A quest to find good primers for gene expression analysis of Candida albicans from clinical samples

Highlights

• Published primers for genes SAP1 and HWP1 were specific for C. albicans.

• Published primers for SOD1 gene reacted with C. albicans and C. dubliniensis.

• Newly designed primers for ACT1, ALS1 and HWP1 genes were specific for C. albicans.

• Primers for CAP1, CAT1, EFG1, LIP3 and PLB1 reacted with C. albicans and C. dubliniensis.

• After standardization, the primers can be used in clinical samples.

Abstract

Biofilm production contributes to several human diseases, including oral candidiasis. Among the Candida species, Candida albicans is the most prevalent. The expression of virulence genes is implicated in the pathogenic potential of Candida biofilms. However, the evaluation of microbial gene expression from in vivo biofilm samples is not trivial, specifically, assessment via quantitative PCR (qPCR) can be a challenge because of several species present in clinical samples. Hence, the necessity of primers specificity. The aim of this study was to evaluate through in silico and in vitro analyses the specificity of published primers and newly designed primers for C. albicans virulence genes: ALS1, CAP1, CAT1, EFG1, HWP1, LIP3, PLB1, SAP1, SAP4, SOD1, SOD5 and ACT1 (normalizing gene). In silico analysis was performed through a PubMed search of articles with primer sequences that evaluated gene expression of C. albicans. Then, the sequence similarity of twenty-eight primers was checked through BLASTn and ClustalW2. The analysis of secondary structures was performed using mfold. When the primers did not present satisfactory characteristics (absence of secondary structures, not discrepant Tm of forward and reverse sequences and specificity) following in vitro analysis (i.e., end point PCR), new primers were designed using Beacon Designer™ and sequences obtained from the “Candida Genome Database”. The selected primers were tested in vitro by end point PCR using a panel of genomic DNA from five different Candida species (C. albicans, Candida glabrata, Candida dubliniensis, Candida krusei, and Candida tropicalis). The resulting PCR products were visualized on agarose gel. qPCR reactions were performed to determine primers' optimal concentration and PCR efficiency. End point PCR demonstrated that published primers for the SAP1 and HWP1 were specific for C. albicans and the one for SOD1 reacted with C. albicans and C. dubliniensis. The sequence of primers designed for ACT1, ALS1 and HWP1 genes were specific for C. albicans, while the ones for CAP1, CAT1, EFG1, LIP3, and PLB1 were detected in C. albicans and C. dubliniensis. After optimization, all primers presented a single peak on melt curves, correlation coefficient of ≅1 and qPCR reaction efficiency of 90–110%, with slope of ≅−3.3. Therefore, these primers should be suitable for future gene expression analyses from clinical samples.

Introduction

Oral candidiasis is the most common soft tissue fungal infection of the oral cavity in humans caused by Candida spp. (Akpan and Morgan, 2002; Sardi et al., 2013). An overgrowth of these fungi can cause superficial, cutaneous, mucosal, and invasive infections (Sardi et al., 2013). Candida albicans is considered the most prevalent pathogenic species in the oral microbiota (Kulak et al., 1994), growing as yeast, pseudohyphal or hyphal forms. The yeast morphology of C. albicans is considered commensal in healthy humans but can cause systemic infection in immunocompromised patients, mainly because of their ability to adapt to different niches (Sardi et al., 2013). Even though the yeast form is considered a less harmful morphology, there is an increase in resistant of this fungus to antifungal drugs, contributing to human disease once the immune system is repressed, C. albicans can prevail and act as an opportunistic fungus, causing infection especially when the host microbiota is modified by certain predisposing factors (Akpan and Morgan, 2002; Nobile and Johnson, 2015). Besides the high prevalence of C. albicans, other species have been detected in human infections. The most commonly described are: Candida glabrata, Candida dubliniensis, Candida krusei, and Candida tropicalis (Lyon et al., 2006). In addition to these species, Candida auris is an emerging multidrug resistant fungal pathogen (Chatterjee et al., 2015).

A major virulence factor of C. albicans is its capacity to form biofilms on biotic or abiotic surfaces (Gulati and Nobile, 2016; Mayer et al., 2013). Biofilms can be described as surface-associated communities of microorganisms embedded within an extracellular matrix (Silva et al., 2011). The biofilm formation by Candida spp. is an important virulence factor because it protects microbial cells from host immune responses, limits the penetration of substances through the matrix, thereby conferring significant resistance to conventional antifungal therapy (Gulati and Nobile, 2016; Silva et al., 2011). In addition to the ability to form biofilms, there are others important factors to the virulence of C. albicans: the morphological transition between yeast and hyphal forms; the expression of adhesins and invasins on the cell surface; thigmotropism; phenotypic switching; secretion of hydrolytic enzymes; resistance to changes in environmental pH; metabolic flexibility; powerful nutrient acquisition systems (glucose, lipids, proteins and amino acids); and response to oxidative stress (Mayer et al., 2013).

The increased incidence of superficial and systemic infections caused by Candida spp. has been attributed to resistance to antifungals and expression of many virulence factors that these fungi present after exposure to antifungals to infection treatment (Haynes, 2001; Sardi et al., 2013). Therefore, understanding the virulence and resistance mechanisms associated with these species is relevant. Moreover, the knowledge about the expression of virulence factors genes and how therapeutic approaches affect the expression of those genes can be an indicator of treatment/intervention effectiveness.

There are a few of methods for gene expression analysis to identify changes of expression of important virulence factors responsible for the onset and development of infection in the host and for resistance to therapeutics. Among them is the qPCR (quantitative Polymerase Chain Reaction) technique, which can quantify mRNA expression of virulence genes. An ideal PCR reaction shows high specificity, yield and fidelity (Cha and Thilly, 1993). Therefore, it is important that the qPCR primers have satisfactory characteristics (i.e., absence of secondary structures, resulting PCR product size…) and specifically anneal to improve the sensitivity of PCR. Although transcriptomic analysis using next-generation sequencing (NGS) approaches can provide an overall expression profile, subsequent validation of critical genes is almost invariably carried out by qPCR. A summary of considerations can be found in Bustin (2004) and Thornton and Basu (2011).

Therefore, to evaluate the expression of C. albicans genes in in vivo biofilm samples from patients, we first analyzed whether the published primers were suitable for use. Biofilms or clinical sample analysis can be challenging because several species may be present in a clinical sample and the primers need to be specific. In this context, the purpose of this study was to find and standardize published or newly designed primers for target C. albicans virulence genes, these primers needed to display satisfactory characteristics to be used in future in vivo studies. The primers pairs were tested through in silico and later with the in vitro tests, end point PCR and quantitative PCR.