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.
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 [
Different SAP genes appear to be essential for mucosal (SAP1-SAP3) [23,24], and systemic infections (SAP4- SAP6) [
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.
Procedures followed in this study were in accordance with the Ethical Committee of each hospital.
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.
Samples were collected from the pure cultures grown in Sabouraud Agar and were identified by colony and microscopic 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 [
The primers and amplification conditions for SAP1 were described by Hube et al. [
Both C. 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. albicans ATCC32354 strain was used as a positive control. The following strains from our collection, which are each missing one SAP gene, were used as negative controls: C. albicans 4, C. albicans 7, C. albicans 22, Staphylococcus epidermidis ATCC35984, C. albicans 27, C. albicans 50, and C. albicans 28. A total of 2 × 106 C. albicans cells suspended in 50 µl of PBS were inoculated onto the surface of the RHVE A431 (SkinEthic Laboratory, Nice, France) and were incubated at 37˚C for 72 hours with 5% CO2 and saturated humidity. The maintenance media was changed every 24 hours.
C. albicans cells were harvested from the RHVE, suspended in 200 µl of Y1 buffer containing lyticase (50 U/107 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.
The SAP primers that were used for the endpoint PCR were also used for the real-time PCR assay (
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 c2 test with a significance threshold of P < 0.005.
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 (
The SAP4, SAP5, SAP6, SAP2, SAP8, SAP1, and SAP10 genes were detected at a higher frequency in C. albicans strains than were the SAP9, SAP3, and SAP7 genes (
Sixteen different association patterns of SAP genes were found among these strains (
To determine the SAP virulence markers, 11 clinical strains carrying all of the SAP genes (Pattern 1,
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 [
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 (
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 (
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.
This work was supported by the grant PAPIME PE200209 from Universidad Nacional Autónoma de México.