Proteomic Analysis of the Action of the Mycobacterium ulcerans Toxin Mycolactone: Targeting Host Cells Cytoskeleton and Collagen

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone''s cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.     

Interfacial and micellar behavior of glucose lipid

Summary Glucose lipid of notable surfactant properties was produced by using hydrocarbon assimilating bacterium of the bacterial strain MM1. Its surface active properties were notable in spite of ,-hydrophilic and bulky molecule. The critical micelle concentration (cmc) was small (0.165 M); surface and interfacial tension for hexadecane at 0.1 % (pH 7.35) solution, 24.6 and 13.3 mN/m, respectively. The emulsifying action was excellent and comparable to those of rhamnolipids. The micelle has the outer radius. 28.5 Å and inner core, 6.7 Å on the basis of the concentric spherical shell model by means of the small angle X-ray solution scattering measurement.Dedicated to Professor Fritz Wagner on the occasion of his 65th birthday.     

t-Complex-Associated Embryonic Surface Antigen Homologous to mLAMP-1

Previously we described an embryonic cell surface glycoprotein, ESGp, associated with the t-embryonic lethal alleles of the mouse t complex. This antigen is expressed on the cell surface of both early mouse embryos and embryonal carcinoma (EC) cell lines. The antigen is localized to areas of cell–cell contact in EC lines and redistributes to the outer edges of the blastomeres during compaction, thereby indicating a potential role in embryonic cell–cell interaction. We now report that this t-complex-associated ESGp is homologous to the mouse lysosomal-associated membrane protein-1 (LAMP-1). Limited protein sequence analyses of the amino terminal and an internal peptide indicate considerable homology with the LAMP-1 protein. Biochemical parameters such as protein core size, sulfation and phosphorylation status, and resistance to proteolysis also demonstrate homology. While we detect only a single message with a mouse LAMP-1 cDNA probe via Northern blotting, Southern analyses indicate the existence of at least two homologous LAMP-1 genes. Additionally, we present evidence suggesting that ESGp/LAMP-1 serves as a substrate which may be differentially glycosylated by the activities of the gene products of the different t-lethal alleles.     

Sporogenesis in Eusporangiate Ferns: II. Polyplastidic Meiosis in Ophioglossum (Ophioglossales)

Ophioglossum petiolatum . Unlike Angiopteris (Marattiales), which is monoplastidic, Ophioglossum undergoes polyplastidic meiosis like members of the fern-seed plant clade. The meiotic spindle is distinctly multipolar in origin and is consolidated into a bipolar spindle that is variously twisted and curved to accommodate the large number of chromosomes. Although a phragmoplast forms after first meiosis, no wall is deposited. Instead, an organelle band consisting of intermingled plastids and mitochondria is formed in the equatorial region between the dyad domains. Following second meiosis, a complex of phragmoplasts forms among sister and non-sister nuclei. Cell plates are deposited first between sister nuclei and then in the region of the organelle band resulting in a tetrad of spores each with a equal allotment of organelles. Received 30 January 2001/ Accepted in revised form 24 April 2001     

Movement,connectivity and population structure of the intertidal fish Lipophrys pholis as revealed by otolith oxygen and carbon stable isotopes

The shanny Lipophrys pholis is an intertidal fish commonly found in Portuguese coastal waters. Spawning takes place from early autumn to mid spring, after which demersal eggs hatch and larvae disperse along the coast. Two to three months later, young juveniles return to the tide pools to settle. However, information on fish movement, habitat connectivity and population structure is scarce for this species. One hundred and twenty early juveniles (16–35?mm) were collected in April 2014 from six rocky beaches along the western and south Portuguese coasts (Agudela, Cabo do Mundo, Boa Nova, Peniche, Sines and Olhos de Água). δ¹⁸O and δ¹³C were determined by isotope-ratio mass spectrometry. Data were analysed to determine whether isotopic signatures could be used to assess the degree of separation between individuals collected from different locations. Mean δ¹³C and δ¹⁸O values ranged from ?0.02‰ to 1.14‰ and ?7.77‰ to ?6.62‰, respectively. Both seawater temperature and salinity caused differences in otolith δ¹⁸O among the four main sampling areas. The variation among areas in δ¹³C was most likely related to slight differences in the diet, growth and metabolism of fish. The distinct isotopic signatures, at least for the northern and central areas, suggested low levels of connectivity across large spatial scales during the juvenile stage. Furthermore, similar isotopic signatures within the same area indicated some degree of larval oceanic retention at short spatial scales. This study suggests that stable isotope records in otoliths could provide information about the home residency, movements and habitat connectivity of intertidal fishes.     

