Argos satellite tags were deployed on 15 whales (7 males and 6 females) during the peak of the breeding season in Reunion Island. A switching-state-space model was applied to the telemetry data, in order to discriminate between "transiting" and "localized" movements, the latter of which relates to meandering behavior within putative breeding habitats, and a kernel density analysis was used to assess the spatial scale of the main putative breeding sites. RESULTS: Whales were tracked for up to 71 days from 31/07/2013 to 16/10/2013. The mean transmission duration was 25.7 days and the mean distance travelled was 2125.8 km. The tracks showed consistent movement of whales from Reunion to Madagascar, demonstrating a high level of connectivity between the two sub-regions, and the use of yet unknown breeding sites such as underwater seamounts (La Perouse) and banks (Mascarene Plateau). A localized movement pattern occurred in distinct bouts along the tracks, suggesting that whales were involved in breeding activity for 4.3 consecutive days on average, after which they resume transiting for an average of 6.6 days. Males visited several breeding sites within the SWIO, suggesting for the first time a movement strategy at a basin scale to maximize mating. Unexpectedly, females with calf also showed extensive transiting movement, while they engaged in localized behavior mainly off Reunion and Sainte-Marie (East Madagascar). CONCLUSIONS: The results indicated that whales from Reunion do not represent a discrete population. Discrete breeding sites were identified, thereby highlighting priority areas for conservation. The study is a first attempt to quantify movement of humpback whales within the southwestern Indian Ocean breeding range. We demonstrate a wandering behavior with stopovers at areas that likely represent key breeding habitat, a strategy which may enhance likelihood of individual reproductive success.

A system for deletion and complementation of Candida glabrata genes amenable to high-throughput application.

We describe a method for deleting or modifying genes from the pathogenic fungus Candida glabrata as well as a companion vector for complementation or ectopic expression experiments. A linear deletion fragment generated by polymerase chain reaction was used to replace a gene of interest with the C. glabrata gene encoding imidazoleglycerol-phosphate dehydratase (HIS3). As test cases, the chromosomal loci of the C. glabrata genes encoding aminoimidazole ribonucleotide carboxylase (ADE2) and encoding isopropylmalate dehydrogenase (LEU2) were deleted. To facilitate application of the deletion technique to essential genes, we also constructed vectors to allow expression of a complementing copy of the wildtype gene under control of the copper-inducible C. glabrata metallothionein I (MT-1) promoter. One version of the vector carried the Saccharomyces cerevisiae centromere (CEN) and autonomously-replicating sequence (ARS) regions. The C. glabrata ADE2 and LEU2 genes and a transposon-derived neomycin/kanamycin resistance gene were successfully expressed from this vector, with expression of the ADE2 and LEU2 genes complementing the ADE2 and LEU2 deletion mutations, respectively. However, this vector showed regulated expression only for the ADE2 gene. A second version of the vector, which carried an additional C. glabrata CEN and ARS region for stable plasmid maintenance, did show regulated expression for the LEU2 and neomycin/kanamycin resistance genes. This deletion and expression system is potentially applicable to any C. glabrata gene and is amenable to high-throughput application. We anticipate that these tools will have broad utility in deletion or modification of specific C. glabrata genes. This approach is also applicable to other yeast fungi.

Genomics strategies for antifungal drug discovery--from gene discovery to compound screening.

The use of genomics tools to discover new genes, to decipher pathways or to assign a function to a gene is just beginning to have an impact. Genomics approaches have been applied to both antibacterial and antifungal target discovery in order to identify a new generation of antibiotics. This review discusses genomics approaches for antifungal drug discovery, focusing on the areas of gene discovery, target validation, and compound screening. A variety of methods to identify fungal genes of interest are discussed, as well as methods for obtaining full-length sequences of these genes. One approach is well-suited to organisms having few introns (Candida albicans), and another for organisms with many introns (Aspergillus fumigatus). To validate broad spectrum fungal targets, the yeast Saccharomyces cerevisiae was used as a model system to rapidly identify genes essential for growth and viability of the organism. Validated targets were then exploited for high-throughput compound screening.

Systematic discovery of new genes in the Saccharomyces cerevisiae genome.

We used genome-wide comparative analysis of predicted protein sequences to identify many novel small genes, named smORFs for small open reading frames, within the budding yeast genome. Further analysis of 117 of these new genes showed that 84 are transcribed. We extended our analysis of one smORF conserved from yeast to human. This investigation provides an updated and comprehensive annotation of the yeast genome, validates additional concepts in the study of genomes in silico, and increases the expected numbers of coding sequences in a genome with the corresponding impact on future functional genomics and proteomics studies.

Identification of potential cell-surface proteins in Candida albicans and investigation of the role of a putative cell-surface glycosidase in adhesion and virulence.

Cell-surface proteins are attractive targets for the development of novel antifungals as they are more accessible to drugs than are intracellular targets. By using a computational biology approach, we identified 180 potential cell-surface proteins in Candida albicans, including the known cell-surface adhesin Als1 and other cell-surface antigens, such as Pra1 and Csa1. Six proteins (named Csf1-6 for cell-surface factors) were selected for further biological characterization. First, we verified that the selected CSF genes are expressed in the yeast and/or hyphal form and then we investigated the effect of the loss of each CSF gene on cell-wall integrity, filamentation, adhesion to mammalian cells and virulence. As a result, we identified Csf4, a putative glycosidase with an apparent orthologue in Saccharomyces cerevisiae (Utr2), as an important factor for cell-wall integrity and maintenance. Interestingly, deletion of CSF4 also resulted in a defect in filamentation, a reduction in adherence to mammalian cells in an in vitro adhesion assay, and a prolongation of survival in an immunocompetent mouse model of disseminated candidiasis. A delay in colonization of key organs (e.g. kidney) was also observed, which is consistent with a reduction in virulence of the csf4-deletion strain. These data indicate a key role for extracellular glycosidases in fungal pathogenesis and represent a new site for therapeutic intervention to cure and prevent fungal disease.

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus.

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.