Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

BACKGROUND Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. RESULTS The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate. CONCLUSION The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.Huiping Wang, Fanrong Kong, Tania C Sorrell, Bin Wang, Paul McNicholas, Namfon Pantarat, David Ellis, Meng Xiao, Fred Widmer and Sharon CA Che

Heterocyclic dithiocarbazate iron chelators: Fe coordination chemistry and biological activity

The iron coordination and biological chemistry of a series of heterocyclic dithiocarbazate Schiff base ligands is reported with regard to their activity as Fe chelators for the treatment of Fe overload and also cancer. The ligands are analogous to tridentate heterocyclic hydrazone and thiosemicarbazone chelators we have studied previously which bear NNO and NNS donor sets. The dithiocarbazate Schiff base ligands in this work also are NNS chelators and form stable low spin ferric and ferrous complexes and both have been isolated. In addition an unusual hydroxylated ligand derivative has been identified via an Fe-induced oxidation reaction. X-ray crystallographic and spectroscopic characterisation of these complexes has been carried out and also the electrochemical properties have been investigated. All Fe complexes exhibit totally reversible Fe couples in mixed aqueous solvents at potentials higher than found in analogous thiosemicarbazone Fe complexes. The ability of the dithiocarbazate Schiff base ligands to mobilise Fe from cells and also to prevent Fe uptake from transferrin was examined and all ligands were effective in chelating intracellular Fe relative to known controls such as the clinically important Fe chelator desferrioxamine. The Schiff base ligands derived from 2-pyridinecarbaldehyde were non-toxic to SK-N-MC neuroepithelioma (cancer) cells but those derived from the ketones 2-acetylpyridine and di-2-pyridyl ketone exhibited significant antiproliferative activity

Localized adoptive cellular therapy of Ex Vivo expanded Gamma Delta T cells for solid tumors

In recent years, adoptive cellular immunotherapy has emerged as a promising approach to treat cancers, particularly haematological cancers. However, its clinical utility in solid tumours is limited partly due to the requirement of transferred T cells to travel from the intravenous injection site to the tumour bed. Local administration of transferred T cells directly into the tumour mass or in the vicinity of a freshly resected tumour may be better suited to capitalize the full therapeutic potential of T cell adoptive therapy. This study explored the therapeutic potential of local delivery of gamma delta (Vγ9Vδ2) T cells (effector cells) in combination with zoledronate (cancer cell sensitiser) when embedded into biomaterial scaffolds for the local control of solid tumours using various cellular and animal models of breast cancer and glioblastoma. In vitro, ex vivo expanded Vγ9Vδ2 T cells maintained cell viability in Matrigel scaffolds and time-lapse video fluorescence imaging demonstrated progressive release of large numbers of Vγ9Vδ2 T cells from the scaffolds into surrounding area within just a few hours, rapidly killing cancer cells in their path. However, unlike the strong Vγ9Vδ2 T cell-mediated cytotoxicity observed in vitro, the effects seen in vivo using xenograft models of both breast cancer and glioblastoma were highly disappointing with only marginal anticancer efficacy observed. To explore the reasons for this, FACS was used to analyse the tumours at 72 hrs after the co-delivery of fluorescently labelled Vγ9Vδ2 T cells and fluorescent ZOL in Matrigel scaffolds. The data showed that, whereas Vγ9Vδ2 T cells were abundantly present in the tumour tissue, ZOL, on the other hand, could not be detected in cancer cells but rather accumulated selectively in the tumour resident macrophages and was preferentially rooted to the skeleton of mice due to its calcium binding characteristics. Collectively the data presented in this thesis clearly indicate that ZOL given locally, is unlikely to be of sufficient benefit in potentiating Vγ9Vδ2 T cell function in vivo and is therefore destined to fail clinically despite the overwhelming cytotoxicity observed in many cellular models. In this regard, the use of nanotechnology to improve cancer cell uptake of ZOL may open up a new therapeutic scenario for ZOL and Vγ9Vδ2 cell-based local immunotherapy.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202

Near-Infrared Photoimmunotherapy Using a Small Protein Mimetic for HER2-Overexpressing Breast Cancer

Near-infrared photoimmunotherapy (NIR-PIT) is a new and promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer which is activated by near-infrared light irradiation, causing cell death. We investigated NIR-PIT using a small protein mimetic (6–7 kDa), Affibody molecules, instead of a monoclonal antibody for HER2-overexpressing cancer. Because of its small size, the Affibody has rapid clearance, high imaging contrast, and good tumor penetration. Due to the small size of the Affibodies, which can cross the blood–brain barrier, NIR-PIT using Affibodies has the potential to extend the target cancer of NIR-PIT, including brain metastases. In vitro, NIR-PIT using HER2 Affibody–IR700Dye conjugates induced the selective destruction of HER2-overexpressing breast cancer cells without damage to control cells having low level expression of HER2. HER2-overexpressing cancer cells showed necrotic cell death and their viability maintained at low levels, even 5 days after NIR-PIT. In contrast, treatment with high concentration of HER2 Affibody–IR700Dye conjugate alone or irradiation with high dose of NIR light alone was without effect on cell viability. Affibody and IR700Dye are currently used clinically, and therefore, we would expect the current formulation to be safely and quickly transitioned into clinical trials

