Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species

<p>Abstract</p> <p>Background</p> <p>The virulence of <it>Candida </it>species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various <it>Candida </it>species has not been studied so far.</p> <p>Results</p> <p>The tolerance/sensitivity of four pathogenic <it>Candida </it>species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na⁺/H⁺antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, <it>C. dubliniensis </it>and <it>C. parapsilosis </it>respectively, were identified, cloned and functionally expressed in the plasma membranes of <it>Saccharomyces cerevisiae </it>cells lacking their own cation exporters. Both <it>Cp</it>Cnh1 and <it>Cd</it>Cnh1 antiporters had broad substrate specificity and transported Na⁺, K⁺, Li⁺, and Rb⁺. Their activity in <it>S. cerevisiae </it>cells differed; <it>Cp</it>Cnh1p provided cells with a much higher salt tolerance than the <it>Cd</it>Cnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters.</p> <p>Conclusion</p> <p>We have cloned two genes encoding putative Na⁺/H⁺antiporters in <it>C. parapsilosis </it>and <it>C. dubliniensis</it>, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na⁺/H⁺antiporters is one of the factors determining the tolerance of pathogenic <it>Candida </it>species to high external concentrations of alkali metal cations.</p

Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast <it>Saccharomyces cerevisiae </it>the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the α-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal <it>kex2 </it>deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates.</p> <p>Results</p> <p>In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal <it>kex2 </it>deletion mutants by <it>in vitro </it>digestion of recombinant substrates from <it>Candida albicans </it>and <it>C. glabrata</it>. We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates.</p> <p>Conclusion</p> <p>Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from <it>S. cerevisiae</it>, <it>P. pastoris</it>, <it>C. albicans </it>and <it>C. glabrata</it>, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.</p

Plasma-membrane alkali-metal-cation transporters involved in salt tolerance of pathogenic Candida species

of Ph.D.Thesis Conclusions All the aims of the thesis were achieved.The toleranceto alkali metalcationsof four pathogenic Candida species was studied in detail and revealed differencesamong the yeasts. These differences in sall tolerance remained the same under various growth conditions.For the first time, the internalsodium and potassiumconcentrationsof several Candidaspecieswere estimatedunder highsalt-stressgrolvth.The internalK./Na* ratiowas not in relationwith the salt tolerancerevealingdifferentadaptationmechanismsto salt stress in Candida species. A first study on combinatoryuse of fluconazoleand NaCl revealed severe synergisticeffects of both compounds, leading to grovvthinhibitionand increased internalNa* concentrationsin C. albicans.The molecularbasis of this synergismremainsto be established. Ihe C. dubliniensis, C. glabrata and C. parapsilosis Cnhl NalH- antiporters were cloned and functionallycharacterizedupon heterologousexpression in S. cerevlslae to understandthe mechanismsinvolvedin the differentsalttolerancesof Candidaspecies.The three antiportersdifferedin theiractivityfor alkalimetalcations,which roughlycorrelatedwith the observed differences in salt tolerance among the species. Additionally,during the characterizationof heteroiogouslyexpressed antiporters,two antiporter chimeras..

Úloha transportéru kationtů alkalických kovů plasmatické membrány pathogenních druhů kvasinek v toleranci vůči solím

