New synthetic sulfone derivatives inhibit growth, adhesion and the leucine arylamidase APE2 gene expression of Candida albicans in vitro

Article abstract of DOI:10.1016/j.bmc.2014.11.038

The successful preventing and effective treatment of invasive Candida albicans infections required research focused on synthesis of new classes of agents and antifungal activity studies. Bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (named compound 6); dichloromethyl-4-chloro-3-nitrophenyl sulfone (named 7); and chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (named 11) on inhibition of planktonic cells' growth, leucine arylamidase APE2 gene expression, and adhesion to epithelial cells were investigated. In vitro anti-Candida activities were determined against wild-types, and the morphogenesis mutants: Δefg1 and Δcph1. MICs of compounds 6, 7 and 11 (concentrated at 0.25-16 μg/ml) were determined using the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document). APE2 expression was analyzed using RT-PCR; relative quantification was normalized against ACT1 in cells growth in YEPD and on Caco-2 cell line. Adherence assay of C. albicans to Caco-2 was performed in 24-well-plate. The structure activity relationship suggested that sulfone containing hydrazine function at C-1 (compound 11) showed higher antifungal activity (cell inhibition% = 100 at 1-16 μg/ml) than the remaining sulfones with chlorine at C-1. Δcph1/Δefg1 was highly sensitive to compound 11, while the sensitivity was reduced in Δcph1/Δefg1::EFG1 (% = 100 at 16-fold higher concentration). Compound 11 significantly affected adherence to epithelium (P ≤0.05) and hyphae formation. The APE2 up-regulation plays role in sulfones' resistance on MAP kinase pathway. Either CPH1 or EFG1 play a role in the resistance mechanism in sulfones. The strain-dependent phenomenon is a factor in the sulfone resistance mechanism. Sulfones' mode of action was attributed to reduced virulence arsenal in terms of adhesiveness and pathogenic potential related to the APE2 expression and morphogenesis.

Full text of DOI:10.1016/j.bmc.2014.11.038

Accepted Manuscript
New Synthetic Sulfone Derivatives Inhibit Growth, Adhesion and the Leucine
Arylamidase APE2 Gene Expression of Candida Albicans in Vitro
Monika Staniszewska, Małgorzata Bondaryk, Zbigniew Ochal
PII:
S0968-0896(14)00833-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.11.038
BMC 11927
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 August 2014
19 November 2014
27 November 2014
Please cite this article as: Staniszewska, M., Bondaryk, M., Ochal, Z., New Synthetic Sulfone Derivatives Inhibit
Growth, Adhesion and the Leucine Arylamidase APE2 Gene Expression of Candida Albicans in Vitro, Bioorganic
& Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.11.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New Synthetic Sulfone Derivatives Inhibit Growth, Adhesion and the Leucine
Arylamidase APE2 Gene Expression of Candida Albicans in Vitro
Monika Staniszewskaa*, Małgorzata Bondaryka, Zbigniew Ochalb
aNational Institute of Public Health-National Institute of Hygiene, Chocimska 24, 00-791
Warsaw, Poland
bFaculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw,
Poland
*Correspondence: Monika Staniszewska, Independent Laboratory of Streptomyces and Fungi
Imperfecti, National Institute of Public Health – National Institute of Hygiene, 24 Chocimska,
00-791 Warsaw, Poland
Phone: +48 22 54 21 228. Fax: + 48 22 849 74 84
E-mail: mstaniszewska@pzh.gov.pl

Abstract
The successful preventing and effective treatment of invasive Candida albicans infections
required research focused on synthesis of new classes of agents and antifungal activity
studies. Bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (named Compound 6);
dichloromethyl-4-chloro-3-nitrophenyl sulfone (named 7); and chlorodibromomethyl-4-
hydrazino-3-nitrophenyl sulfone (named 11) on inhibition of planktonic cells’ growth, leucine
arylamidase APE2 gene expression, and adhesion to epithelial cells were investigated. In vitro
anti-Candida activities were determined against wild-types, and the morphogenesis mutants:
∆efg1 and ∆cph1. MICs of Compounds 6, 7 and 11 (concentrated at 0.25-16 µg/ml) were
determined using the Clinical and Laboratory Standards Institute Broth Microdilution Method
(M27-A3 Document). APE2 expression was analyzed using RT-PCR; relative quantification
was normalized against ACT1 in cells growth in YEPD and on Caco-2 cell line. Adherence
assay of C. albicans to Caco-2 was performed in 24-well-plate.
The structure activity relationship suggested that sulfone containing hydrazine function at C-1
(Compound 11) showed higher antifungal activity (cell inhibition %=100 at 1-16 µg/ml) than
the remaining sulfones with chlorine at C-1. ∆cph1/∆efg1 was highly sensitive to Compound
11, while the sensitivity was reduced in ∆cph1/∆efg1::EFG1 (%=100 at 16-fold higher
concentration). Compound 11 significantly affected adherence to epithelium (P≤0.05) and
hyphae formation. The APE2 up-regulation plays role in sulfones’ resistance on MAP kinase
pathway. Either CPH1 or EFG1 play a role in the resistance mechanism in sulfones. The
strain-dependent phenomenon is a factor in the sulfone resistance mechanism. Sulfones’ mode
of action was attributed to reduced virulence arsenal in terms of adhesiveness and pathogenic
potential related to the APE2 expression and morphogenesis.
Keywords: Candida albicans, sulfone derivatives, anti-virulence agents, APE2, adhesion,
morphogenesis
2

