Valerie J. Smith

Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential

Amongst the diverse and potent biological activities of free fatty acids (FFAs) is the ability to kill or inhibit the growth of bacteria. The antibacterial properties of FFAs are used by many organisms to defend against parasitic or pathogenic bacteria. Whilst their antibacterial mode of action is still poorly understood, the prime target of FFA action is the cell membrane, where FFAs disrupt the electron transport chain and oxidative phosphorylation. Besides interfering with cellular energy production, FFA action may also result from the inhibition of enzyme activity, impairment of nutrient uptake, generation of peroxidation and auto-oxidation degradation products or direct lysis of bacterial cells. Their broad spectrum of activity, non-specific mode of action and safety makes them attractive as antibacterial agents for various applications in medicine, agriculture and food preservation, especially where the use of conventional antibiotics is undesirable or prohibited. Moreover, the evolution of inducible FFA-resistant phenotypes is less problematic than with conventional antibiotics. The potential for commercial or biomedical exploitation of antibacterial FFAs, especially for those from natural sources, is discussed.

A comparison of phenoloxidase activity in the blood of marine invertebrates

A range of marine invertebrates were screened for prophenoloxidase (a marker protein of the prophenoloxidase activating system) in the coelomic fluid or hemolymph. The crustaceans and the ascidian, Ciona intestinalis, displayed strongest activity, with low levels found in the starfish, Asterias rubens, the sea urchin, D. antillarum, and the brachiopod, Liothyrella uva. The enzyme appeared to be absent from the isopod, Glyptonotus antarcticus. Further analyses of the blood cells of selected species revealed that prophenoloxidase tends to reside in the granular-type cells or their equivalent, and in the crustaceans and C. intestinalis is activated by lipopolysaccharides. In arthropods, prophenoloxidase is known to represent the terminal component of a complex cascade of enzymes that functions in non-self-recognition and host defence. The present study establishes that the enzyme is present in the blood cells of most, but not all, crustaceans and occurs in certain other invertebrate species, notably the urochordate, C. intestinalis.

β-l, 3 GLUCAN ACTIVATION OF CRUSTACEAN HEMOCYTES IN VITRO AND IN VIVO

The effects of β-1,3 glucans on the hemocytes of the freshwater crayfish, Astacus astacus, and the shore crab, Carcinus maenas, were studied in vitro and in vivo to determine the role of the prophenoloxidase activating system, in the cellular defense reactions of crustaceans.In vitro, phagocytosis of the bacterium, Moraxella sp. was significantly raised by addition of laminarin, a β-l,3 glucan, simultaneously with the test particles to the hemocytes in monolayer cultures. Both the proportion of cells ingesting one or more bacterial particles and the number of bacteria taken up by individual cells were increased, and the responses were found to be time dependent and dose related. Glucose, dextran, cellulose, and chitin had no stimulatory influence on the cells, and the agglutination of erythrocytes by crab hemocytes or serum was unchanged by glucan incubation. Examination of the monolayers under phase contrast microscopy, revealed that the glucans induced degranulation and occasionally lysis in the crayfis...

In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.)

This study examines the phagocytic hyaline cells of the shore crab, Carcinus maenas, for a respiratory burst in vitro. Following stimulation of the cells with phorbol myristate acetate (PMA), ferricytochrome c was reduced at 550 nm. Addition of superoxide dismutase (SOD) to the reaction mixture decreased this reduction, confirming that superoxide ions are produced by the stimulated hemocytes. Phytohemagglutinin, con A, and LPS were also shown to stimulate the cells although laminarin failed to elicit a burst. This is the first demonstration of a respiratory burst for crustacean hemocytes.

Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans

Crustins are antibacterial proteins of ca. 7-14 kDa with a characteristic four-disulphide core-containing whey acidic protein (WAP) domain, expressed by the circulating haemocytes of crustaceans. Over 50 crustin sequences have been now reported from a variety of decapods, including crabs, lobsters, shrimp and crayfish. Three main types seem to occur but all possess a signal sequence at the amino terminus and a WAP domain at the carboxyl end. Differences between types lie in the structure of the central region. Those crustins purified as the native protein or expressed recombinantly all kill Gram-positive bacteria, and gene studies have shown that they are constitutively expressed, often at high levels, but show no consistent patterns of change in expression following injection of bacteria. This variable response to infection is enigmatic but indicates that these proteins could perform additional functions, perhaps as immune regulators in recovery from wounding, trauma or physiological stress.

Non-cellular immunity in crustaceans

Following the tradition established for vertebrates, the host defences of invertebrates are often categorised as either humoral or cellular, i.e. mediated by factors in the plasma or brought about by activities of intact blood cells. This distinction, while convenient, may be inappropriate for some invertebrate groups and may cloud our understanding of the defence processes in lower animals. The present review considers the so-called humoral strategies in crustaceans and attempts to assess the current state of knowledge in this area. In addition, we explore the validity of separating cellular from non-cellular defence reactivity in these invertebrates. The presence, biological activity, biochemical features and origin of the agglutinins, killing factors, lysins, preciptins, cytokine-like molecules and clotting agents in the blood of various crustacean species are described, and their relationship to the cellular defences discussed. It is concluded that while some of these factors appear to represent components of a genuine humoral defence system, others are (or may be) derived from the circulating haemocytes, with several operating effectively only in conjunction with the cellular network. In view of the central role played by the cells in mediating immune capability in crustaceans we suggest that the acquisition of the complex clotting pathway in arthropods might have provided the means for the development of the cellular messenger systems characteristic of crustacean immunity. However, much still needs to be done to clarify the status, biological roles and source of the ‘humoral’ factors in crustaceans, particularly with respect to their biochemical identity and role in protection of the host. Until a better understanding of these defences is achieved, the prospects for the development of effective vaccines for commercially valuable species will be limited and the potential of the constituent factors as useful indicators of environmental stress will remain obscure.

Isolation and characterisation of oncorhyncin II, a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss.

A potent antimicrobial peptide, tentatively named oncorhyncin II, was isolated from an acid extract of rainbow trout skin secretions. Amino acid sequencing showed that the first 17 residues of oncorhyncin II are identical to residues 138-154 of histone H1 from rainbow trout. Matrix-assisted laser desorption ionization mass spectrometry revealed that the purified peptide has a molecular mass of 7195.3Da. Taken together, these data indicate that oncorhyncin II is a 69-residue C-terminal fragment of histone H1, probably phosphorylated at two residues. Oncorhyncin II has minimal inhibitory concentrations in the submicromolar range against Gram-(+) as well as Gram-(-) bacteria and it does not display significant haemolytic activity towards trout erythrocytes. The purified peptide was found to induce a marked destabilisation of planar lipid bilayers without the formation of stable ion channels. Oncorhyncin II is possibly a cleavage product of histone H1 with a potentially important role in mucosal defence of rainbow trout.

A Fatty Acid from the Diatom Phaeodactylum tricornutum is Antibacterial Against Diverse Bacteria Including Multi-resistant Staphylococcus aureus (MRSA)

Pathogenic bacteria, such as multidrug-resistant Staphylococcus aureus (MRSA), which are not susceptible to most conventional antibiotics, are causing increased concern in healthcare institutions worldwide. The discovery of novel antibacterial compounds for biomedical exploitation is one avenue that is being pursued to combat these problematic bacteria. Marine eukaryotic microalgae are known to produce numerous useful products but have attracted little attention in the search for novel antibiotic compounds. Cell lysates of the marine diatom, Phaeodactylum tricornutum Bohlin, have been reported to display antibacterial activity in vitro, but the compounds responsible have not been fully identified. In this paper, using column chromatography and reversed-phase high-performance liquid chromatography, we report the isolation of an antibacterial fatty acid. Mass spectrometry and 1H-nuclear magnetic resonance spectroscopy revealed it to be the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). We show that EPA is active against a range of both Gram-positive and Gram-negative bacteria, including MRSA, at micromolar concentrations. These data indicate that it could find application in the topical and systemic treatment of drug-resistant bacterial infections.

Conventional and Unconventional Antimicrobials from Fish, Marine Invertebrates and Micro-algae

All eukaryotic organisms, single-celled or multi-cellular, produce a diverse array of natural anti-infective agents that, in addition to conventional antimicrobial peptides, also include proteins and other molecules often not regarded as part of the innate defences. Examples range from histones, fatty acids, and other structural components of cells to pigments and regulatory proteins. These probably represent very ancient defence factors that have been re-used in new ways during evolution. This review discusses the nature, biological role in host protection and potential biotechnological uses of some of these compounds, focusing on those from fish, marine invertebrates and marine micro-algae.

Host Defence Reactions of the Shore Crab, Carcinus Maenas (L.), in Vitro

The Crustacea play an important economic role in the marine and aquatic environments not only as a food source but also in the productivity of the fisheries. Exploitation of these resources has led to a need for intensive culture methods which impose physiological stress on the animals and consequently increase the incidence of disease. The need to reduce the lethal and debilitating effects of pathogens has stimulated a renewed interest in the defence mechanisms of the Crustacea.