The Role Of Candida Albicans In Root Caries Biofilms: An .

Original 78The role of Candida albicans in rootcaries biofilms: an RNA-seq analysisAbstractLaís Daniela EV¹Nailê DAMÉ-TEIXEIRA²Thuy DO3Marisa MALTZ¹Clarissa Cavalcanti Fatturi PAROLO¹Objective: This study sought to analyze the gene expression of Candidaalbicans in sound root surface and root caries lesions, exploring its role inroot caries pathogenesis. Methodology: The differential gene expression ofC. albicans and the specific genes related to cariogenic traits were studiedin association with samples of biofilm collected from exposed sound rootsurface (SRS, n 10) and from biofilm and carious dentin of active rootcarious lesions (RC, n 9). The total microbial RNA was extracted, and thecDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500.Unique reads were mapped to 163 oral microbial reference genomesincluding two chromosomes of C. albicans SC5314 (14,217 genes). Theputative presence of C. albicans was estimated (sum of reads/total numberof genes 1) in each sample. Count data were normalized (using the DESeqmethod package) to analyze differential gene expression (using the DESeq2Rpackage) applying the Benjamini-Hochberg correction (FDR 0.05). Results:Two genes (CaO19.610, FDR 0.009; CaO19.2506, FDR 0.018) were upregulated 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 relatedto metabolic activity, sugar transport, stress tolerance, invasion and pHregulation. The use of alternative carbon sources, including lactate, and theability to form hypha may be a unique trait of C. albicans influencing biofilmvirulence. Conclusions: C. albicans is metabolically active in SRS and RCbiofilm, with different roles in health and disease.Keywords: Sequence analysis. RNA. Candida albicans. Root caries.Transcriptome.Submitted: October 1, 2019Modification: November 20, 2019Accepted: January 19, 2020Corresponding address:Laís Daniela EvDepartamento de Odontologia Preventiva e Social- Faculdade de Odontologia Universidade Federal do Rio Grande do Sul.Rua Ramiro Barcelos 2492 - Santa Cecília - 90035004 - Porto Alegre - RS - Brasil.Phone: 55 51 33085193 - Fax: 55 1 3308 5023e-mail: laisdanielaev@gmail.com¹Universidade de Federal do Rio Grande do Sul, Faculdade de Odontologia, Departamento deOdontologia Preventiva e Social, Porto Alegre, Rio Grande do Sul, Brasil.²Universidade de Brasília, Faculdade de Ciências da Saúde, Departamento de Odontologia, Brasília,Distrito Federal, Brasil.³University of Leeds, Faculty of Medicine & Health, School of Dentistry, Leeds, United Kingdom.J Appl Oral Sci. 1/10 2020;28:e20190578

The role of Candida albicans in root caries biofilms: an RNA-seq analysisMethodologyIntroductionThe bacterial biofilm associated with root cariesThis study is part of the project “metatranscriptomelesions must harbor microorganisms that can produceof root caries”.10 Briefly, volunteers to this studyacid from carbohydrates (acidogenicity) and must bewere divided into two groups: sound exposed root1able to growth in a low-pH environment (aciduricity).surface group (SRS; n 10) and root caries groupDiverse bacteria are prevalent and involved in the(RC; n 30). Participants were allocated to the SRSetiology of root caries, albeit to date, little hasgroup (n 10) if they had an exposed root surface onbeen explored regarding other microorganismsat least one tooth and no root caries lesions. Dentaldomains, such as archea, fungi and virus, and theirbiofilms were collected with sterilized Gracey curetterole in biofilms. Previous studies demonstratedfrom all available exposed root surfaces. The numberthat Streptococcus mutans, Lactobacillus speciesof exposed root surfaces varied among individuals.(spp.), and Veillonella spp., as well as C. albicans,Participants recruited to the root caries (RC) groupare present in major proportion in root caries than(n 30) had one primary cavitated root lesion inin sound root surface. Actinomyces spp., Veillonellaneed of restorative treatment. All lesions showedspp., Streptococcus spp., Bifidobacterium spp., Rothia,characteristics of present activity (soft and yellowEnterococcus, Staphylococcus spp., Capnocytophagadentin). Biofilm and carious dentin samples (of softspp., Prevotella spp. and Candida spp., were alsoand infected tissue) were collected from patientscultivated from root caries.1,3,4during the restorative treatment. All participants2Candida species has been associated with dentalwere asked to refrain from tooth brushing for atcaries, especially with early childhood caries and rootleast 12 hours prior to the sampling, to allow forcaries. A strong association was found between thedental biofilm accumulation, and were also asked to5prevalence of C. albicans and dental caries. Severalrefrain from eating and drinking for at least 1 hourauthors showed that the proportion of Candidaprior to the sampling. After collection, biofilm andspecies was higher in individuals with caries than incarious dentin were immediately placed in 1 mL ofindividuals without caries. Furthermore, C. albicans isRNA protect reagent (Qiagen, Hilden, North Rhine-an important colonizer of carious lesions and has beenWestphalia, Germany). The total RNA was extractedfound frequently in dentin caries lesions rather than inusing the UltraClean Microbial RNA Isolation (Mo-bio,biofilm or saliva.4 Lower salivary flow rate, a commonSan Diego, Califórnia, USA) with on-column DNaseoccurrence in older adults, is one of the factors thatdigestion (Qiagen, Hilden, North Rhine-Westphalia,promote favorable conditions for a presence of C.Germany). Samples with total RNA concentration 30albicans in these sites.7 However, it is still unknownng/RNA were pooled, leading to a final sample sizewhether the yeast acts as caries pathogen or playsof 10 SRS and 9 RC. The Ribo-Zero Meta-Bacteriaa role as a commensal microbe. C. albicans possessKit (Illumina, Madison, Wisconsin, USA) was usedsome important properties that can characterize itfor mRNA enrichment and Illumina TruSeq libraryas an important root caries pathogen. It is capableprep protocols (Illumina, San Diego, Califórnia, USA)of adhering to saliva-coated hydroxyapatite andwere used to library preparation and sequencing waspossesses strong adherence to collagen. It is as acidperformed with Illumina HiSeq2500 (Illumina, Santolerant and acidogenic as S. mutans and Lactobacilli,Diego, Califórnia, USA). RNA sequencing data arewhich are both well-established cariogenic pathogens.9available in the National Center for BiotechnologyTo determine the role of C. albicans in root caries, aInformation (NCBI) Sequence Read Archive, underhigh-throughput sequencing of mRNA (RNA-Seq) wasthe accession numbers SRS779973 and SRS796739.applied in clinical biofilms samples from two distinctFASTQ files were obtained for each sample andconditions: sound root-surface biofilms and rootimported into the CLC Genomics Workbench 7.5.1carious lesions biofilms. This technique may be helpfulsoftware (CLC bio, Aarhus, Denmark) for mappingto investigate Candida’s role in a carious biofilm.against 163 oral microbial genomes.10 The number of68sequence reads that have been assigned to each geneis considered as the read count data.J Appl Oral Sci. 2/10 2020;28:e20190578

