SAP Expression in Candida albicans Strains Isolated from Mexican Patients with Vaginal Candidosis *

To determine the frequency and expression of the ten SAP (secreted aspartyl protease) genes in a group of Candida albicans strains isolated from Mexican women suffering from vaginal candidosis, a group of 264 women (age 18 57 years) with vaginal infections, predisposed by diabetes mellitus or contraceptive consumption, were evaluated. C. albicans was identified using PCR to amplify the rRNA internal transcribed spacer regions ITS1 and ITS2. The presence of the SAP genes was determined using conventional PCR, and their expression levels were determined using real-time PCR after the C. albicans strains had been grown in reconstituted human vaginal epithelium (RHVE). C. albicans was identified in the samples from 50 women (18.9%). The genotyping frequencies of the SAP genes were as follows: SAP1, 94%; SAP2, 98%; SAP3, 80%; SAP4, 100%; SAP5, 100%; SAP6, 100%; SAP7, 63%; SAP8, 96%; SAP9, 70%; and SAP10, 88%. The most frequently expressed genes in the strains harboring all of the genes were SAP1, 90%; SAP2, 90%; SAP3, 90%; SAP4, 100%; SAP5, 90%; SAP6, 90%; SAP7, 100%; SAP8, 90%; SAP9, 100%; and SAP10, 100%. SAP genes were expressed in the RHVE, suggesting that the Sap proteins play an important role in the pathogenesis of infection.


Introduction
Candida albicans are classified as commensal fungi that inhabit the human gastrointestinal tract.It grows as round cells in smooth, white colonies.Additionally, C. albicans can cause oral and vaginal infections as well as systemic diseases [1].Vaginal candidosis (VC) is one of the most common infections that afflicts women of reproductive age [2,3].Approximately 75% of women will experience at least one episode of VC during their lives [3,4].C. albicans possesses several virulence factors that are involved in hyphae formation [5], phenotype switching [6][7][8], cell adhesion [9][10][11][12], and extracellular production of hydrolytic enzymes [13,14].Secreted aspartyl proteases (Saps) are enzymes that are secreted by C. albicans and are coded for by the SAP gene family (SAP1-SAP10) [15].The proteolytic activity of the Sap proteins is involved in the degradation of the host's barriers during infection [16], immune response evasion [17], and adhesion to the host's cells [18].This family of proteases can be differentially expressed and regulated under a variety of growth conditions in the laboratory [19,20], such as during experimental infections using reconstituted human oral epithelium (RHE) [21], or in vivo [22].
The purpose of this work was to use a reconstituted human vaginal epithelium (RHVE) model to determine the frequency and expression of the ten SAP genes in C. albicans strains that had been isolated from women with VC.
This study included 264 women who presented with vulvovaginitis-associated symptoms, such as burning, itching, dysuria, and curd-like discharge; these women visited gynecologic services at public hospitals located in the State of Mexico, Mexico.The age range of the women in this study was between 18 and 57 years.Patients presenting with cervical cancer, pregnancy, or who had undergone antibiotic or antimycotic treatments within the last 30 days were excluded from the study.After obtaining informed consent from each patient, two samples were taken from the vaginal cavity using sterile cotton swabs.One of these samples was used to confirm the presence of pseudohyphae or hyphae by direct examination under a microscope.The second sample was used to inoculate brain hearth infusion (BHI) culture media (BD Bioxon, Cuatitlán Izcalli, State of Mexico, Mexico), and the cultures were then incubated at 37˚C for 24 hours.After 24 hours, the cultures were plated on Sabouraud Agar (BD Bioxon, Cuatitlan Izcalli, State of Mexico, Mexico) containing 50 µg/ml chloramphenicol and were incubated at 37˚C for 72 hours.

Identification of C. albicans
Samples were collected from the pure cultures grown in Sabouraud Agar and were identified by colony and mi-croscopic morphologies and using a germ tube test in BHI that was supplemented with 10% horse serum and the API 20 C AUX system (BioMerieux, Durham, NC, USA).The C. albicans ATCC32354 strain was used as a positive control.
C. albicans was also identified using PCR by amplifying the internal transcribed spacers (ITS1 and ITS2) from the rRNA gene [28].C. albicans genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA).PCR amplification was performed in a Corbette Research Thermocycler using PuReTaq Ready-To-Go PCR Beads (GE Healthcare, Piscataway, New Jersey, USA).The amplicons were stained with ethidium bromide after electrophoresis in a 2% agarose gel and were visualized with UV illumination using a GEL LOGIC 100 (KODAK).The C. albicans ATCC32354 strain was used as a positive control.

C. albicans RNA Purification and Reverse Transcription
C. albicans cells were harvested from the RHVE, suspended in 200 µl of Y1 buffer containing lyticase (50 U/10 7 cells), and incubated at 30˚C for 20 minutes using gentle shaking to facilitate the formation of spheroplasts.
The extraction and purification of the total RNA was performed using an RNeasy MiniKit (Qiagen, Hilden, Germany).The RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer.To obtain cDNA, a QuantiTec Reverse transcription kit (Qiagen) was used according to the manufacturer's instructions.

