Filipe Sansonetty

Hyperosmotic stress induces metacaspase - and mitochondria - dependent apoptosis in Saccharomyces cerevisiae

During the last years, several reports described an apoptosis‐like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Δcyc7Δ and cyc3Δ, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis‐like programmed cell death that is mediated by a caspase‐dependent mitochondrial pathway partially dependent on cytochrome c.

Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species.

Ibuprofen, a non-steroidal anti-inflammatory drug, exhibited antimicrobial activity against Candida albicans and non-albicans strains. At 10 mg/ml, ibuprofen showed a rapid cidal activity against exponential growth phase C. albicans, accompanied by rapid and extensive leakage of intracellular K+, permeation to propidium iodide, lysis of spheroplasts and severe membrane ultrastructural alterations. These results indicate that the killing of Candida cells is due to direct damage to the cytoplasmic membrane. At 5 mg/ml, ibuprofen inhibited growth; however, it did not kill the yeasts and did not directly affect the cytoplasmic membrane. Evaluation of yeast metabolic vitality with the fluorescent probe FUN-1 showed that growth inhibition induced by the fungistatic drug concentration was due to metabolic alterations. The combination of ibuprofen with fluconazole resulted in synergic activity with eight of the 12 Candida strains studied, including four of the five fluconazole-resistant strains. The MICs of fluconazole for the fluconazole-resistant strains decreased 2-128-fold when the drug was associated with ibuprofen. When in combination with fluconazole, MICs for ibuprofen decreased by up to 64-fold for all the 12 strains studied. These results point to the practicability of using ibuprofen, alone or in combination with azoles, in the treatment of candidosis, particularly when applied topically, taking advantage of the drugs antifungal and anti-inflammatory properties.

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli

BackgroundEfflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems.ResultsEtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 ± 0.0057 min-1; = 0.0106 ± 0.0033 min-1; and = 0.0230 ± 0.0075 min-1.ConclusionThe combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall.

Flow Cytometric Kinetic Assay of Calcium Mobilization in Whole Blood Platelets Using Fluo-3 and CD41

BACKGROUND Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. METHODS Anticoagulated blood was diluted in Tyrodes buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. RESULTS In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. CONCLUSIONS Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy.

Antifungal activity of local anesthetics against Candida species.

OBJECTIVE: To evaluate the activity of benzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism of activity. METHODS: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains). The fungistatic activity was studied with the fluorescent probe FUN-1 and observation under epifluorescence microscopy and flow cytometry. The fungicidal activity of the three drugs was assayed by viability counts. Membrane alterations induced in the yeast cells were evaluated by staining with propidium iodide, by quantitation of intracellular K+ leakage and by transmission electron microscopy of intact yeast cells and prepared spheroplasts. RESULTS: The MIC ranged from 12.5-50.0 microg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively. The inhibitory activity of these concentrations could be detected with the fluorescent probe FUN-1 after incubation for 60 minutes. A very fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL of benzydamine, lidocaine, and bupivacaine, respectively. CONCLUSIONS: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while at higher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane.

Flow cytometric analysis of chronic and acute toxicity of copper(II) on the marine dinoflagellate Amphidinium carterae

BACKGROUND Copper(II) is a heavy metal whose levels have increased in some marine ecosystems to polluting levels. Dinoflagellates, an important phytoplankton group, are at the base of aquatic food chains and bioaccumulation of copper by these microorganisms can result in complex ecosystem alterations, so we investigated how copper disturbs those cells. METHODS Cytotoxic effects of sublethal and lethal copper concentrations ranging from 4.2 nM (control condition) to 3.13 microM estimated labile copper were studied in batch cultures of Amphidinium carterae. Cell morphology, motility, autofluorescence, and fluorescein diacetate (FDA)-dependent fluorescence generation were evaluated by flow cytometry (FCM) and microscopy. RESULTS Exposure of A. carterae to toxic levels of copper impaired cell mobility, delayed cell proliferation, led to increased green autofluorescence, and at 3.13 microM labile copper also induced encystment and death. Chlorophyll fluorescence, however, was not affected. Kinetic FCM assay of FDA-dependent fluorescence generation showed a dose-dependent enhancement of fluorescein fluorescence immediately after copper addition and in cultures with sustained exposure to this toxicant. CONCLUSIONS Our data suggest that copper toxicity occurs quickly at the membrane level in relation to oxidative stress generation. Based on fluorescence kinetic studies, the Na(+)/H(+) antiporter seemed to be affected by copper, thereby affecting intracellular pH.

