The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5.
The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.
Baev
Didi
D
Department of Oral Biology, School of Dental Medicine, State University of New York, Buffalo, NY 14214, USA.
Rivetta
Alberto
A
Vylkova
Slavena
S
Sun
Jianing N
JN
Zeng
Ge-Fei
GF
Slayman
Clifford L
CL
Edgerton
Mira
M
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000483:Alleles; D000838:Anions; D000935:Antifungal Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry; D015153:Blotting, Western; D002176:Candida albicans / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D018118:Chloride Channels / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D017353:Gene Deletion; D005816:Genetic Complementation Test; D054808:Histatins; D006639:Histidine / Q000737:chemistry; D008956:Models, Chemical; D008957:Models, Genetic; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012413:Rubidium / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors
D000838:Anions; D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D027682:Cation Transport Proteins; D002412:Cations; D018118:Chloride Channels; D002712:Chlorides; D017931:DNA Primers; D018076:DNA, Complementary; C068075:HTN3 protein, human; D054808:Histatins; D009841:Oligonucleotides; D011480:Protease Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D012471:Salivary Proteins and Peptides; C496533:Trk1 protein, Candida albicans; C068664:TRK1 protein, S cerevisiae; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D006639:Histidine; D012313:RNA; D000255:Adenosine Triphosphate; D012413:Rubidium; D011188:Potassium