Professor Grace Spatafora – India Drummond | How Do Small Regulatory RNAs Promote Tooth Decay?

Tooth decay (known as ‘dental caries’) is a global health problem. The key pathogen is Streptococcus mutans, a hardy tooth-colonising bacterium. An overlooked factor in caries is manganese (Mn²⁺). India Drummond and Professor Grace Spatafora of Middlebury College are investigating the effect of Mn²⁺ on Streptococcus mutans traits controlled by the SloR metalloregulator and small RNAs. Their research has important implications for dental health.   

Formation of Dental Caries

One of the world’s most prevalent chronic diseases is tooth decay, also known as caries. This widespread disease is driven by plaque, a multi-species biofilm on the tooth surface. Bacteria colonise teeth by secreting an extracellular polysaccharide (EPS) matrix. With a high-sugar diet, acid-forming bacteria metabolise sucrose into organic acids, and various bacteria thrive under the resulting low-pH conditions. The acidity causes enamel de-mineralisation and decay, leading to caries (cariogenesis). The main caries-forming pathogen is Streptococcus mutans (S. mutans), an oval-shaped, gram-positive bacterium with EPS-forming, acid-forming, and acid-tolerant traits.

Manganese as a Factor in Caries Formation

There is a growing focus on the importance of metal ions to caries-forming bacteria. Oral bacteria require several trace metals – including iron, zinc, nickel, and manganese – for enzymatic and structural roles. However, ion supply is variable. So, oral bacteria must limit uptake when ions are abundant (during meals) and scavenge ions when scarce (between meals). This is known as nutritional immunity.

An overlooked factor in caries formation is manganese. In fact, manganese is an absolute requirement for Streptococci. As a divalent cation, manganese (Mn²⁺) is a cofactor for several enzymes, including the antioxidant enzyme superoxide dismutase. Moreover, Mn²⁺ is involved in EPS production needed for biofilm formation. The functions of Mn²⁺ in S. mutans are turning out more diverse than was thought possible. India Drummond and colleagues at Professor Grace Spatafora’s laboratory group at Middlebury College, Vermont, are investigating Mn²⁺ in S. mutans’ transcriptional gene regulation.

Probing into the SloR Regulon

Manganese is pivotal to an important metalloregulator, SloR, in S. mutans. SloR is a 25-kilodalton manganese-binding protein of the DtxR metalloregulatory family. SloR activity depends on the availability of Mn²⁺, so it is regarded as a manganese-centric sensor. SloR regulates the transcription of numerous genes, including those dictating nutritional immunity, biofilm formation, and stress tolerance.

Deploying a combination of in silico tools and ‘wet lab’ experiments, Professor Spatafora’s group is probing into this diversity of SloR-regulated genes – known as the SloR regulon. The regulon includes small RNAs (sRNAs) – short (18–500 nt) non-coding transcripts. They discovered that three Mn²⁺ ions bind to SloR, allowing dimerisation. The SloR dimer N-termini then bind to the DNA, likely in the major groove, at sequences known as SloR recognition elements (SRE).

The Role of SloR in Manganese Homeostasis

S. mutans has at least two SloR-regulated Mn²⁺ uptake systems – SloABC and MntH. Modulation of the SloABC system by SloR is a major focus of the group’s research. SloABC consists of three components expressed from a single operon: an ATP-binding protein (SloA), integral membrane protein (SloB), and lipoprotein receptor protein (SloC). The sloR gene, with its independent promoter, is immediately downstream of the sloABC operon. When Mn²⁺ is plentiful (during meals), SloR is bound to Mn²⁺ and binds to the sloA promoter, blocking RNA polymerase and repressing transcription. When Mn²⁺ is scarce (between meals), SloR, no longer bound to Mn²⁺, disengages from the sloABC promoter, allowing access to RNA polymerase and consequent transcription. The SloABC manganese-import machinery is then expressed, enabling the scavenging of Mn²⁺. The group discovered three SREs in the sloA promoter region, indicating that SloR binds here.

Investigating sRNA-SloR Interactions

Although well-studied in eukaryotes, sRNAs are under-researched in prokaryotes. Emerging research shows that sRNAs are regulators of fitness and virulence, with particular relevance to pathogenic bacteria. sRNAs can enhance or repress gene expression via complementary base pairing, often at the 3’- or 5’-untranslated region. From a bacterial standpoint, sRNAs represent an adaptive, low-energy control system, as they are usually untranslated and can facilitate rapid responses. The group is exploring S. mutans’ surprisingly diverse ‘sRNAome.’

