Essential oils obtained by hydrodistillation of seeds, pericarps, leaves and rhizomes of Aframomum dalzielii, A letestuianum and A. pruinosum grown in Cameroon were analyzed by GC-FID and GC-MS. The seed oils of the three species were characterized by a high content of ( E)-( R)-nerolidol (>88.0 %), which was fully characterized by NMR spectroscopy and chiral GC analysis. The main constituents of the pericarp and rhizome oils were monoterpene hydrocarbons, mainly β-pinene (0.8%–22.9%) and sabinene (29.0%–42.3%), along with 1,8-cineole (4.5%–23.7%); leaf oils were characterized by sesquiterpenes, namely ( E)-β-caryophyllene (18.4%–82.4%) and caryophyllene oxide (4.5%–23.7%). The antibacterial activities of these essential oils and of nine pure compounds (sabinene, β-pinene, 1,8-cineole, linalool, racemic ( E)-nerolidol, ( E)-( R)-nerolidol, ( E)-β-caryophyllene, α-humulene and caryophyllene oxide) were assessed against Micrococcus luteus and Escherichia coli. The strongest activities were observed against E. coli. The seed essential oils and their major component, ( E)-( R)-nerolidol, exhibited the lowest MIC values (0.19-0.39 μL/mL), justifying their traditional use and their potential application as natural food preservatives.

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded β-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.

A new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gram-negative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm ×100 μm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-L-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-L-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications.

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera , require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg -EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that ( i ) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and ( ii ) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg -EcSOD.

A repeatable preconcentration electrophoretic methodology for the analysis of bacteria was developed. This method is based on an isotachophoretic mode coupled with a simultaneous hydrodynamic-electrokinetic injection in conditions of field-amplified sample injection. This electrophoretic method allows the quantification of Enterobacter cloacae (studied as a model of Gram negative bacteria) with a limit of detection of 2 × 104 cells/mL. With the optimized conditions, a preconcentration factor of about 500-fold was obtained as compared to a standard hydrodynamic injection. The RSD (n = 5) on the migration time and on the peak area were 3% and 5%, respectively. This capillary electrophoretic methodology has been applied for the quantification of microbes in natural water (river and natural spring waters). Filtration of the sample prior to injection was required to remove ions present in the water and to keep the field-amplified sample injection condition at the injection. Filtrated bacteria were then recovered in terminating electrolyte diluted 10 times with water. Good agreements were obtained between cellular ATP measurements and the proposed CE methodology for the quantification of bacteria in waters.

An isotachophoretic method has been developed for mobilizing and focusing bacteria. This allows quantification and detection of bacteria in a narrow zone. Very good linearity was obtained for Micrococcus lysodeikticus (also called Micrococcus luteus, studied as a model of Gram+ bacteria) in the range of 0.4 × 10(8) cells/mL to 2.9 × 10(8) cells/mL, with correlation coefficients for peak height and peak area as a function of cell concentration of 0.999 and 0.998, respectively. This method is usable on both bare and hydroxypropyl cellulose-coated fused silica capillaries. The best results were obtained using 13.6 mM Tris, 150 mM boric acid as terminating electrolyte, and 4.5 mM Tris, 50 mM boric acid, and 3.31 mM HCl as leading electrolyte. With a 33.5 cm ×100 μm i.d. capillary, short migration times were obtained while maintaining very low electrical current in order to minimize any Joule heating and lysis of the bacteria. A UV area imaging detector (ActiPix D100, Paraytec) was used with a 109 cm × 100 μm i.d. capillary having three loops and four detection windows to monitor the migration behavior of M. luteus and to show the stability of the zone of the focused bacteria along the capillary. Similar results were obtained for Erwinia carotovora (a model of Gram- bacteria), and for Enterobacter cloacae and Vibrio splendidus.

