David P. Overy

Antimicrobial organometallic dendrimers with tunable activity against multidrug-resistant bacteria

Multidrug-resistant pathogens are an increasing threat to public health. In an effort to curb the virulence of these pathogens, new antimicrobial agents are sought. Here we report a new class of antimicrobial organometallic dendrimers with tunable activity against multidrug-resistant Gram-positive bacteria that included methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Mechanistically, these redox-active, cationic organometallic dendrimers induced oxidative stress on bacteria and also disrupted the microbial cell membrane. The minimum inhibitory concentrations, which provide a quantitative measure of the antimicrobial activity of these dendrimers, were in the low micromolar range. AlamarBlue cell viability assay also confirms the antimicrobial activity of these dendrimers. Interestingly, these dendrimers were noncytotoxic to epidermal cell lines and to mammalian red blood cells, making them potential antimicrobial platforms for topical applications.

An assessment of natural product discovery from marine (sensu strictu) and marine-derived fungi

The natural products community has been investigating secondary metabolites from marine fungi for several decades, but when one attempts to search for validated reports of new natural products from marine fungi, one encounters a literature saturated with reports from ‘marine-derived’ fungi. Of the 1000+ metabolites that have been characterized to date, only approximately 80 of these have been isolated from species from exclusively marine lineages. These metabolites are summarized here along with the lifestyle and habitats of their producing organisms. Furthermore, we address some of the reasons for the apparent disconnect between the stated objectives of discovering new chemistry from marine organisms and the apparent neglect of the truly exceptional obligate marine fungi. We also offer suggestions on how to reinvigorate enthusiasm for marine natural products discovery from fungi from exclusive marine lineages and highlight the need for critically assessing the role of apparently terrestrial fungi in the marine environment.

CLONAL EXPANSION OF THE PSEUDOGYMNOASCUS DESTRUCTANS GENOTYPE IN NORTH AMERICA IS ACCOMPANIED BY SIGNIFICANT VARIATION IN PHENOTYPIC EXPRESSION

Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition - dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

Sea foam as a source of fungal inoculum for the isolation of biologically active natural products

Due to a rate increase in the resistance of microbial pathogens to currently used antibiotics, there is a need in society for the discovery of novel antimicrobials. Historically, fungi are a proven source for antimicrobial compounds. The main goals of this study were to investigate the fungal diversity associated with sea foam collected around the coast of Prince Edward Island and the utility of this resource for the production of antimicrobial natural products. Obtained isolates were identified using ITS and nLSU rDNA sequences, fermented on four media, extracted and fractions enriched in secondary metabolites were screened for antimicrobial activity. The majority of the isolates obtained were ascomycetes, consisting of four recognized marine taxa along with other ubiquitous genera and many ‘unknown’ isolates that could not be identified to the species level using rDNA gene sequences. Secondary metabolite isolation efforts lead to the purification of the metabolites epolones A and B, pycnidione and coniothyrione from a strain of Neosetophoma samarorum; brefeldin A, leptosin J and the metabolite TMC-264 from an unknown fungus (probably representative of an Edenia sp.); and 1-hydroxy-6-methyl-8-hydroxymethylxanthone, chrysophanol and chrysophanol bianthrone from a Phaeospheria spartinae isolate. The biological activity of each of these metabolites was assessed against a panel of microbial pathogens as well as several cell lines.

Antimicrobial activity of non-natural prodigiosenes

Tripyrrolic prodigiosenes, derivatives of the natural product prodigiosin, have been produced via multi-step synthesis beginning with 2-formyl pyrroles bearing various functionalities at the 4-position. Two tin complexes are also reported, and these feature a prodigiosene ligand bearing a conjugated benzyl-ester. Antimicrobial activities of prodigiosenes are evaluated against Gram positive and Gram negative bacterial strains, as well as a yeast.

Phylogeny and intercontinental distribution of the pneumocandin-producing anamorphic fungus Glarea lozoyensis

Glarea lozoyensis is an anamorphic ascomycete that produces pneumocandin B0, the starting molecule for the synthesis of the antifungal drug caspofungin (CANCIDAS™). Glarea lozoyensis was first isolated in 1985 from a water sample from Madrid, Spain. Until now, only the original strain was known, but we have discovered new strains from Argentina and the USA. Molecular phylogenetic reference to a 28S rDNA database of antibiotic-producing fungi quickly identified these strains as being conspecific with G. lozoyensis. Bayesian inference phylogeny of ITS, 28S rDNA and α-actin gene fragments revealed that G. lozoyensis is related to species of the genus Cyathicula (Helotiales). Glarea lozoyensis was not conspecific with any of the Cyathicula species sequenced, although it appears to share a common ancestor. Glarea lozoyensis and Cyathicula strains were fermented on nutritional microarrays in 96-well plates. Cyathicula extracts did not show antifungal activity and did not produce pneumocandins, whereas potent antifungal activity and pneumocandin A0 production were confirmed for the four G. lozoyensis isolates. Also, culture morphology differed, with G. lozoyensis strains producing a dark brown, profusely sporulating mycelium with pigmented multicellular conidia accumulating in conidial masses, while all Cyathicula species tested in culture formed hyaline to light brown mycelia and lacked conidia. The chemistry and taxonomic distribution of the echinocandin class of antifungals is comprehensively reviewed.

