Genotypic analysis of secreted aspartyl proteinases in vaginal Candida albicans isolates

Introduction: Candida albicans is the most common etiologic agent of fungal vaginitis. These yeasts produce secreted aspartyl proteinases encoded by a family of 10 genes (SAP1-10). Objective: The purpose of this study was to analyze the presence of genes SAP1-7 in vulvovaginal C. albicans. Materials and method: The study included 26 C. albicans vaginal isolates. Detection of aspartyl proteases genes (SAP1-7) was performed by polymerase chain reaction (PCR). Results: The most frequent gene in C. albicans isolated from colonization was SAP6 (93.33%), and from infection, SAP7 (100%). We observed a statistical difference (p = 0.049) in SAP1 gene frequency between isolates from vulvovaginal colonization and infection. Conclusion: High frequency of SAP genes was observed in vulvovaginal C. albicans. The results suggest SAP1 participation in vulvovaginal candidiasis infection.


introDuCtion
Vulvovaginal candidiasis (VVC) is an endogenous infection caused by yeasts of the genus Candida.The disease commonly develops when conditions exist favoring fungal growth, such as alterations in normal microbiota or lowered host immune response (1)(2)(3)(4)(5) .The species of Candida are microorganisms commonly found in the microbiota of the gastrointestinal and urogenital tracts without causing disease, but when the balance between fungus and host is disturbed, there is an increase in colonization, and the fungus invades tissues, initiating the infectious process (6,7) .
VVC is one of the most frequent diagnoses in clinical practice, with a rising incidence, becoming the second most common genital infection in the United States and in Brazil (1,8,9) .Among vulvovaginitides, VVC accounts for 39% of the cases, just behind bacterial vaginosis.Candida species can be found in up to 50% of healthy women without causing symptoms; however, around 70%-75% of women in childbearing age develop at least one episode of fungal vulvovaginitis during life.Among these, 50% will present two or more episodes, and approximately 6%-9% are likely to present recurrent VVC (RVVC), characterized by the presence of four or more symptomatic episodes in a year (10)(11)(12)(13)(14) .
Candida albicans is the most prevalent etiologic agent in VVC, accounting for 85%-90% of the cases.The virulence of this species is due to several mechanisms and abilities inherent in the fungus, as yeast-to-hypha transition, phenotypic switching, expression of adhesins and invasins on the cell surface, biofilm formation, and secretion of hydrolytic enzymes (3,11,(15)(16)(17) .
The most studied hydrolases related to Candida virulence are proteases, phospholipases, and lipases.Secreted aspartyl proteinases (Sap) form a family of 10 isoenzymes (Sap1-10) that participate in the infection process by degrading several host cell proteins, such as immunoglobulins, proteins of the complement system and extracellular matrix, contributing to tissue damage and the resulting invasion by the microorganism (18)(19)(20)(21) .These enzymes play different roles depending on the environmental stimuli and are involved in the host inflammatory response to fungus.The activation of these proteins is a well-regulated process at specific time points, what increases the infection potential of C. albicans (6,16,22,23) .Among the SAP gene family, the most frequently studied in the literature and associated with pathogenicity of C. albicans are SAP1-7 genes.The objective of this study was to evaluate the presence of these genes in vulvovaginal C. albicans cell lines.

Studied microorganisms
Twenty-six C. albicans cell lines were isolated from the vaginal mucosa of asymptomatic (n = 15) and symptomatic (n = 11) women with VVC.The yeasts were obtained from vaginal swabs as described by Goulart et al. (2016) (24) .The clinical samples were collected from women seen at basic health-care units in the municipality of Rondonópolis (MT), Brazil, regardless VVC symptoms.Participants were divided into two groups: 1) symptomatic patients, characterized by the presence of curdlike vaginal discharge, pruritus, edema and erythema of vulva and vagina; and 2) asymptomatic patients, which did not present the mentioned characteristics.Yeasts were stored at Sabouraud agar at 4°C.Previously, microorganisms were grown in Sabouraud broth at 37°C, under agitation [200 revolutions per minute (rpm)] for 24 h.Then the culture was centrifuged, the supernatant was discharged, and deoxyribonucleic acid (DNA) was extracted from the cell sediment with a kit (Nucleo Spin Tissue, Macherey-Nagel GmbH & Co. KG, Duren, Germany), following the instructions by the manufacturer.The yeast species was determined by the speciesspecific polymerase chain reaction (PCR) based on the protocol of Liguori et al. (2010) (25) , with modifications.

