The Rate of Osmotic Downshock Determines the Survival Probability of Bacterial Mechanosensitive Channel Mutants

Mechanosensitive (MS) channels allow cells to sense and respond to environmental changes. In bacteria, these channels are believed to protect against an osmotic shock. The physiological function of these channels has been characterized primarily by a standardized assay, where aliquots of batch-cultured cells are rapidly pipetted into a hypotonic medium. Under this method, it has been inferred many types of MS channels (MscS homologs in Escherichia coli) demonstrate limited effectiveness against shock, typically rescuing less than 10% of the cells when expressed at native levels. We introduce a single-cell-based assay which allows us to control how fast the osmolarity changes, over time scales ranging from a fraction of a second to several minutes. We find that the protection provided by MS channels depends strongly on the rate of osmotic change, revealing that, under a slow enough osmotic drop, MscS homologs can lead to survival rates comparable to those found in wild-type strains. Further, after the osmotic downshift, we observe multiple death phenotypes, which are inconsistent with the prevailing paradigm of how cells lyse. Both of these findings require a reevaluation of our basic understanding of the physiology of MS channels.     

The Zeamine Antibiotics Affect the Integrity of Bacterial Membranes

The zeamines (zeamine, zeamine I, and zeamine II) constitute an unusual class of cationic polyamine-polyketide-nonribosomal peptide antibiotics produced by Serratia plymuthica RVH1. They exhibit potent bactericidal activity, killing a broad range of Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Examination of their specific mode of action and molecular target revealed that the zeamines affect the integrity of cell membranes. The zeamines provoke rapid release of carboxyfluorescein from unilamellar vesicles with different phospholipid compositions, demonstrating that they can interact directly with the lipid bilayer in the absence of a specific target. DNA, RNA, fatty acid, and protein biosynthetic processes ceased simultaneously at subinhibitory levels of the antibiotics, presumably as a direct consequence of membrane disruption. The zeamine antibiotics also facilitated the uptake of small molecules, such as 1-N-phenylnaphtylamine, indicating their ability to permeabilize the Gram-negative outer membrane (OM). The valine-linked polyketide moiety present in zeamine and zeamine I was found to increase the efficiency of this process. In contrast, translocation of the large hydrophilic fluorescent peptidoglycan binding protein PBD_KZ-GFP was not facilitated, suggesting that the zeamines cause subtle perturbation of the OM rather than drastic alterations or defined pore formation. At zeamine concentrations above those required for growth inhibition, membrane lysis occurred as indicated by time-lapse microscopy. Together, these findings show that the bactericidal activity of the zeamines derives from generalized membrane permeabilization, which likely is initiated by electrostatic interactions with negatively charged membrane components.     

Insights into the Structure and Dynamics of Measles Virus Nucleocapsids by 1H-detected Solid-state NMR

¹H-detected solid-state nuclear magnetic resonance (NMR) experiments are recorded on both intact and trypsin-cleaved sedimented measles virus (MeV) nucleocapsids under ultra-fast magic-angle spinning. High-resolution ¹H,¹⁵N-fingerprints allow probing the degree of molecular order and flexibility of individual capsid proteins, providing an exciting atomic-scale complement to electro microscopy (EM) studies of the same systems.     

Meta-analyses of human gut microbes associated with obesity and IBD

Recent studies have linked human gut microbes to obesity and inflammatory bowel disease, but consistent signals have been difficult to identify. Here we test for indicator taxa and general features of the microbiota that are generally consistent across studies of obesity and of IBD, focusing on studies involving high-throughput sequencing of the 16S rRNA gene (which we could process using a common computational pipeline). We find that IBD has a consistent signature across studies and allows high classification accuracy of IBD from non-IBD subjects, but that although subjects can be classified as lean or obese within each individual study with statistically significant accuracy, consistent with the ability of the microbiota to experimentally transfer this phenotype, signatures of obesity are not consistent between studies even when the data are analyzed with consistent methods. The results suggest that correlations between microbes and clinical conditions with different effect sizes (e.g. the large effect size of IBD versus the small effect size of obesity) may require different cohort selection and analysis strategies.     

Diversity of Rickettsiales in the Microbiome of the Lone Star Tick,Amblyomma americanum

Ticks are important vectors for many emerging pathogens. However, they are also infected with many symbionts and commensals, often competing for the same niches. In this paper, we characterize the microbiome of Amblyomma americanum (Acari: Ixodidae), the lone star tick, in order to better understand the evolutionary relationships between pathogens and nonpathogens. Multitag pyrosequencing of prokaryotic 16S rRNA genes (16S rRNA) was performed on 20 lone star ticks (including males, females, and nymphs). Pyrosequencing of the rickettsial sca0 gene (also known as ompA or rompA) was performed on six ticks. Female ticks had less diverse microbiomes than males and nymphs, with greater population densities of Rickettsiales. The most common members of Rickettsiales were “Candidatus Rickettsia amblyommii” and “Candidatus Midichloria mitochondrii.” “Ca. Rickettsia amblyommii” was 2.6-fold more common in females than males, and there was no sequence diversity in the sca0 gene. These results are consistent with a predominantly vertical transmission pattern for “Ca. Rickettsia amblyommii.”     

The long-term immune response after HPV16 peptide vaccination in women with low-grade pre-malignant disorders of the uterine cervix: a placebo-controlled phase II study

The capacity of a low-dose HPV16 synthetic long-peptide vaccine (HPV16-SLP) to induce an HPV16-specific T-cell response as well as to establish long-term immunologic memory in patients with low-grade abnormalities of the cervix was determined in a placebo-controlled, double-blinded phase II study. In addition, the effect of a booster vaccination after 1 year was evaluated. Patients received either the HPV16-SLP or a placebo at the start of the study. After 1 year, the vaccinated patients were again randomized to receive the HPV16-SLP or a placebo. Patients were followed for 2 years. HPV16-specific T-cell responses were determined in pre- and post-vaccination blood samples by ELISPOT, proliferation assay and cytokine assays. We show that the HPV16-specific T-cell responses detected after vaccination are clearly due to vaccination and that reactivity was maintained for at least 2 years. Interestingly, a booster vaccination after 1 year especially augmented the HPV16-specific Th2 response. Furthermore, pre-existing immunity to HPV16 was associated with a stronger response to vaccination and with more side effects, reflected by flu-like symptoms. We conclude that two low-dose injections of HPV16-SLP can induce a strong and stable HPV16-specific T-cell response that lasts for at least 1 year. If booster vaccination is required, then polarizing adjuvant should be added to maintain the Th1 focus of the vaccine-induced T-cell response.     

The impact of river regulation on the biodiversity intactness of floodplain wetlands

Alteration of natural flow regime is considered a major threat to biodiversity in river floodplain ecosystems. Measurements of quantitative relationships between flow regime change and biodiversity are, however, incomplete and inconclusive. This hampers the assessment of human impact on riverine floodplain wetlands in global biodiversity evaluations. We systematically reviewed the scientific literature and extracted information from existing data sets for a meta-analysis to unravel a general quantitative understanding of the ecological consequences of altered flow regimes. From 28 studies we retrieved both ecological and hydrological data. Relative mean abundance of original species (mean species abundance, MSA) and relative species richness were used as effect size measures of biodiversity intactness. The meta-analysis showed that alteration of a natural flow regime reduces the MSA by more than 50 % on average, and species richness by more than 25 %. Impact on species richness and abundance tends to be related to the degree of hydrological alteration. These results can be used in strategic quantitative assessments by incorporating the relationships into global models on environmental change and biodiversity such as GLOBIO-aquatic.