Identification and characterization of lipases from Malassezia restricta, a causative agent of dandruff

Dandruff, a skin disorder affecting 50% of the world population, is linked with proliferation of lipophilic yeasts of the genus Malassezia (particularly Malassezia globosa and M. restricta). Most Malassezia species show a unique lipid dependency and require external lipids for growth. Genome mining of the incomplete M. restricta genome led to the identification of eight lipase sequences. Sequences representing the class 3 and LIP lipase families were used to clone the lipases MrLip1, MrLip2 and MrLip3, recombinantly expressed in Pichia pastoris, and tested for their activity using mono-, di- and triacylglycerol substrates. Hydrolysis by the M. restricta lipase MrLip1 and MrLip2 (family class 3) was limited to the mono- and diacylglycerol, while MrLip3 (family LIP) hydrolyzed all three substrates. This result confirms that Malassezia family LIP lipases are responsible for the hydrolysis of triacylglycerols, the main component of human sebum. Furthermore, the information regarding lipases from M. restricta presented here might aid in the search for anti-dandruff agents.

Highly Variable Bacterial Communities Associated with the Octocoral Antillogorgia elisabethae

Antillogorgia elisabethae (synonymous with Pseudopterogorgia elisabethae) is a common branching octocoral in Caribbean reef ecosystems. A. elisabethae is a rich source of anti-inflammatory diterpenes, thus this octocoral has been the subject of numerous natural product investigations, yet relatively little is known regarding the composition, diversity and the geographic and temporal stability of its microbiome. To characterize the composition, diversity and stability of bacterial communities of Bahamian A. elisabethae populations, 17 A. elisabethae samples originating from five sites within The Bahamas were characterized by 16S rDNA pyrosequencing. A. elisabethae bacterial communities were less diverse and distinct from those of surrounding seawater samples. Analyses of α- and β-diversity revealed that A. elisabethae bacterial communities were highly variable between A. elisabethae samples from The Bahamas. This contrasts results obtained from a previous study of three specimens collected from Providencia Island, Colombia, which found A. elisabethae bacterial communities to be highly structured. Taxa belonging to the Rhodobacteriales, Rhizobiales, Flavobacteriales and Oceanospiralles were identified as potential members of the A. elisabethae core microbiome.

Westerdykella reniformis sp. nov., producing the antibiotic metabolites melinacidin IV and chetracin B

Westerdykella reniformis Ebead & Overy sp. nov. is described based on morphology and phylogenetic analyses using ITS, nLSU rDNA, and β-tubulin gene sequences. Westerdykella reniformis is characterized by the production of cleistothecioid ascomata, containing small globose to subglobose asci with 32, aseptate, dark colored, pronouncedly reniform ascospores having a concave central groove. The isolate was obtained from a red alga (Polysiphonia sp.) collected from the tidal zone in Canada at low tide. Organic extracts enriched in extrolites, obtained from fermentation on a rice-based media, inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), S. warneri, and Proteus vulgaris. Presented here is the identification of the compounds responsible for the observed antimicrobial activity, the taxonomic description of W. reniformis, and a dichotomous key to the known species of Westerdykella based on macro- and micromorphological characters.

Redox-active cationic organoiron complex: a promising lead structure for developing antimicrobial agents with activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium

We report a new class of antimicrobial agent, a redox-active, cationic organometallic, η6-arene–η5-cyclopentadienyliron(II) complex, with activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Structure–property relationship investigations revealed that the antimicrobial activity against these pathogens, especially methicillin-resistant Staphylococcus aureus, is tunable. The ability of this new class of antimicrobial agent to induce cellular oxidative stress was confirmed using dichlorodihydrofluorescein assay. We attributed the induction of oxidative stress as a mechanism that contributes to the overall antimicrobial activity of these compounds. Generally, this antimicrobial agent was non-toxic to BJ fibroblast cell lines at ≤128 μg mL−1. The η6-arene–η5-cyclopentadienyliron(II) complex represents a potential lead structure for the development of topical antimicrobial therapeutics to combat resistant strains of Gram-positive bacteria.