Flow cytometric assessment of cell structural and functional changes induced by acetic acid in the yeasts Zygosaccharomyces bailii and Saccharomyces cerevisiae

Flow cytometry (FCM) was used with different viability dyes to assess changes in cell structure and function induced by acetic acid (AA) in populations of Zygosaccharomyces bailii (AA resistant) and Saccharomyces cerevisiae (AA sensitive). Kinetic changes in esterase activity, intracellular dye processing, and membrane integrity were monitored, and to detect those changes we used three assays involving fluorescein diacetate hydrolysis, FUN-1 processing, and propidium iodide exclusion, respectively. In S. cerevisiae, the decrease in the ability to process FUN-1 preceded the decrease in esterase activity, and there was loss of cell membrane integrity after incubation with AA. In Z. bailii, with higher AA concentrations, there was a similar decrease in the ability to process FUN-1, which also preceded the loss of cell membrane integrity. Changes in esterase activity in this yeast induced by AA treatment could not be monitored because the changes occurred independently of the presence of the acid. For control samples (untreated cells killed with 10% v/v of AA), the percentages of nonaltered cells as estimated by FCM and percentages of viable cells as estimated by colony forming unit (CFU) counts were identical. However, for cell samples treated for short periods with 3% (v/v) or less of AA, none of the dyes produced FCM results comparable to those produced by CFU counts.

Energy conversion coupled to cyanide-resistant respiration in the yeasts Pichia membranifaciens and Debaryomyces hansenii

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.

Susceptibility to fluconazole of Candida clinical isolates determined by FUN-1 staining with flow cytometry and epifluorescence microscopy.

The susceptibility of clinical Candida isolates to fluconazole was assayed by flow cytometry (FCM) and epifluorescence microscopy (EFM), with FUN-1 staining. In all, 25 clinical isolates of Candida spp. (12 sensitive, 3 dose-dependently sensitive and 10 resistant to fluconazole according to the NCCLS M27-A protocol) were treated with increasing concentrations of fluconazole during 1 or 2 h staining with FUN-1 for 30 min and analysed, respectively, by FCM at 575 nm (FL2) and by EFM. Fluconazole-susceptible strains showed an increased accumulation of FUN-1 in comparison with controls as determined by FCM and a reduced metabolic processing of the probe, confirmed by EFM. Conversely, resistant strains showed decreased FUN-1 staining and were able to process the probe. The fluconazole minimal inhibitory concentrations (MICs) determined by FCM or EFM after FUN-1 staining compared very well with the corresponding values determined by the M27-A protocol, indicating that FUN-1 staining can be used as an alternative to the conventional method. MIC values of resistant strains, with the exception of C. krusei, were lower when treatment with fluconazole followed pre-incubation with 0.1 mM sodium azide, a concentration known to inhibit the activity of efflux pumps. These results show that FUN-1 staining can be used as an alternative and rapid method for the assessment of susceptibility of Candida clinical isolates to fluconazole. Furthermore, the results suggest that resistance of Candida cells to fluconazole, with the exception of C. krusei strains, is likely to be due to the activity of efflux pumps.

Rapid detection of efflux pumps and their relation with drug resistance in yeast cells

BACKGROUND Cell drug resistance can be due to the presence of active efflux pumps (AEP). Identification of yeast cells with a resistance phenotype is important either from a clinical, agricultural or biotechnological point of view. Rapid and reliable methods to detect AEP can be therefore very useful. METHODS Some yeast cells change their staining by calcein-AM, BCECF-AM, rhodamine 123 and DiOC(5), when pretreated with verapamil, CCCP or ATP depletion, or when pretreated with specific antimicrobial agents. This fact may be interpreted as an indication of the presence/absence of AEP. Six yeast species were tested with a flow cytometric method (FCM) and an epifluorescence microscopic method (EFM), and ten other species were evaluated only by EFM. The minimum inhibitory concentration (MIC) of penconazol, benomyl and cycloheximide for Saccharomyces cerevisiae and Kluyveromyces marxianus, were determined by growth inhibition on solid medium and were compared to the staining changes detected by FCM. RESULTS The FCM and the EFM allowed the detection of AEP in all the yeast species tested. High MIC values for a drug were related with the presence of at least one AEP indicated by the cytometric data. CONCLUSIONS The FCM revealed to be a robust assay whereas the EFM can be used as a preliminary test. It is possible to identify resistance/sensitivity patterns in yeast cells through cytometric detection methods of different efflux pumping systems.