Single-Cell Census of Mechanosensitive Channels in Living Bacteria

Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering

Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal \u3b2-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and \u3b2-catenin. A pharmacological activator of the WNT/\u3b2-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and \u3b2-catenin, and evidence for targeted activation of the WNT/\u3b2-catenin pathway as a potential treatment for this disease

LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP(HA) was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP(HA) was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP(HA) was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP(HA) and IcsA showed that IcsP(HA) preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP(HA) in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP(HA) was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP(HA) detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.Elizabeth Ngoc Hoa Tran, Matthew Thomas Doyle, Renato Moron

Membrane-mediated interactions

Interactions mediated by the cell membrane between inclusions, such as membrane proteins or antimicrobial peptides, play important roles in their biological activity. They also constitute a fascinating challenge for physicists, since they test the boundaries of our understanding of self-assembled lipid membranes, which are remarkable examples of two-dimensional complex fluids. Inclusions can couple to various degrees of freedom of the membrane, resulting in different types of interactions. In this chapter, we review the membrane-mediated interactions that arise from direct constraints imposed by inclusions on the shape of the membrane. These effects are generic and do not depend on specific chemical interactions. Hence, they can be studied using coarse-grained soft matter descriptions. We deal with long-range membrane-mediated interactions due to the constraints imposed by inclusions on membrane curvature and on its fluctuations. We also discuss the shorter-range interactions that arise from the constraints on membrane thickness imposed by inclusions presenting a hydrophobic mismatch with the membrane.Comment: 38 pages, 10 figures, pre-submission version. In: Bassereau P., Sens P. (eds) Physics of Biological Membranes. Springer, Cha

Defining motility in the Staphylococci

The ability of bacteria to move is critical for their survival in diverse environments and multiple ways have evolved to achieve this. Two forms of motility have recently been described for Staphylococcus aureus, an organism previously considered to be non-motile. One form is called spreading, which is a type of sliding motility and the second form involves comet formation, which has many observable characteristics associated with gliding motility. Darting motility has also been observed in Staphylococcus epidermidis. This review describes how motility is defined and how we distinguish between passive and active motility. We discuss the characteristics of the various forms of Staphylococci motility, the molecular mechanisms involved and the potential future research directions

On the validity of the geometrical theory of diffraction by convex cylinders

In this paper we consider the scattering of a wave from an infinite line source by an infinitely long cylinder C. The line source is parallel to the axis of C , and the cross section C of this cylinder is smooth, closed and convex. C is formed by joining a pair of smooth convex arcs to a circle C 0 , one on the illuminated side, and one on the dark side, so that C is circular near the points of diffraction. By a rigorous argument we establish the asymptotic behavior of the field at high frequencies, in a certain portion of the shadow S that is determined by the geometry of C in S. The leading term of our asymptotic expansion is the field predicted by the geometrical theory of diffraction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46181/1/205_2004_Article_BF00248157.pd

Dynamic changes in human-gut microbiome in relation to a placebo-controlled anthelminthic trial in Indonesia

Background: Microbiome studies suggest the presence of an interaction between the human gut microbiome and soil-transmitted helminth. Upon deworming, a complex interaction between the anthelminthic drug, helminths and microbiome composition might occur. To dissect this, we analyse the changes that take place in the gut bacteria profiles in samples from a double blind placebo controlled trial conducted in an area endemic for soil transmitted helminths in Indonesia. Methods: Either placebo or albendazole were given every three months for a period of one and a half years. Helminth infection was assessed before and at 3 months after the last treatment round. In 150 subjects, the bacteria were profiled using the 454 pyrosequencing. Statistical analysis was performed cross-sectionally at pre-treatment to assess the effect of infection, and at post-treatment to determine the effect of infection and treatment on microbiome composition using the Dirichlet-multinomial regression model. Results: At a phylum level, at pre-treatment, no difference was seen in microbiome composition in terms of relative abundance between helminth-infected and uninfected subjects and at post-treatment, no differences were found in microbiome composition between albendazole and placebo group. However, in subjects who remained infected, there was a significant difference in the microbiome composition of those who had received albendazole and placebo. This difference was largely attributed to alteration of Bacteroidetes. Albendazole was more effective against Ascaris lumbricoides and hookworms but not against Trichuris trichiura, thus in those who remained infected after receiving albendazole, the helminth composition was dominated by T. trichiura. Discussion: We found that overall, albendazole does not affect the microbiome composition. However, there is an interaction between treatment and helminths as in subjects who received albendazole and remained infected there was a significant alteration in Bacteroidetes. This helminth-albendazole interaction needs to be studied further to fully grasp the complexity of the effect of deworming on the microbiome. Trial registration: ISRCTN Registy, ISRCTN83830814

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity