C. albicans Flu1 mediated efflux of salivary Histatin 5 reduces its cytosolic concentration and fungicidal activity.
ABSTRACT Histatin 5 is a salivary human antimicrobial peptide that is toxic to the opportunistic yeast Candida albicans. Fungicial 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 S. cerevisiae TPO1 that encodes a major spermidine efflux transporter, indicating the products of these two genes may be involved in efflux of Hst 5. We found flu1Δ/Δ cells, but not tpo2Δ/Δ cells, had significant reduction in their rate of Hst 5 efflux, and had significantly higher cytoplasmic Hst 5 and Hst 5 susceptibility compared to wild type. We also found that flu1Δ/Δ 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 over-expression 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.
- SourceAvailable from: Dr. Bheru S. Kitawat[Show abstract] [Hide abstract]
ABSTRACT: Azoles have consistently been recognized as the mainstays of antifungal drugs, amongst which ergosterol prevails as an integral component of fungal plasma membrane. It is most commonly produced via demethylation of lanosterol by the cytochrome P450-dependent enzyme lanosterol 14α-demethylase. Azoles exert their antifungal activities via binding to lanosterol 14 α-demethylase and thereby preventing the demethylation of lanosterol. This leads to the depletion of demethylated lanosterol (ergosterol) with the concomitant accumulation of methylated sterol precursors (lanosterol, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol) and deterioration of the membrane integrity, resulting in fungal growth inhibition. Resistance to azoles is a concern, particularly during the long-term treatment of fungus mediated cellular complications. To combat azole resistance and to extend the spectrum of treatable pathogens, the development of novel and more potent azoles, with alteration in active sites has attracted worldwide scientific attention. With such an insight, this review focuses on antifungal potentials of azole compounds with an emphasis on the corresponding drug resistance episodes complemented with novel strategies for the development of new generation of azole compounds.The Natural Products Journal. 01/2014; 4(2).
- [Show abstract] [Hide abstract]
ABSTRACT: Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral and fungal pathogens. The medically important fungus Candida albicans colonizes different host niches as part of the normal human microbiota. Proliferation of C. albicans is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host, C. albicans has developed a three-phase AMP evasion strategy including secretion of peptide effectors, AMP efflux pumps and regulation of signaling pathways. These mechanisms prevent C. albicans from the antifungal activity of the major AMP classes including cathelicidins, histatins and defensins leading to a basal resistance. This review summarizes human AMP attack and C. albicans resistance mechanisms, as well as current developments in the use of AMPs as antifungals.Eukaryotic Cell 06/2014; · 3.18 Impact Factor
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ABSTRACT: Candida albicans is a major fungal pathogen in humans. Antimicrobial peptides (AMPs) are critical components of the innate immune response in vertebrates and represent the first line of defense against microbial infection. LL-37 is the only member of the human family of cathelicidin AMPs and is commonly expressed by various tissues and cells, including surfaces of epithelia. The candidacidal effects of LL-37 have been well documented, but the mechanisms by which LL-37 kills C. albicans are not completely understood. In this study, we examined the effects of LL-37 on cell wall and cellular responses in C. albicans. Using transmission electron microscopy, carbohydrate analyses, and staining for β-1,3-glucan, changing of C. albicans cell wall integrity was detected upon LL-37 treatment. In addition, LL-37 also affected cell wall architecture of the pathogen. Finally, DNA microarray analysis and quantitative PCR demonstrated that sub-lethal concentrations of LL-37 modulated the expression of genes with a variety of functions, including transporters, regulators for biological processes, response to stress or chemical stimulus, and pathogenesis. Together, LL-37 induces complex responses in C. albicans, making LL-37 a promising candidate for use as a therapeutic agent against fungal infections.The Journal of Microbiology 05/2014; · 1.53 Impact Factor
Candida albicans Flu1-Mediated Efflux of Salivary Histatin 5 Reduces
Its Cytosolic Concentration and Fungicidal Activity
Rui Li, Rohitashw Kumar, Swetha Tati, Sumant Puri, Mira Edgerton
Department of Oral Biology, University at Buffalo, Buffalo, New York, USA
Histatin 5 (Hst 5) is a salivary human antimicrobial peptide that is toxic to the opportunistic yeast Candida albicans. Fungicidal
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. albi-
cans FLU1 and TPO2 were found to be the closest homologs of Saccharomyces cerevisiae TPO1, which encodes a major spermi-
Tpo2, mediates efflux of Hst 5 and is responsible for reduction of its toxicity in C. albicans.
