Identification and analysis of fluoride-resistant Streptococcus mutans genomic mutation based on silicon solid nanopore

Abstract

This aim of this study was to identify the genomic mutations present in fluoride-resistant Streptococcus mutans (strain UA159-FR) using next generation sequencing, and to establish the role of these mutations by comparing fluoride-sensitive (UA159) and UA159-FR strains. In this paper, the genome of UA159-FR strains was sequenced by PacBio RS and IIIumina sequencing platform using silica-based nanopore. The genomic alignment to the reference S. mutans strain UA159 (NC_004350) was conducted using MUMmer 3.22 to list all the structural variations (SVs), single nucleotide polymorphisms (SNPs) and indels. A second round of Sanger sequencing of all regions in which the relevant mutations (SVs, indels, and SNPs) were identified was conducted to confirm these findings. In addition, blast comparison of virulence factor sequence was carried out. In order to explore the effect of the mutant gene against S. mutans, the KEGG pathway, COG function and go enrichment of the annotated gene were analyzed. Expressions of these variants were compared between S. mutans UA159 and UA159-FR through RT-qPCR. Genome sequencing revealed the presence of a single 2,031,692 bp circular chromosome, including 1940 ORFs, of which the G+C content was 37.46%. In addition, 35 genomic SNPs and 5 SVs type in UA159-FR were identified through an extensive bioinformatics analysis. The results of indel analysis showed that there was no insertion or deletion of genes in UA159 and UA159- FR; Synteny analysis showed that the gene of UA159 was collinear with UA159-FR, which further indicated that the main type of gene mutation was single base mutation. By comparing the amino acid sequences of UA159-FR samples with virulence factors, blast obtained 92 virulence factors, among which, ciaH, eno and pykF had gene mutations. The mutations of ciaH, eno and pykF may be responsible for the functional changes of Streptococcus. At the same time, enrichment analysis showed that the expression genes of S. mutans were mainly enriched in KEGG pathway such as glycolysis/gluconeogenesis, citric acid cycle (TCA cycle), pentose phosphate pathway, involved in DNA replication, and related to biological processes such as cell development and cell metabolism. ciaH, eno and pykF were involved in these processes. In summary, this study highlights new mutations in fluoride-resistant bacteria that may enhance our current understanding of fluoride-resistant pathways in microorganisms. It has been proved that silicon based nanopores and nanopores arrays can detect gene sequences sensitively and can be used as the main means of gene sequencing in the next generation.