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“Though she be but little, she is fierce!” – William Shakespeare: Exploring sRNAs in the S. mutans regulon

thesis
posted on 2024-06-28, 19:23 authored by India Drummond

Dental caries is one of the most prevalent diseases worldwide. In the United States, 92% of adults aged 20-64 have experienced dental caries in their permanent teeth, with this rate increasing in less developed countries. Caused by demineralization of the enamel, caries arise when conditions in the human mouth favor the survival and persistence of acid-producing bacteria in the plaque biofilm. Chief among them is Streptococcus mutans, a homolactic fermenter of dietary sucrose that shifts the plaque environment from one of health to one that is associated with disease. S. mutans harbors a 25kDa manganese-dependent global regulator, SloR, that regulates a variety of virulence attributes. In some instances, SloR regulation is direct, with SloR binding to recognition elements on the DNA and enhancing or repressing their expression at the transcriptional level. In other instances, the impact of SloR on gene expression is indirect and may therefore involve an intermediate protein or RNA regulator at a transcriptional or post-transcriptional level. Among regulatory RNAs are small regulatory RNAs (sRNAs), which are 18-500nt, often non-coding, and can enhance or repress gene expression at the level of transcription or translation. Reports in the literature describe micro-sized (ms)RNAs (18-30nt) as having an emerging role in the bacterial response to stress and adaptation. In the present study, we seek to determine whether msRNAs play a role in the S. mutans SloR regulon and, if so, how they contribute to SloR's regulation of genes important to the bacteria's fitness and virulence potential. In silico work was performed to extract sRNA sequences in S. mutans and other streptococcal species, as well as Staphylococcus aureus from the literature. NCBI blastn was used to reveal the identities of sRNAs that are present in the S. mutans UA159 genome. Those that mapped to the genome were used to create a small database. At the bench, we identified sRNA intermediates as possible mediators of the project by Illumina sequencing. Specifically, msRNAs 18-30nt in size were analyzed in RNA-seq experiments performed in quadruplicate for differential expression in the SloR-proficient UA159 wild-type strain of S. mutans, and its isogenic SloR-deficient mutant GMS584. msRNAs that localized to intergenic regions as independent transcripts in alignment files were considered further if they had a p-value <0.05 and a log2 fold change greater than ±2.0 in UA159 versus GMS584. These msRNAs of interest were then compared against the database of sRNAs from the literature that mapped to the S. mutans genome to assess conservation of sRNAs across species. For select sRNAs, secondary structure was examined using mFold and target mRNAs were predicted using TargetRNA2 and IntaRNA. The sRNA targets were explored using KEGG Mapper, termination sequences were predicted with ARNold freeware, and promoter sequences were assessed manually. The extraction of sRNAs from the literature yielded 13,354 sequences, 655 of which mapped to the S. mutans genome using blastn. The results of RNA-seq reveal several differentially expressed msRNAs, including SmsR882 and SmsR1532 which can assume predicted stem-loop structures typical of most sRNAs. SmsR882 and SmsR1532 are novel and likely involved in the regulation of important cellular processes such as genetic competence, metal ion transport, carbohydrate metabolism, and oxidative and acid stress. Accumulating evidence supports SmsR1532 as a trans-acting 3' UTR-derived sRNA in S. mutans. Specifically, SmsR1532 could be a type I sRNA that is transcribed from an internal promoter in the same direction as the adjacent sloR or SMU_187c (hypothetical protein) genes, or it could be a type II sRNA generated via transcription from the SMU_187c promoter followed by RNase processing. These findings support a role for sRNAs in the SloR regulon and assign a potential mechanism for sRNA-mediated gene regulation in an important oral cariogen.

History

Institution

  • Middlebury College

Department or Program

  • Molecular Biology and Biochemistry

Degree

  • Bachelor of Arts, Honors

Academic Advisor

Dr. Grace Spatafora

Conditions

  • Restricted to Campus

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