Candida albicans Flu1-Mediated Efflux of Salivary Histatin 5 Reduces Its Cytosolic Concentration and Fungicidal Activity

Abstract

Histatin 5 (Hst 5) is a salivary human antimicrobial peptide that is toxic to the opportunistic yeast Candida albicans. Fungicidal activity of Hst 5 requires intracellular translocation and accumulation to a threshold concentration for it to disrupt cellular processes. Previously, we observed that total cytosolic levels of Hst 5 were gradually reduced from intact cells, suggesting that C. albicans possesses a transport mechanism for efflux of Hst 5. Since we identified C. albicans polyamine transporters responsible for Hst 5 uptake, we hypothesized that one or more polyamine efflux transporters may be involved in the efflux of Hst 5. C. albicans FLU1 and TPO2 were found to be the closest homologs of Saccharomyces cerevisiae TPO1, which encodes a major spermidine efflux transporter, indicating that the products of these two genes may be involved in efflux of Hst 5. We found that flu1⌬/⌬ cells, but not tpo2⌬/⌬ cells, had significant reductions in their rates of Hst 5 efflux and had significantly higher cytoplasmic Hst 5 and Hst 5 susceptibilities than did the wild type. We also found that flu1⌬/⌬ cells had reduced biofilm formation compared to wild-type cells in the presence of Hst 5. Transcriptional levels of FLU1 were not altered over the course of treatment with Hst 5; therefore, Hst 5 is not likely to induce FLU1 gene overexpression as a potential mechanism of resistance. Thus, Flu1, but not Tpo2, mediates efflux of Hst 5 and is responsible for reduction of its toxicity in C. albicans. Candida albicans is a human fungal pathogen that causes serious infections in immunocompromised populations (1-3). In hospitalized patients, this organism can disseminate hematogenously and infect virtually all organs (4-6). The incidence and mortality rates associated with this infectious disease have remained unchanged for more than a decade despite major advances in the field of antifungal therapy (6, 7).Azoles are one of the most widely used groups of antifungal drugs used to treat candidiasis patients and represent a class of five-membered, nitrogen-containing, heterocyclic compounds (8, 9). The azole drugs enter C. albicans cells by facilitated diffusion (10) and inhibit the biosynthesis of ergosterol, which is a major component of C. albicans membranes (11-13). However, the widespread use of azoles coupled with the fungistatic nature of these drugs has led to the emergence of resistance in clinical isolates (8,14,15). Mechanisms of azole resistance include alteration (either by mutation or by overexpression) of the drug target enzyme lanosterol demethylase (encoded by ERG11) and/or constitutive overexpression of multidrug transporters (16).There are two main classes of multidrug transporters that are found to be upregulated in drug-resistant clinical isolates and experimentally evolved populations of C. albicans; these include transporters containing ATP-binding cassettes (ABC) and transporters of the major facilitator superfamily (MFS) (17-20). The ABC transporter superfamily, including Cdr1 and Cdr2, consists of membran...

Interplay between Candida albicans and the Antimicrobial Peptide Armory

“…The polyamine efflux transporter Flu1, a member of the MDR (multidrug resistance) family, was found to mediate efflux of Hst 5 in C. albicans, resulting in reduction of AMP toxicity (60). Concordantly, deletion of FLU1 reduced the efflux of Hst 5 and therefore increased sensitivity to the AMP.…”

Membrane Proteome-Wide Response to the Antifungal Drug Clotrimazole in Candida glabrata: Role of the Transcription Factor CgPdr1 and the Drug:H+ Antiporters CgTpo1_1 and CgTpo1_2

“…Interestingly, the Tpo1 homolog in C. albicans, CaFlu1, was found to complement fluconazole hypersusceptibility in a S. cerevisiae ⌬pdr5 mutant but not to have a significant role in fluconazole resistance in C. albicans (23). More recently, Flu1 was shown to confer resistance to the salivary human antimicrobial peptide histatin 5, playing a direct role in its efflux from C. albicans cells, thus reducing histatin 5 toxicity (24). Based on the proteomics data, CgTPO1_1 and CgTPO1_2 were further analyzed, in this study.…”

A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

“…On the basis of the findings of previous studies with oral and systemic isolates, long-term azole treatment and long-term exposure select for azole-resistant C. albicans isolates (11)(12)(13). Several factors can contribute to drug resistance in this organism.…”