The present study reports the characterization of Ls-Stylicin1, a novel antimicrobial peptide from the penaeid shrimp, Litopenaeus stylirostris. The predicted mature peptide of 82 residues is negatively charged (theoretical pI=5.0) and characterized by a proline-rich N-terminal region and a C-terminal region containing 13 cysteine residues. The recombinant Ls-Stylicin1 has been isolated in both monomeric and dimeric forms. Both display strong antifungal activity against Fusarium oxysporum (1.25μM<MIC<2.5μM), a pathogenic fungus of shrimp, but lower antimicrobial activity against Gram (−) bacteria, Vibrio sp. (40μM<MIC<80μM). However, rLs-Stylicin1 is able to agglutinate Vibrio penaeicidae in vitro in agreement with its potent LPS-binding activity on immobilized LPS of V. penaeicidae (dissociation constant (Kd) of 9.6×10−8M). This molecule with no evident homology to other hitherto described antimicrobial peptides but identified herein several species of penaeid shrimp is thought to be the first member of a shrimp antimicrobial peptide family, which we termed stylicins.

An easily synthesized DendriGraft poly-lysine DGL-G3 (third generation) was shown to act as an efficient carrier for raising antibodies directed against small molecules. The immunological properties of three different forms of DGL-G3 were investigated: the native form (molecular weight 22 kDa bearing a mean number of 123 surface amino groups as TFA salts), a form modified at the C-terminus by fluorescein (fluorescein-DGL-G3), and last a surface-modified form bearing histamine (DGL-G3-Histamine). Our studies demonstrate the native DGL-G3 to be inefficient in eliciting antibody production in rabbits. Immunizations of rabbits using the core-modified fluorescein-DGL-G3 or the surface-modified DGL-G3-histamine conjugate failed in eliciting antibody production. Conversely, following a primary immunization using a BSA-histamine conjugate, a second immunization with DGL-G3-histamine conjugate improved the production of specific hapten-directed antibodies, which demonstrates the utility of DGL-G3 as a carrier for the production of highly specific antibody against haptens.

Vibrio splendidus, strain LGP32, is an oyster pathogen associated with the summer mortalities affecting the production of Crassostrea gigas oysters worldwide. Vibrio splendidus LGP32 was shown to resist to up to 10 microM Cg-Def defensin and Cg-BPI bactericidal permeability increasing protein, two antimicrobial peptides/proteins (AMPs) involved in C. gigas immunity. The resistance to both oyster Cg-Def and Cg-BPI and standard AMPs (polymyxin B, protegrin, human BPI) was dependent on the ompU gene. Indeed, upon ompU inactivation, minimal bactericidal concentrations decreased by up to fourfold. AMP resistance was restored upon ectopic expression of ompU. The susceptibility of bacterial membranes to AMP-induced damages was independent of the ompU-mediated AMP resistance. Besides its role in AMP resistance, ompU proved to be essential for the adherence of V. splendidus LGP32 to fibronectin. Interestingly, in vivo, ompU was identified as a major determinant of V. splendidus pathogenicity in oyster experimental infections. Indeed, the V. splendidus-induced oyster mortalities dropped from 56% to 11% upon ompU mutation (Kaplan-Meier survival curves, P < 0.01). Moreover, in co-infection assays, the ompU mutant was out competed by the wild-type strain with competitive indexes in the range of 0.1-0.2. From this study, ompU is required for virulence of V. splendidus. Contributing to AMP resistance, conferring adhesive properties to V. splendidus, and being essential for in vivo fitness, the OmpU porin appears as an essential effector of the C. gigas/V. splendidus interaction.

Series of perfluoroalkanesulfonamides 1, sodium salt of perfluoroalkanesulfonamides 2 and polyfluoroalkanesulfonamides 3 derivatives were synthesized and characterized by (1)H NMR, (13)C NMR, (19)F NMR, IR and HRMS. Inhibition effects of these compounds on bovine carbonic anhydrase (bCA) and human carbonic anhydrase isoenzyme II (hCA) have been investigated. Comparing IC(50) values of the synthesized molecules 1, 2 and 3, it has been found that compound 2b is a more potent inhibitor than acetazolamide on hCA. Moreover 2b does not present cellular toxicity on sheep red globules.