Identification of SAP1-7 genes
Genes were identified by PCR based on the method proposed by Bassyouni et al. (2015) (26) , with some modifications.Reactions were performed at a final volume of 25 μl, containing approximately 20 ng of DNA, 12.5 μl of GoTaq Hot Start Green Master Mix (Promega, Madison, Wisconsin, USA) and 0.75 μl (20 pmol/μl) of each specific primer.Amplification conditions for SAP1, 3, 4 and 7 genes were: initial denaturation at 94°C for 3 minutes, 30 denaturation cycles at 94°C for 30 seconds, annealing at 46°C for 30 seconds, extension at 72°C for 30 seconds and final extension at 72°C for 10 minutes.PCR for SAP2, 5 and 6 genes was standardized from denaturation at 92°C for 3 minutes, 30 denaturation cycles at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 30 seconds and final extension at 72°C for 10 minutes.Amplification products were analyzed on 1% agarose gel electrophoresis containing DNA stain (Promega, Madison, Wisconsin, USA) and visualized under ultraviolet light.The oligonucleotide sequences used in the study are described in Table 1.

Statistical analysis
Data were recorded in Excel 2016 spreadsheets and assessed in the statistical software Epi-info 7.2.0.Data analysis was carried out by descriptive statistics and non-parametric Fisher exact test, adopting a 5% significance level.We evaluated the correlation of the presence of SAP1-7 genes with the infection process and vulvovaginal colonization.

rESuLtS
By means of PCR method, it was possible to detect the presence of SAP1-7 genes in clinical isolates of C. albicans.After molecular analysis of C. albicans isolated in women with symptoms of VVC, the presence of SAP1 gene was identified in 90.9% (10/11) When comparing the frequency of genes encoding aspartyl proteases (SAP1-7) among C. albicans isolated in asymptomatic and symptomatic women with VVC, we found a statistical difference just for SAP1 gene (p = 0.049), with this being more prevalent in isolates associated with the infectious process.Although genes SAP2-7 present distribution profiles distinct among the groups, this difference did not represent statistical difference.These pieces of data are shown in Table 2.
The results revealed genetic variability for the members of the SAP family in the studied cell lines, with 19 different genotypic patterns being found; just three isolates presented all the studied genes (Table 3).