1. Introduction
Candida albicans is the most common etiological agent of invasive fungal infections in
immunocompromised hosts with cancer or AIDS and in organ-transplant patients.1 The
capacity of C. albicans to rapidly acquire resistance to antifungal drugs, such as amphotericin
B, flucytosine, and azoles,1 means that further studies are needed to examine the effect of new
compounds on virulence factors of C. albicans.
The virulence factors, such as adhesion of the fungus to host cells and hyphae
formation2 have been suggested as attractive antifungal targets. Moreover, leucine
arylamidase Ape2 is also believed to contribute to the invasion and to play a role in C.
albicans virulence by removing the N-terminal L-leucine from peptide substrates.3 Thus Ape2
facilitates the penetration of C. albicans into the host tissues.
Adherence to host tissues and morphological versatility are thought to be important in C.
albicans virulence.4, 5 Efg1 and Cph1 play a major role in promoting filamentous growth and
regulates the expression of several genes with a crucial function in the invasion of host cells
or in biofilm formation.6, 7 As adhesion and morphogenesis are crucial for biofilm formation
(difficult to eradicate with conventional antifungal therapy), it is fundamental to develop new
approaches to managing the factors (EFG1, CPH1, APE2) associated with morphogenesis,
adhesion, and tissue invasion. Thus the inhibition of morphogenesis factors represents a
promising strategy for the design of anti-virulence drugs, especially in view of the alarming
rise in life-threatening systemic fungal infections.8 Moreover, potent degradative enzymes’
inhibitors have been discussed as a novel antimycotic agent for the treatment of candidiasis.8
Sulfone derivatives provide an example of an important class of bioactive compounds
with a wide spectrum of activities, as the sulfone group is an important core found in
numerous biologically active compounds with a wide range of biological activity including
antifungal properties.9 There is evidence that the key feature of these compounds is a 6-
3

member heterocyclic ring attached to a sulfone, and additional modification of the benzene
ring has been considered. Among these derivatives, phenyl trihalomethyl sulfone with
10
different halogens: fluorine, chlorine and bromine prepared by Borys et al. exhibited good
11
inhibitory activity against plant pathogenic fungi. Moreover, Jha et al. reported that 2-(5-
sulfanyl-1,3,4-oxadiazol-2-yl)phenylacetate and 5-(pyridin-3-yl)-1,3,4-oxadiazole-2-thiol
exhibit good antibacterial activities against Escherichia coli (MTCC 443). As part of our
ongoing search for novel sulfone compounds displaying activity against the pathogenic C.
albicans strains, new derivatives of bromodichloromethyl-4-chloro-3-nitrophenyl sulfone
(named Compound 6) 12 and dichloromethyl-4-chloro-3-nitrophenyl sulfone (Compound 7),12
and chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (Compound 11)10 were tested
on some series of C. albicans strains. Moreover, studying Candida mutants lacking
morphological transitionality (EFG1 and/ or CPH1) may provide a deeper insight into the
new compounds antifungal mechanisms under the epithelial growth model.
The goal of the present study was to investigate the in vitro susceptibility of planktonic
cells of C. albicans wild-type strains and morphogenesis mutants to synthetic sulphones
(named Compound 6, 7, 11 respectively). Furhtermore, we tested the effect of their activity on
C. albicans adhesion to a monolayer cell culture of colorectal carcinoma of Caco-2 (ATCC).
The next goal was to assess whether the expression of APE2 is inhibited by the new
Compound 11 (as the most effective from all the tested ones) during the development phase
(adhesion) of the biofilm formation in in vitro mucosal infections. We evaluated the role that
APE2 as well as EFG1 and CPH1 play in the processes by inhibiting the enzymatic activity
prior to the adhesion assay.
2. Materials and Methods
2.1. The sulfone synthesis, purification, and analytical data
4

Bromodichloromethyl-4-chloro-3-nitrophenyl sulfone 6 was synthesized according to the
described procedure,12 starting from chlorobenzene 1, which was chlorosulfonated by
chlorosulfonic acid and obtained 4-chlorobenzenesulfochloride 2 was transformed into
natrium salt of 4-chlorobenzene sulfinic acid 3 by alkaline reduction with natrium sulfite.
Obtained sodium 4-chlorobenzenesulfinate 3 was converted into dichloromethyl-4-
chlorophenyl sulfone 4 by reaction with chloroform in alkaline solution. Sulfone 4 was then
brominated by bromine chloride, fresh prepared with bromine and chlorine as 50% solution in
carbon tetrachloride. The resulting bromodichloromethyl-4-chlorophenyl sulfone 5 was
converted into nitrocompound 6 in the next step applying the mixture of the concentrated
sulfuric acid and nitric acid (Fig. 1). Dichloromethyl-4-chloro-3-nitrophenyl sulfone 7 was
obtained by nitration of dichloromethyl-4-chlorophenyl sulfone 4 under similar conditions to
the previous synthesis of sulfone 6 (Fig. 1). Chlorodibromomethyl-4-hydrazino-3-nitrophenyl
sulfone 11 was synthesized starting from natrium salt of 4-chlorobenzene sulfinic acid 3,
which was subjected to the reaction with dichloroacetic acid in alkaline solution. Obtained
chloromethyl-4-chlorophenyl sulfone 8 was then brominated by sodium hypobromite
according to the described procedure.10 Chlorodibromomethyl-4-chlorophenyl sulfone 9 was
nitrated to yield nitrosulfone 10, followed by transformation with the reaction of nucleophilic
substitution with hydrazine to obtain the final product (Fig. 1).
2.2. Strains and Media
Candida albicans strains used in the current study are listed in Table 1.6, 13, 14 All the strains
used in the present study were stored on ceramic beads in Microbank tube (Prolab
Diagnostics, Richmond Hill, ON, Canada) at -70°C. Prior to the respective examinations,
routine culturing of strains for growth was conducted at 30°C for 18 h in YEPD.15
2.3. Anticandidal activity against planktonic growth
5

Antifungal susceptibility of the compounds was determined for each strain using the method
M27-A3 (CLSI).16 The final inoculum: from 0.5x102 to 2.5x103 cells/ml saline was prepared
in synthetic RPMI medium (Sigma, USA). The compound test wells (CTW) were prepared
with stock solution of the compound (1600 µg/ml) dissolved in water with 9% (v/v) addition
of DMSO. Subsequently, serial two-fold dilutions were made, using RPMI as a solvent. Then,
the fungal blastoconidial suspension and the compound (final dilution 1:100) were dispensed
into 96-well microplates. The compound was tested at seven concentrations that ranged from
0.25 to 16 µg/ml. The DMSO concentration was maintained at 0.09% (v/v) in all the
experiments, including the control ones. At this concentration, DMSO was not able to inhibit
the growth of C. albicans. Growth control wells (GCW) (containing medium, inoculum, the
same amount of DMSO used in CTW, but compound-free) and sterility control wells (SCW)
(sample, medium, and sterile water replacing inoculum) were included for each strain tested.
The microplates were read with the Infinite M200 PRO NANOQuant (Tecan Group Ltd.,
Austria). MIC was read spectrophotometrically (optical density OD405) and the end point was
calculated as a 100% reduction in OD405 as compared to the growth in the control well.
Growth reduction for each compound concentration was calculated as follows: % of
inhibition: 100 - (OD405 CTW - OD405 SCW)/(OD405 GCW - OD405 SCW). Amphotericin B
(Sigma-Aldrich, USA) was diluted in DMSO (1600 µg/ml) to be subsequently used in the
assay as a reference antifungal at the concentration of 1 µg/ml (100% cell inhibition).
2.4. Cultivation and Infection of Caco-2 Cell Line (ATCC HTB27, LGC, Poland)
Following the supplier’s guidelines, monolayers of the colon adenocarcinoma derived cell
line were maintained in a humidified incubator at 37°C in 5% CO2. For the experiment 1.2 x
105 of Caco-2 cells per milliliter were seeded into 24-well-plates (Corning, USA) and cultured
in the EMEM medium (10% FCS, 1mM pyruvic acid, without antibiotics or antifungal
agents) up to 18 h. Next, after 18-h post seeding the Caco-2 monolayers were inoculated with
6