Induction of Attachment of the Mussel Perna perna by Natural Products from the Brown Seaweed Stypopodium zonale

Marine invertebrates settle, attach, and/or metamorphose in response to signals from several sources, including seaweeds. In response to the aquaculture challenge of producing constant numbers of juveniles from cultured species, natural inducers have been screened for their ability to improve those processes. However, few chemical inducers of attachment of invertebrates have been identified, and even less of these were secondary metabolites. The goal of this work was to isolate the natural products responsible for induction activity using bioassay-guided fractionation of the organic extract of the brown seaweed Stypopodium zonale and the attachment of juveniles of the common brown mussel, Perna perna, as a model. The meroditerpene epitaondiol, identified by comparison of spectral data with the literature, promoted as much as 4.7 times more mussel attachment compared to controls at the natural concentration found in this alga (0.041% of the crude extract or 0.012% of algal dry weight). This is the first report showing that a seaweed produces terpenoid compounds as cues for invertebrate attachment, and future studies evaluating this action on settlement of mussels in the field are expected to improve aquaculture technology by increasing mussel spat production.     

Trypanosoma cruzi: in vitro activity of Epoxy-α-Lap, a derivative of α-lapachone, on trypomastigote and amastigote forms

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-α-Lap, an oxyran derivative of α-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-α-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-α-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-α-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-α-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.     

Dynamic proteomic overview of glioblastoma cells (A172) exposed to perillyl alcohol

Perillyl alcohol (POH) is a naturally occurring terpene and a promising chemotherapeutic agent for glioblastoma multiform; yet, little is known about its molecular effects. Here we present results of a semi-quantitative proteomic analysis of A172 cells exposed to POH for different time-periods (1′, 10′, 30′, 60′, 4 h, and 24 h). The analysis identified more than 4000 proteins; which were clustered using PatternLab for proteomics and then linked to Ras signaling, tissue homeostasis, induction of apoptosis, metallopeptidase activity, and ubiquitin-protein ligase activity. Our results make available one of the most complete protein repositories for the A172. Moreover, we detected the phosphorylation of GSK3β (Glycogen synthase kinase) and the inhibition of ERK's (extracellular signal regulated kinase) phosphorylation after 10′, which suggests a new mechanism of POH's activation for apoptosis.     

Escherichia coli expression and purification of four antimicrobial peptides fused to a family 3 carbohydrate-binding module (CBM) from Clostridium thermocellum

Antimicrobial peptides (AMPs) are molecules that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. Several hundreds of AMPs have been identified and characterized. These molecules are presently gaining increasing importance, as a consequence of their remarkable resistance to microorganism adaptation. Carbohydrate-binding modules (CBMs) are non-catalytic domains that anchor glycoside hydrolases into complex carbohydrates. Clostridium thermocellum produces a multi-enzyme complex of cellulases and hemicellulases, termed the cellulosome, which is organized by the scaffoldin protein CipA. Binding of the cellulosome to the plant cell wall results from the action of CipA family 3 CBM (CBM3), which presents a high affinity for crystalline cellulose. Here CipA family 3 CBM was fused to four different AMPs using recombinant DNA technology and the fusion recombinant proteins were expressed at high levels in Escherichia coli cells. CBM3 does not present antibacterial activity and does not bind to the bacterial surface. However, the four recombinant proteins retained the ability to bind cellulose, suggesting that CBM3 is a good candidate polypeptide to direct the binding of AMPs into cellulosic supports. A comprehensive characterization of the antimicrobial activity of the recombinant fusion proteins is currently under evaluation.