DiSCuSSion
SAP genes greatly stand out in the pathogenesis of candidiasis, once they encode proteins able to degrade collagen, keratin, and peptides found in the surface of mucosas, ensuring, thus, an important and efficient proteolytic system to C. albicans, from the colonization process to active infection (20,23,(27)(28)(29)(30) .
In this study, we observed that the most prevalent SAP genes in C. albicans isolated from vulvovaginal infection and colonization were SAP7 (100%) and SAP6 (93.33%), respectively.Monroy-Pérez et al. (2013) (31) determined the frequency and the expression of SAP1-10 genes isolates from women with VVC in Mexico and identified that SAP4-6 genes were the most frequent (100%), besides observing that all SAP genes were expressed in a model of reconstituted human vaginal epithelium (RHVE), suggesting that aspartyl proteases play an important role in the pathogenesis of the infection.Bassyouni et al. (2015) (26) investigated the presence of SAP1-8 genes in C. albicans isolated from vaginal mucosa of diabetic and non-diabetic women and identified that SAP1 and SAP2 were the most frequently detected genes, followed by SAP5 in both groups; there was no difference in the distribution of genes between diabetic and non-diabetic women.Kalkanci et al. (2005) (32) verified that SAP1-3 genes were the most prevalent in vaginal isolates of C. albicans, with a 92.5% rate, followed by SAP6, with 12.5%, and SAP4-5, with 7.5%.
The most frequent SAP genes in the present research and in other studies are available in Table 4.
Predominance of the SAP6 gene in the studied colonization isolates and SAP7 in the studied infection isolates suggests the necessity of further studies to evaluate the expression of these genes in vivo.The SAP6 gene participates with the SAP4-6 subfamily in the development of hyphae, an essential process for the fungus (33,34) capacity of invasion.Besides, Sap6 protein was associated with integrity maintenance of cell surface and capacity of producing inflammatory response in the host (33,35,36) .The SAP7 gene encodes the most divergent protein within the Sap family and can be preferably associated with infections of human mucosas (36) .The expression of this gene was associated with the initial adaptation of C. albicans to human cells of the intestinal tract, while transcripts of SAP6-7 were associated with tissue damage in the early phase of infection, after 24 h, at a RHVE (37,38) model.This study revealed high (90.9%)presence of SAP4 gene in vaginal cell lines of C. albicans involved in infection.The proteins encoded by these genes are associated with adherence of the fungus to human cells and alterations in the morphogenesis of the yeast, playing an important role in biofilm formation.The SAP4 gene has also been implicated in evasion of phagocytosis (30,39,40) .The expression of SAP4 genes was demonstrated in vivo in the vaginal mucosa of women who were pregnant (40%), post-menopausal (50%), and in childbearing age (33%) (41) .
The statistical analysis showed significant difference (p = 0.049) for the SAP1 gene frequency among the C. albicans cell lines isolated from infection (90.9%) and colonization (53.33%).This suggests a probable participation of SAP1 in the infectious process of VVC and points to a possible aim of further studies to distinguish colonization from active infection.This result can also contribute to researches aimed at developing new diagnostic methods for VVC.Sap1 protein is known to be linked to the capacity to cause lesions in the mucosa and to the development of systemic infections (30) .At a previous work, the expression of SAP1 in C. albicans from patients with VVC was observed in 80% of the studied isolates (42) .The SAP1-7 genes can be expressed by C. albicans in the vaginal mucosa, both in colonization processes and in infections, but a differential expression of these genes is observed when comparing the transcript levels in isolates of carriers and of active VVC, besides a predominance of certain members of the SAP family during vaginal infection (43) .
and women with symptomatic and recurrent VVC.The authors observed that the transcripts for SAP1 and SAP3 genes were present only in isolates of VVC and RVVC.Lin et al. (2007) (44) determined by real-time PCR (RT-PCR) that SAP2 and SAP5 were the most commonly expressed genes in the vaginal mucosa of women with acute VVC.The expression of SAP genes in the different studies is shown in Table 5.
Medeiros et al. (2017) (45) did not find differences in the expression of virulence factors between C. albicans isolated from patients with sporadic VVC and those obtained from patients with RVVC, suggesting that the ability to express virulence factors is important in the pathogenesis of VVC, but it seems not to be crucial for the transition from colonization to infection.We did not identify statistical difference for the presence of SAP2-7 genes in isolates from infection and vulvovaginal colonization.This suggests that other factors that control the genic fungal expression and characteristics inherent in the host are likely to influence in the infection/colonization process.Also, further studies comprising a larger number of samples must be carried out.

ConCLuSion
C. albicans isolated from symptomatic women with VVC and asymptomatic women presented different patterns of distribution for SAP1-7 genes, with predominance of the SAP6 gene in colonization and SAP7 gene in infection.The SAP1 gene was associated with the process of vaginal infection, suggesting its participation in the pathogenesis of VVC.

taBLE 2 −
Frequency of SAP1-7 genes in C. albicans isolated from vulvovaginal infection and colonization

taBLE 3 −
Genotypic patterns identified in vaginal isolates of C. albicans

taBLE 5 −
Expression of SAP genes in different studies