The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm

LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar K_d determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.     

Restoration of stratum corneum with nacre lipids

To discover potential new products for the atopic dermatitis treatment, lipids extracted from nacre from the oyster Pinctada margaritifera were tested on artificially dehydrated skin explants. Expression of filaggrin and transglutaminase 1 was investigated after treatment of dehydrated skin with P. margaritifera lipid extracts according to light microscopy after labelling with specific monoclonal antibodies. The lipids were extracted from the nacre with methanol/chloroform mixture at room temperature and the extract composition was determined according to TLC and densitometry measures. Relative to the dry nacre material, a yield of extraction in lipids of 0.54% (w/w) was determined. Fatty acids, triglycerides, cholesterol and ceramides were in low abundance. Then, application of lipid formulations on skin explants previously dehydrated gave after 3 h an overexpression of filaggrin and a decrease of transglutaminase expression as shown by light microscopy. Using immunofluorescence labelling, we showed that lipids extracted from the mother of pearl of P. margaritifera induced a reconstitution of the intercellular cement of the stratum corneum. The signaling properties of the nacre lipids could be used for a development of new active product treatment against the symptoms of the dermatitis.     

Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones

ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2′-deoxythymidine-5′-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation.     

From analgesia to myopathy: When local anesthetics impair the mitochondrion

The expanding utilization of local anesthesia and analgesia revealed the occurrence of myopathies induced by local anesthetics. Such iatrogenic effect encouraged anesthesiologists to study the toxicity of local anesthetics and to reevaluate their protocols in order to reduce muscle pain and dysfunction. Studies performed in rats and human cells showed that bupivacaine induces muscle histological damages with sarcomers disruption along with structural alteration of mitochondria, the powerplant of the cell. Bupivacaine-induced myopathies (BIM) are underestimated as patients are not examined by the anesthesiologist after the surgery. Biochemical analyses indicate that BIM could be explained both by the alteration of mitochondrial energetics with consecutive oxidative stress and mitophagy, and the modification of sarcoplasmic reticulum activity with perturbations of calcium homeostasis. BIM is time-dependent, local anesthetic concentration-dependent, enhanced by preexisting metabolism alteration or young age, and could be prevented in part by antioxidant agents and rhEPO. These observations suggest that adapted changes in postoperative analgesia protocols, including the adjustment of LA concentration and volume, a more precise delivery of the drug and an adapted duration of analgesia, may prevent myopathies consecutive to local anesthesia.     

Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization

Shed membrane microparticles (MPs) are microvesicles generated from the plasma membrane when cells are submitted to stress conditions. Although MPs reflect the cell state (at least in vitro), little is known on their protein composition. We describe the first set of experiments aiming to characterize the MP proteome. Two ways of triggering MP formation from a T-lymphocytic cell line were analyzed using a 1-D gel approach coupled with LC-MS/MS and the results were compared with those obtained from a classic membrane preparation. In total, 390 proteins were identified in MPs, among which 34% were localized to the plasma membrane. The MPs revealed a broad representation of plasma membrane proteins including 17 hematopoietic clusters of differentiation. This approach was successfully applied to one human chronic B-cell lymphoid malignancy. In all, 413 proteins were identified, including 117 membrane proteins, many of them being pathology associated. The sequence coverage in identified proteins was improved combining both nano-LC-MS/MS and MALDI-MS data. The suppression effect, observed on very complex peptide mixtures, was remediated by chromatographic fractionation. MPs may represent a new tool for studying plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination, and being potentially applicable to most cell types.     

Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.     

Keep in touch: Does spatial overlap correlate with contact rate frequency?

Quantifying animal contact rate is crucial both in fundamental and applied studies to understand the evolution of sociality and predict the spread of infectious diseases. Researchers typically use home-range overlap among individuals as a proxy of contact rate, assuming a positive correlation. However, very few studies have assessed how the correlation between home-range overlap and contact rate may vary with ecological context. We used proximity loggers to quantify intraspecific contact rate among raccoons (Procyon lotor) and explored the correlation between contact rate and home-range overlap in different seasons. We monitored 15 female raccoons that formed 121 dyads during summer 2010 and winter 2011. We compared contact rate with the 5 most common overlap indices: home-range overlap proportion, home-range overlap probability, utilization distribution overlap index (UDOI), volume of intersection index, and Bhattacharyya's affinity index. Our results generally supported the contention of a positive and significant correlation between home-range overlap and intraspecific contact rate in raccoons. The strength of the relationship differed among seasons and indices, being weaker during winter than summer for home-range overlap proportion and home-range overlap probability. When contact rates were high, their frequency had stronger correlations with the UDOI and volume of intersection index indices than with the other indices. Our results suggest that the UDOI performs better than other indices, as we obtained a good contact rate–home-range overlap correlation with this index with animals both aggregated and randomly distributed in space. © 2012 The Wildlife Society.     

Ion-Abrasion Scanning Electron Microscopy Reveals Surface-Connected Tubular Conduits in HIV-Infected Macrophages

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of ∼150–200 nm, and lengths of up to ∼5 µm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.