The role of Candida albicans in root caries biofilms: an RNA-seq analysis

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.


Introduction
The bacterial biofilm associated with root caries lesions must harbor microorganisms that can produce acid from carbohydrates (acidogenicity) and must be able to growth in a low-pH environment (aciduricity). 1 Diverse bacteria are prevalent and involved in the etiology of root caries, albeit to date, little has been explored regarding other microorganisms domains, such as archea, fungi and virus, and their role in biofilms. Previous studies demonstrated that Streptococcus mutans, Lactobacillus species (spp.), and Veillonella spp., as well as C. albicans, are present in major proportion in root caries than in sound root surface. 2 Actinomyces spp., Veillonella spp., Streptococcus spp., Bifidobacterium spp., Rothia, Enterococcus, Staphylococcus spp., Capnocytophaga spp., Prevotella spp. and Candida spp., were also cultivated from root caries. 1,3,4 Candida species has been associated with dental caries, especially with early childhood caries and root caries. 5 A strong association was found between the prevalence of C. albicans and dental caries. 6 Several authors showed that the proportion of Candida species was higher in individuals with caries than in individuals without caries. Furthermore, C. albicans is an important colonizer of carious lesions and has been found frequently in dentin caries lesions rather than in biofilm or saliva. 4 Lower salivary flow rate, a common occurrence in older adults, is one of the factors that promote favorable conditions for a presence of C. albicans in these sites. 7 However, it is still unknown whether the yeast acts as caries pathogen or plays a role as a commensal microbe. C. albicans possess some important properties that can characterize it as an important root caries pathogen. It is capable of adhering to saliva-coated hydroxyapatite and possesses strong adherence to collagen. 8 It is as acid tolerant and acidogenic as S. mutans and Lactobacilli, which are both well-established cariogenic pathogens. 9 To determine the role of C. albicans in root caries, a high-throughput sequencing of mRNA (RNA-Seq) was applied in clinical biofilms samples from two distinct conditions: sound root-surface biofilms and root carious lesions biofilms. This technique may be helpful to investigate Candida's role in a carious biofilm.

Methodology
This study is part of the project "metatranscriptome of root caries". 10 Briefly, volunteers to this study were divided into two groups: sound exposed root surface group (SRS; n=10) and root caries group (RC; n=30). Participants were allocated to the SRS group (n=10) if they had an exposed root surface on at least one tooth and no root caries lesions. Dental biofilms were collected with sterilized Gracey curette from all available exposed root surfaces. The number of exposed root surfaces varied among individuals.
Participants recruited to the root caries (RC) group (n=30) had one primary cavitated root lesion in need of restorative treatment. All lesions showed characteristics of present activity (soft and yellow dentin). Biofilm and carious dentin samples (of soft and infected tissue) were collected from patients during the restorative treatment. All participants were asked to refrain from tooth brushing for at least 12 hours prior to the sampling, to allow for dental biofilm accumulation, and were also asked to refrain from eating and drinking for at least 1 hour prior to the sampling. After collection, biofilm and carious dentin were immediately placed in 1 mL of RNA protect reagent (Qiagen, Hilden, North Rhine-Westphalia, Germany Candida albicans genome and data analysis The C. albicans SC5314 was the genome selected for this study. This strain was chosen for being largely studied and its genome has been fully sequenced as well. After mapping, a count table was generated containing the read count for 14,217 oral C. albicans SC5314 genes.
The putative presence of the organism in the sample was estimated by the sum of reads assigned to C. albicans divided by the total number of genes for each sample. Samples with ≥14,217 reads were considered as valid; then samples with less than 30% of genes with at least one read were excluded from the analysis.
The number of reads and the relative median expression (RME) (25 th -75 th ) level for genes were estimated for each of the sample groups, as previously described. 11 Then, the RME was ranked to observe the most highly expressed transcripts in RC and SRS samples. To draw a profile of gene expression, the median of RME of transcripts in SRS and RC conditions were considered low expression RME between 0-10, medium 11-100, and high above 100 (percentile 10 of RME distribution). RME was calculated from the median values of normalized read counts using DE-Seq algorithm. Genes related to C. albicans virulence factors were analyzed: invasion, biofilm formation and co-aggregation, adherence and damage, morphogenesis, acid production, acid tolerance and stress response.
All RME medians for SRS and RC were ranked and all genes with median RME values ≥100 per group were analyzed for an overview of the most prevalent genes.
Differential gene expression was inferred between sample groups by applying the R package DESeq2. 12 The cut-off for designating a gene as being differentially All volunteers signed an informed consent form and received clinical dental assistance.

Results
According to the cut-off point chosen to determine the putative presence of a mapped organism in each sample, C. albicans was present in n=4 biofilms from SRS and in n=6 biofilm from RC, as shown in Figure   1. Table 1 shows that the number of reads distribution in sound and disease samples were equal (p=0.522).  Table 2.
Expression of genes related to possible cariogenic traits C. albicans genes associated with possible virulence factors (RME and percentiles of these genes) were evaluated in both conditions ( Table 2). We found transcript of 51 out of 67 genes related to virulence traits that are presented in the literature as important factors. None of these genes had significant differential expression.

Differential expression analysis (DE)
The DE analysis has shown the overexpressed genes in root biofilms with and without caries ( Figure 2     Continued from previous page   (i.e., drought, salinity, cold, heat, low temperature, etc). 20,21 The STT4 codes for a hypothetical protein phosphatidylinositol-4-kinase.
The gene STT4 is essential for viability and plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity. 22 Therefore, the up-regulated genes DHN6 and STT4 could be related to the ability to survive in an extreme environment with several stress factors (low pH, carbohydrate viability, for example) such as the ones found in root cavitated caries lesions.
The UTP20 codes for a potential U3 small nucleolar RNAs (snoRNA) protein. UTP20 has been reported as a component of U3 snoRNA protein complex and has been implicated in 18S rRNA processing, being essential for 18 rRNA function. 23,24 The ITR1 codes for a potential active sugar transporter, potential Myo-inositol transporter, similar to S. cerevisiae ITR1 (YDR497C). Myo-inositol is an essential substrate for C. albicans, and it can be used as carbon source. For its survival, C. albicans must be able to synthesize the essential metabolite inositol or acquire it from the host. The overexpressed genes in RC were related to sugar transport (ITR1 -Myo-inositol) and to carbon source regulation (GUT1 -Glycerol kinase), that were related to the use of alternative carbon sources ( Figure   2). The use of lactate by C. albicans could be related to the pH regulation (neutralization) in biofilm, which is important for the microbiome survival. 27

Conclusions
Our data shows that Candida albicans SC5314 have an active metabolism in biofilm of SRS and biofilm of carious dentin of RC as well. The differential expression analysis shows that, in healthy individuals, the upregulated genes were related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. C. albicans may have a role in root caries progression.

Conflicts of interest statement
The authors declare no conflicts of interest in the subject matter discussed in this manuscript.