Sigmund Sperstad

Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.).

Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.

Antimicrobial peptides from marine invertebrates: Challenges and perspectives in marine antimicrobial peptide discovery

The emergence of pathogenic bacteria resistance to conventional antibiotics calls for an increased focus on the purification and characterization of antimicrobials with new mechanisms of actions. Antimicrobial peptides are promising candidates, because their initial interaction with microbes is through binding to lipids. The interference with such a fundamental cell structure is assumed to hamper resistance development. In the present review we discuss antimicrobial peptides isolated from marine invertebrates, emphasizing the isolation and activity of these natural antibiotics. The marine environment is relatively poorly explored in terms of potential pharmaceuticals, and it contains a tremendous species diversity which evolved in close proximity to microorganisms. As invertebrates rely purely on innate immunity, including antimicrobial peptides, to combat infectious agents, it is believed that immune effectors from these animals are efficient and rapid inhibitors of microbial growth.

Hyastatin, a glycine-rich multi-domain antimicrobial peptide isolated from the spider crab (Hyas araneus) hemocytes

Marine invertebrates are a rich source for the discovery of novel antimicrobial peptides, compounds regarded as important defense components in the host defense system. Here we report the purification and characterization of an 11.7kDa Gly-rich peptide, named hyastatin, from the hemocytes of Hyas araneus. It consists of three distinctly different domains: an N-terminal region enriched in Gly residues, a short Pro/Arg-rich region, and a C-terminal region containing six Cys residues with a Cys pattern resembling the one found in penaeidins. The C-terminus of the mature peptide is presumably amidated. The hyastatin transcript is constitutively expressed and mainly found in hemocytes. Hyastatin shows antimicrobial activity against yeasts, and Gram-positive and Gram-negative bacteria. The N-terminal region, devoid of the Cys-containing region, was recombinantly expressed in Escherichia coli cells, and shows only weak activity against the Gram-positive bacteria Corynebacterium glutamicum. Both hyastatin and the N-terminal region had the ability to bind chitin. Conclusively, this indicates the importance of the Cys-containing region for the antimicrobial activity, and a possible multifunctional character of hyastatin.

Capsaspora owczarzaki is an independent opisthokont lineage

Abstract Transitions from unicellularity to multicellularity have occurred several times in the history of life. Two of the most conspicuous multicellular kingdoms, Metazoa and Fungi, form a monophyletic clade, known as the opisthokonts, which comprises some unicellular lineages, such as Choanoflagellata, Nucleariidae, Ichthyosporea (also known as the DRIPs or Mesomycetozoea), Ministeria and Corallochytrium [1,2]. Among these unicellular opisthokonts we should expect to find the closest extant relatives of the multicellular Metazoa and Fungi. Choanoflagellates have traditionally been considered to be closely related to metazoans [1–6] and the Ichthyosporea are proposed to be the next closest outgroup [5]. By contrast, the position of the nucleariids is controversial [7–10]. Nuclearia sp. (ATCC 30864), recently renamed as Capsaspora owczarzaki, a filose amoeboid symbiont of a pulmonate snail, has never been confidently assigned to any opisthokont lineage [7–9]. Here, we report large subunit ribosomal and actin sequences of Capsaspora owczarzaki and an actin sequence of the novel ichthyosporean species Sphaeroforma arctica [11]. We test the phylogenetic position of Capsaspora owczarzaki , and the phylogenetic position of the nucleariids within the opisthokonts.

Characterization of crustins from the hemocytes of the spider crab, Hyas araneus, and the red king crab, Paralithodes camtschaticus.

Crustins are distributed across the decapods and are believed to play a significant part in the humoral defense system of their host. In this study, two crustin isoforms from Hyas araneus hemocytes were purified and tested for antimicrobial activity against selected microorganisms. They show both antibacterial and antifungal activity, with highest activity against the Gram-positive bacteria Corynebacterium glutamicum. Sequencing of the transcripts showed them to have a mature peptide of 90 amino acids and differing in three positions in the mature peptide. They were named CruHa1 and CruHa2. Real-time RT-PCR revealed that they mainly are expressed in hemocytes. Screening a cDNA library detected a crustin sequence in Paralithodes camtschaticus hemocytes, coding for a mature peptide of 98 amino acids. It was named CruPc. Based on phylogenetic inference and primary structure, CruHa1 and CruHa2 were placed within the Type I group of crustins, while CruPc belongs to the Type II.

