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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 1  |  Page : 28-33

Differential expression levels of agglutinin-like sequence, lipase, and secreted aspartyl protease genes in Candida tropicalis treated with fluconazole alone and in combination with clotrimazole


1 Department of Biology, Gachsaran Branch, Islamic Azad University, Gachsaran, Iran
2 Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj, Iran

Date of Web Publication28-Mar-2019

Correspondence Address:
Fahimeh Alizadeh
Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrptps.jrptps_22_18

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  Abstract 

Background: The frequency of opportunistic fungal infections in immunocompromised patients, especially by Candida species, has sharply increased in the last few decades. As the number of antifungal drugs available for the treatment of candidiasis is limited, combination therapy has been employed as one of the most commonly used techniques to alleviate this problem. Aims: The main aim of this study was to explore the antifungal activity of fluconazole in combination with clotrimazole on expression levels of virulence genes, agglutinin-like sequences (ALS1 and ALS2), lipases (LIP1 and LIP4) and secreted aspartyl proteases (SAP2 and SAP4) in Candida tropicalis. Methods: Ten infected clinical isolates obtained from recurrent vulvovaginal candidiasis patients were used in this study. The broth microdilution assay was utilized to investigate antifungal susceptibilities to fluconazole alone and in combination with clotrimazole and the synergistic effects were interpreted with reference to the fractional inhibitory concentration (FIC) index model. The expression levels of ALS1, ALS2, LIP1, LIP4, SAP2 and SAP4 genes were quantified by real-time RT–PCR. Results: Antifungal susceptibility results showed that isolates were resistant to at least one type of azole antifungals. The combination of fluconazole with clotrimazole revealed synergistic effects against C. tropicalis isolates with FIC90 index ranging from 0.011 to 0.43. The results indicated that combination of fluconazole with clotrimazole could cause a down-regulation of gene expression of ALS1, SAP2, LIP4, SAP4, LIP1 and ALS2 genes, respectively. Conclusions: Fluconazole in combination with clotrimazole may diminish the virulence properties of C. tropicalis.

Keywords: Antifungals, Candida tropicalis, clotrimazole, fluconazole


How to cite this article:
Khodavandi A, Alizadeh F, Abdolahi M, Jahangiri M. Differential expression levels of agglutinin-like sequence, lipase, and secreted aspartyl protease genes in Candida tropicalis treated with fluconazole alone and in combination with clotrimazole. J Rep Pharma Sci 2019;8:28-33

How to cite this URL:
Khodavandi A, Alizadeh F, Abdolahi M, Jahangiri M. Differential expression levels of agglutinin-like sequence, lipase, and secreted aspartyl protease genes in Candida tropicalis treated with fluconazole alone and in combination with clotrimazole. J Rep Pharma Sci [serial online] 2019 [cited 2019 Aug 23];8:28-33. Available from: http://www.jrpsjournal.com/text.asp?2019/8/1/28/255050


  Introduction Top


Candida tropicalis has been identified as one of the most virulent species of the Candida-non-albicans group. The distribution and frequency of Candida-non-albicans species vary geographically. C. tropicalis has been widely considered the second to fourth among the most commonly isolated species.[1],[2],[3],[4],[5]

Various factors have been reported to contribute to the virulence of C. tropicalis including adhesion to medical devices and host surfaces, biofilm formation, yeast-to-hyphae transition (morphogenesis), phenotypic switching, thigmotropism and the secretion of hydrolytic enzymes, including secreted aspartyl proteases (SAPs), esterases, lipases, phospholipases, and hemolysins.[5],[6],[7],[8]

As an important step in pathogenesis, adhesion leads to tissue damage and invasive infections. The agglutinin-like sequence (ALS) protein family form important adhesion molecules. The composition of the ALS family of C. tropicalis includes 16 genes which encode cell-surface glycoproteins contribute to adhesion.[5],[8],[9] In addition, the activity of hydrolytic enzymes has been associated with infections thought to facilitate the disruption of host tissues, degrade defense proteins, and enhance the efficiency of acquisition nutrients. Moreover, the family of Saps comprising one subfamily of four genes (SAPT1-4) is recognized as an important virulence factor to the pathogenesis of C. tropicalis. In C. tropicalis, the family of lipases consists of 10 members (LIP1-10), which are involved in the triacylglycerols hydrolysis and synthesis.[5],[10],[11],[12]

Various imidazole antifungal drugs such as clotrimazole have been claimed to inhibit Candida growth. Moreover, triazoles such as fluconazole represent the wide spectra of antifungal activity against fungi. Although azole antifungals are members belonging to the same class of antifungal drugs, they represent largely different chemical properties which impact the pharmacokinetics and spectrum of activities. All azole antifungals inhibit the function of the cytochrome P450 system to some degree of specificity.[13],[14] The increasing rate of azole drug-resistance of C. tropicalis has been documented. The number of antifungal agents is limited and their mostly fungistatic activity facilitates selection of antibiotic-resistant strains.[1],[3],[15] Thus, advances in the development of candidiasis therapies have focused on the use of antifungal agents in combination.[16] In this study, we first investigated the antifungal susceptibilities to fluconazole alone and in combination with clotrimazole using the broth microdilution method interpreting the synergistic effects by the fractional inhibitory concentration (FIC) index model. Finally, the antifungal effect of fluconazole in combination with clotrimazole on expression levels of ALS1, ALS2, LIP1, LIP4, SAP2, and SAP4 genes in C. tropicalis were examined.


  Materials and Methods Top


Candida tropicalis and growth conditions

Candida tropicalis ATCC 750 and ten infected clinical isolates obtained from recurrent (defined as 3 or more per year) vulvovaginal candidiasis patients who had previously used clotrimazole in the past 5 years were used in this study. All isolates were maintained on Sabouraud Dextrose Broth (Merck Research Laboratories, Darmstadt, Germany). Before experiments, the isolates were grown overnight at 35°C on Sabouraud Dextrose Agar (Merck Research Laboratories) plates.[17]

Antifungal agents

The antifungal agents used in the present study, i.e., fluconazole and clotrimazole, were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The antifungal agents were then dissolved and the stock solutions were diluted based on CLSI M27-A3 guidelines.[18]

Preparation of the Candida tropicalis cell inocula

The C. tropicalis cell density of five colonies more than 1 mm in diameter of propagated cells, suspended in 5 ml sterile phosphate-buffered saline was estimated through measurement of the optical density at 530 nm. The cell suspension measured at OD530 was made in Roswell Park Memorial Institute-1640 medium (Sigma-Aldrich) and prepared in accordance with CLSI M27-A3 guidelines. Briefly, the cell suspension (a concentration of 1–5 × 106 colony-forming units (CFU)/ml) was diluted to 5 × 102–2.5 × 103 CFU/ml and the viability of the yeast cells was measured through the viable pour plate counting method.[18]

Broth microdilution assay

The broth microdilution assay of fluconazole alone and in combination with clotrimazole was performed in accordance with the CLSI (M27-A3, M27-S4) guidelines with a few adaptations.[18],[19] Briefly, 5 × 102–2.5 × 103 CFU/ml inoculum was added to the wells of a U-bottom polystyrene 96-well microdilution plates. One hundred μl of the two-fold dilution of each antifungal agent (alone or in combination) dilution was added to the respective wells with the cell suspension. Plates were incubated for 24 h at 35°C. The minimal inhibitory concentrations (MICs) were determined using a Stat Fax 303 Reader (Awareness Technology, Inc., USA) as the lowest concentration of antifungal agents required for 50% and 90% growth inhibition compared to the controlled growth. Clearly, the antifungal agent interactions were interpreted by the FIC index model.[20],[21]

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).

The qRT-PCR analysis was based on the procedure previously described.[22] The inoculum of 1-5 × 106 CFU/ml C. tropicalis ATCC 750 strain was treated with fluconazole alone and in combination with clotrimazole at concentrations of 2 × MIC and 1 × MIC. Next, the mixture under study was pelleted at 3000 rpm for 10 min. For each treatment, the total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's recommended protocol. The RNA quality was observed using the formaldehyde-denaturing agarose gel electrophoresis. The OD260/280 and OD260/230 were measured with a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies Inc., Wilmington, DE). RNAse-free DNase I (Fermentas, USA) was used for the removal of genomic DNA contamination. Single-stranded cDNA was synthesized using M-MuLV reverse transcriptase and random hexamer oligonucleotides (Fermentas, USA) in accordance with the manufacturer's instructions. The synthesized cDNA formed in each treatment was amplified with primers [Table 1] by PCR using ™SYBR Green qPCR Master Mix (Fermentas, EU) in a Bio-Rad MiniOpticon™ system (USA). The cycling conditions included an initial step at 50°C for 2 min; holding at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s and subsequently annealing at 55°C for 1 min. Finally, the melting reaction occurred at 72°C–99°C. The results from amplifications were quantified by the Pfaffl method.[22]
Table 1: Oligonucleotide primers used for quantitative real-time polymerase chain reaction

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Ethical issues

The Research Ethics Committees of our institute, Islamic Azad University of Yasooj, Iran (Ethical code 1213342) approved the study. The study protocol conformed to the ethical guidelines of the 2008 Declaration of Helsinki. Informed consent was obtained from patients.

Statistical analysis

Results represent the mean of the three independent experiments ± standard deviations. Data were subjected to analysis of variance. The comparison of two means was calculated using the Tukey's post hoc test. Value of P ≤ 0.05 was considered statistically significant. Statistical analyses were performed using the software SPSS 21.0 for Windows (SPSS Inc. Chicago, IL, USA).


  Results Top


The age range of studied patients was 23–48 years old. The overall isolation rate of Candida species from vulvovaginal samples was 93.50%. The incidence rate of C. tropicalis was found in 10 of 187 (5.35%) of Candida species.

The antifungal effect assay setup was based on CLSI guidelines for the broth microdilution assay and the MICs of fluconazole alone and in combination with clotrimazole was determined against C. tropicalis [Table 2]. From among the 10-infected clinical isolates of C. tropicalis, 6 (60%) were resistant to fluconazole (MIC ≥8 μg/ml) and 10 (100%) were resistant to clotrimazole (MIC ≥1 μg/ml). A comparison between these values for fluconazole alone and in combination with clotrimazole against C. tropicalis showed that the combination of fluconazole with clotrimazole can be decreased from its MIC value. It is also noteworthy to mention that the FIC index values of all C. tropicalis tested (100%) were synergistic effects (FIC ≤ 0.5) in the presence of fluconazole in combination with clotrimazole. Fluconazole exhibits synergy with clotrimazole with a FIC90 index ranged from 0.011 to 0.43 in C. tropicalis isolates.
Table 2: Minimal inhibitory concentration (μg/ml) and fractional inhibitory concentration values of fluconazole alone and in combination with clotrimazole against Candida tropicalis

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The analysis of the effect of fluconazole alone and in combination with clotrimazole against C. tropicalis ATCC 750 on their expression levels of ALS1, ALS2, LIP1, LIP4, SAP2, and SAP4 genes revealed that the overall genes expression levels of the treatments differed significantly (P < 0.05). The results of the qRT-PCR analysis showed that fluconazole alone caused down-regulation in the expression levels of SAP2, SAP4 and LIP4 (P < 0.05) and the expression levels of ALS1, ALS2, and LIP1 represented no significant changes (P = 0.20). The expression of LIP1, LIP4, SAP2, and SAP4 genes was decreased on clotrimazole challenge. The combination of fluconazole with clotrimazole showed down-regulation of genes compared to the untreated control (P < 0.05; [Figure 1]).
Figure 1: Expression analysis of agglutinin-like sequence 1, agglutinin-like sequence 2, lipase 1, lipase 4, secreted aspartyl protease 2 and secreted aspartyl protease 4 genes in Candida tropicalis ATCC 750 treated with 2 × minimal inhibitory concentration and 1 × minimal inhibitory concentration of fluconazole alone and in combination with clotrimazole by quantitative reverse transcription polymerase chain reaction. (*) means significant reduction of gene expression to untreated control at level <0.05

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The results showed that fluconazole in combination with clotrimazole at concentrations of 2 × MIC and 1 × MIC down-regulated the expression levels of ALS1 by 3.75- and 3.13-fold and ALS2 by 2.00- and 1.89-fold, respectively (P < 0.05). The expression levels of LIP1 and LIP4 genes were down-regulated by 2.18- and 1.95-fold and 2.90- and 1.55-fold in C. tropicalis treated with 2 × MIC and 1 × MIC of fluconazole in combination with clotrimazole, respectively (P < 0.05). The expression level of SAP2 was down-regulated by 2.91- and 2.74-fold and SAP4 down-regulated by 2.51- and 2.038-fold with 2 × MIC and 1 × MIC of fluconazole in combination with clotrimazole treatment, respectively (P < 0.05). However, the combination of fluconazole with clotrimazole could cause a down-regulation of gene expression of ALS1, SAP2, LIP4, SAP4, LIP1, and ALS2 genes, respectively (P < 0.05).


