Peter D. Steinberg

Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip® microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein.

Acylated homoserine lactone (AHL)-mediated gene expression controls phenotypes involved in colonization, often specifically of higher organisms, in both marine and terrestrial environments. The marine red alga Delisea pulchra produces halogenated furanones which resemble AHLs structurally and show inhibitory activity at ecologically realistic concentrations in AHL bioassays. Evidence is presented that halogenated furanones displace tritiated OHHL [N-3-(oxohexanoyl)-L-homoserine lactone] from Escherichia coli cells overproducing LuxR with potencies corresponding to their respective inhibitory activities in an AHL-regulated bioluminescence assay, indicating that this is the mechanism by which furanones inhibit AHL-dependent phenotypes. Alternative mechanisms for this phenomenon are also addressed. General metabolic disruption was assessed with two-dimensional PAGE, revealing limited non-AHL-related effects. A direct chemical interaction between the algal compounds and AHLs, as monitored by 1H NMR spectroscopy, was shown not to occur in vitro. These results support the contention that furanones, at the concentrations produced by the alga, can control bacterial colonization of surfaces by specifically interfering with AHL-mediated gene expression at the level of the LuxR protein.

Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover

N-acyl-L-homoserine lactones (AHLs) are co-regulatory ligands required for control of the expression of genes encoding virulence traits in many Gram-negative bacterial species. Recent studies have indicated that AHLs modulate the cellular concentrations of LuxR-type regulatory proteins by binding and fortifying these proteins against proteolytic degradation (Zhu & Winans, 2001 ). Halogenated furanones produced by the macroalga Delisea pulchra inhibit AHL-dependent gene expression. This study assayed for an in vivo interaction between a tritiated halogenated furanone and the LuxR protein of Vibrio fischeri overproduced in Escherichia coli. Whilst a stable interaction between the algal metabolite and the bacterial protein was not found, it was noted by Western analysis that the half-life of the protein is reduced up to 100-fold in the presence of halogenated furanones. This suggests that halogenated furanones modulate LuxR activity but act to destabilize, rather than protect, the AHL-dependent transcriptional activator. The furanone-dependent reduction in the cellular concentration of the LuxR protein was associated with a reduction in expression of a plasmid encoded P(luxI)-gfp(ASV) fusion suggesting that the reduction in LuxR concentration is the mechanism by which furanones control expression of AHL-dependent phenotypes. The mode of action by which halogenated furanones reduce cellular concentrations of the LuxR protein remains to be characterized.

Bacterial community assembly based on functional genes rather than species

The principles underlying the assembly and structure of complex microbial communities are an issue of long-standing concern to the field of microbial ecology. We previously analyzed the community membership of bacterial communities associated with the green macroalga Ulva australis, and proposed a competitive lottery model for colonization of the algal surface in an attempt to explain the surprising lack of similarity in species composition across different algal samples. Here we extend the previous study by investigating the link between community structure and function in these communities, using metagenomic sequence analysis. Despite the high phylogenetic variability in microbial species composition on different U. australis (only 15% similarity between samples), similarity in functional composition was high (70%), and a core of functional genes present across all algal-associated communities was identified that were consistent with the ecology of surface- and host-associated bacteria. These functions were distributed widely across a variety of taxa or phylogenetic groups. This observation of similarity in habitat (niche) use with respect to functional genes, but not species, together with the relative ease with which bacteria share genetic material, suggests that the key level at which to address the assembly and structure of bacterial communities may not be “species” (by means of rRNA taxonomy), but rather the more functional level of genes.

Broad spectrum effects of secondary metabolites from the red alga delisea pulchra in antifouling assays

In this study the antifouling activity was investigated of a series of chemically related, halogenated furanones isolated from Delisea pulchra (Greville) Montagne, a red alga which is rarely fouled in the field. The metabolites were tested in laboratory assays against representatives of the three major groups of fouling organisms, the barnacle Balanus amphitrite amphitirite Darwin, the macroalga Ulva lactuca Linnaeus and a marine bacterium (strain SW 8). Settlement of barnacle cyprid larvae was strongly inhibited, with an EC50 of < 25 ng.ml‐1 (25 ppb) for some compounds. The settlement and growth of algal gametes was also strongly inhibited, in some cases at concentrations as low as 25 ngcm‐2. Growth of the marine bacterium SW8 was inhibited more strongly than by the common antibiotic gentamicin. Overall, activity of the D. pulchra metabolites was comparable to that of the heavy metals and biocides currently used in antifouling paints. However, no single compound was most active in all tests and some meta...

The tropicalization of temperate marine ecosystems: climate-mediated changes in herbivory and community phase shifts

Climate-driven changes in biotic interactions can profoundly alter ecological communities, particularly when they impact foundation species. In marine systems, changes in herbivory and the consequent loss of dominant habitat forming species can result in dramatic community phase shifts, such as from coral to macroalgal dominance when tropical fish herbivory decreases, and from algal forests to ‘barrens’ when temperate urchin grazing increases. Here, we propose a novel phase-shift away from macroalgal dominance caused by tropical herbivores extending their range into temperate regions. We argue that this phase shift is facilitated by poleward-flowing boundary currents that are creating ocean warming hotspots around the globe, enabling the range expansion of tropical species and increasing their grazing rates in temperate areas. Overgrazing of temperate macroalgae by tropical herbivorous fishes has already occurred in Japan and the Mediterranean. Emerging evidence suggests similar phenomena are occurring in other temperate regions, with increasing occurrence of tropical fishes on temperate reefs.