Low cytotoxic trace element selenium nanoparticles and their differential antimicrobial properties against S. aureus and E. coli

Antimicrobial agents that have no or low cytotoxicity and high specificity are desirable to have no or minimal side effects. We report here the low cytotoxicity of polyvinyl alcohol-stabilized selenium (Se) nanoparticles and their differential effects on growth of S. aureus, a gram-positive bacterium and E. coli, a gram-negative bacterium. The nanoparticles were synthesised through redox reactions in an aqueous environment at room temperature and were characterised using UV visible spectrophotometry, transmission electron microscopy, dynamic light scattering and x-ray photoelectron spectroscopy. The nanoparticles showed low toxicity toward fibroblasts which remained more than 70% viable at Se concentrations as high as 128 ppm. The nanoparticles also exhibited very low haemolysis with only 18% of maximal lysis observed at a Se concentration of 128 ppm. Importantly, the nanoparticles showed strong growth inhibition toward S. aureus at a concentration as low as 1 ppm. Interestingly, growth of E. coli was unaffected at all concentrations tested. This study therefore strongly suggests that these nanoparticles should be investigated further to understand this differential effect as well as for potential advanced antimicrobial applications such as S. aureus infection - resisting, non-cytotoxic coatings for medical devices. © 2016 IOP Publishing Ltd

Membrane transport and intracellular sequestration of novel thiosemicarbazone chelators for the treatment of cancer

Iron is a critical nutrient for DNA synthesis and cellular proliferation. Targeting iron in cancer cells using specific chelators is a potential new strategy for the development of novel anticancer agents. One such chelator, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), possesses potent and selective anticancer activity (J Med Chem 50:3716-3729, 2007). To elucidate the mechanisms of its potent antitumor activity, Bp4eT was labeled with ¹⁴C. Its efficacy was then compared with the 14C-labeled iron chelator pyridoxal isonicotinoyl hydrazone (PIH), which exhibits low anticancer activity. The ability of these ligands to permeate the cell membrane and their cellular retention was examined under various conditions using SK-N-MC neuroepithelioma cells. The rate of [ ¹⁴C]PIH uptake into cells was significantly (p < 0.001) lower than that of [ ¹⁴C]Bp4eT at 37°C, indicating that the increased hydrophilicity of [ 14C]PIH reduced membrane permeability. In contrast, the efflux of [ ¹⁴C]PIH was significantly (p < 0.05) higher than that of [ ¹⁴C]Bp4eT, leading to increased cellular retention of [ ¹⁴C]Bp4eT. In addition, the uptake and release of the 14C-labeled chelators was not reduced by metabolic inhibitors, indicating that these processes were energy-independent. No significant differences were evident in the uptake of [ ¹⁴C]Bp4eT at 37 or 4°C, demonstrating a temperature-independent mechanism. Furthermore, adjusting the pH of the culture medium to model the tumor microenvironment did not affect [ ¹⁴C]Bp4eT membrane transport. It can be concluded that [ ¹⁴C]Bp4eT more effectively permeated the cell membrane and evaded rapid efflux in contrast to [ ¹⁴C]PIH. This property, in part, accounts for the more potent anticancer activity of Bp4eT relative to PIH.10 page(s

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy

MIC and MBC of melittin for <i>F</i>. <i>nucleatum</i> and <i>S</i>. <i>mutans</i> and comparison with the membrane disrupting concentration (MDC) determined at different peptide/bacteria incubation times.

<p>MIC and MBC of melittin for <i>F</i>. <i>nucleatum</i> and <i>S</i>. <i>mutans</i> and comparison with the membrane disrupting concentration (MDC) determined at different peptide/bacteria incubation times.</p

Increasing concentration of the antimicrobial peptide Melittin increases bacterial membrane permeability to propidium iodide.

<p>(a) Schematic representation of the work flow for determining MIC, MBC and MDC. (b) Flow cytometry dot blots of <i>F</i>. <i>nucleatum</i> incubated with increasing concentrations of Melittin and stained with Syto9 dye (membrane permeable) and propidium iodide dye (membrane impermeable). (c) Percent propidium iodide positive <i>F</i>. <i>nucleatum</i> cells correlates with increasing peptide concentration. Insert shows the reciprocal plot of percent membrane disrupted cells (PI+) and peptide concentration to determine MDC.</p

Antimicrobial activity of Magainin analogues against <i>S</i>. <i>mutans</i>, and <i>F</i>. <i>nucleatum</i>.

<p>Antimicrobial activity of Magainin analogues against <i>S</i>. <i>mutans</i>, and <i>F</i>. <i>nucleatum</i>.</p