Structural Bases for Stability-Function Tradeoffs in Antibiotic Resistance

Preorganization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein; consequently, enzymes can be dramatically stabilized by substitutions that attenuate the size and preorganization “strain” of the active site. How this stability-activity tradeoff constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities or how these new activities manifest structurally. These questions are both germane and easy to study in β-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of β-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C β-lactamases, which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100-fold and 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability (from 1.7 kcal mol^− 1 to 4.1 kcal mol^− 1), consistent with the stability-function hypothesis. Intriguingly, several of the substitutions were 10-20 Å from the catalytic serine; the question of how they conferred extended-spectrum activity arose. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by X-ray crystallography. Distinct mechanisms of action, including changes in the flexibility and ground-state structures of the enzyme, are revealed for each mutant. These results explain the structural bases for the antibiotic resistance conferred by these substitutions and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.     

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant–pathogen interactions.     

A stable meta‐carborane enables the generation of boron‐rich peptide agonists targeting the ghrelin receptor

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron‐containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein‐coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor‐directed boron uptake. In this study, we present the generation of short, boron‐rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super‐agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta‐carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron‐loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.     

Physiological and behavioral indices of short-term stress in wild vicuñas (Vicugna vicugna) in Jujuy Province,Argentina

The management of wild vicuñas can trigger a stress response that may compromise welfare. In Santa Catalina, Jujuy Province, Argentina, indices of short-term stress associated with capture, handling, and shearing were studied in 105 wild vicuñas (Vicugna vicugna). The study included 2 groups (n = 59 and n = 46) of wild vicuñas captured in 2 consecutive days. Independent variables analyzed included sex, restraint time, and groups. Cortisol, creatine kinase, glucose, white blood cells, temperature, heart rate, and respiratory frequency were higher than published values. Respiratory rate increased during handling and correlated with holding time and group size, while heart rate decreased. Packed cell volume was higher in females. Cortisol concentrations differed between restraint groups and sex and inversely correlated with agonistic behavior. The most common behavior was increased vigilance. Sternal recumbency increased over holding time. During handling procedures, frequency of sudden movements like kicking and attempts to stand increased as restraint time increased. Females vocalized more than males. In conclusion, the methods used triggered measurable changes suggestive of short-term stress that appeared to be physiologically tolerated by the vicuñas.     

Estimating minke whale (Balaenoptera acutorostrata) boing sound density using passive acoustic sensors

Density estimation for marine mammal species is performed primarily using visual distance sampling or capture‐recapture. Minke whales in Hawaiian waters are very difficult to sight; however, they produce a distinctive “boing” call, making them ideal candidates for passive acoustic density estimation. We used an array of 14 bottom‐mounted hydrophones, distributed over a 60 × 30 km area off Kauai, Hawaii, to estimate density during 12 d of recordings in early 2006. We converted the number of acoustic cues (i.e., boings) detected using signal processing software into a cue density by accounting for the false positive rate and probability of detection. The former was estimated by manual validation, the latter by applying spatially explicit capture‐recapture (SECR) methods to a subset of data where we had determined which hydrophones detected each call. Estimated boing density was 130 boings per hour per 10,000 km² (95% CI 104–163). Little is known about the population's acoustic behavior, so conversion from boing to animal density is difficult. As a demonstration of the method, we used a tentative boing rate of 6.04 boings per hour, from a single animal tracked in 2009, to give an estimate of 21.5 boing‐calling minke whales per 10,000 km².     

Protecs,a comprehensive and powerful storage and analysis system for OMICS data,applied for profiling the anaerobiosis response of Staphylococcus aureus COL

Broad functional genomic studies call for comprehensive and powerful data repositories for storage of genome sequences, experimental information, protein identification data, protein properties and expression values. The better such data repositories can integrate and display complex data in a clear and structured way the more biologically meaningful conclusions or novel hypotheses can be derived from extensive omics data sets. This work presents the web accessible database system Protecs and how it was used to support analysis of 50 samples drawn from four Staphylococcus aureus cultivations under anaerobiosis. Protecs incorporates findings from visualization science, e.g. micro charts and heat maps in the user interface. Its integrated tools for expression data analysis in combination with TIGR Multi Experiment Viewer were used to highlight similar gene expression profiles in single or multiple experiments based on the continuously updated S. aureus master gel. Raw data analysis results are available online at www.protecs.uni‐greifswald.de . Our meta‐study revealed that S. aureus responds in different anaerobiotic experimental setups (growth without oxygen; growth without oxygen but with supplemental pyruvate and uracil; growth without oxygen but with NO; growth without oxygen but with NO and without functional nreABC genes) with a general anaerobiosis response. Among others, this response is characterized by an induction of fermentation enzymes (PflB, Ldh1, SACOL0135, SACOL0660) as well as the response regulator SrrA. Interestingly, especially genes with a high codon adaptation index highly overlap with anaerobically induced genes.     

DNA extraction from bovine faeces: current status and future trends

There is an increasing interest in the detection and enumeration of micro‐organisms pathogenic for human and present in bovine faeces. This interest is because pollution of the environment by animal faeces may affect the safety of food and of drinking or recreational water. Detection and quantification of microbial pathogens carried out using DNA extracted from the faecal matrix are affected by the quality and the quantity of the DNA extracts, which are critical factors that limit the accuracy and sensitivity of molecular studies. This review compares published methods on DNA extraction from bovine faeces, focusing on the extent to which the success of DNA amplification is affected by issues related to the faeces. Following a general discussion on the DNA extraction methods used for faeces, we focus particularly on issues related to the faecal environment itself. The objective is to identify information that can be used to improve the sensitivity of those PCR methods used after direct DNA extraction.