Molecular-phylogenetic, structural and biochemical features of a cold-adapted, marine ichthyosporean near the animal-fungal divergence, described from in vitro cultures

A hitherto undescribed eukaryotic microorganism, provisionally named Sphaeroforma arctica, was isolated from the arctic marine amphipod Gammarus setosus. Phylogenetic inference based on a partial sequence of the small subunit ribosomal RNA (SSU-rRNA) gene, grouped the organism with members of the Ichthyophonida sub-clade of the recently established class Ichthyosporea. Its closest relative by SSU-rDNA analysis was Pseudoperkinsus tapetis, a Perkinsus-like clam pathogen of the Iberian peninsula. S. arctica and P. tapetis were found to share 99.3% sequence similarity. Under the in vitro growth conditions employed, the organism showed a continuous vegetative growth, manifested as steady proliferation of cells 5–7 m in diameter within a spherical wall derived from its parent cell. After approximately 48 h at 12 °C, the wall ruptured with the release of 120–150 individual cells, and the cycle was repeated. No budding, hyphal, amoeboid, sporal or flagellated growth stages were observed. The mitochondrial cristae of S. arctica were flattened. The cell wall of cells cultured in vitro was composed almost exclusively of carbohydrates, with N-acetyl-galactosamine as the major monomer, constituting close to 70% of the dry weight. Polyunsaturated fatty acids, mostly eicosapentaenoic and docosahexaenoic acid, constituted approximately 70% of total fatty acids, supporting an evolutionary adaptation to a life at permanently low temperatures. We conclude that S. arctica is a new species belonging to a new genus within the Ichthyosporea class of protists.

Expression of antimicrobial peptides from Hyas araneus haemocytes following bacterial challenge in vitro

Circulating haemocytes play major roles in the host defense reactions of decapods, including the synthesis and release of antimicrobial peptides (AMPs). Unlike the AMPs from insects, those in decapods are constitutively expressed. This study aims to establish primary cell cultures of the three main haemocyte types in Hyas araneus haemocytes, and to measure the in vitro expression of AMP genes in the cells following microbial challenge. The haemocyte populations were separated on Percoll gradients and cultured in modified L-15 medium. Expression analysis by real-time RT-PCR showed that the granular cells are the main producers of crustin, hyastatin and arasin 1 AMPs, but the hyaline cells and semigranular cells also show some expression of these genes. Incubating the cell populations with Aerococcus viridans var. homari (a Gram-positive bacterium) or Listonella anguillarum (a Gram-negative pathogen) provoked no dramatic changes in the gene expression of any of the AMP, and although there was a small (single doubling) significant increase in expression of the crustin gene in granular cells 24h after exposure to L. anguillarum, it is unclear if this is biologically relevant under in vitro conditions. The results presented in this study are in accordance with several in vivo studies.

Bacterial Community of Koumiss from Mongolia Investigated by Culture and Culture-Independent Methods

The bacterial community of fermented horse milk (koumiss) from Mongolia was studied using three methods: cultivation, direct identification by 16S rRNA clone library and denaturing gradient gel electrophoresis (DGGE). Ninety-eight strains were isolated by traditional cultivation and 61 of those were randomly selected for further identification by 16S rRNA gene sequencing. The strains were dominated by lactic acid bacteria (LAB; six different lactobacilli), Acinetobacter, Bacillus and Psychrobacter. Construction of the clone library analysis revealed that 16S sequences of 220 clones, genus Lactobacillus was dominant, but Streptococcus thermophilus, Acetobacter pasteurianus and uncultured clones were also detected. Ten unique bands were sequenced from the DGGE and revealed: Lactococcus lactis, Lactococcus lactis subsp. lactis, Clostridium acidurici, Acinetobacter johnsonii, Dickeya sp., Enterobacter sp., Pseudomonas sp., Raoultella sp., and Ruminococcus sp.. In vitro growth inhibition of three human pathogens, Escherichia coli, Enterobacter sakazakii and Staphylococcus aureus by 14 culturable bacteria displayed that only three of the isolates tested inhibit growth of E. sakazakii while most of the other bacteria delayed growth of the target bacteria.

Paralithocins, Antimicrobial Peptides with Unusual Disulfide Connectivity from the Red King Crab, Paralithodes camtschaticus

As part of an ongoing exploration of marine invertebrates as a source of new antimicrobial peptides, hemocyte extracts from the red king crab, Paralithodes camtschaticus, were studied. Three cationic cysteine (Cys)-rich peptides, named paralithocins 1-3, were isolated by bioassay-guided purification, and their amino acid sequences determined by Edman degradation and expressed sequences tag analysis. Disulfide bond mapping was performed by high-resolution tandem mass spectrometry. The peptides (38-51 amino acids in length) share a unique Cys motif composed of eight Cys, forming four disulfide bridges with a bond connectivity of (Cys relative position) Cys1-Cys8, Cys2-Cys6, Cys3-Cys5, and Cys4-Cys7, a disulfide arrangement that has not been previously reported among antimicrobial peptides. Thus, paralithocins 1-3 may be assigned to a previously unknown family of antimicrobial peptides within the group of Cys-rich antimicrobial peptides. Although none of the isolated peptides displayed antimicrobial activity against the target strains Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus, they inhibited the growth of several marine bacterial strains with minimal inhibitory concentrations in the 12.5-100 μM range. These findings corroborate the hypothesis that marine organisms are a valuable source for discovering bioactive peptides with new structural motifs.