hospitalized patients, this organism can disseminate hematog-
enously and infect virtually all organs (4–6). The incidence and
mortality rates associated with this infectious disease have re-
mained unchanged for more than a decade despite major ad-
vances 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 diffu-
sion (10) and inhibit the biosynthesis of ergosterol, which is a
major component of C. albicans membranes (11–13). However,
these drugs has led to the emergence of resistance in clinical iso-
(either by mutation or by overexpression) of the drug target en-
zyme lanosterol demethylase (encoded by ERG11) and/or consti-
tutive 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 ex-
perimentally evolved populations of C. albicans; these include
transporters containing ATP-binding cassettes (ABC) and trans-
porters of the major facilitator superfamily (MFS) (17–20). The
ABC transporter superfamily, including Cdr1 and Cdr2, consists
of membrane proteins that have two membrane-spanning do-
mains and two nucleotide-binding domains that utilize ATP to
drive substrates across the membrane (21, 22). PDR5 encodes an
romyces cerevisiae. Pdr5 has a nucleotide-binding domain fol-
lowed by a transmembrane domain, which is repeated in the sec-
ond half of the protein. CDR1 and CDR2 were both identified
based on their functional complementation of an S. cerevisiae
Pdr5 mutant (19, 23). Other studies showed that a C. albicans
CDR1 homozygous deletion mutant was hypersensitive to azoles,
whereas a CDR2 homozygous deletion mutant did not have al-
andida albicans is a human fungal pathogen that causes seri-
tered sensitivity (24). However, the combined deletion of both
CDR1 and CDR2 resulted in an increased hypersensitivity com-
pared to that resulting from the deletion of CDR1 alone, suggest-
ing that the Cdr2 protein also plays a role in azole resistance (24–
26). MFS drug pumps, including Mdr1 and Flu1, have no
nucleotide-binding domain but instead use the proton motive
force of the membrane as an energy source (21, 22). The Mdr1
albicans, which exchanges H?with antifungal compounds (27–
29). Both MDR1 and FLU1 were overexpressed specifically in flu-
conazole-resistant C. albicans isolates (23, 30–32). Like the CDR
genes, FLU1 was also identified during genomic library screening
for complementation of fluconazole hypersusceptibility in the S.
cerevisiae Pdr5 mutant strain (33). Flu1 can mediate fluconazole
resistance when expressed in S. cerevisiae; however, disruption of
tibility (33). Disruption of FLU1 in a background of C. albicans
mutants with deletions in several multidrug efflux transporter
genes, including CDR1, CDR2, and MDR1, resulted in enhanced
susceptibility to several azole derivatives, indicating that FLU1
also contributes to azole resistance (30). Since a BLAST search of
available databases with C. albicans FLU1 revealed high similarity
with C. albicans MDR1 (33, 34), there has been a focus on its
FLU1 gene product also has high homology with S. cerevisiae
Tpo1, a major plasma membrane polyamine efflux transporter
(35), suggesting other functions for this protein.
Received 13 November 2012 Returned for modification 10 December 2012
Accepted 28 January 2013
Published ahead of print 4 February 2013
Address correspondence to Mira Edgerton, firstname.lastname@example.org.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
aac.asm.orgAntimicrobial Agents and Chemotherapyp. 1832–1839April 2013 Volume 57 Number 4
Histatin 5 (Hst 5) is a histidine-rich, antifungal cationic pro-
displayed by Hst 5 compared with that of azoles or polyenes may
be beneficial when resistance to conventional antifungals has oc-
curred. Unlike other cationic peptides, the fungicidal mechanism
of Hst 5 is not a result of cytolysis or membrane disruption. In-
from yeast cells, resulting in gradual cell death similar to osmoti-
cally induced cell death (38, 39). These cytotoxic effects are initi-
ated once Hst 5 translocates into the intracellular compartment
gradually reduced while cells were still intact (as shown by the
inability of propidium iodide to enter cells) and metabolically
anism for efflux of Hst 5. Since we found that Hst 5 utilizes the C.
albicans polyamine influx transporters Dur3 and Dur31 (both
plasma membrane permeases) for its uptake (41), we hypothesize
efflux of Hst 5.
