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Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR.

ABSTRACT: Streptococcus mutans, a primary bacterium associated with dental caries, has four known clinical serotypes (c, e, fand k). Certain serotypes, the presence of multiple serotypes and strains with collagen-binding proteins (CBP, Cnm and Cbm) have been linked with systemic disease. Evaluation of S mutans serotype distribution and caries association is needed in the United States. The purpose of this study was to evaluate the prevalence of S mutans serotypes from two cohorts of African-American children in rural Alabama using three sample types (saliva, plaque and individual S mutans isolates) by PCR detection for association with caries. Detection of CBP was also performed by PCR. In total, 129 children were evaluated and overall prevalence of serotypes were: serotype c(98%), e(26%), f(7%) and k(52%). Serotype c was statistically associated with higher caries scores in older children (P < 0.001) and serotype k was statistically more likely in females (P = 0.004). Fourteen per cent of children had CBP. Thirteen S mutans isolates from five children tested positive for both CBP. This study is the first to report on the prevalence of S mutans serotypes in a US population using the PCR-based approach. The frequency of serotype k in this study is the highest reported in any population, illustrating the need for further study to determine the prevalence of this clinically relevant serotype in the US. This is the first study to report S mutans isolates with both Cnm and Cbm in the same strain, and further analysis is needed to determine the clinical significance of these strains.

SUBMITTER: Momeni SS

PROVIDER: S-EPMC7192136 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR.

Momeni Stephanie S SS Ghazal Tariq T Grenett Hernan H Whiddon Jennifer J Moser Stephen A SA Childers Noel K NK

Molecular oral microbiology 20190226 2

Streptococcus mutans, a primary bacterium associated with dental caries, has four known clinical serotypes (c, e, fand k). Certain serotypes, the presence of multiple serotypes and strains with collagen-binding proteins (CBP, Cnm and Cbm) have been linked with systemic disease. Evaluation of S mutans serotype distribution and caries association is needed in the United States. The purpose of this study was to evaluate the prevalence of S mutans serotypes from two cohorts of African-American child ...[more]

PMID: 30667593

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Project description:The cnm gene, coding for the glycosylated collagen- and laminin-binding surface adhesin Cnm, is found in the genomes of approximately 20% of Streptococcus mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans, such as P1 and WapA, have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the β-sheet-rich N-terminal collagen-binding domain (CBD) of Cnm has a propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids in either its native glycosylated or recombinant nonglycosylated form and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro, ex vivo, and in vivo assays to characterize the amylogenic properties of Cnm. In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates, whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, amyloid formation by Cnm might act as a negative regulatory mechanism to modulate collagen-binding activity within S. mutans biofilms and warrants further investigation. IMPORTANCE Streptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor of S. mutans. Expression of Cnm by S. mutans is hypothesized to contribute to niche expansion, allowing colonization of multiple sites in the body, including collagen-rich surfaces such as dentin and heart valves. Here, we suggest that Cnm function might be modulated by its aggregation status. As a monomer, its primary function is to promote attachment to collagenous substrates via its collagen-binding domain (CBD). However, in later stages of biofilm maturation, the same CBD of Cnm could self-assemble into amyloid fibrils, losing the ability to bind to collagen and likely becoming a component of the biofilm matrix. Our findings shed light on the role of functional amyloids in S. mutans pathobiology and ecology.

Project description:Cnm is a surface-associated protein present in a subset of Streptococcus mutans strains that mediates binding to extracellular matrices, intracellular invasion, and virulence. Here, we showed that cnm transcription is controlled by the global regulators CovR and VicRKX. In silico analysis identified multiple putative CovR- and VicR-binding motifs in the regulatory region of cnm as well as in the downstream gene pgfS, which is associated with the posttranslational modification of Cnm. Electrophoretic mobility shift assays revealed that CovR and VicR specifically and independently bind to the cnm and pgfS promoter regions. Quantitative real-time PCR and Western blot analyses of ?covR and ?vicK strains as well as of a strain overexpressing vicRKX revealed that CovR functions as a positive regulator of cnm, whereas VicRKX acts as a negative regulator. In agreement with the role of VicRKX as a repressor, the ?vicK strain showed enhanced binding to collagen and laminin and higher intracellular invasion rates. Overexpression of vicRKX was associated with decreased rates of intracellular invasion but did not affect collagen or lamin binding activities, suggesting that this system controls additional genes involved in binding to these extracellular matrix proteins. As expected, based on the role of CovR in cnm regulation, the ?covR strain showed decreased intracellular invasion rates, but, unexpectedly collagen and laminin binding activities were increased in this mutant strain. Collectively, the results presented here expand the repertoire of virulence-related genes regulated by CovR and VicRKX to include the core gene pgfS and the noncore gene cnm IMPORTANCE Streptococcus mutans is a major pathogen associated with dental caries and also implicated in systemic infections, in particular, infective endocarditis. The Cnm adhesin of S. mutans is an important virulence factor associated with systemic infections and caries severity. Despite its role in virulence, the regulatory mechanisms governing cnm expression are poorly understood. Here, we describe the identification of two independent regulatory systems controlling the transcription of cnm and the downstream pgfS-pgfM1-pgfE-pgfM2 operon. A better understanding of the mechanisms controlling expression of virulence factors like Cnm can facilitate the development of new strategies to treat bacterial infections.

Project description:Early childhood caries (ECC) is a chronic disease affecting the oral health of children globally. This disease is multifactorial, but a primary factor is cariogenic microorganisms such as Streptococcus mutans. Biosynthetic gene clusters (BGCs) encode small molecules with diverse biological activities that influence the development of many microbial diseases, including caries. The purpose of this study was to identify BGCs in S. mutans from a high-caries risk study population using whole-genome sequencing and assess their association with ECC. Forty representative S. mutans isolates were selected for genome sequencing from a large-scale epidemiological study of oral microbiology and dental caries in children from a localized Alabama population. A total of 252 BGCs were identified using the antiSMASH BGC-mining tool. Three types of BGCs identified herein-butyrolactone-like, ladderane-like, and butyrolactone-ladderane-like hybrid (BL-BGC)-have not been reported in S. mutans. These 3 BGCs were cross-referenced against public transcriptomics data, and were found to be highly expressed in caries subjects. Furthermore, based on a polymerase chain reaction screening for core BL genes, 93% of children with BL-BGC had ECC. The role of BL-BGC was further investigated by examining cariogenic traits and strain fitness in a deletion mutant using in vitro biofilm models. Deletion of the BL-BGC significantly increased biofilm pH as compared to the parent strain, while other virulence and fitness properties remained unchanged. Intriguingly, BL-BGC containing strains produced more acid, a key cariogenic feature, and less biofilm than the model cariogenic strain S. mutans UA159, suggesting the importance of this BL-BGC in S. mutans-mediated cariogenesity. The structure of any BL-BGC derived metabolites, their functions, and mechanistic connection with acid production remain to be elucidated. Nevertheless, this study is the first to report the clinical significance of a BL-BGC in S. mutans. This study also highlights pangenomic diversity, which is likely to affect phenotype and virulence.

Dataset Information

Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR.

Publications

Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR.

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