Expression and Activity of a Novel Cathelicidin from Domestic Cats

Cathelicidins are small cationic antimicrobial peptides found in many species including primates, mammals, marsupials, birds and even more primitive vertebrates, such as the hagfish. Some animals encode multiple cathelicidins in their genome, whereas others have only one. This report identifies and characterizes feline cathelicidin (feCath) as the sole cathelicidin in domestic cats (Felis catus). Expression of feCath is predominantly found in the bone marrow, with lower levels of expression in the gastrointestinal tract and skin. By immunocytochemistry, feCath localizes to the cytoplasm of neutrophils in feline peripheral blood. Structurally, the mature feCath sequence is most similar to a subgroup of cathelicidins that form linear α-helices. feCath possesses antimicrobial activity against E. coli D31, Salmonella enterica serovar Typhimurium (IR715), Listeria monocytogenes and Staphylococcus pseudintermedius (clinical isolate) similar to that of the human ortholog, LL-37. In contrast, feCath lacks the DNA binding activity seen with LL-37. Given its similarity in sequence, structure, tissue expression, and antimicrobial activity, the cathelicidin encoded by cats, feCath, belongs to the subgroup of linear cathelicidins found not only in humans, but also non-human primates, dogs, mice, and rats

Snake Cathelicidin from Bungarus fasciatus Is a Potent Peptide Antibiotics

Background: Cathelicidins are a family of antimicrobial peptides acting as multifunctional effector molecules of innate immunity, which are firstly found in mammalians. Recently, several cathelicidins have also been found from chickens and fishes. No cathelicidins from other non-mammalian vertebrates have been reported. Principal Findings: In this work, a cathelicidin-like antimicrobial peptide named cathelicidin-BF has been purified from the snake venoms of Bungarus fasciatus and its cDNA sequence was cloned from the cDNA library, which confirm the presence of cathelicidin in reptiles. As other cathelicidins, the precursor of cathelicidin-BF has cathelin-like domain at the N terminus and carry the mature cathelicidin-BF at the C terminus, but it has an atypical acidic fragment insertion between the cathelin-like domain and the C-terminus. The acidic fragment is similar to acidic domains of amphibian antimicrobial precursors. Phylogenetic analysis revealed that the snake cathelicidin had the nearest evolution relationship with platypus cathelicidin. The secondary structure of cathelicidin-BF investigated by CD and NMR spectroscopy in the presence of the helicogenic solvent TFE is an amphipathic α-helical conformation as many other cathelicidins. The antimicrobial activities of cathelicidin BF against forty strains of microorganisms were tested. Cathelicidin-BF efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. It was especially active against Gram-negative bacteria. Furthermore, it could exert antimicrobial activity against some saprophytic fungus. No hemolytic and cytotoxic activity was observed at the dose of up to 400 µg/ml. Cathelicidin-BF could exist stably in the mice plasma for at least 2.5 hours. Conclusion: Discovery of snake cathelicidin with atypical structural and functional characterization offers new insights on the evolution of cathelicidins. Potent, broad spectrum, salt-independent antimicrobial activities make cathelicidin-BF an excellent candidate for clinical or agricultural antibiotics

Characterizing the Role of Cell-Wall β-1,3-Exoglucanase Xog1p in Candida albicans Adhesion by the Human Antimicrobial Peptide LL-37

Candida albicans is the major fungal pathogen of humans. Its adhesion to host-cell surfaces is the first critical step during mucosal infection. Antimicrobial peptides play important roles in the first line of mucosal immunity against C. albicans infection. LL-37 is the only member of the human cathelicidin antimicrobial peptide family and is commonly expressed in various tissues, including epithelium. We previously showed that LL-37 significantly reduced C. albicans adhesion to plastic, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. The inhibitory effect of LL-37 on cell adhesion occurred via the binding of LL-37 to cell-wall carbohydrates. Here we showed that formation of LL-37–cell-wall protein complexes potentially inhibits C. albicans adhesion to polystyrene. Using phage display and ELISA, we identified 10 peptide sequences that could bind LL-37. A BLAST search revealed that four sequences in the major C. albicans cell-wall β-1,3-exoglucanase, Xog1p, were highly similar to the consensus sequence derived from the 10 biopanned peptides. One Xog1p-derived peptide, Xog1p90–115, and recombinant Xog1p associated with LL-37, thereby reversing the inhibitory effect of LL-37 on C. albicans adhesion. LL-37 reduced Xog1p activity and thus interrupted cell-wall remodeling. Moreover, deletion of XOG1 or another β-1,3-exoglucanase-encoding gene EXG2 showed that only when XOG1 was deleted did cellular exoglucanase activity, cell adhesion and LL-37 binding decrease. Antibodies against Xog1p also decreased cell adhesion. These data reveal that Xog1p, originally identified from LL-37 binding, has a role in C. albicans adhesion to polystyrene and, by inference, attach to host cells via direct or indirect manners. Compounds that target Xog1p might find use as drugs that prevent C. albicans infection. Additionally, LL-37 could potentially be used to screen for other cell-wall components involved in fungal cell adhesion