The Spatafora lab hypothesised that S. mutans’ ability to cause cavities is, in part, the result of interactions between sRNAs and SloR-mediated gene expression. They grew two S. mutans strains, a wild-type (UA159) and a mutant SloR-deficient strain (GMS584), under high vs low manganese conditions. With RNA sequencing, they generated sRNA libraries, and compared transcript expression levels between wild-type vs SloR-deficient strains, and between high vs low manganese (differential expression). From these transcripts, they identified two S. mutans small RNAs of interest – SmsR1532 and SmsR1785. SmsR1532 is a 30-nt sRNA in an intergenic region downstream of sloABCR (i.e., the sloABC operon with adjacent sloR gene). SmsR1785 is a 66-nt sRNA mapping to genes encoding a toxin-antitoxin system.

Genomics Reveal Different Ancestral Origins

The group undertook in silico genomics analyses of SloR, SmsR1532 and SmsR1785 – phylogenetics (using Anvi’o), NCBI BLASTN searching, and covariance modelling (using LocARNA). Like a time machine, these analyses reveal evolutionary history – as well as family relationships! They found that SloR is highly conserved across the Streptococcus genus. SmsR1532 emerged more recently as a common ancestor of S. mutans and S. troglodytae. In contrast, the analyses revealed no clear inheritance of SmsR1785, suggesting that it was acquired via horizontal gene transfer.

Elucidating Transcription with Northern Blotting

Are the sRNAs transcribed from an independent promoter (Type I)? Or are they co-transcribed as one multi-gene transcript (Type II)? The group carried out northern blotting to find out. The results revealed Type I precursor transcripts of both SmsR1532 (106 nt) and SmsR1785 (73 nt and 70 nt), indicating that they undergo post-transcription processing to yield their smaller final forms (30 and 66 nt, respectively).

Promoter-proximal and Promoter-distal – What’s the Difference?

Differences in sRNA expression between the SloR-deficient and wild-type strains showed that SloR repressed SmsR1532 but enhanced SmsR1785 transcription. But how? Electromobility shift assays revealed SloR binding to SREs in the promoter regions of both sRNAs – showing that SloR binds directly to their promoters. The SmsR1532 SRE is promoter-proximal, overlapping with the -10 promoter element. This location indicates that SloR binds here and blocks RNA polymerase, preventing transcription. In contrast, the SmsR1785 SRE is promoter-distal, being 95 nt upstream of the transcription start site. SloR evidently encourages transcription of SmsR1785, and the group speculates that DNA bending is involved.

Predicting sRNA Behaviour with Bioinformatics

The group used in silico tools to predict sRNA behaviour. This analysis involved predicting sRNA secondary structure (using mFold V3.6), mRNA binding targets (using IntaRNA and TargetRNA2), and pathways potentially involving the two sRNAs (using KEGG Mapper). SmsR1532 and SmsR1785 were both predicted to form hairpin structures. Predicted targets of SmsR1532 included sloABC, and genes involved with sugar transport and acid tolerance. Among the predicted targets of SmsR1785 were the Fst-Sm toxin, genes involved with hydrophobicity, intracellular polysaccharides, and acid stress response. Interestingly, both SmsR1532 and SmsR1785 were predicted to target genes involved in oxidative stress, as well as EPS and biofilm formation – traits associated with S. mutans hardiness.

Why is This Important?

India Drummond, Professor Spatafora, and their group have demonstrated manganese-sensing by S. mutans via SloR and sRNAs. This work provides unique insights into phenotypes involved in cariogenesis. Using ‘systems biology’ approaches, they want to delve deeper into S. mutans’ SloR regulon and sRNAome – with implications for dental health. While toothpaste and dental hygiene go so far, there is a need for effective anti-caries therapeutics. Professor Spatafora’s research suggests that SloR is a good therapeutic target. SloR is expressed in bacteria and not humans, and has a druggable binding pocket. As a next step, the lab wants to carry out rational drug design to identify potential therapeutics. This research would involve investigating small molecules that bind SloR and facilitate its ability to repress S. mutans virulence.