Structure-activity relationships of piscidin 4, a piscine antimicrobial peptide.

Piscidin 4, an antimicrobial peptide recently isolated from mast cells of hybrid striped bass (Morone chrysops female × Morone saxatilis male), is unusual in that it is twice as long (44 amino acids) as the typical members of the piscidin family. We previously showed that native piscidin 4 had a modified amino acid at position 20, but synthetic piscidin 4 (having an unmodified Trp at position 20) had similar potent activity against a number of both human and fish bacterial pathogens. In this study, the structure and membrane topology of synthetic piscidin 4 were examined using liposomes as model bilayers. Circular dichroism analyses revealed that it had a disordered structure in aqueous solution and folded to form a relatively weak α-helical structure in both membrane-mimetic trifluoroethanol solutions and liposome suspensions. Fluorescence data (piscidin 4 embedded in liposomes) and leakage experiments indicated that piscidin 4 interacted strongly with the hydrophobic part of the liposome. Binding of piscidin 4 to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp20). Quenching of Trp20 by water-soluble quencher (either acrylamide or I-) indicated that the fluorescence of Trp20 decreased more in the presence of liposomes than in buffer solution, thus revealing that Trp20 is less accessible to the quenchers in the presence of liposomes. The relative leakage abilities of piscidin 4 (1 µM) with liposomes were in the following order: DPPC (100%)≥EYPC (94%)>DPPC/DPPG (65%)>EYPC/EYPG (0%). This high activity against DPPC and EYPC liposomes was contrary to our data suggesting that piscidin 4 has a much weaker tendency to form an α-helix than other piscidins, such as piscidin 1. However, the structural similarity of protozoan membranes to EYPC liposomes might explain our discovery of the potent activity of piscidin 4 against the important skin/gill parasite ich (Ichthyophthirius multifiliis), but its negligible hemolytic activity against vertebrate membranes (hybrid striped bass or human erythrocytes). It also suggests that other conformation(s) in addition to the α-helix of this peptide may be responsible for its selective activity. This differential toxicity also suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass and may be capable of functioning extracellularly.

Evidence for widespread distribution of piscidin antimicrobial peptides in teleost fish.

Antimicrobial peptides (AMPs) are increasingly recognized as a critical component of the host's defense against infection. Several types of AMPs have been recently identified from mucosal tissues or immune cells of a number of teleosts. Among these are the piscidins, which are 22 residue, alpha-helical AMPs that were originally isolated from mast cells of hybrid striped bass Morone saxatilis male x Morone chrysops female. Using an antibody specific for the conserved N-terminal amino acid sequence of piscidin 1, we used immunohistochemistry to probe skin, gill, and gastrointestinal tract of 39 teleosts representing 7 different orders. Nine fish species were piscidin-positive, with all of these species being in the Perciformes, the largest and most evolutionarily advanced order of teleosts. Piscidin-positive cells were identified in species belonging to the families Moronidae, Serranidae, Sciaenidae, Siganidae and Belontidae. Immunopositive cells were usually most consistent with mast cells, although in some species, the granule appearance and tinctorial properties diverged somewhat from those of a typical piscine mast cell. In addition, rodlet cells were piscidin-positive in one member of the family Cichlidae; to our knowledge, it is the first time that a host-associated chemical biomarker has been identified in rodlet cells. Our data suggest that piscidins are present in many evolutionarily advanced teleosts. Piscidin-immunoreactive cells were most common at sites of pathogen entry, including the skin, gill and gastrointestinal tract. These results strongly suggest that piscidins are a widespread and important component of many fishes' defense against disease.

Effects of nonimmunized egg yolk powder-supplemented feed on Salmonella enteritidis prevention and elimination in broilers.

Chicken consumption is a newly identified risk factor in Salmonella enterica serovar Enteritidis (SE) infection in humans. SE is widely distributed in commercial chicken flocks and high levels of cecal carriage and shedding may lead to broiler meat contamination. In the present study, the preventive and eliminative effect of nonimmunized freeze-dried egg yolk powder (EYP) on SE in broilers was investigated. In the prevention trial, reduced SE counts were observed in liver (P < or = 0.05), cecal contents, and fecal shedding (P < or = 0.05) in birds fed 10% or 5% EYP. Histological examination of cecal wall and cecal tonsils at 23 days postinfection indicated a lesser degree of intestinal pathology. In the elimination trial, a significantly lower (P < or = 0.05) number of SE reached the liver and spleen, and a reduction in cecal carriage and fecal shedding was observed. The histological changes in the cecal mucosa and cecal tonsils reflected an apparent inflammation and mucosal repair and also suggested that the infection had not completely resolved, confirming SE bacterial isolations in the cecal tissue. The present study indicates that supplementing the diets of broilers with 5% nonimmunized EYP, at the early stages of the growing period, reduces preharvest Salmonella load with a minimal degree of intestinal pathology.

Histone-like proteins from fish are lethal to the parasitic dinoflagellate Amyloodinium ocellatum.

Antimicrobial proteins were purified from acid extracts of rainbow trout (Oncorhynchus mykiss) and sunshine bass (Morone saxatilis male x M. chrysops female) skin, gill and spleen by reverse-phase HPLC. Mass spectrometry and amino acid sequence data suggest that these proteins are closely related to histone H2B and histone H1 and thus they were designated histone-like proteins (HLPs). These proteins were lethal to Amyloodinium ocellatum, which is one of the most important parasitic agents affecting fish. Antibiotic concentrations as low as 12.5 microg/ml were inhibitory. Activity was directed against the trophont (feeding) stage of the parasite, while the disseminative (dinospore) stage was unaffected. Thus, HLPs act unlike typical drugs used to treat amyloodiniosis, which usually target the dinospore. Both the ability of the parasite to infect host cells, as well as the ability to grow and differentiate after infection were severely inhibited. This is in contrast to magainin 2, which was similarly toxic to both the dinospore and trophont stages. These findings provide further evidence that histone-like proteins may be important defensive molecules in fish.

Cladosporiosis in a tomato clownfish (Amphiprion frenatus).

Infection with Cladosporium sp., a deuteromycete, caused a deep dermal ulcer that extended to bone in a cultured tomato clownfish (Amphiprion frenatus). The infection may have been secondary to immunosuppression resulting from transport or confinement-induced stress or may have resulted from chronic exposure to copper. Reports of fungal infections in tropical marine fish are rare.