Real-Time PCR Amplification of the SAP Genes
The SAP primers that were used for the endpoint PCR were also used for the real-time PCR assay (Table 1).
The Rotor-Gene SYBR Green PCR kit (Qiagen) was used for the real-time PCR experiments.A final volume of 25 µl was used for each reaction and contained 12.5 µl of SYBR Green Master Mix, 1 µl of forward primer (1 µm), 1 µl of reverse primer (1 µm), 2 µl of cDNA (20 ng), and 8.5 µl of RNase-free water.The amplification conditions were 95˚C for 5 minutes followed by 95˚C for 5 seconds with an annealing/extension combination step at 60˚C for 10 seconds for 40 cycles.The same positive and negative controls that were used for the endpoint PCR experiments were also used for the real-time PCR assay.

Statistical Analysis
The frequency of the SAP family genes among the C. albicans strains isolated from women within the age ranges of 18 -29 (n = 13), 29 -39 (n = 14), 40 -49 (n = 11), and 50 -57 (n = 12) years were analyzed using the  2 test with a significance threshold of P < 0.005.

C. albicans Positive Samples
Infection with C. albicans was determined using microbiologic criteria and by PCR.The ITS were amplified from 18.9% (n = 50) of the vaginal samples (Figure 1).
Sixteen different association patterns of SAP genes were found among these strains (Table 3).Twenty strains carried all of the studied SAP genes, and ten of the strains had only nine of the SAP genes (Table 3).

SAP Gene Expression Determined by Real-Time PCR
To determine the SAP virulence markers, 11 clinical strains carrying all of the SAP genes (Pattern 1, Table 3) were inoculated onto RHVE and were then analyzed using real-time PCR.The Tm was recorded for each realtime assay to differentiate specific from non-specific PCR products (data not shown).Constitutive rRNA expression was used as a control (data not shown).All of the SAP genes were expressed.The genes SAP4, SAP7, SAP9, and SAP10 were expressed in 100% of the isolated C. albicans strains, whereas SAP1, SAP2, SAP3, SAP5, SAP6, and SAP8 were expressed in 90% of the strains (Table 2, Figure 2).

Discussion
Candida albicans was identified in vaginal samples of 18.9% (n = 50) of the women in this study.This result is in agreement with previously reported frequencies for VC, which occurs in 20% -25% of infectious vaginitis cases, which is just below the 40% -50% of cases that develop bacterial vaginitis [4].Among Mexican women, VC is the ninth most common cause of disease (incidence of 529 in 100,000 women; SUIVE/DGE/Ministry of Health/United Mexican States-2008).Candidosis is not common during puberty; however, by 25 years of age, 50% of women have had at least one clinically diagnosed VC episode [34,35].C. albicans causes 85% -95% of VC cases [4,36].External factors that predispose patients to VC are pregnancy, the use of oral contraceptives, diabetes mellitus, and antibiotics usage [3,37].The high frequency of SAP genes (Table 2) and the high number of association patterns in C. albicans strains (Table 3) suggest that multiple Sap expression profiles exist during VC pathogenesis.For example, the SAP4-SAP6 genes, which were present in all of the analyzed C. albicans strains (Table2), are associated with hyphae formation.In addition to being involved in cell adhesion, hyphae can penetrate individual cells or intercellular spaces [38].The Sap2 protein, which was detected in 98% of the studied strains (Table2), can degrade many human proteins, including those that protect the mucosal surfaces, such as mucine [39,40] and secretory immunoglobulin A (sIgA) [41], and molecules from the extracellular matrix, such as keratin, collagen, and vimentin [42][43][44].The ability of Sap2 to degrade these proteins may facilitate the dissemination of C. albicans throughout the circulatory system [45].
In this study the genes SAP4, SAP7, SAP9, and SAP10 were expressed in 100% of the isolated C. albicans strains, whereas SAP1, SAP2, SAP3, SAP5, SAP6, and SAP8 were expressed in 90% of the strains (Table 2).Similar data were described by Schaller et al. [46], who used an in vitro candidosis RHVE model.In their study, the genes that were most frequently expressed during the late infection stage were SAP1, SAP2, SAP4, SAP7, SAP9, and SAP10, whereas in studies performed on vaginal infections in vivo, the most frequently expressed genes were SAP1, SAP3, and SAP6-SAP8 [22].In vulvovaginal candidosis (VVC) and recurrent vulvovaginal candidosis (RVVC), the expression of SAP2 and SAP4-SAP7 [47] was reported.It has been suggested that the SAP4-SAP6 genes, which are frequently expressed in vaginal infection samples, play an important role in the evasion of the immune response during the active infection stage, which partially protects C. albicans from macrophage phagocytosis [17].It has also been reported that the expression of the SAP4-SAP6 genes is associated with hyphae development in vitro [48], which suggests that Sap4-Sap6 production in the vaginal lumen enables C. albicans to attach to the vaginal mucosa.
Results presented in this study showed that all of the SAP genes were expressed in the RHVE, suggesting that the Sap proteins play an important role in the pathogenesis of infection.

Figure 2 .
Figure 2. Representative figure of real-time PCR experiments.Detection of SAP1 (a), SAP2 (b), SAP3 (c), and SAP4 (d) expression by real time PCR in Candida albicans strains isolated from women suffering from vaginal candidosis.C. albicans ATCC32354 (a-d) was used as a positive control.Negative controls were as follows: Ca4 for a, Ca7 for b, Ca22 for c and Staphylococcus epidermidis ATCC 35984 for d.NTC control without DNA.

RHVE Inoculation with C. albicans
[16,21] albicans strains tested positive for the expression of all of the SAP genes, and the control strains were grown as described by Schaller et al.[16,21].The C.