Although little is known about efflux of polyamines in C. albi-
is carried out by the proteins Tpo1 to Tpo4 (42, 43). S. cerevisiae
TPO2 and TPO3 encode transporters specific for spermine,
whereas TPO1- and TPO4-encoded pumps utilize putrescine,
spermidine, and spermine as the substrates (43). The S. cerevisiae
TPO1 gene product is the major plasma membrane-bound ex-
porter involved in the detoxification of excess levels of intracellu-
lar spermidine (44). However, Tpo1 can excrete various sub-
drug mycophenolic acid (45) and mediates growth resistance to
cycloheximide and quinidine (46). The S. cerevisiae Tpo1 poly-
amine transporter is pH dependent, so that polyamine uptake
occurs at pH 8 while excretion occurs at acidic pH (pH 5). Both
processes are catalyzed by activity of a polyamine-H?antiporter
functionally similar with respect to pH-dependent excretion of
polyamines and other small cationic substances, including Hst 5.
in efflux of polyamines and Hst 5. However, we report here that
for reduction of its toxicity in C. albicans.
MATERIALS AND METHODS
Yeast strains and peptides. C. albicans strains used in this study are de-
scribed in Table 1. C. albicans cdr1?/?, mdr1?/?, and cdr1/cdr2?/?
strains were generously provided by D. Sanglard (University of Lausanne
and University Hospital Center, Lausanne, Switzerland). Knockout mu-
tants of C. albicans Flu1 (flu1?/?) and C. albicans Tpo2 (tpo2?/?) were
to generate Ura?constructs. For restoration strain construction (flu1?/
FLU1 or tpo2?/TPO2), a copy of wild-type (WT) FLU1 or TPO2 was
introduced into the RPS10 locus of the respective flu1?/? or tpo2?/?
strain using the vector CIp10 containing URA3 (41). Yeast cells were
transformed with purified PCR products using the Frozen-EZ Yeast
Transformation II kit (Zymo Research, CA). Cells were maintained in
yeast extract-peptone-dextrose (YPD; Difco) medium with the addition
of uridine when required and stored at ?80°C. Spermidine was obtained
from Sigma. Hst 5 and N-terminally biotin-labeled Hst 5 (BHst 5) were
synthesized by Genemed Synthesis Inc. (San Antonio, TX). BHst 5 was
verified to have biological activity similar to that of unlabeled Hst 5 by
candidacidal assays (38, 48).
Phylogenetic analysis. Candidate polyamine excretion proteins in C.
albicans were identified by homology with TPO1 to TPO4 in S. cerevisiae
using CLUSTAL 2.0.12 and the Candida Genome Database. CLUSTAL
2.0.12 was used to calculate a phylogenetic tree with use of the neighbor-
Spermidine efflux assays. Spermidine was labeled (BODIPY-Spd) as
described previously using boron dipyrromethene difluoride (BODIPY)
?, tpo2?/?, flu1?/FLU1, and tpo2?/TPO2) were grown in YPD broth
overnight at 30°C with shaking and then subcultured and regrown to
mid-log phase. Harvested cells were washed twice in 100 mM sodium
106) were incubated with 100 ?M BODIPY-Spd in 100 mM NaPB for 15
min, allowing BODIPY-Spd to enter the cells (41). After 15 min of incu-
bation, cells were washed with 100 mM NaPB (pH 5.0/pH 7.4), resus-
plate to a concentration of 2 ? 106cells/well. A standard curve for
of 3.5 nmol to 10 nmol spermidine. Released BODIPY-Spd was detected
using a Bio-Tek multifunction plate reader and Gen5 software and was
quantified by reference to the standard curve. Statistical analysis and de-
termination of kinetic parameters were performed using Prism version
5.0 (GraphPad Software, San Diego, CA). Each experiment was per-
formed in triplicate in at least two biological replicates.
For assays measuring Hst 5 efflux from C. albicans, cells (1 ? 107) were
mM NaPB (pH 7) and then washed and resuspended in NaPB (pH 5) for
each time point and assayed by a slot blot assay. For the time course assay
reaction was stopped at each time point, and the cell pellet was subjected
TABLE 1 C. albicans strains used in this study
?ura3::imm434/ura3::imm434 flu1/FLU1::FRT/RP10::FLU1 ORFa
?ura3::imm434/ura3::imm434 tpo2/TPO2::FRT/RP10::TPO2 ORF
aORF, open reading frame.