How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1

Halogenated furanones produced by the benthic marine macroalga Delisea pulchra inhibit swarming motility of Serratia liquefaciens MG1. This study demonstrates that exogenously added furanones control transcription of the quorum sensing regulated gene swrA in competition with the cognate signal molecule N:-butanoyl-L-homoserine lactone. This in turn results in reduced production of the surface-active compound serrawettin W2, which is crucial for surface translocation of the differentiated swarm cells. It is demonstrated that furanones interfere with interspecies communication during swarming of mixed cultures and that the mode of interference in quorum-sensing control and interspecies communication is not through inhibition of autoinducer synthesis.

Tolerance of Marine Invertebrate Herbivores to Brown Algal Phlorotannins in Temperate Australasia

Estes and Steinberg hypothesized that the intensity of sublittoral marine plant—herbivore interactions among different temperate regions has differed over the past 5—10 million yr, due to differences in the influence of diving mammals that prey on marine herbivores. They argued that because diving predators have not occurred in temperate Australasia, densities of sublittoral herbivores have been historically higher there than in comparable North American systems. This has resulted in marine algae and herbivores in temperate Australasia having strong selective effects on each other. Estes and Steinberg predicted that one result of these strong interactions would be that invertebrate herbivores in temperate Australasia should have evolved greater tolerance to brown algal phlorotannins (polyphenolic compounds that are the dominant secondary metabolites in temperate brown seaweeds) than similar herbivores in North America. We tested this prediction for five invertebrate herbivores in temperate Australasia. Mo...

Phlorotannin-protein interactions

Tannins are one of the most broadly distributed types of plant secondary compounds, and have been the focal point for many studies of plant/herbivore interactions. Tannins interact strongly with proteins, so that the fate and effects of ingested tannins are in part dependent on the mode of interaction of the tannin with dietary and endogenous proteins in an herbivores gut. We investigated the factors affecting the precipitation of proteins by phlorotannins from three species of marine brown algae:Carpophyllum maschalocarpum, Ecklonia radiata, andLobophora variegata. Phlorotannins were precipitated by proteins in a pH-dependent and concentration-dependent fashion. Precipitation also varied as a function of the presence of reducing agent, the type of phlorotannin or protein used, and the presence of organic solvents such as hydrogen bond inhibitors. Of particular significance was the ability of some phlorotannins to oxidize and form covalent bonds with some proteins. In contrast, under similar experimental conditions three types of terrestrial tannins (procyanidins, profisetinidins, and gallotannins) apparently did not form covalent complexes with proteins. Our results suggest several ways in which the biological activity of phlorotannins may vary as a function of the properties of the gut environment of marine herbivores. Moreover, we identify specific structural characteristics of phlorotannins which affect their tendency to oxidize, and thus, their potential effects on marine herbivores.

FEEDING PREFERENCES OF TEGULA FUNEBRALIS AND CHEMICAL DEFENSES OF MARINE BROWN ALGAE

I examined the effects of phenolic compounds in marine brown algae on the feeding behavior of the intertidal gastropod Tegula funebralis and other marine herbivores. The effects of algal nitrogen content and thallus toughness were also investigated. T. funebralis showed consistent feeding preferences in the laboratory and in the field in pairwise tests of 13 species of marine brown algae (Phaeophyta) from central California. Relative levels of phenolic compounds, which are known to deter feeding by herbivorous snails, differed greatly between preferred and nonpreferred species of algae. The total phenolic content of the six most preferred species of algae averaged 0.83% of dry mass; the phenolic content of the seven least preferred species averaged 4.53%. Differences in relative tanning ability were similarly large. Tannic acid, a terrestrial plant tanin, when experimentally added to one kelp species, deterred feeding by T. funebralis. These results and analyses of studies of additional species of brown algae also showed consistent differences in the phenolic content between species in the order Laminariales (typically low) and those in the order Fucales (uniformly high). Feeding preferences of other gastropod, echinoid, and crustacean algivores from the northeast Pacific Ocean are strikingly similar to those of T. funebralis; this supports the contention that high levels of phenolic compounds in brown algae are a generally effective feeding deterrent against many kinds of marine invertebrate herbivores. Organic nitrogen content of the algae was positively correlated with the feeding preferences of T. funebralis. However, this was most likely an indirect effect of the significant negative correlation between algal nitrogen and phenolic contents. Thallus toughness of the algae did not seem important in determining overall feeding preferences of Tegula, but may have affected choices between algae that were low in phenolics. I suggest that feeding preferences of Tegula funebralis for brown algae are primarily due to avoidance of algal defenses; positive aspects of food quality (such as attractants or nutritional content) probably play a lesser role in determining preference.