  Discussion Top


In the past decades, there has been renewed interest in investigating the combination therapy effect of antifungal agents for the treatment of candidal infections that are difficult to treat. Combination therapy is the potential strategy used to reduce resistance development, minimize their side effects/toxicities, increase the rate or extent of killing by synergy and may broaden the spectrum of activity.[24],[25],[26]

Azoles are the initial treatment of choice for antifungal treatment of candidiasis. Nevertheless, the development of antifungal resistance, drug-drug interactions, and severe side effects/toxicities has limited their effective therapeutic for fungal diseases. Therefore, the development of the effectiveness and acceptability therapeutic strategies for fungal diseases is warranted to overcome antifungal agent resistance and its side effect/toxicity.[24],[27],[28] In the present study, we observed significant results of resistance to clotrimazole in infected clinical isolates of C. tropicalis obtained from recurrent vulvovaginal candidiasis patients with previous exposure to clotrimazole. Similar results were obtained by Pelletier et al.[29] for the HIV-infected pediatric population receiving this azole. Diaz et al.[30] showed resistance to clotrimazole of vaginal C. tropicalis isolates.

The results from the present research show that fluconazole in combination with clotrimazole exhibit potent antifungal synergy against all clinical isolates of C. tropicalis. Combination therapy of fluconazole with clotrimazole was as effective as single dose fluconazole in vulvovaginal mycoses.[31] Gharibi et al.[32] evaluated the effectiveness of fluconazole and clotrimazole combination in the treatment of recurrent vaginal candidiasis caused by C. albicans. They observed that for the treatment of patients with recurrent vulvovaginal candidiasis, fluconazole in combination with clotrimazole can be more effective than other treatments. Our results show that in vitro combination of fluconazole with clotrimazole could be effective against C. tropicalis.

The gene expression analysis of C. tropicalis treated with fluconazole alone and in combination with clotrimazole revealed that fluconazole in combination with clotrimazole could down-regulate the expression levels of genes. The results from the present research also revealed that the expression levels of ALS1, ALS2, LIP1, LIP4, SAP2, and SAP4 were affected by fluconazole in combination with clotrimazole in C. tropicalis which is in agreement with the recent experimental results of Khodavandi et al.[33] The findings revealed that fluconazole in combination with amphotericin B significantly down-regulated the expression of PLB and SAP2 genes in C. tropicalis. Moreover, the expression levels of ALS1 and ALS2 differed significantly in C. tropicalis treated with the combination of the two azoles. This may occur due to distinct adherence and biological functions of ALS family genes. Research suggests that the ALS family form important adhesion molecules for C. tropicalis pathogenicity.[5],[8],[9] Roudbarmohammadi et al.[34] investigated the expression of ALS1 and ALS3 genes in C. albicans isolated from vulvovaginal candidiasis. The results indicated that the expression of ALS1 and ALS3 genes was greater than that of the control group.

Our results showed that fluconazole alone and in combination with clotrimazole can induce changes in the expression levels of hydrolytic enzymes (LIP1, LIP4, SAP2, and SAP4), which are recognized as important virulence factors for C. tropicalis pathogenicity.[5],[10],[11],[12] Our results consistent with those obtained by Gu et al.,[35] who showed that the combinations of fluconazole with fluoxetine cause a down-regulation of gene expression of SAP1-4 C. albicans strains. The expression of virulence genes (ALST1-3, LIP1, LIP4, and SAPT1-4) was investigated in C. tropicalis strains with diverse virulence. RT-PCR analysis showed that the expression of virulence genes was significantly different in the corresponding genes for most C. tropicalis.[8] Stehr et al.[36] investigated the expression pattern of LIP1–LIP10 genes in C. albicans during experimental infections and in samples of patients with oral candidiasis. The findings of Stehr et al.[36] showed that individual lipase genes were differentially expressed in a mouse model of systemic candidiasis and in human specimens. Khodavandi et al.[37] showed that the expression of SAP4 gene was down-regulated in C. albicans treated with fluconazole.


  Conclusions Top


This study illustrates the potent synergist activity of fluconazole in combination with clotrimazole against C. tropicalis demonstrating the down-regulation of ALS1, ALS2, LIP1, LIP4, SAP2, and SAP4 genes in C. tropicalis treated with fluconazole in combination with clotrimazole, as assessed in the qRT-PCR assay. Further research needs to be conducted to ascertain whether these events reflect the potential of fluconazole in combination with clotrimazole for the inhibition of virulence factors of C. tropicalis which differentially expresses specific gene. However, the present research demonstrated that the antifungal effect of fluconazole in combination with clotrimazole could be of great significance for the development of therapeutic strategies against resistant C. tropicalis.

Acknowledgment

The authors would like to thank the Islamic Azad University of Yasooj for financial support. The results presented in this study are part of the Master thesis (1213342).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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