C. albicans Flu1 Mediates Efflux of Histatin 5
April 2013 Volume 57 Number 4 aac.asm.org 1833
tein from each extract were subjected to SDS-PAGE using 12.5% acryl-
amide gels. Extracellular and cytoplasmic BHst 5 (?3 kDa) was detected
using streptavidin conjugated with horseradish peroxidase (Pierce).
Quantitative analysis of BHst 5 proteins was performed using a Bio-Rad
(version 4.2). Concentrations of cytoplasmic BHst 5 from at least three
using Prism 5.0 software. Differences between experimental groups were
evaluated for significance by unpaired t test, using Prism 5.0 software.
Total RNA isolation, cDNA synthesis, and real-time reverse tran-
scription-PCR (RT-PCR). Log-phase cultures (1 ? 107cells/ml) of C.
albicans (wild-type and flu1?/? and tpo2?/? mutant strains) were ex-
90 min; total RNA isolation was performed using the RNeasy minikit
PCR amplification of the 18S rRNA housekeeping gene. One microgram
of total RNA was used per reaction for the first-strand synthesis (cDNA)
were as follows: 5=-GACCAATGACTGCTGCTGAA-3= and 5=-GCCAGT
TTTGACGTTTGGAT-3= for DUR3, 5=-GATCATCTGTGCTGCTGGA
A-3= and 5=-AGCAGCTGAAGCCAATGT-3= for DUR31, 5=-CAACGAT
ATTGCTCCTGAAG-3= and 5=-TGGCTCTTCTCGATAATTCA-3= for
FLU1, and 5=-CGATGGAAGTTTGAGGCAATA-3= and 5=-CTCTCGGC
CAAGGCTTATACT-3= for 18S RNA. Amplifications (amplicons) were
150 to 200 bp in length, and the annealing temperatures were 54°C. Am-
plification and detection were carried out in 96-well plates on an iCycler
iQ real-time detection system (Bio-Rad). All samples contained 10 ?l iQ
SYBR Green supermix (2? concentration), 1 ?l forward primer, 1 ?l
reverse primer, 1 ?l template (cDNA), and 17 ?l nuclease-free water.
Fluorescent data were collected and analyzed with iCycler iQ software.
The threshold cycle (?CT) value was obtained by calculating the differ-
ence between CTvalues of the target gene and the normalizer (18S). The
?CTat 1 min was used as a reference (baseline). The ??CTvalues
the baseline and were transformed to absolute values (2???CT) to calcu-
calculated from at least three independent biological replicates. Differ-
ences between experimental groups were analyzed with Expert qPCR
Candidacidal assays of Hst 5. The susceptibility of C. albicans cells to
described (50). Briefly, 10 ml of YPD medium with uridine (50 ?g/ml)
was inoculated with single colonies of each strain. Cells were grown over-
night at room temperature. Overnight cultures were diluted to an A600of
at 30°C for 30 min and diluted in 10 mM NaPB, and aliquots of 500 cells
were spread onto YPD agar plates and incubated for 36 to 48 h until
colonies could be visualized. Cell survival was expressed as a percentage
compared with untreated control cells, and the percent killing was calcu-
lated as [1 ? (number of colonies from peptide-treated cells/number of
for each strain.
Biofilm formation upon treatment with Hst 5. Overnight-grown
cells were diluted to an A600of 0.3 to 0.4 and were incubated at 30°C with
to an A600of ?1 in phosphate-buffered saline (PBS; pH 7.4). One milli-
liter of cells was added to each well of a 12-well tissue culture plate (Bec-
h, then nonadherent cells were removed by gently washing the wells with
PBS, and fresh medium (1 ml yeast nitrogen base [YNB]) was added and
incubated for 24 h at 37°C. After 24 h, all samples were washed with 1 ml
PBS twice. For control groups, 1 ml YNB was added to each well after
washing. For test groups, medium was removed and replaced with 500 ?l
Hst 5 (60 ?M) for 1 h, after which 500 ?l YNB medium was added. All
removed mechanically and collected into preweighed microcentrifuge
tubes, and dry weights of cells per well were calculated. Experiments were
conducted in triplicate. The data represent mean dry weight of cells with
(dry weight of Hst 5-treated cells/dry weight of control cells)] ? 100%.