105 log phase yeast cells of C. albicans wild types and mutants. After 18 h of incubation the
Caco-2 was lysed by adding sterile water in the result of which the C. albicans cells were
recovered.
2.5. Assay of Adherence to Human Line Caco-2 Epithelial Cells
Adherence of C. albicans to the Caco-2 cell line (ATCC HTB-37TM) was performed as
described previously.5 Briefly, the Caco-2 cell line was cultivated as described above 2.4.
Subsequently, the blastoconidia were grown overnight in the YEPD medium at 30°C. Then,
final density 104/ml was added to each well of the epithelial cells to be afterwards incubated
for 90 min (adhesion phase). Next, the non-adherent cells were removed by rinsing with PBS.
Then, Caco-2 was lysed by adding sterile water resulting with C. albicans cells recovery.
After 18-h growth on Sabouraud dextrose agar plates at 30°C, the number of adherent cells
was determined by colony counting. Adherence was expressed as a percentage of the total
number of cells added (control cells).
2.6. Inhibition of Candida Albicans Adhesion: Macrowell- Based Assay
The cells grown for 18 h in YEPD at 30 °C were then pre-treated with the Compounds 6, 7
and 11. Briefly, 200 µl of the blastoconidial suspension (at the final density of 104/ml) were
preincubated with 1800 µl of the RPMI medium (containing selected concentration of the
sulfones) for 2 h on a shaker at 35°C. Then, the cells were washed twice with phosphate-
buffered saline (PBS) and resuspended in 2000 µl of fresh RPMI medium. The wells of the
plate containing adherent endothelial Caco-2 cells were washed twice with PBS and then
incubated with the blastoconidial suspension (pre-treated with the sulfones) for 2 h at 37°C at
5% (v/v) CO2. The C. albicans cells recovery and the assassing of adherent cells were
conducted as described above 2.5.
2.7. RT-PCR Analyses to Assess the Effect of the Chlorodibromomethyl-4-hydrazino-3-
nitrophenyl sulfone 11 on the APE2 Gene Expression
7

17
Total RNA from cells was extracted as described Amberg et al. RNA was isolated from
cells after 18-h growth in YEPD at 30°C. Simultaneously, the cells after having been grown
in the YEPD medium were washed with water and then 200 µl of the suspension was added to
1800 µl of the RPMI medium (the final density of 104 cfu/ml) and inoculated onto the Caco-2
monolayer. Incubation was conducted for 18 h at 37°C until the RNA extraction.
For the cells pre-treated with Compound 11, blastoconidia grown in YEPD (104 cfu/ml) after
having been washed with water were suspended in the YEPD medium containing 16 µg/ml of
Compound 11. Then, after 2-h incubation with Compound 11 the cells were washed with
water and resuspended in 2000 µl YEPD for 18 h at 30°C. Simultaneously, the blastoconidial
cells pre-icubated with 16 µg/ml of Compound 11 in RPMI after washing were resuspended in
2000 µl of RPMI and inoculated onto the Caco-2 monolayer for 18 h at 37°C. Prior to further
examinations C. albicans total RNA was stored at -20°C.
First-strand cDNA synthesis was performed using the Enhanced Avian HS RT-PCR kit
(Sigma-Aldrich, USA) according to the manufacturer’s instructions. Appropriate Taq-Man
primer set was designed for APE2 by using Primer3 (Primer-BLAST, NCBI, Table 2). The
primer set of ACT1 (Table 2) was used as described previously by Naglik et al. 18 cDNA was
quantified using the FastStart Essential DNA Green Master (Roche, Germany) according to
the manufacturer’s instructions. Each reaction mixture (15 µl) contained FastStart Taq DNA
polymerase, reaction buffer, dNTP mix, SYBR Green I dye, 2 µl (250 nM) of each forward
and reverse primer, water PCR grade, and 5 µl of template 60 ng cDNA.18. For reliable
normalization of the APE2 gene expression data in C. albicans cells grown for 18 h in both
media we used the housekeeping gene ACT1 as a reference gene. The real time PCR reactions
were performed as described previously by Naglik et al.:18 at 95°C for 15 min, followed by 45
cycles of 15 s at 94°C and 1 min at 60°C with the LightCycler 96 Instrument (Roche
Diagnostics GmbH, Germany). A dissociation curve was generated at the end of each PCR
8

cycle to very that a single product was amplified. The CT values were provided from RT-PCR
instrumentation and were imported into a spreadsheet Microsoft Excel 2010. The relative
quantification was calculated using Eq.,19 where ∆CT = Avg. APE2 CT – Avg. ACT1 CT and
∆∆CT = ∆CT - ∆CT parental strain. Finally, 2 - Ct was calculated.
ꢀꢀ
2.8. Phase-contrast Microscopy (Docuval, Carl Zeiss, Germany)
The cells of 90028 pre-treated with Compound 11 at 16 µg/ml for 18 h according to the CLSI
document M27-A316 were examined under the phase-contrast microscope. Washed once with
disitilled water, the cells were then suspended in 250 µl of water. Fifty µl of the suspension
were pipetted onto microscope glass slides with coverslips on top to be later examined under
the microscope. The control morphologies of 90028 (untreated with Compound 11) were
generated in GCW wells (as described above 2.3.)
2.9. Statistical Analysis
Each experiment was performed in triplicate on three separate occasions. The percentage of
cell growth inhibition as well as inhibition of adhesion and the APE2 expression were
formulated as a mean standard deviation. Statistical differences were evaluated through
comparison with the non-parametric Wilcoxon test, P≤ 0.05 was considered significant.
3. Results
3.1. Antifungal Activity
At each concentration tested the % of inhibition displayed by the compounds was determined
and compared with MIC of amphotericin B, with values summarised in Tables 3, 4, and 5.
Compounds 6 and 7, having bromodichloromethylsulfonyl and dichloromethylsulfonyl group
respectively, at the para position of the phenyl rings attached to C-1, C-2 and C-4 chlorine,
nitro and sulfone moiety respectively showed moderate activity against all the strains tested
even at the maximum concentration of 16 µg/ml. Thus, MIC90=16 µg/ml was assessed as a
supra-MIC concentration (the highest concentration with incomplete killing). Interestingly,
9

the introduction of the bromodichloromethylsulfonyl group at the C-4 position of the benzene
ring (Compound 6) exerted moderate activity against the null ∆efg1 mutant and the rescued
∆cph1::CPH1 strain (P≤0.05, Table 3). On the contrary, dichloromethylsulfonyl substituted
compound at the C-4 position of the benzene ring (Compound 7) was more active against the
following strains: ꢀcph1 andꢀcph1/ꢀefg1 (P≤0.05, Table 4).
Compound 11 with hydrazino function at the C-1 position of the phenyl ring attached to C-2
and C-4 of the nitro and sulfone moiety respectively along with chlorodibromomethyl
substituent at the C-4 position of the phenyl ring exerted a greater anti-Candidal potential.
The Table 5 shows, Compound 11 which was more effective against ꢀcph1, exhibited 100%
of cell inhibition at 2 µg/ml (P≤0.05). In the case of ꢀefg1, the same level of cell inhibition
was observed at a two-fold higher concentration (P≤0.05). Moreover, 100% of cell inhibition
was noted for ꢀcph1/ꢀefg1 at the range of 1-16 µg/ml. Compound 11 out of all the
compounds screened was the most effective (Table 5), with a clear end point (100% cell
reduction) at 8 µg/ml against 90028. On the other hand, Compound 11 did not show MIC50
against 90028 at the lowest concentration tested of 0.25 µg/ml. Contrariwise, compounds 6
and 7 were found comparably effective, yet neither Compound 6 nor 7 displayed 100% of
cell inhibition.
3.2. Impact of Sulfone Derivatives on Adhesive Properties of the C. albicans Species
Pre-treating the cells with sulfone 6 (8-16 µg/ml) did not significantly affect adhesion of all
the Candida strains tested compared to their non-treated counterparts (P≥0.05, Table 6).
Attachment of Candida cells to the Caco-2 monolayer was inhibited (P≥0.05) at the highest
concentration tested (16 µg/ml). Interestingly, in half of the strains tested cell adhesion
increased after 90-min pre-treatment with two-fold lower concentration (8 µg/ml). On the
contrary, the remaining strains displayed decreased adhesion at 8 µg/ml. At the concentration
of 8 µg/ml, 90028 exhibited increase in attachment to Caco-2 compared to the non-treated
10

counterparts. In the case of the resued ꢀcph1::CPH1 the same phenomenon was noted, yet
the increase in adhesion did not go beyond the adhesion level of the non-treated cells
(observed in 90028). The adhesion level was 5.8-fold higher at 8 µg/ml versus to 16 µg/ml.
Moreover, this feature rose three- and five-fold at 4 µg/ml and 2 µg/ml respectively compared
to 16 µg/ml. The similar trend was observed for ꢀefg1 and ꢀcph1/ꢀefg1, i.e., 2.4- and 2.0-
fold at 4 and 2 µg/ml respectively compared to the highest concentration tested.
Sulfone 7 did not significantly (P≥0.05) affect Candida cells’ adhesion to epithelium at 2-16
µg/ml. Generally, the concentration of 16 µg/ml inhibited Candida cells’ attachment to Caco-
2, except for the rescued ꢀcph1/ꢀefg1::EFG1 strain (increased adhesion, P≥0.05). The
latter strain and 90028 also displayed increased adhesion ability at 8 µg/ml. The mutants
(ꢀcph1, ꢀefg1 and ꢀcph1/ꢀefg1) were inhibited in the attachment to the Caco-2
monolayer from 1.5- to 3.4-fold in comparison to the non-treated cells. Moreover, there was
no considerable increase in adhesion at lower concentrations compared to 16 µg/ml.
Compound 11 altered adhesion of the strains significantly (P≤0.05) at all the concentrations
tested. In 62% (5 strains) of all the strains tested the concentration of 16 µg/ml inhibited
attachment of cells to Caco-2. The exceptions are as follows: 90028, ꢀcph1/ꢀefg1, and the
latter one with one copy of EFG1 reintroduced, in which adhesion increased under the 16
µg/ml treatment. In the case of 90028 the increase observed at 8 µg/ml was 1.7-fold higher
compared to non-treated counterparts and cells treated with 16 µg/ml. Additionally, slightly
increased adhesion at the concentration 8 µg/ml was noted for ꢀcph1 and its counterpart
reverted with one copy CPH1. In the remaining strains, the level of attachment was reduced at
the latter concentration compared to the control cells. Moreover as for the concentration of 4
µg/ml, an increased adhesion was seen for ꢀcph1/ꢀefg1. This feature was higher at 2 µg/ml
in the following strains: ꢀcph1 reverted with CPH1, ꢀefg1, and ꢀcph1/ꢀefg1 (2.3-, 1.6-
and 1.3-fold compared to 16 µg/ml respectively).
11

