Lena Gerwick

The acute phase response and innate immunity of fish.

Tissue trauma or invasion by pathogens or parasites induce changes in the quantities of several macromolecules in animal body fluids. These changes comprise one aspect of the acute phase response (APR), which in toto involves metabolic changes in several organ systems. One clear indication of the response is the increase in synthesis and secretion by the liver of several plasma proteins, with simultaneous decreases in others. These acute phase proteins (APP) function in a variety of defense-related activities such as limiting the dispersal of infectious agents, repair of tissue damage, inactivation of proteases, killing of microbes and other potential pathogens, and restoration of the healthy state. Some APP are directly harmful to microbes, while others modify targets thus marking them for cell responses. Some work alone while others contribute to cascades. Proteins that are APP in mammals, and that have been identified in both teleosts and elasmobranchs include C-reactive protein, serum amyloid P, and several components of the Complement system. Others reported in teleosts include transferrin and thrombin. Of these, only CRP has been reported to increase in acute phase plasma. In trout, a precerebellin-like protein is an APP with unknown functions. A cDNA library enriched in fragments of transcripts that were more abundant in livers from fish undergoing an APR recently yielded sequences resembling 12 additional known APP, and as many others either not known to be APP, or not similar to others yet in public databases. It appears that, as in mammals, hepatocytes are the prime source of APP in fish, and that pro-inflammatory cytokines induce transcription of their genes.

Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization

To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to injection with a Vibrio bacterin emulsified in Freunds Incomplete Adjuvant. The resulting cDNA library contains 300-600bp long fragments of 25 or more immune-relevant genes. Fifteen were previously unreported for salmonids, and 12 were not known from any fish species. Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein. Components of both the complement system (n=5) and the clotting system (n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library. One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor. Three cDNAs appear to represent complete open reading frames.

Metamorphic enzyme assembly in polyketide diversification

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a β-branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide β-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH1s), decarboxylases (ECH2s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide β-branching pathway was modified by introduction of a γ-chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), α-ketoglutarate-dependent enzyme. In a divergent scheme, Cur ECH2 was found to catalyse formation of the α,β enoyl thioester, whereas Jam ECH2 formed a vinyl chloride moiety by selectively generating the corresponding β,γ enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH2 instead of the canonical α,β C = C saturation reaction. Thus, the combination of chlorination and polyketide β-branching, coupled with mechanistic diversification of ECH2 and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.

Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

Marine natural product drug discovery: Leads for treatment of inflammation, cancer, infections, and neurological disorders

Natural products, secondary metabolites, isolated from plants, animals and microbes are important sources for bioactive molecules that in many cases have been developed into treatments for diseases. This review will focus on describing the potential for finding new treatments from marine natural products for inflammation, cancer, infections, and neurological disorders. Historically terrestrial natural products have been studied to a greater extent and such classic drugs as aspirin, vincristine and many of the antibiotics are derived from terrestrial natural products. The need for new therapeutics in the four areas mentioned is dire. Within the last 30 years marine natural products, with their unique structures and high level of halogenation, have shown many promising activities against the inflammatory response, cancer, infections and neurological disorders. The review will outline examples of such compounds and activities.

Genomic insights into the physiology and ecology of the marine filamentous cyanobacterium Lyngbya majuscula.

Filamentous cyanobacteria of the genus Lyngbya are important contributors to coral reef ecosystems, occasionally forming dominant cover and impacting the health of many other co-occurring organisms. Moreover, they are extraordinarily rich sources of bioactive secondary metabolites, with 35% of all reported cyanobacterial natural products deriving from this single pantropical genus. However, the true natural product potential and life strategies of Lyngbya strains are poorly understood because of phylogenetic ambiguity, lack of genomic information, and their close associations with heterotrophic bacteria and other cyanobacteria. To gauge the natural product potential of Lyngbya and gain insights into potential microbial interactions, we sequenced the genome of Lyngbya majuscula 3L, a Caribbean strain that produces the tubulin polymerization inhibitor curacin A and the molluscicide barbamide, using a combination of Sanger and 454 sequencing approaches. Whereas ∼293,000 nucleotides of the draft genome are putatively dedicated to secondary metabolism, this is far too few to encode a large suite of Lyngbya metabolites, suggesting Lyngbya metabolites are strain specific and may be useful in species delineation. Our analysis revealed a complex gene regulatory network, including a large number of sigma factors and other regulatory proteins, indicating an enhanced ability for environmental adaptation or microbial associations. Although Lyngbya species are reported to fix nitrogen, nitrogenase genes were not found in the genome or by PCR of genomic DNA. Subsequent growth experiments confirmed that L. majuscula 3L is unable to fix atmospheric nitrogen. These unanticipated life history characteristics challenge current views of the genus Lyngbya.

The unique mechanistic transformations involved in the biosynthesis of modular natural products from marine cyanobacteria

Cyanobacteria are abundant producers of natural products well recognized for their bioactivity and utility in drug discovery and biotechnology applications. In the last decade, characterization of several modular gene clusters that code for the biosynthesis of these compounds has revealed a number of unusual enzymatic reactions. In this article, we review several mechanistic transformations identified in marine cyanobacterial biosynthetic pathways, with an emphasis on modular polyketide synthase(PKS)/non-ribosomal peptide synthetase (NRPS) gene clusters. In selected instances, we also make comparisons between cyanobacterial gene clusters derived from marine and freshwater strains. We then provide an overview of recent developments in cyanobacterial natural products biosynthesis made available through genome sequencing and new advances in bioinformatics and genetics.

Polyketide Decarboxylative Chain Termination Preceded by O-Sulfonation in Curacin A Biosynthesis

Biosynthetic innovation in natural product systems is driven by the recruitment of new genes and enzymes into these complex pathways. Here, an unprecedented decarboxylative chain termination mechanism is described for the polyketide synthase of curacin A, an anticancer lead compound isolated from the marine cyanobacterium Lyngbya majuscula. The unusual chain termination module containing adjacent sulfotransferase (ST) and thioesterase (TE) catalytic domains embedded in CurM was biochemically characterized. The TE was proved to catalyze a hydrolytic chain release of the polyketide chain elongation intermediate. Moreover, a selective ST-mediated sulfonation of the (R)-beta-hydroxyl group was found to precede TE-mediated hydrolysis, triggering a successive decarboxylative elimination and resulting in the formation of a rare terminal olefin in the final metabolite.

Characterization of cyanobacterial hydrocarbon composition and distribution of biosynthetic pathways

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.

Real-time metabolomics on living microorganisms using ambient electrospray ionization flow-probe.

Microorganisms such as bacteria and fungi produce a variety of specialized metabolites that are invaluable for agriculture, biological research, and drug discovery. However, the screening of microbial metabolic output is usually a time-intensive task. Here, we utilize a liquid microjunction surface sampling probe for electrospray ionization-mass spectrometry to extract and ionize metabolite mixtures directly from living microbial colonies grown on soft nutrient agar in Petri-dishes without any sample pretreatment. To demonstrate the robustness of the method, this technique was applied to observe the metabolic output of more than 30 microorganisms, including yeast, filamentous fungi, pathogens, and marine-derived bacteria, that were collected worldwide. Diverse natural products produced from different microbes, including Streptomyces coelicolor , Bacillus subtilis , and Pseudomonas aeruginosa are further characterized.