Anahit Penesyan

Development of Novel Drugs from Marine Surface Associated Microorganisms

While the oceans cover more than 70% of the Earth’s surface, marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds (“bioactives”) to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactives because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surfaces of marine eukaryotes. Chemically driven interactions are also important for the establishment of cross-relationships between microbes and their eukaryotic hosts, in which organisms producing antimicrobial compounds (“antimicrobials”), may protect the host surface against over colonisation in return for a nutrient rich environment. As is the case for bioactive discovery in general, progress in the detection and characterization of marine microbial bioactives has been limited by a number of obstacles, such as unsuitable culture conditions, laborious purification processes, and a lack of de-replication. However many of these limitations are now being overcome due to improved microbial cultivation techniques, microbial (meta-) genomic analysis and novel sensitive analytical tools for structural elucidation. Here we discuss how these technical advances, together with a better understanding of microbial and chemical ecology, will inevitably translate into an increase in the discovery and development of novel drugs from marine microbial sources in the future.

Marine Biofilm Bacteria Evade Eukaryotic Predation by Targeted Chemical Defense

Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.

Antibiotic Discovery: Combatting Bacterial Resistance in Cells and in Biofilm Communities

Bacterial resistance is a rapidly escalating threat to public health as our arsenal of effective antibiotics dwindles. Therefore, there is an urgent need for new antibiotics. Drug discovery has historically focused on bacteria growing in planktonic cultures. Many antibiotics were originally developed to target individual bacterial cells, being assessed in vitro against microorganisms in a planktonic mode of life. However, towards the end of the 20th century it became clear that many bacteria live as complex communities called biofilms in their natural habitat, and this includes habitats within a human host. The biofilm mode of life provides advantages to microorganisms, such as enhanced resistance towards environmental stresses, including antibiotic challenge. The community level resistance provided by biofilms is distinct from resistance mechanisms that operate at a cellular level, and cannot be overlooked in the development of novel strategies to combat infectious diseases. The review compares mechanisms of antibiotic resistance at cellular and community levels in the light of past and present antibiotic discovery efforts. Future perspectives on novel strategies for treatment of biofilm-related infectious diseases are explored.

Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment

Background Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions. Methodology/Principal Findings To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicatas oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions. Conclusions/Significance The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.

Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs

The surfaces of marine eukaryotes provide a unique habitat for colonizing microorganisms where competition between members of these communities and chemically mediated interactions with their host are thought to influence both microbial diversity and function. For example, it is believed that marine eukaryotes may use their surface-associated bacteria to produce bioactive compounds in defence against competition and to protect the host against further colonization. With the increasing need for novel drug discovery, marine epibiotic bacteria may thus represent a largely underexplored source of new antimicrobial compounds. In the current study, 325 bacterial isolates were obtained from the surfaces of marine algae Delisea pulchra and Ulva australis. Thirty-nine showed to have antimicrobial activity and were identified via 16S rRNA gene sequencing. The majority of those isolates belonged to Alpha- and Gammaproteobacteria. Interestingly, the most commonly isolated bacterial strain, Microbulbifer sp., from the surface of D. pulchra has previously been described as an ecologically significant epibiont of different marine eukaryotes. Other antimicrobial isolates obtained in this study belonged to the phyla Actinobacteria, Firmicutes and Bacteroidetes. Phylogenetically, little overlap was observed among the bacteria obtained from surfaces of D. pulchra and U. australis. The high abundance of cultured isolates that produce antimicrobials suggest that culturing remains a powerful resource for exploring novel bioactives of bacterial origin.

Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins

Significance Drug resistance is an increasing problem in clinical settings with some bacterial pathogens now resistant to virtually all available drugs. Chlorhexidine is a commonly used antiseptic and disinfectant in hospital environments, and there is increasing resistance to chlorhexidine seen in some pathogenic bacteria, such as Acinetobacter baumannii. This paper examines the global gene expression of A. baumannii in response to chlorhexidine exposure and identifies a gene that we demonstrate to mediate chlorhexidine resistance. Biochemical investigation reveals that this gene encodes a previously uncharacterized type of drug efflux pump that actively transports chlorhexidine out of the cell. Chlorhexidine is widely used as an antiseptic or disinfectant in both hospital and community settings. A number of bacterial species display resistance to this membrane-active biocide. We examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeAB. The next most highly overexpressed gene under chlorhexidine stress was annotated as encoding a hypothetical protein, named here as AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species. Expression of aceI or its orthologs from several other γ- or β-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [14C]-chlorhexidine determined that AceI was able to mediate the energy-dependent efflux of chlorhexidine. An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. Taken together, these data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial drug efflux transporters.

Identification of the Antibacterial Compound Produced by the Marine Epiphytic Bacterium Pseudovibrio sp. D323 and Related Sponge-Associated Bacteria

Surface-associated marine bacteria often produce secondary metabolites with antagonistic activities. In this study, tropodithietic acid (TDA) was identified to be responsible for the antibacterial activity of the marine epiphytic bacterium Pseudovibrio sp. D323 and related strains. Phenol was also produced by these bacteria but was not directly related to the antibacterial activity. TDA was shown to effectively inhibit a range of marine bacteria from various phylogenetic groups. However TDA-producers themselves were resistant and are likely to possess resistance mechanism preventing autoinhibition. We propose that TDA in isolate D323 and related eukaryote-associated bacteria plays a role in defending the host organism against unwanted microbial colonisation and, possibly, bacterial pathogens.

The effect of zinc limitation on the transcriptome of Pseudomonas protegens Pf-5

Zinc is an important nutrient but can be lacking in some soil environments, influencing the physiology of soil-dwelling bacteria. Hence, we studied the global effect of zinc limitation on the transcriptome of the rhizosphere biocontrol strain Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens). We observed that the expression of the putative zinc uptake regulator (Zur) gene was upregulated, and we mapped putative Zur binding sites in the Pf-5 genome using bioinformatic approaches. In line with the need to regulate intracellular zinc concentrations, an array of potential zinc transporter genes was found to be zinc-regulated. To adapt to low-zinc conditions, a gene cluster encoding non-zinc-requiring paralogues of zinc-dependent proteins was also significantly upregulated. Similarly, transcription of genes encoding non-zinc-requiring paralogues of ribosomal proteins L31 and L36 was increased by zinc limitation. A strong transcriptional downregulation of the putative copper chaperone gene (copZ) was also observed, suggesting interplay between zinc and copper homeostasis. Importantly, zinc also affected biocontrol attributes in Pf-5, most notably reducing the expression of the gene cluster responsible for biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (DAPG) under zinc limitation. This study clearly defines changes to the molecular physiology of Pf-5 that enable it to survive under zinc limitation.

Genetically and Phenotypically Distinct Pseudomonas aeruginosa Cystic Fibrosis Isolates Share a Core Proteomic Signature.

The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of “ready-made” nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such extrapolations may be fraught with peril.

Phenotypic profiling of Scedosporium aurantiacum, an opportunistic pathogen colonizing human lungs.

Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains.