Effect of progesterone on Candida albicans biofilm formation under acidic conditions: a transcriptomic analysis

Vulvovaginal candidiasis (VVC) caused by Candida albicans is a common disease worldwide. A very important C. albicans virulence factor is its ability to form biofilms on epithelium and/or on intrauterine devices promoting VVC. It has been shown that VVC has a hormonal dependency and that progesterone affects virulence traits of C. albicans cells. To understand how the acidic environment (pH 4) and progesterone (either alone and in combination) modulate C. albicans response during formation of biofilm, a transcriptomic analysis was performed together with characterization of the biofilm properties. Compared to planktonic cells, acidic biofilm-cells exhibited major changes in their transcriptome, including modifications in the expression of 286 genes that were not previously associated with biofilm formation in C. albicans. The vast majority of the genes up-regulated in the acidic biofilm cells (including those uniquely identified here) are known targets of Sfl1, and the expression of this regulator impaired formation of the acidic biofilm. Under the acidic conditions used, progesterone treatment reduced C. albicans biofilm biomass, structural cohesion, matrix quantity and susceptibility to fluconazole. Transcriptomic analysis of progesterone-exposed biofilms led to the identification of 65 down-regulated genes including, among others, the regulator Tec1 and several of its target genes suggesting that the function of this transcription factor is inhibited by the presence of the hormone. Overall, the results of this study show that progesterone modulates C. albicans biofilm formation and genomic expression under acidic conditions, which may have implications for C. albicans pathogenicity in the vaginal environment. Author summary Vulvovaginal candidiasis (VVC) is an infection of the vaginal tract that affects millions of women every year. It is caused by fungi of the genus Candida, mainly Candida albicans. Several C. albicans virulence factors contribute to the establishment of this infection, including the ability to form biofilms on vaginal walls and intrauterine devices. Candida species belong to vaginal microflora, however under certain conditions they can cause infection. It has been shown that conditions that prompt VVC include those leading to high progesterone levels, as pregnancy. Here we show that progesterone impairs the ability of C. albicans cells to form biofilms but causes a potential protective stress response. Indeed, we reveal an increased fluconazole resistance of biofilm cells grown in the presence of the hormone. Additionally, our results suggest that biofilm cells have a specific response to acidic conditions, as those established in the vaginal environment. Deepening the knowledge on the modulation of C. albicans virulence by vaginal conditions is essential for a full understanding of the pathogenesis of this species in the vaginal tract and contribute to the disclosure of new targets to treat VVC.


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Vulvovaginal candidiasis (VVC) affects millions of women every year and is considered 62 to be an important public health problem. It is estimated that approximately 70-75% of women 63 will experience an episode of VVC in their lifetime [1]. Although VVC is not usually a life-64 threatening condition, the vaginal tract constitutes a main access route to the bloodstream. microbes from the vaginal lumen thus contributing to the recurrence of VVC [10].

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The development of VVC has been associated with the disturbance of the hormonal 81 vaginal environment resulting from behavioral or other host-related factors such as pregnancy,

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hormone replacement therapy, and use of oral contraceptives or IUDs [1]. It is thought that 83 progesterone contributes to VVC development by stimulating the production of glycogen by 84 epithelial cells [13,14] and inhibiting certain traits of the innate and adaptive immune response 85 [15][16][17]. Besides these effects on the host it has been also shown that progesterone has direct

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Specifically, we observed a decrease in the number of cultivable cells of approximately 2 117 orders of magnitude [Log (CFUs/ml)/cm 2 ] ( Fig 1A) and a decrease in biofilm biomass of 118 approximately 20% (Fig 1B). These results are consistent with those reported by Alves et al.

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[21]. It is important to stress that the study of Alves et al. [21] was undertaken using two strains 120 (ATCC 90028 and 558234, a reference strain from the American Type Culture Collection

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(ATCC) and a vaginal isolate, respectively), different from the one used in this study, thereby 122 leading us to conclude that the inhibitory effect of progesterone on biofilm formation by C.
123 albicans is independent of the genetic background of the strains used.

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The effect of progesterone on the metabolic activity of C. albicans biofilm's cells was 125 also evaluated, using XTT reduction assay. The results obtained ( Fig 1C) show a slight 126 decrease in the metabolic activity of biofilm cells cultivated in the presence of progesterone, 127 compared to the absence of the hormone; however, this difference is not statistically significant 128 (p-value >0.05). The effect exerted by progesterone on the structure and morphological cultivable cells ( Fig 1A) and total biomass ( Fig 1B) of C. albicans SC5314 biofilms.

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Effect of progesterone on biofilm matrix production 137 One of the most important characteristics of Candida biofilms is the presence and 138 composition of the extracellular matrix. The functions of the matrix are not entirely clear, but it 139 is thought that it controls the desegregation of the biofilm and protects against antifungal 140 agents and the host immune system [23]. In order to analyze the effect of progesterone on C.
141 albicans biofilm matrix production and composition, the total protein and carbohydrate content 142 of the extracellular matrix was determined in the absence and presence of progesterone.
143 Table 1 shows that progesterone led to a significant reduction (p-value ≤0.05) of the amount 144 of biofilm matrix to less than half of that formed in its absence. Furthermore, progesterone also 145 led to an alteration of matrix composition, especially the amount of total carbohydrate, which 146 decreases to almost half of the amount detected in the matrix of biofilm formed without 147 progesterone (p-value ≤0.05). There is also a slight decrease in the protein level of the biofilm 148 matrix but it was not statistically significant (p-value >0.05) ( Transcriptome-wide alterations in C. albicans biofilms in the absence of 182 progesterone. Transcriptional profiling of C. albicans biofilms formed after 24h of cultivation 616 genes were up-regulated in biofilm cells, while 397 genes were down-regulated. A subset 186 of these genes is listed in Table 2 and the full list is available in S1

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A subset of genes whose expression was found to increase or decrease (above or below 2-fold) in C.

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albicans SC5314 biofilms grown 24 h in RPMI at pH 4, in comparison with the transcript levels registered 205 in planktonic cells cultivated in the same conditions, was selected and are herein shown, while the full 206 list is available on S1 Table. Genes whose transcription was found to be biofilm-induced or -repressed 207 specifically at the acidic conditions used in this study, are highlighted in grey and among them are 208 highlighted in bold those previously described as essential to biofilm formation. The biological function

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indicated is based on the information available at Candida Genome Database. energy", "protein with binding function", "transport", "stress response" and "interaction with the 218 environment" (Fig 2). In general, the functional clustering of the genes found to be differently 219 expressed in our acidic biofilms is similar to those found in other studies [25][26][27][28][29] something 220 that could be attributable to the fact that most of the genes that we found differentially 221 expressed in our acidic biofilms have a poorly or even uncharacterized function.

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To further understand the transcriptional regulatory network active in the formation of  Table). Figure  biofilms without progesterone than in its presence (87 genes); iii) genes more strongly down-278 regulated in biofilms formed without progesterone (12 genes); iv) genes more strongly down-279 regulated in the progesterone-exposed biofilms (79 genes). A subset of these progesterone-

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responsive genes is shown in Table 3 and the full list is available in supplementary S4 Table. 281 282

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Similarly, we observed that progesterone-exposed biofilm cells were significantly more 302 tolerant to fluconazole than biofilm cells grown in the absence of the hormone (MIC of 1.5 303 µg/ml compared with 0.25 µg/ml).

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The inhibitory effect of progesterone in biofilm formation may be related to reduced 305 expression of genes required for this process. Expression of 166 progesterone-responsive 306 genes is reduced in biofilm cells grown with progesterone (87 clustered in group ii and 79 in 307 group iv (S4 Table). These include several key regulators of biofilm formation, including the shown to be required for biofilm formation (all highlighted in grey in S4 Table). The

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Several genes involved in the process of biofilm formation that had a reduced expression 314 in the presence of progesterone are documented targets of Tec1 ( Fig 5A) and Brg1, according

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to the PathoYeastract database [36]. We therefore tested whether these two regulators are 316 required for biofilm formation in the presence of the hormone. Deleting the TEC1 reduced the 317 formation of acidic biofilms, which was further aggravated in the presence of progesterone 318 (Fig 5C; data not shown for BGR1 deletion). Consistently, Tec1 was required for maximal exposed biofilm cells. However, in the absence of the hormone expression of HYR1 is which can be greatly shaped by the environmental conditions.

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Our study examined, for the first time, the alterations occurring in the genomic 328 expression of C. albicans during biofilm formation in acidic conditions (RPMI at pH 4), which 329 could potentially lead to the identification of novel players required for maximal biofilm 330 formation in the acidic vaginal tract. Indeed, we identified 286 genes whose transcription was 331 changed during biofilm formation that have not previously been associated with this process.

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These genes and their regulators represent thus an interesting cohort to search for new 333 players involved in biofilm formation in the acidic vaginal tract. Using this rationale, we showed 334 that Sfl1 is essential for maximal biofilm formation and that is also likely to play a significant presence may contribute to the disclosure of new targets to treat VVC infections. respective parent. All the strains are listed in Table 4.  presented as matrix dry weight (g) per biofilm's cells dry weight (g) (g/g biofilm.cells ).

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Protein and carbohydrate quantification. The supernatants obtained in the previous 448 step were used to measure protein and carbohydrate contents in the matrices of the biofilms 449 grown with and without progesterone. The protein content was measured using the BCA Kit the standard [49]. The results were normalized with the matrix dry weight previously matrix dry weight and presented as mg of carbohydrate per g of matrix dry weight (mg/g matrix ).

Biofilm cells antifungal susceptibility testing 458
The susceptibility of C. albicans biofilm cells to fluconazole was tested using the 459 reference protocol for broth microdilution antifungal susceptibility testing of yeasts, according The effect of progesterone on the transcriptome of C. albicans biofilms was assessed 471 using species-specific DNA microarrays [53]. For this, the transcriptomes of C. albicans 472 SC5314 cells present in biofilms grown in RPMI at pH 4 for 24 h in absence and presence of 473 progesterone (2 µM) were compared with the transcriptome of planktonic cells cultivated for 474 the same time in hormone-free RPMI medium (pH 4). The experimental setups used to 475 cultivate the cells in planktonic and biofilm life styles were the same as described above and 476 using 24-well polystyrene microtiter plates (1 ml per well). Developed biofilms obtained after supernatants were rejected and pellets were used for RNA extraction.

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The filters were transferred to fresh collection tubes and RNA was eluted in two times by

PCR)
530 In order to validate some of the results obtained in the microarray analysis, the manufacturer's instructions. cDNA synthesis was performed at 70°C for 5 min followed by

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Primers for the target genes were designed using Primer3 web software and their sequences 572 are provided in S5 Table. 573 574

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Results concerning the determination of biofilm cultivable cells, total biomass, metabolic 576 activity, biofilm matrix quantity and of its components (carbohydrate and protein) and also the 577 results of the qRT-PCR were statistically analyzed using t tests implemented in GraphPad 578 Prism 6 software. All tests were performed with a confidence level of 95 %.  Table. List of progesterone-responsive genes in C. albicans SC5314 biofilms grown 24 h at 752 pH4. Were selected and listed here C. albicans SC5314 genes found to be up-and down-regulated in 753 biofilms grown 24 h in RPMI at pH 4, in the presence (+ PRG) and/or absence (-PRG) of 2 µM of 754 progesterone, and whose expression differed more than 40% between the two biofilm-forming 755 conditions. Progesterone-responsive genes are presented in four groups i) genes up-regulated in 756 biofilms formed in the presence or absence of progesterone, but showing a stronger induction in the 757 presence of the hormone (42 genes); ii) genes more strongly up-regulated in biofilms without 758 progesterone than on its presence (87 genes); iii) genes more strongly down-regulated in biofilms 759 formed without progesterone (12 genes); iv) genes more strongly down-regulated in the progesterone-760 exposed biofilms (79 genes). Progesterone-decreased genes (groups ii and iv) required for biofilm 761 formation (bold) and/or analyzed by qRT-PCR ( Fig 5B) are highlighted in grey. The biological function

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indicated is based on the information available at Candida Genome Database.  Table) that were found to have a reduced expression in progesterone-826 exposed biofilm cells, in comparison with the level attained in biofilms not exposed to the hormone. This the hormone (-PRGST) and in progesterone-exposed biofilm cells (+PRGST). Consistent with these 833 results, Tec1 was found to be essential for formation of acidic biofilms and this phenotype was 834 aggravated in the presence of progesterone (C). Error bars represent standard deviation. Asterisks