EV LD, DAMÉ-TEIXEIRA N, DO T, MALTZ M, PAROLO CCCandida albicans genome and data analysisall genes with median RME values 100 per group wereThe C. albicans SC5314 was the genome selectedanalyzed for an overview of the most prevalent genes.for this study. This strain was chosen for being largelyDifferential gene expression was inferred betweenstudied and its genome has been fully sequenced assample groups by applying the R package DESeq2.12well. After mapping, a count table was generatedThe cut-off for designating a gene as being differentiallycontaining the read count for 14,217 oral C. albicansexpressed was a change in transcript levels of at leastSC5314 genes.2-fold change (Log2FoldChange 1) and false discoveryThe putative presence of the organism in therate (FDR) 0.05 (padj value 0.05, Benjamini &sample was estimated by the sum of reads assignedHochberg). Functions and putative pathogenicity into C. albicans divided by the total number of genesroot caries of genes up-regulated in SRS and RC werefor each sample. Samples with 14,217 reads wereanalyzed.considered as valid; then samples with less than 30%Regarding to ethics considerations, this study wasof genes with at least one read were excluded fromapproved by the Federal University of Rio Grande dothe analysis.Sul research ethics committee (process n 427.168)The number of reads and the relative medianand by the research ethics committee of the Nationalexpression (RME) (25th-75th) level for genes wereResearch Ethics Service Committee Yorkshire & Theestimated for each of the sample groups, as previouslyHumber – Leeds West (protocol no. 2012002DD).Then, the RME was ranked to observeAll volunteers signed an informed consent form anddescribed.11the most highly expressed transcripts in RC and SRSreceived clinical dental assistance.samples. To draw a profile of gene expression, themedian of RME of transcripts in SRS and RC conditionswere considered low expression RME between 0-10,medium 11-100, and high above 100 (percentile 10of RME distribution). RME was calculated from themedian values of normalized read counts using DESeq algorithm. Genes related to C. albicans virulencefactors were analyzed: invasion, biofilm formationand co-aggregation, adherence and damage,morphogenesis, acid production, acid tolerance andstress response.ResultsAccording to the cut-off point chosen to determinethe putative presence of a mapped organism in eachsample, C. albicans was present in n 4 biofilms fromSRS and in n 6 biofilm from RC, as shown in Figure1. Table 1 shows that the number of reads distributionin sound and disease samples were equal (p 0.522).All RME medians for SRS and RC were ranked andFigure 1- Relative median expression (RME; log10) of genes in the Sound Root Surfaces (SRS; n 4) and Root Caries (RC; n 6) samples.RME was calculated from the median values of normalized read counts. The top median RME values for SRS and RC were selected andsorted, and indicate the most expressed genes by C. albicans SC5413J Appl Oral Sci. 3/10 2020;28:e20190578