3.3. Compound 11 Modulates the APE2 mRNA Expression
The APE2 expression significantly differed (P≤0.05) between the cells treated with
Compound 11 and untreated and subsequently growing for 18 h both in the in the YEPD
medium (Table 7) as well as on the Caco-2 monolayer (Table 8).
In the case of SC5314, the expression of APE2 in the cells grown either in YEPD or on Caco-
2 (pre-treated with Compound 11) was down-regulated (P≤0.05). APE2 up-regulation was
observed in the cells of ꢀcph1 treated with Compound 3 grown both in YEPD and on Caco-
2. In the cells of ꢀefg1 up-regulation of APE2 was noted under both tested conditions. The
ꢀefg1 strain reverted with one copy of EFG1, showed differences in the APE2 expression
between the cells preincubated with Compound 11 grown in YEPD and those grown on the
epithelial monolayer. In the cells of ꢀcph1/ꢀefg1 treated with Compound 11 an up-
regulation of APE2 in YEPD and a significant down-regulation (10-fold compared to the
untreated cells) on Caco-2 (P≤0.05) was noted.
3.4. Microscopy Study. Compound 11 Perturbs C. albicans Morphogenesis
To visualize the disturbed hyphae formation under Compound 11, we performed a phase-
contrast microscopy on C. albicans 90028 cells exposed to 16 µg/ml for 18 h (Fig. 2). Images
revealed long true-hyphal morphologies of the untreated cells (Fig. 2(A)), and spherical
blastoconidia with polarly located budding without filament formation under Compound 11
(Fig. 2(B)).
4. Discussion
The inhibition of degradative enzymes,8 adhesion and morphogenesis factors8, 18, 20 represents
a promising strategy for the design of new compounds with effective antifungal abilities
targeting Candida spp. In our study, Compounds 6 , 7, and 11 first tested for their activity
against the pathogenic C. albicans planktonic cells differed in the inhibition abilities of yeast
growth depending on their derivatives (Table 3, 4, and 5). By using the genetic alternations in
12

EFG1 and CPH1 we tested whether these morphogenesis factors could have an impact on C.
albicans resistance to the tested sulfones.
The lack of both CPH1 and/or EFG1 linked to the dysfunction of the cell wall integrity and
filamentous growth of C. albicans significantly increased the fungicidal potential of
21
Compound 11. As described Zavrel et al.
Efg1 and Cph1, strongly impact cell wall
thickness as well as polysaccharide composition even if only one copy of the EFG1 or CPH1
genes is removed. In our study (Table 5), ∆cph1/∆efg1 was highly sensitive to the sulfone
containing hydrazine function at benzene ring (Compound 11), while the sensitivity was
reduced in the rescued ∆cph1/∆efg1::EFG1 strain (%=100 at a 16-fold higher concentration).
Although deletion of the CPH1 gene reduces hyphal growth on solid medium6 we showed
(unpublished data) that it still forms hyphae during adhesion to a polarized monolayer of
Caco-2 epithelial cells. In stark contrast, the ∆efg1 mutant was slightly attenuated in the
developing filaments.22 In our study, deletion of either CPH1 or EFG1 provoked excellent
adhesion to the epithelial cells relative to 90028 and SC5314 (Table 6). Interestingly, a 90-
min pre-treatment of the planktonic cells of the strains tested with Compound 11 significantly
affected adherence to epithelial cells (P≤0.05) compared with the non-treated counterparts.
Conversely, for the strains: 90028, ∆cph1/∆efg1, and ∆cph1/∆efg1::EFG1, the number of the
adherent cells on the Caco-2 surface was not reduced significantly with Compound 11 at 16
µg/ml (short 90-min pre-treatment induced adherence) relative to the control cells. The
development of Compound 11 tolerance was also likely to have impelled the remodelling of
the cell wall components in ∆cph1/∆efg1.21 Moreover, under Compound 11 the compensation
of adhesins recognizing ligands (ALS3, HWP1) can occur in the initial attachment to Caco-
2.23 Thus suggesting their contribution to the resistance to sulfone derivatives. Furthermore,
over-expression of the efflux pump MDR gene24, 25 induced by the sulfones tested ought to be
considered. Our results demonstrated that the rescued ∆cph1::CPH1 strain exposed to
13

Compound 11 showed increasing adhesion following decreased sulfone concentrations
(concentration-dependent manner), but still lower than the non-treated cells. In the cells pre-
treated with Compound 11, the loss of the morphogenesis factors either CPH1 or EFG1
decreased the adhesive properties. Conversely, the rescued ∆cph1::CPH1 strain showing a
higher adhesion level compared with ∆cph1 under Compound 11, the differences were not
signifiact (P≥0.05). Our studies not only provided a comprehensive evaluation of
morphogenesis mutants as the determinants of the sulfone resistance, but also showed that
each strain is likely to possess an altered cell wall composition influencing their susceptibility
to these compounds. Thus the strain-dependent phenomenon ought to be considered as a
factor in the sulfone resistance mechanisms. Furthermore, we demonstrated that it was
particularly C. albicans heterozygousity that significantly influences the latter phenomenon
(e.g., in the case of the rescued strains tested).
The reintroduction of an ectopic copy of the wild-type allele back into each mutant reversed
their respective resistance to Compound 11. These results harmonize with the ones8 showing
that heterozygous cells are more resistant to antifungals. Furthermore, we found susceptibility
in the MAP kinase pathway mutant against Compound 11, which confirms sulfones also
targetting cell wall biosynthesis in C. albicans. Thus the cell wall playing a critical role in the
sulfone resistance mechanism requires further investigation.
The exposure to antifungals induced up-regulation of drug targets and genes as contributing to
resistance.25, 26 In our study, the cells exposed to Compound 11 resulted in the APE2 increased
expression, which can imply resistance to this compound. The disruption of CPH1 in C.
albicans cells generated an APE2 down-regulation. Conversely, under Compound 11 in the
∆cph1::CPH1 rescued strain APE2 was downstream of CPH1 on the MAP kinase pathway.
This is in agreement with earlier results27 that growth inhibition resulting from treatment with
antifungals required both a functional histidine kinase and an inact HOG pathway. In our
14

study, Efg1-compromised cells displayed up-regulation of APE2, suggesting an Ras1-cAMP-
independent mechanism of APE2. The activity of transcriptional factors is regulated in many
different ways.27 Cph1 and Efg1 were shown to be transferred to the nucleus and bound to its
up-stream promoter regions of target genes in the absence of inducers.28 Nobile et al.29
showed that Efg1 is both an activator and repressor of its target genes. In compliance with the
latter, APE2 was up- or down-regulated depending on the regulator gene mutant background
combination. It seems likely that these regulators are necessary as activators (Cph1) or
negative regulators (Efg1) for APE2 during the attachment to the Caco-2 monolayer (in stress:
exposure to Compound 11). Conversely, both Cph1 and Efg1 negatively regulated the APE2
expression during the epithelium colonization in vitro (without exposure to Compound 11).
Furthermore, as found Pierce and Kumamoto7 the expression of C. albicans’ virulence factors
was Efg1-dependant or not with respect to growth conditions. Our studies revealed that the C.
albicans sulfone resistance events are associated with differences in the target gene
transcription. Consistently with the previous data,7 while comparing planktonic with sessile
growth we revealed that Cph1 and Efg1 do not regulate adhesion. In order to interpret our
whole data, it should be borne in mind that binding of the regulators is associated with
differential transcription in the biofilm vs planktonic cultures.29 Other possible explanation for
the results obtained here is that Compound 11 can actually be a substrate of Ape2. As Ape2 is
implicated in several facets of biological processes of fungal cells and in the interaction with
the host,3 it offers a promising target for the action of sulfones.
In our study genetic changes in both EFG1 and/or CPH1 enabled Compound 11 to gain
access to intracellular targets, facilitating its membrane transience and increasing its contact
with intracellular targets (transcription of APE2). Under Compound 11, adhesion of the wild-
type cells was not reduced but the cells were defective in hyphae formation (Fig. 2B). Since
hyphal development is an important step in a normal biofilm development in vivo (causing the
15