Differences between experimental groups were evaluated for significance
by an unpaired t test, using Prism 5.0 software.
Identification of C. albicans TPO transporter family members.
To identify likely C. albicans polyamine efflux transporters, we
screened the C. albicans genome database (CGD) for orthologs of
the major S. cerevisiae transporters responsible for polyamine ef-
flux, which include TPO1, TPO2, TPO3, and TPO4. Homology
searching using S. cerevisiae TPO genes showed five related mem-
bers of the C. albicans TPO family, including FLU1 (orf19.6577),
TPO2 (orf19.7148), TPO3 (orf19.4737), orf19.341, and TPO4
efflux pump at the plasma membrane and plays a major role in
albicans FLU1 is a TPO family member also annotated in CGD as
TPO1. C. albicans FLU1 and TPO2 were found to be the closest
homologs to S. cerevisiae TPO1, which encodes a major spermi-
dine efflux transporter. C. albicans TPO3 (orf19.4737) and
orf19.341 were found to be most closely related to S. cerevisiae
TPO2 and TPO3, while C. albicans TPO4 (orf19.473) was homol-
ogous to S. cerevisiae TPO4.
plemented with spermidine had higher resistance to Hst 5 killing
than did cells grown with putrescine and spermine (41), we hy-
pothesized that the candidal homolog of S. cerevisiae Tpo1 would
be a major efflux transporter for spermidine as well as Hst 5. To
performed by optimal alignment of the amino acid sequences us-
ing CLUSTAL2.0 software with both C. albicans Flu1 and Tpo2.
However, both C. albicans Flu1 and Tpo2 proteins showed high
homology to S. cerevisiae Tpo1p, having 60% and 59% similari-
ties, respectively. Hence, we selected both C. albicans Tpo2 and
Flu1 proteins as likely candidates to mediate efflux of Hst 5 in C.
FIG 1 Phylogenetic analysis of C. albicans TPO transporter family members
and their relationship with Saccharomyces cerevisiae Tpo. Members of the C.
albicans TPO transporter family (http://www.candidagenome.org) are shown
with TPO-homologous protein sequences from the yeast S. cerevisiae. C. albi-
cans FLU1 (orf19.6577)- and TPO2 (orf19.7148)-encoded proteins have the
closest phylogenetic relationship to S. cerevisiae polyamine excretion protein
Tpo1, a spermidine efflux transporter.
Li et al.
aac.asm.org Antimicrobial Agents and Chemotherapy
Spermidine efflux in C. albicans is pH dependent and is re-
duced by FLU1 deletion. Rates of spermidine efflux by S. cerevi-
siae Tpo1 are higher at pH 5 due to its polyamine-H?antiport
activity (35). Therefore, we examined whether this was also the
case for C. albicans Tpo1 homologs. C. albicans WT, flu1?/? and
tpo2?/? mutant, and flu1?/FLU1 and tpo2?/TPO2 restoration
strains were incubated with BODIPY-Spd to load cells with la-
BODIPY-Spd after 15 min were detected between these strains.
Cells were then washed and transferred to medium without poly-
amines, and efflux of BODIPY-Spd was measured for 5 min, dur-
ing which efflux was linear, allowing calculation of efflux rates
(Table 2). The total efflux of BODIPY-Spd from WT cells was
increased by nearly 3-fold by placing cells in medium at pH 5
compared to placing them at pH 7.4. Thus, C. albicans Tpo1 ho-
mologs function similarly to S. cerevisiae Tpo1 with respect to pH
dependence. C. albicans tpo2?/? and tpo2?/TPO2 cells had sper-
midine efflux rates identical to those of WT cells under both neu-
tral and acidic conditions, showing that removal of TPO2 had no
impact on spermidine efflux. However, C. albicans flu1?/? cells
had a statistically significant reduction in their rate of spermidine
efflux at pH 5, while no difference was detected under neutral pH
conditions (Table 2). Restoration of FLU1 in flu1?/? (flu1?/
FLU1) cells returned the efflux rate of BODIPY-Spd at acidic pH
to that of WT cells (Table 2). These results suggest a role for Flu1
but not Tpo2 in efflux of spermidine in C. albicans, particularly
under acidic conditions.