Strain specific differences in ribosomal DNA from the ectomycorrhizal fungi Laccaria bicolor (Maire) Orton and Laccaria laccata (Scop ex Fr) Br

The restriction fragment length polymorphism patterns of the ribosomal RNA genes of 14 isolates belonging to various ectomycorrhizal fungus species including the related basidiomycetous ectomycorrhizal fungi Laccaria laccata (Scop ex Fr) Br and Laccaria bicolor (Maire) Orton have been determined. The isolates were obtained from various geographical sources in France, the United Kingdom and North America. Total DNA of vegetative mycelium was cleaved with a series of restriction enzymes, electrophoretically separated and probed with radiolabelled rDNA gene from Coprinus cinereus (Schaeff: Fr) SF Gray. Results indicate that isolates belonging to different species had different restriction enzyme sites in the rDNA. Although distinct patterns were observed within species, a core of common bands could be discerned within each species. Since various patterns were observed within L bicolor and L laccata, rRNA gene restriction patterns may have epidemiological as well as taxonomic interest.Étude du polymorphisme de l'ADN ribosomal chez différentes souches de champignons ectomycorhiziens Laccaria bicolor et Laccaria laccata. Afin de caractériser la diversité génétique au sein des champignons ectomycorhiziens appartenant aux espèces Laccaria bicolor et L laccata, une étude du polymorphisme de l'ADN ribosomal (ADNr) de 14 souches appartenant à plusieurs espèces et de provenances géographiques variées a été entreprise. Dans un premier temps, nous avons développé une méthode d'extraction de l'ADN total du mycélium végétatif simple et rapide. Les régions intergéniques de l'ADNr des champignons présentant des variations importantes à la fois au niveau du nombre de sites de restriction des endonucléases et au niveau de la taille des séquences, une analyse du polymorphisme de longueur des fragments de restriction (RFLP) a été conduite sur l'ADN total de ces champignons mycorhiziens. Il apparaît que le polymorphisme de longueur des fragments de restriction est très important entre des genres différents (fig 1A), modérés entre espèces d'un même genre (figs 2A et B) et restreint avec les isolats d'une même espèce (figs 2A et B). En général, on observe un bonne conservation du nombre de sites de restriction au niveau du gène de l'ADNr des Laccaires. Les fragments de restriction EcoRI de 1.45, 8.0, et 9.4 kpb se rencontrent chez la plupart des souches de Laccaria que nous avons analysées (tableau II). La comparaison des profils de restriction EcoRI des souches de L bicolor et L laccata permet l'attribution aisée d'une souche à l'une ou l'autre de ces deux espèces. De plus, le polymorphisme des fragments de restriction est suffisant pour distinguer les souches de provenances géographiques différentes (figs 2A et B). Il est particulièrement intéressant de noter que le profil de restriction de L laccata S238 que nous avons obtenu est similaire à celui des isolats américains de L bicolor CRBF581 et CRBF569. Ces résultats confirment ceux publiés par Armstrong et al (1989) et conduisent à reclasser la souche américaine L laccata S238 dans l'espèce bicolor. En conclusion, l'étude du polymorphisme des fragments de restriction de l'ADNr des champignons ectomycorhiziens nous a permis de : 1) montrer que le gène codant pour les ARNr de Laccaria présente une homologie élevée avec le gène de Coprinus cinereus confirmant une conservation importante de l'ADNr au sein des Agaricales; 2) démontrer qu'il existe un polymorphisme des fragments de restriction de l'ADNr au sein des isolats des différentes espèces analysées; et 3) discriminer un certain nombre de souches appartenant aux espèces Laccaria bicolor et L laccata. La RFLP de l'ADNr peut donc s'appliquer avec succès à l'étude des divergences génétiques et à l'identification de champignons ectomycorhiziens. L'amplification préalable de l'ADNr à l'aide de la PCR (Polymerase Chain Reaction), en évitant l'emploi de radioisotopes, devrait conduire à une simplifiication considérable de l'analyse du polymorphisme des fragments de restriction

Sintering of anorthite based ceramics prepared from kaolin DD2 and calcite

Abstract In this work, the preparation of anorthite based ceramics using a modified milling system and 80 wt% kaolin (DD2 type) and 20 wt% calcium oxide extracted from CaCO3 is shown. The choice of these raw materials was dictated by their natural abundance. Previous studies have shown that a simple and vibratory multidirectional milling system using a bimodal distribution of highly resistant ceramics can be successfully used for obtaining fine powders. The prepared samples were sintered at different temperatures ranging between 800 and 1100 °C. It has been found that the relative density of samples sintered at 900 °C for 1 h with a heating rate of 5 °C/min was about 96% of the theoretical density of anorthite (2.75 g/cm3). Finally, the prepared samples were also characterized by scanning electron microscopy, X-ray diffraction and Raman spectroscopy