majority of infections in humans), it is worth indicating that we found Compound 11
inhibiting the morphogenesis process. Using our data, we identified the APE2 gene that can
be expected to play an important role in sulfone resistance (Compound 11). The overall
results indicate that the performed antifungal screens are a valid approach to understanding
how C. albicans cells respond to sulfone derivatives.
Performing genetic screens of APE2 regulators is our future challenge allowing to answer
whether APE2 is controlled by more than one regulator under the sulfone influence.
Moreover, sulfones’ interaction with the cell wall biosynthesis pathway will provide an
effective strategy to blocking C. albicans’ invasion of epithelial cells.
5. Conclusions
To our knowledge, ours is the first study of sulfone derivatives’ influence on virulence factors
of C. albicans. Our data provide evidence that the sulfones’ mode of action is associated with
a reduced virulence arsenal in terms of pathogenic potential related to the expression of the
APE2 gene and the morphogenesis factors. The use of sulfone derivatives can successfully
inhibit degradative enzyme production and the induction of hyphal forms constitutively
expressed in C. albicans biofilms difficult to prevent by the current antifungal drugs. The
results obtained may be of great value for the design of new efficient inhibitors of the cell
wall components.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
6. Acknowledgements
We are extremely grateful to Professor Hsiu-Jung Lo from National Health Research Institute in
Zhunan (Taiwan) for providing us with the following strains: Can16, YLO323, HLC52, HLC74 and
HLC84. The work was in part supported by the research project 19/EM.1/2014 by the National
Institute of Public Health-National Institute of Hygiene. The cells adhession and genes quantification
16

studies were funded by the National Science Centre (No. DEC-2011/03/D/NZ7/06198). The sulfones
synthesis was supported by Warsaw University of Technology, Faculty of Chemistry.
Appendix A. Supplementary data
Supplementary material related to this article can be found at http://dx.doi.org/......./j.bmc....
17

References
1.
Zarb, P.; Amadeo, B.; Muller, A.; Drapier, N.; Vankerckhoven, V.; Davey, P.;
Goossens, H. Clin Microbiol Infect 2012, 18, E389.
2.
Mayer, F. L.; Wilson, D.; Hube, B. Virulence 2013, 4, 119.
3.
Staniszewska, M.; Bondaryk, M.; Kurzatkowski, W. Postepy Mikrobiologii 2013, 52,
373.
4.
Naglik, J. R.; Moyes, D. L.; Wachtler, B.; Hube, B. Microbes Infect 2011, 13, 963.
5.
Hashash, R.; Younes, S.; Bahnan, W.; El Koussa, J.; Maalouf, K.; Dimassi, H. I.;
Khalaf, R. A. Mycoses 2011, 54, 491.
6. Lo, H. J.; Kohler, J. R.; DiDomenico, B.; Loebenberg, D.; Cacciapuoti, A.; Fink, G. R.
Cell 1997, 90, 939.
7.
8.
Pierce, J. V.; Kumamoto, C. A. MBio 2012, 3, e00117.
Sardi, J. C.; Scorzoni, L.; Bernardi, T.; Fusco-Almeida, A. M.; Mendes Giannini, M. J.
J Med Microbiol 2013, 62, 10.
9. Hiromichi I; Masakazu T; Ten U; K, S. Preparation of disulfonylthiadiazoles and their
use as agrochemical microbiocides: Tokyo, Japan, 1994;
10.
11.
Borys KM; Korzyński MD; Z, O. Tetrahedron Letters 2012, 53, 6606.
Jha, K. K.; Samad, A.; Kumar, Y.; Shaharyar, M.; Khosa, R. L.; Jain, J.; Kumar, V.;
Singh, P. Eur J Med Chem 2010, 45, 4963.
12.
13.
14.
Ochal, Z.; Kamiński, R. Polish Journal of Applied Chemistry 2005, 49, 215.
Gillum, A. M.; Tsay, E. Y.; Kirsch, D. R. Mol Gen Genet 1984, 198, 179.
Pfaller, M. A.; Bale, M.; Buschelman, B.; Lancaster, M.; Espinel-Ingroff, A.; Rex, J.
H.; Rinaldi, M. G. J Clin Microbiol 1994, 32, 1650.
15. Ness, F.; Prouzet-Mauleon, V.; Vieillemard, A.; Lefebvre, F.; Noel, T.; Crouzet, M.;
Doignon, F.; Thoraval, D. Fungal Genet Biol 2010, 47, 1001.
18

16.
17.
Method M27-A2: CLSI, 2002.
Amberg, D.; Burke, D.; Strathern, J. In Methods in Yeast Genetics; Amberg, D.,
Burke, D., Strathern, J., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor,
2005, pp. 127.
18.
Naglik, J. R.; Moyes, D.; Makwana, J.; Kanzaria, P.; Tsichlaki, E.; Weindl, G.;
Tappuni, A. R.; Rodgers, C. A.; Woodman, A. J.; Challacombe, S. J.; Schaller, M.; Hube, B.
Microbiology 2008, 154, 3266.
19.
20.
21.
22.
Livak, K. J.; Schmittgen, T. D. Methods 2001, 25, 402.
PW, T.; YT, C.; PC, H.; CY, L. BioMedicine 2013, 3, 51.
Zavrel, M.; Majer, O.; Kuchler, K.; Rupp, S. Eukaryot Cell 2012, 11, 129.
Staniszewska, M.; Bondaryk, M.; Swoboda-Kopeć, E.; Siennicka, K.; Sygitowicz, G.;
Kurzątkowski, W. Brazilian Journal of Microbiology 2013, 44, 813.
23.
24.
Brand, A. International Journal of Microbiology 2012, 2012.
Ramage, G.; Rajendran, R.; Sherry, L.; Williams, C. International Journal of
Microbiology 2012, 2012.
25.
26.
Morschhauser, J. Fungal Genet Biol 2010, 47, 94.
Cannon, R. D.; Lamping, E.; Holmes, A. R.; Niimi, K.; Tanabe, K.; Niimi, M.; Monk,
B. C. Microbiology 2007, 153, 3211.
27.
28.
29.
Kaba, H. E.; Nimtz, M.; Muller, P. P.; Bilitewski, U. BMC Microbiol 2013, 13, 16.
Lin, Z.; Wu, W. S.; Liang, H.; Woo, Y.; Li, W. H. BMC Genomics 2010, 11, 581.
Nobile, C. J.; Fox, E. P.; Nett, J. E.; Sorrells, T. R.; Mitrovich, Q. M.; Hernday, A. D.;
Tuch, B. B.; Andes, D. R.; Johnson, A. D. Cell 2012, 148, 126.
19