up via C. albicans Dur3 and Dur31 spermidine uptake transport-
we examined Hst 5 efflux from C. albicans cells loaded with sub-
lethal concentrations of biotin-labeled Hst 5 (BHst 5). C. albicans
wild-type, tpo2?/? and flu1?/? mutant, and flu1?/FLU1 and
tpo2?/TPO2 restoration strains were incubated first with BHst 5
for 20 min, as we have shown that this exposure time results in
cells that have a significant cytosolic load of Hst 5 but still retain
cellular integrity as measured by lack of propidium iodide uptake
(40). After washing to remove extracellular BHst 5, cells were re-
suspended in buffer adjusted to pH 5, cell supernatants were col-
and 30 min. We found that the amount of extracellular BHst 5 in
the flu1?/? strain was reduced compared with wild-type cells,
while there was no difference in concentrations of extracellular
the efflux of Hst 5. We expected that decreased efflux of cytosolic
Hst 5 as a result of deletion of FLU1 would be accompanied by an
flu1?/?, and tpo2?/? strains and their respective restoration
strains. We found that flu1?/? cells had significantly (P ? 0.05)
larger amounts of cytoplasmic BHst 5 than did wild-type,
tpo2?/?, or tpo2?/TPO2 cells (Fig. 2B). Furthermore, levels of
FLU1 restoration strain (Fig. 2B). To determine whether azole
drug transporters might be involved in efflux of Hst 5, the sensi-
transporter-deficient strains (cdr1?/?, mdr1?/?, and cdr1/
the toxicity of Hst 5.
To rule out the possibility that the observed differences in cy-
Hst 5, we examined expression levels of the two major Hst 5 up-
take transporter-encoding genes (DUR3 and DUR31) after expo-
sure to Hst 5. Real-time RT-PCR was performed to measure the
transcript levels of DUR3 and DUR31, as well as FLU1 genes in C.
albicans strains following treatment with sublethal doses of Hst 5
ing gene was used as an internal control. Transcriptional levels of
DUR3 and DUR31 in wild-type (Fig. 4) and flu1?/? and tpo2?/?
to Hst 5, as analyzed by Expert qPCR Analysis software. This sug-
gested that the observed increase in cytosolic levels of Hst 5 in
detect differences in spermidine uptake in flu1?/? cells (data not
shown), pointing toward normal functioning of Dur uptake pro-
teins up to 60 min. However, transcriptional levels of DUR3 but
not DUR31 were increased by 5-fold after 90 min of exposure to
levels of FLU1 did not change over a 90-min course of treatment
with Hst 5 (Fig. 4). Thus, Hst 5 is not likely to induce FLU1 gene
at least within the early exposure times that we examined.
Deletion of FLU1 increases sensitivity of C. albicans plank-
wild-type, flu1?/? and tpo2?/? mutant, and flu1?/FLU1 and
(pH 5) conditions. Interestingly, all strains, including the wild-
type, deletion, and restoration strains, had higher sensitivity to
Hst 5 at pH 5 than at pH 7 (Fig. 5A), indicating the importance of
the H?electrochemical gradient in the Hst 5 candidacidal func-
tion. Deletion of FLU1 resulted in an increase in Hst 5 sensitivity
under both neutral and acidic conditions (Fig. 5A) that was re-
versed upon gene restoration (Fig. 5B). At pH 7, the sensitivity of
5 concentrations (filled circles), while increased sensitivity of the
flu1?/? strain was more evident at lower (?15 ?M) Hst 5 con-
centrations at pH 5 (open circles) due to the overall higher sensi-
tivity. However, there was no difference in Hst 5 sensitivity be-
tween tpo2?/? and wild-type cells. Next, we examined the
Hst 5. Biofilms formed by flu1?/? cells were slightly higher in
total dry weight than those formed by the wild-type strain, show-
ing that loss of Flu1 does not inhibit biofilm formation (Fig. 6).
TABLE 2 Spermidine efflux rates for C. albicans strains
Efflux rate (nmol spermidine/2 ? 106cells/min) for straina:
0.23 ? 0.03
0.63 ? 0.03
0.26 ? 0.03
0.53 ? 0.02b
0.26 ? 0.03
0.62 ? 0.03
0.23 ? 0.04
0.63 ? 0.03
0.25 ? 0.04
0.62 ? 0.02
aData presented are means ? standard deviations of duplicate assays.
bP ? 0.05.