A cDNA sequence that encodes a 61-amino acid polypeptide precursor with homologies to proline-rich antimicrobial peptides (AMPs) was identified in the oyster Crassostrea gigas. After release of a hydrophobic signal peptide, the resulting 37-amino acid peptide, Cg-Prp, is composed of an acidic region and a cationic proline-rich region. To evaluate the biological properties of Cg-Prp, multiple proline-rich peptides corresponding to putative processing of the full-length Cg-Prp were synthesized. A limited antimicrobial activity was observed for two of them, which also showed strong synergistic antimicrobial activity with Cg-Def, a defensin from C. gigas. To our knowledge, this is the first evidence of synergy between a defensin and another AMP in an invertebrate. By in situ hybridization, the expression of Cg-prp was found to be restricted to hemocytes and induced following bacterial challenge. Cg-prp transcripts were also detected in hemocytes infiltrating mantle, where Cg-Def is expressed. Additionally, by immunocytochemistry, we showed that Cg-Prp or one of its variants is present in some hemocytes together with defensins. In conclusion, we described here the first proline-rich AMP from mollusk. From our study, it is likely to provide a first line of defense against bacterial invasion by acting through synergy with defensins.

Essential oil samples of Cinnamosma fragrans from two regions in Madagascar, Tsaramandroso (38 samples) and Mariarano (30 samples), were analysed by GC/MS. Fifty-seven components were identified, accounting from 88.3% to 99.4% of the oils' composition. The major components were linalool (72.5 ± 23.3%) in Tsaramandroso and 1,8-cineole (47.3 ± 10.2%) in Mariarano. Samples B8 (95.8% linalool) from Tsaramandroso and B143 (71.6% 1,8-cineole) from Mariarano containing the highest proportions of the two main components identified, were selected to determine antimicrobial activities against 10 microbial strains. Bacillus subtilis and Staphylococcus aureus were the most sensitive strains to both oils. Minimum inhibitory concentration (MIC) values were lower for B143 against all tested Gram-negative strains than pure 1,8-cineole. B8 showed higher MIC values than pure linalool against Salmonella typhimurium and Vibrio alginolyticus, and similar MIC values to linalool towards the other Gram-negative strains. Both essential oils exhibited higher MIC values towards Fusarium oxysporum than their respective pure major component. These results suggested the occurrence of synergism or antagonism effects between the different oil constituents.

A cDNA sequence with homologies to members of the LPS-binding protein and bactericidal/permeability-increasing protein (BPI) family was identified in the oyster Crassostrea gigas. The recombinant protein was found to bind LPS, to display bactericidal activity against Escherichia coli, and to increase the permeability of the bacterial cytoplasmic membrane. This indicated that it is a BPI rather than an LPS-binding protein. By in situ hybridization, the expression of the C. gigas BPI (Cg-bpi) was found to be induced in hemocytes after oyster bacterial challenge and to be constitutive in various epithelia of unchallenged oysters. Thus, Cg-bpi transcripts were detected in the epithelial cells of tissues/organs in contact with the external environment (mantle, gills, digestive tract, digestive gland diverticula, and gonad follicles). Therefore, Cg-BPI, whose expression profile and biological properties are reminiscent of mammalian BPIs, may provide a first line of defense against potential bacterial invasion. To our knowledge, this is the first characterization of a BPI in an invertebrate.

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. We previously characterized the first AMP from an oyster, a defensin, that was shown to be continuously expressed in the mantle of Crassostrea gigas. In this study, we report the cDNA cloning of two new isoforms of the defensin AMP family (Cg-defh1 and Cg-defh2) from the hemocytes of the oyster. The deduced amino acid sequences reveal two peptides of 73 amino acid residues with a mature portion consisting of 43 amino acid residues. Cg-Defh1 and Cg-Defh2 share 86% amino acid identity and belong to the "arthropod-molluscs defensin family". qRT-PCR analyses indicate that Cg-defh2 is continuously expressed in the hemocytes of C. gigas. In addition, after a bacterial challenge, the level of Cg-defh2 transcripts decreases dramatically in the circulating hemocyte population and this decrease can be correlated with an increase of Cg-defh2 transcripts in the gill and the mantle tissue, suggesting a possible migration of the hemocytes expressing Cg-defh2 towards the tissues implicated in the first defense barrier of the oyster. These results would suggest an important role of Cg-Defh2 in the oyster response to a microbial challenge.