Figure Captions
Fig. 1. The sulphone derivatives synthesis. Detail procedures and characteristic data of
synthesised compounds are given in Supplementary data
Fig. 2. Phase-contrast microscopy of Candida albicans untreated cells (A), and cells treated
with 16 µg/ml of chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (B). (A) After 18
h, note true hyphal forms grown in RPMI medium contained 0.09% DMSO. Oval
blastoconidial mother cell (arrowhead) with polarly filamented true hyphal forms (open
arrow) were discerned. (B) Note clumps of budding blastoconidial cells (arrowhead) upon
exposure to Compound 11
20

21

22

Table 1
Candida albicans strains used in the study
Strain
Parental strain
Relevant characteristics or genotype
Reference
13
14
SC5314
90028
Wild type, reference strain
Wild type, reference strain
ura3::1imm434/ura3::1imm434
cph1::hisG/cph1::hisG-URA3-hisG
ura3::1imm434/ura3::1imm434
cph1::hisG/cph1::hisG (CPH1)
ura3::1imm434/ura3::1imm434
efg1::hisG/efg1::hisG-URA3-hisG
ura3::1imm434/ura3::1imm434
cph1::hisG/cph1::hisG
6
6
6
∆cph1
∆cph1 (CPH1)
∆efg1
CAI4
CAI4
CAI4
6
6
6
∆cph1/∆efg1
CAI4
CAI4
CAI4
efg1::hisG/efg1::hisG-URA3-hisG
ura3::1 imm434/ura3::1 imm434
efg1::hisG/efg1::hisG (EFG1)
ura3::1 imm434/ura3::1 imm434
cph1::hisG/cph1::hisG
∆efg1 (EFG1)
∆cph1/∆efg1
(EFG1)
efg1::hisG/efg1::hisG (EFG1)
23

Table 2
List of primers used in this study
Primers
Sequence (5’-3’)
GCACTGAATTCCCAACCAGT
TGGTTTAGTCGCTGATGCTG
APE2-1
APE2-2
ACT1-1
ACT1-2
GACAATTTCTCTTTCAGCACTAGTAGTGA
GCTGGTAGAGACTTGACCAACCA
24

Table 3
Antifungal activity (cells inhibition %) of bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (Compound 6) against Candida albicans strains after 48 h
16b
8
4
2
1
0.5
0.25
1a
Candida albicans
90028
98.90
98.90
98.75
97.75
98.72
98.53
99.2
98.72
97.42
99.60
99.63
99.80
0.32
0.1
99.51
99.32
99.80
99.55
99.40
99.41
99.80
1.44
1.6
99.40
98.74
99.72
99.00
0.25
0.1
0.23
0.61
6.84
0.07
3.83
0.3
0.35
0.14
5.8
0.15
0.1
98.63
97.40
96.70
98.40
99.0
0.60
0.25
2.53
0.30
0.30
0.6
100
100
100
100
100
100
100
∆cph1
∆cph1 (CPH1)
∆efg1
2.40
0.40
5.60
7.7
0.34
0.8
1.12
0.07
2.60
0.32
3.4
99.20
98.91
99.3
97.10
98.73
99.00
98.62
98.80
2.5
99.61
99.45
99.52
0.41
1.4
0.1
∆efg1 (EFG1)
∆cph1/∆efg1
5.14
8.4
99.30
99.00
99.52
1.22
0.4
98.75
98.81
98.90
0.06
2.95
98.31
96.50
∆cph1/∆efg1 (EFG1) 99.80
1.55
1.25
99.30
1.05
4.64
2.34
a, Amphotericin B in concentration of 1 µg ml-1 was used as a control (% inhibition=100). b, MIC90 - the highest concentration with incomplete killing for the
reference strain 90028. Values in bold indicate significant inhibtion (P≤0.05) of cells growth compared to the reference strain 90028
26

Table 4
Antifungal activity (cells inhibition %) of dichloromethyl-4-chloro-3-nitrophenyl sulfone (Compound 7) against Candida albicans strains after 48 h
16b
8
4
2
1
0.5
0.25
0.0006
1a
Candida albicans
90028
99.50
99.24
99.35
98.85
99.42
96.05
99.10
99.01
99.14
99.42
99.60
99.38
0.05
99.30
0.003 99.20
0.004 99.12
0.004 98.71
0.003
0.007
0.003
0.15
0.01
0.01
0.01
0.02
0.008
0.002
0.01
0.02
0.01
0.02
0.01
0.003 99.11
100
100
100
100
100
100
100
99.00
99.12
∆cph1
0.003 99.24
0.006 99.36
0.006 99.42
0.001 98.90
0.002 99.40
0.002 99.32
0.005
0.008
0.01
0.01
0.01
0.01
0.01
97.7
91.83
99.10
95.2
0.005
0.01
0.01
0.01
0.01
0.02
∆cph1 (CPH1)
∆efg1
0.045 98.63
96.74
99.36
95.41
99.10
99.01
99.49
99.40
99.51
99.20
99.72
0.002
0.003
98.10
0.03
∆efg1 (EFG1)
∆cph1/∆efg1
0.004 98.53
99.10
0.001 99.25
0.004
99.05
98.8
99.22
∆cph1/∆efg1 (EFG1) 99.40
0.01
99.30
0.002
a, Amphotericin B in concentration of 1 µg ml-1 was used as a control (% inhibition=100). b, MIC90 - the highest concentration with incomplete killing for the
reference strain 90028. Values in bold indicate significant inhibition (P≤0.05) of cells growth compared to the reference strain 90028
27

Table 5
Antifungal activity (cells inhibition %) of chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (Compound 11) against Candida albicans strains after 48h
16
8
4b
2
1
0.5
0.25
1a
Candida albicans
90028
99.6
100
97.40
99.85
98.8
99.5
98.80
100
81.75
96.6
100
100
0.5
0.15
1.7
100
100
15.1
0.7
99.73
100
0.2
0.3
0.2
0.8
0.1
0.4
1.44
1.9
17.0
20.0
98.6
70.80
98.6
70.0
72.0
0.7
0.5
100
100
100
100
100
100
100
∆cph1
99.3
99.74
99.1
100
98.75
97.9
∆cph1 (CPH1)
∆efg1
99.81
100
99.7
100
2.0
1.5
2.0
0.3
0.9
99.1
100
1.3
0.95
3.95
1.3
1.0
0.5
0.15
1.4
0.62
1.7
3.0
1.40
1.0
2.23
0.5
98.75
97.0
∆efg1 (EFG1)
∆cph1/∆efg1
99.81
100
99.80
100
99.31
100
0.95
2.44
0.9
0.15
0.40
2.10
0.11
0.85
0.23
1.05
0.35
0.81
0.64
1.0
98.8
98.8
∆cph1/∆efg1 (EFG1) 100
99.44
99.1
99.3
a, Amphotericin B in concentration of 1 µg ml-1 was used as a control (% inhibition=100); b, MIC90 - the highest concentration with incomplete killing for the
reference strain 90028. Values in bold indicate significant inhibtion (P≤0.05) of cells growth compared to the reference strain 90028
28

Table 6
The percentage of adhesion of C. albicans cells to Caco-2 cell line after pre-treatment with the bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (named
Compound 6), dichloromethyl-4-chloro-3-nitrophenyl sulfone named (Compound 7), and chlorodichloromethyl-4-hydrazino-3-nitrophenyl sulfone
(Compound 11). Adhesion data calculated for cells grown on Sabouraud agar of 24-well-plate. Adherence was expressed as a percentage of the total number
of cells added (control cells non-treated). Data are expressed as the mean SD of three independent experiments. Values in bold indicate significant reduction
of cells adhesive properties compared to non-treated counterparts (P≤0.05)
Antifungal
Compounds
concentrations
(µg/ml)
90028
0.68 0.46
SC5314
ꢀcph1
ꢀcph1(CPH1)
ꢀefg1
ꢀefg1/ꢀcph1
ꢀefg1(EFG1)
ꢀcph1/ꢀefg1 (EFG1)
16
8
0.71 0.00
0.56 0.51
0.35 0.11
0.55 0.46
0.43 0.26
1.03 0.49
0.80 0.47
0.99 0.54
0.80 0.43
0.37 0.12
0.56 0.89
0.22 0,08
0.51 0.23
0.48 0.22
2.78 4.77
1.39 0.06
2.38 1.09
2.28 0.52
0.71 0.00
1.28 0.24
1.43 1.01
0.78 0.75
0.90 0.59
0.76 0,05
1.86 0.35
0.99 0.24
0.49 0.33
1.14 1.16
0.90 0.74
1.06 0.08
0.69 0.14
0.97 0.84
1.10 0.28
0.81 0.36
0.63 0.40
0.69 0,17
1.32 0.36
0.43 0.29
0.38 0.08
1.02 0.37
0.78 0.16
0.42 0.21
0.48 0.10
0.36 0.09
0.47 0.11
2.25 1.12
1.66 0,17
2.74 1.03
2.94 0.59
0.77 0.25
1.13 0.27
nt
0.75 0.49
2.20 1.33
nt
0.36 0.00
1.26 0.62
Compound 6
Compound 7
Compound 11
nt
nt
4
nt
nt
nt
nt
2
1.19 0.74
2.05 0.87
nt
0.68 0.17
0.79 0.34
1.18 0.83
nt
1.68 0.32
16
8
nt
2.24 0.50
nt
nt
4
nt
nt
nt
nt
2
16
8
1.60 1.59
2.66 2.00
0,91 0.44
nt
0.80 0.40
0.89 0.42
nt
1.50 0.74
0.45 0.17
0.75±0,50
nt
nt
nt
nt
4
0.91 0.44
0.51 0.23
2
1.58 0.67
1.10 1.10
1.60 0.65
5.37 12.40
2.10 3.52
1.22 0.78
2.60 2.33
1.24 1.06
Non treated cells
nt, not tested
29

Table 7
Analysis of the APE2 gene relative expression compared to the ACT1 reference gene in C. albicans cells untreated and after treatment with
chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (Compound 11). The cells were grown for 18 h in YEPD medium at 30°C.
Not treated cells
CtACT1
Cells treated with sulfone 3
C. albicans
CtAPE2
∆C(t)
-9,04
-5,23
-6,95
-11,10
-6,23
-7,15
-10,89
-4,74
2-∆∆C(t)
CtAPE2
CtACT1
∆C(t)
-2,84
-2,82
-0,11
-4,71
-3,69
-4,05
-3,44
-3,30
2-∆∆C(t)
SC5314
90028
21.68 3,05
23.04 1,75
24.85 2.92
20.46 0.03
22.34 2.52
23.79 1.82
21.39 0.49
25.35 3.14
30.72 1.38
28.27 0.73
31.15 11.99
31.56 2.24
28.57 3.29
30.94 2.03
32.28 3.4
3,81
26.55 3.05
26.79 1.35
26.53 2.47
26.51 2.84
26.87 2.85
27.24 3.30
26.38 2.33
26.88 3.05
29.39 0.50
29.61 0.45
26.64 3.80
31.22 2.04
30.56 2.74
31.29 1.86
29.82 0.01
30.18 1.38
0,02
1
-
1
-
1,73
4,15
-4,87
0,92
3,74
-6,16
-2,71
4,60
∆cph1
∆cph1 (CPH1)
∆efg1
-1,02
0,36
∆cph1/∆efg1
∆efg1 (EFG1)
∆cph1/∆efg1 (EFG1)
-0,61
-0,14
30.09 3.94
Ct- mean for three independent experiment SD; 1strain 90028 – kalibrator in 2-∆∆C(t)
31