C. albicans Flu1 Mediates Efflux of Histatin 5
April 2013 Volume 57 Number 4aac.asm.org 1835
Reduction of biofilm formation in the presence of Hst 5 was de-
tected in both wild-type and flu1?/? strains. The flu1?/? cells,
of biofilm reduction (21.6 ? 2.2) than did the wild type (14.6 ?
1.2) (Fig. 6). Thus, the presence of Flu1p also reduces Hst 5 tox-
icity in C. albicans biofilms.
for oral candidiasis because of its potent candidacidal and candi-
dastatic activities while being nontoxic to humans (51). Previ-
ously, we observed that the concentration of cytosolic Hst 5 was
gradually reduced from intact C. albicans cells (40), suggesting
that C. albicans possesses a transport mechanism for efflux of Hst
5 in order to reduce its toxicity. Therefore, identification of the
route of Hst 5 efflux is crucial for understanding the mechanism
for possible Hst 5 resistance.
Polyamines are essential aliphatic polycations needed for nor-
mal growth and morphogenesis in fungi, so that their depletion
FIG 2 Efflux of BHst 5 is reduced, resulting in increased cytoplasmic accumulation in the C. albicans flu1?/? strain. (A) C. albicans (wild-type, flu1?/?
and tpo2?/? mutant, and flu1?/FLU1 and tpo2?/TPO2 restoration strains) were loaded with biotin-labeled Hst 5 (BHst 5) (31 ?M) for 20 min and then
resuspended in NaPB (pH 5) for 0 min, 2 min, 10 min, 20 min, and 30 min. Supernatants were collected at each time point and immunoblotted to detect
extracellular concentrations of BHst 5 by densitometry for each time point. Only the flu1?/? mutant had significantly (P ? 0.01) reduced BHst 5 in cell
supernatants. (B) Cytosolic concentrations of BHst 5 were quantified following incubation of cells with BHst 5 (31 ?M) for 1 min, 30 min, and 60 min.
Cytoplasmic fractions were isolated at each time point, resolved using 12.5% SDS-PAGE, immunoblotted, and quantified as described above. Only the
at least three independent experiments.
FIG 3 C. albicans drug transporter-deficient strains are not altered in sensi-
tivity to Hst 5. C. albicans deletion mutants (cdr1?/?, mdr1?/?, and cdr1/
cdr2?/?) were exposed to Hst 5 (7.5 ?M to 31 ?M) using microdilution plate
assays, and percent killing was calculated. There was no significant difference
of sensitivity to Hst 5 between wild-type and mutant strains.
Li et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
results in growth cessation while high intracellular accumulation
may be cytotoxic (52). Thus, fungi must strictly regulate their
intracellular polyamine pools using membrane-localized poly-
amine transporter systems that facilitate internalization of exoge-
The major polyamines in fungi are putrescine, spermidine, and
spermine, and all three have a net positive charge (pKavalues of 9
transporters as major carriers for Hst 5 entry into C. albicans cells
and found that Hst 5 uptake is competitive with the polyamine
spermidine (41). Since energy-dependent uptake of Hst 5 into C.
likely that Hst 5 is also subject to excretion by C. albicans cells by
polyamine efflux transporters.
plays an important role in polyamine detoxification. We found
that C. albicans Tpo2 is similar to its close homolog, S. cerevisiae
Tpo1, in terms of its ability to transport spermidine; however,
rates similar to those of the wild type under both neutral and
acidic conditions (Table 2). Deletion of C. albicans TPO2 did not
affect cytosolic Hst 5 levels and Hst 5 excretion or toxicity, thus
albicans Tpo2, in contrast to C. albicans flu1?/? cells. These re-
sults highlight the substrate specificity of polyamine transporters
as described previously for S. cerevisiae Tpo proteins (43). For
example, Glu-201, Glu-324, and Glu-574 residues of S. cerevisiae
FIG 4 Expression of C. albicans FLU1 is not altered following treatment of
cells with Hst 5. Hst 5 (31 ?M) was added to cultures at 0 min, and aliquots
were removed for RNA extraction at 1, 30, 60, and 90 min as indicated. Tran-
by quantitative real-time RT-PCR, and expression levels were normalized to
pared to wild-type cells.
FIG 5 The C. albicans flu1?/? mutant has increased sensitivity to Hst 5. C.
albicans deletion mutants (flu1?/? and tpo2?/? and restoration strains) were
7 (filled circles) using microdilution plate assays, and percent killing was cal-
under both neutral and acidic conditions, while the tpo2?/? strain did not
differ from the parental WT strain in its sensitivity to Hst 5. (B) Restoration
the parental strains under both neutral and acidic conditions.
well were calculated. Reduction of biofilm formation in the presence of Hst 5
was detected in both the wild type (P ? 0.0165) and the flu1?/? strain (P ?
0.0133). The flu1?/? cells had a significantly higher percentage of biofilm
reduction (21.6 ? 2.2) than did wild-type cells (14.6 ? 1.2).
C. albicans Flu1 Mediates Efflux of Histatin 5
April 2013 Volume 57 Number 4aac.asm.org 1837
Tpo1 are important for spermine transport, while Ser-342 is re-
acids on C. albicans Flu1 may be responsible for the transport of
both polyamine and Hst 5 as the substrates and may be lacking in
Tpo2. Although efflux of Hst 5 was reduced in flu1?/? cells
(Fig. 2A), it was not completely stopped, suggesting that addi-
tional mechanisms or yet-to-be-identified transporters may be
involved in the efflux of Hst 5.
Similarly, C. albicans Flu1 also has high homology to CaMdr1,
which is responsible for fluconazole resistance and functions as a
transporters (e.g., Cdr1, Mdr1, and Cdr2) would also be involved
in efflux of Hst 5 in C. albicans. However, we did not find any
significant difference in sensitivity to Hst 5 between the wild type
and the transporter-deficient cdr1?/?, mdr1?/?, and cdr1/
porters appear to possess high substrate specificity, perhaps
through their transmembrane segments, which are envisaged to
peptides such as Hst 5. Although ABC transporters provide the
greatest level of resistance to antimicrobial peptides (AMPs) in
drug transporters (e.g., Cdr1, Mdr1, and Cdr2) are involved in
resistance to AMPs in yeast cells. The fact that Hst 5 has been
lates of C. albicans supports this concept (55).
Our finding that transcriptional levels of FLU1 did not change
over a 90-min course of treatment with Hst 5 has important im-
genes like MDR1 and FLU1 that are involved in azole resistance
(56), we found no evidence of Hst 5-induced FLU1 gene overex-
pression, suggesting that C. albicans is not likely to develop resis-
tance to Hst 5. Fluconazole resistance can occur without overex-
pression of CDR1/MDR1 due to alteration of the drug target
fungi might develop resistance to Hst 5 through mechanisms
other than FLU1 overexpression, such as changes in transporter
affinity for Hst 5 with long-term exposure. However, to date no
stable Hst 5-resistant C. albicans isolate has been identified. Sur-
prisingly, transcriptional levels of DUR3 were increased signifi-
compensatory responses upon accumulation of critical levels of
cytosolic Hst 5. Intracellular Hst 5 may be treated as a polyamine
by being localized within fungal cellular compartments (57) with
excess levels trafficked to the vacuole for detoxification. Indeed,
we have noted considerable vacuolar localization of Hst 5 (40).
However, Hst 5 may overload vacuolar capacity, so that levels of
trafficked Hst 5 (like its polyamine counterpart ) exceed the
same time, accumulation of excess cytosolic Hst 5 may create an-
ion channel-like pathways in the cell membrane, leading to
changes in the membrane potential. The altered membrane po-
tential may energize secondary transporters such as cations and
polyamine transporters, which in turn increase the expression of
genes encoding transport proteins such as Dur3.
In conclusion, this is the first report demonstrating that C.
albicans utilizes the fungal polyamine efflux transporter Flu1 to
pump Hst 5 out of the cell and thus reduce its toxicity. Although
efflux of Hst 5 by Flu1 reduces its toxicity, Hst 5 treatment does
not induce FLU1 gene overexpression during short-term expo-
vivo research is needed to develop specific Flu1 inhibitors to in-
crease the candidacidal activity of Hst 5.
This work was supported by grant R01DE010641 (M.E.) from the Na-
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