Two generations of poly(l-lysine) dendrigrafts (DGLs) were studied with regard to their ability to interact with and translocate through liposomal and cellular membranes. Partial guanidinylation of the surface amino groups of the starting dendrigrafts afforded the guanidinylated derivatives whose membrane translocation properties were also assessed. Mixed liposomes, consisting of dihexadecyl phosphate, phosphatidylcholine, and cholesterol, were employed as model membranes, while A549 human lung carcinoma cells were used for cellular uptake studies. At high surface group/liposomal phosphate molar ratios and depending on the structure of the DGL, the interaction led to aggregation. Dendrigraft liposomal internalization was achieved, however, at low molar ratios. Thus translocation of the second generation dendrigrafts was rather limited at 25 degrees C, which, however, was enhanced when the bilayer was in the liquid-crystalline phase. In contrast, third-generation counterparts exhibited minor translocational ability. Furthermore, the introduction of a guanidinium group to dendrigrafts was found to enhance their transport through liposomal membranes. On the other hand, cellular uptake by A549 cells was monitored up to 3 h incubation time via fluorescence registration employing fluorescein-labeled dendrigrafts. The efficiency of dendrigraft internalization was enhanced by the presence of the guanidinium groups, while DGLs were preferentially localized in the nucleus and nuclear membrane, as revealed by fluorescence microscopy.

We have characterized in the oyster Crassostrea gigas an extracellular superoxide dismutase (Cg-EcSOD) which appears to bind lipopolysaccharides (LPS). The protein has been purified from the oyster plasma and identified as a Cu/ZnSOD according to its N-terminal sequencing and biological activity. Cg-EcSOD expression and synthesis are restricted to hemocytes as revealed by in situ hybridization and immunocytochemistry. Cg-EcSOD-expressing hemocytes were seen in blood circulation, in connective tissues, and closely associated to endothelium blood vessels. Cg-EcSOD presents in its amino acid sequence a LPS-binding motif found in the endotoxin receptor CD14 and we show that the protein displays an affinity to Escherichia coli bacteria and with LPS and Lipid A. Additionally, an RGD motif known to be implicated in the association to membrane integrin receptor is present in the amino acid sequence. The purified Cg-EcSOD was shown to bind to oyster hemocytes and to be immunocolocalized with a beta-integrin-like receptor.

Anti-lipopolysaccharide factors (ALFs), originally characterized from horseshoe crabs, have been recently identified from hemocytes of the black tiger shrimp, Penaeus monodon, by a genomic approach. In order to characterize the properties and biological activities of this immune effector in shrimp, ALFPm3, the most abundant isoform found in P. monodon, was expressed in the yeast Pichia pastoris. Large-scale production in fermentor provided 262mg/l of recombinant ALFPm3 which was purified to homogeneity by single chromatography step on expanded-bed Streamline SP6XL. The rALFPm3 was further characterized in terms of N-terminal sequencing and mass spectrometry. Anti-microbial assays demonstrated that rALFPm3 has a broad spectrum of anti-fungal properties against filamentous fungi, and anti-bacterial activities against both Gram-positive and Gram-negative bacteria, associated with a bactericidal effect. Interestingly, rALFPm3 is highly efficient against various Vibrio species including strains pathogenic for shrimp. Finally, a synthetic peptide corresponding to a part of the putative LPS-binding site of ALFPm3 was shown to display activities mainly directed against Gram-positive bacteria indicating the involvement of the full molecule to the anti-microbial activity for Gram-negative bacteria.