Synthetic molecular mimics of naturally occurring cyclopentenones exhibit antifungal activity towards pathogenic fungi

The naturally occurring reactive electrophilic species 12-oxo-phytodienoic acid (12-oxo-PDA) is a potent antifungal agent, whereas the plant growth regulator jasmonic acid, which is synthesized from 12-oxo-PDA, is ineffective. To address what structural features of the molecule endow it with antifungal activity, we synthesized a series of molecular mimics of 12-oxo-PDA varying in the length of the alkyl chain at its C-4 ring position. The octyl analogue (4-octyl cyclopentenone) was the most effective at suppressing spore germination and subsequent mycelial growth of a range of fungal pathogens and was particularly effective against Cladosporium herbarum and Botrytis cinerea, with minimum fungicidal concentrations in the range 100-200 µM. Introduction of a carboxyl group to the end of the chain, mimicking natural fatty acids, markedly reduced antifungal efficacy. Electrolyte leakage, indicative of membrane perturbation, was evident in both C. herbarum and B. cinerea exposed to 4-octyl cyclopentenone. Lipid composition analysis of the fungal spores revealed that those species with a high oil content, namely Fusarium oxysporum and Alternaria brassicicola, were less sensitive to 4-octyl cyclopentenone. The comparable hydrophobicity of 4-octyl cyclopentenone and 12-oxo-PDA accounts for the similar spore suppression activity of these two compounds. The relative ease of synthesis of 4-octyl cyclopentenone makes it an attractive compound for potential use as an antifungal agent. © 2011 SGM.

Aluminium and Alzheimer's disease: review of possible pathogenic mechanisms

Chronic exposure to aluminium (Al) remains a controversial possible cause of sporadic forms of Alzheimer's disease (AD). This article reviews the evidence that once Al enters the brain and individual brain cells, it may be involved in three pathological processes: (1) the production of abnormal forms of tau leading to the formation of cellular neurofibrillary tangles and neuropil threads; (2) the processing of the amyloid precursor protein, resulting in the formation of beta-amyloid deposits and senile plaques, and (3) that via the mutual histocompatibility system, Al could be involved in the initiation of the immune response observed in AD patients. Despite recent evidence that Al could be involved in these processes, a conclusive case that exposure to Al initiates the primary pathological process in sporadic AD remains to be established.

EPIPOX:immunoinformatic characterization of the shared T-cell epitome between variola virus and related pathogenic orthopoxviruses

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Structural and Biochemical Dissection of the Trehalose Biosynthetic Complex in Pathogenic Fungi

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

Diffusible Signal Factor (DSF)-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell-to-cell signals of the Diffusible Signal Factor (DSF) family are cis-2-unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signaling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.

Identification of potentially pathogenic variants in candidate breast and ovarian cancer susceptibility genes in BRCAX patients

Mutations within the BRCA1 and BRCA2 genes account for approximately 20% of hereditary breast cancers, with a further 10%–15% being attributable to rare mutations in moderate-risk genes and common variants in low-risk genes. The genes harbouring mutations in the remaining ∼65% of hereditary breast cancers are unknown. The identification of mutation carriers in hereditary breast and ovarian cancer (hboc) families is critical for determining who is most at risk of developing the disease and therefore who should be offered risk-reducing procedures or more intensive screening, or both.

Many of the high- and moderate-risk genes for hereditary breast cancers encode proteins that work in concert to maintain genomic stability and in dna damage signalling and repair. A novel BRCA1 protein complex identified within the research group whose target genes are involved in dna repair provided novel candidates for hboc susceptibility genes. These 12 candidate genes were sequenced in a cohort of 675 affected individuals from the Kathleen Cunningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) with hereditary breast or ovarian cancer, but with no mutations in known susceptibility genes (BRCAx patients). This analysis identified 20 individuals (each from a different BRCAx family) with different potentially pathogenic variants across 6 of the candidate hboc susceptibility genes. The family members of each BRCAx index case were tested for the presence of the specific mutation identified in the proband to examine segregation with disease. To further expand on the potential role of the novel candidate hboc susceptibility genes identified in this study, the genetic variation of a second cohort of 520 Northern Irish BRCAx patients is being characterized using a 61-gene panel.

A Loop-mediated Isothermal Amplification Method for Rapid Direct Detection and Differentiation of Non-pathogenic and Verocytotoxigenic Escherichia coli in Beef and Bovine Faeces

The aim of this study was to develop a multiplex loop-mediated isothermal amplification (LAMP) method capable of detecting Escherichia coli generally and verocytotoxigenic E. coli (VTEC) specifically in beef and bovine faeces. The LAMP assay developed was highly specific (100%) and able to distinguish between E. coli and VTEC based on the amplification of the phoA, and stx1 and/or stx2 genes, respectively. In the absence of an enrichment step, the limit of detection 50% (LOD50) of the LAMP assay was determined to be 2.83, 3.17 and 2.83-3.17 log CFU/g for E. coli with phoA, stx1 and stx2 genes, respectively, when artificially inoculated minced beef and bovine faeces were tested. The LAMP calibration curves generated with pure cultures, and spiked beef and faeces, suggested that the assay had good quantification capability. Validation of the assay, performed using retail beef and bovine faeces samples, demonstrated good correlation between counts obtained by the LAMP assay and by a conventional culture method, but suggested the possibility of false negative LAMP results for 12.5-14.7% of samples tested. The multiplex LAMP assay developed potentially represents a rapid alternative to culture for monitoring E.coli levels in beef or faeces and it would provide additional information on the presence of VTEC. However, some further optimisation is needed to improve detection sensitivity.

Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.

Pathogenic effect of microorganisms on loggerhead eggs

[EN] Different types of fungi and bacteria have been isolated from hatched and non-hatched as well as failed and non-failed eggs in natural sea turtles nests (Marco et al. 2006, Phillott and Parmenter, 2001, Phillott et al. 2001). Microbiota infections are common in artificial incubation activities and they seem to have an important negative impact on embryo development (Phillott, 2002). However, no clear evidences of their pathogenic effects have been described. The aim of this study was to investigate whether fungi and bacteria represent pathogenic agents to sea turtle eggs, and to assess whether there exists a specific period during incubation in which eggs are more susceptible to microorganisms.