of Ph.D.Thesis Conclusions All the aims of the thesis were achieved.The toleranceto alkali metalcationsof four pathogenic Candida species was studied in detail and revealed differencesamong the yeasts. These differences in sall tolerance remained the same under various growth conditions.For the first time, the internalsodium and potassiumconcentrationsof several Candidaspecieswere estimatedunder highsalt-stressgrolvth.The internalK./Na* ratiowas not in relationwith the salt tolerancerevealingdifferentadaptationmechanismsto salt stress in Candida species. A first study on combinatoryuse of fluconazoleand NaCl revealed severe synergisticeffects of both compounds, leading to grovvthinhibitionand increased internalNa* concentrationsin C. albicans.The molecularbasis of this synergismremainsto be established. Ihe C. dubliniensis, C. glabrata and C. parapsilosis Cnhl NalH- antiporters were cloned and functionallycharacterizedupon heterologousexpression in S. cerevlslae to understandthe mechanismsinvolvedin the differentsalttolerancesof Candidaspecies.The three antiportersdifferedin theiractivityfor alkalimetalcations,which roughlycorrelatedwith the observed differences in salt tolerance among the species. Additionally,during the characterizationof heteroiogouslyexpressed antiporters,two antiporter chimeras...of Ph.D. Thesis Conclusions All the aims of the thesis were achieved. The tolerance to alkali metal cations of four pathogenic Candida species was studied in detail and revealed differences among the yeasts. These differences in sall tolerance remained the same under various growth conditions. For the first time, the internal sodium and potassium concentrations of several Candida species were estimated under high salt-stress grolvth. The internal K./Na* ratio was not in relation with the salt tolerance revealing different adaptation mechanisms to salt stress in Candida species. A first study on combinatory use of fluconazole and NaCl revealed severe synergistic effects of both compounds, leading to grovvth inhibition and increased internal Na* concentrationsi n C. albicans.T he molecularb asis of this synergismr emains to be established. Ihe C. dubliniensis, C. glabrata and C. parapsilosis Cnhl NalH- antiporters were cloned and functionally characterized upon heterologous expression in S. cerevlslae to understand the mechanisms involved in the different salttolerances of Candida species. The three antiporters differed in their activity for alkali metal cations, which roughly correlated with the observed differences in salt tolerance among the species. Additionally, during the characterization of...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Functional comparison of plasma-membrane Na/Hantiporters from two pathogenic species-3

Phase and proteins extracted. After separation of proteins via SDS-PAGE they were transferred to nitrocellulose membrane and detected with anti-GFP antibody.<p><b>Copyright information:</b></p><p>Taken from "Functional comparison of plasma-membrane Na/Hantiporters from two pathogenic species"</p><p>http://www.biomedcentral.com/1471-2180/8/80</p><p>BMC Microbiology 2008;8():80-80.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2424070.</p><p></p

Functional comparison of plasma-membrane Na/Hantiporters from two pathogenic species-4

or expressing Cnh1p (■), Cnh1p (▲), Cnh1p (♦) antiporters was measured. The initial concentrations of cations in cells (corresponding to 100%) were as follows: Na, 110.5 ± 6.8 nmol (mg dry wt), K, 549.5 ± 25.7 nmol (mg dry wt)(representative results are shown).<p><b>Copyright information:</b></p><p>Taken from "Functional comparison of plasma-membrane Na/Hantiporters from two pathogenic species"</p><p>http://www.biomedcentral.com/1471-2180/8/80</p><p>BMC Microbiology 2008;8():80-80.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2424070.</p><p></p

Growth of various species and on YPD medium supplemented with salts at 30°C

<p><b>Copyright information:</b></p><p>Taken from "Functional comparison of plasma-membrane Na/Hantiporters from two pathogenic species"</p><p>http://www.biomedcentral.com/1471-2180/8/80</p><p>BMC Microbiology 2008;8():80-80.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2424070.</p><p></p

Functional comparison of plasma-membrane Na/Hantiporters from two pathogenic species-1

At 30°C. Cells with empty YEp352 vector (-) were used as negative, cells expressing Nha1p as positive control, respectively. Pictures were taken after 4 (YNB) or 7 days (YNB + salts) of incubation.<p><b>Copyright information:</b></p><p>Taken from "Functional comparison of plasma-membrane Na/Hantiporters from two pathogenic species"</p><p>http://www.biomedcentral.com/1471-2180/8/80</p><p>BMC Microbiology 2008;8():80-80.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2424070.</p><p></p

Green cannabigerol purification through simulated moving bed chromatography

Cannabigerol (CBG) is a minor cannabinoid present in Cannabis sativa L. This molecule is gaining increasing popularity thanks to its antibacterial, antimicrobial, antidepressant and antitumoral properties. In parallel, there is growing attention towards the search of efficient, cost-effective, rapid, high-throughput, and green purification techniques.In this work, CBG has been purified from a real cannabis extract by means of simulated moving bed chromatography. The proposed application is very promising, allowing to achieve a CBG extract free of tetrahydrocannabinol (a psychoactive cannabinoid) with 100% recovery and 97% final purity by using a faster and greener method if compared to traditionally used ones