The collagen binding protein Cnm contributes to oral colonization and cariogenicity of Streptococcus mutans OMZ175.
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ABSTRACT:
SUBMITTER: Miller JH
PROVIDER: S-EPMC4399078 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Streptococcus mutans, a significant contributor to dental caries, is occasionally isolated from the blood of patients with infective endocarditis. We previously showed that S. mutans strains expressing collagen-binding protein (Cnm) are present in the oral cavity of approximately 10-20% of humans and that they can effectively invade human umbilical vein endothelial cells (HUVECs). Here, we investigated the potential molecular mechanisms of HUVEC invasion by Cnm-positive S. mutans. The ability of Cnm-positive S. mutans to invade HUVECs was significantly increased by the presence of serum, purified type IV collagen, and fibrinogen (p < 0.001). Microarray analyses of HUVECs infected by Cnm-positive or -negative S. mutans strains identified several transcripts that were differentially upregulated during invasion, including those encoding the small G protein regulatory proteins ARHGEF38 and ARHGAP9. Upregulation of these proteins occurred during invasion only in the presence of serum. Knockdown of ARHGEF38 strongly reduced HUVEC invasion by Cnm-positive S. mutans. In a rat model of infective endocarditis, cardiac endothelial cell damage was more prominent following infection with a Cnm-positive strain compared with a Cnm-negative strain. These results suggest that the type IV collagen-Cnm-ARHGEF38 pathway may play a crucial role in the pathogenesis of infective endocarditis.
| S-EPMC7645802 | biostudies-literature
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.
| S-EPMC8516039 | biostudies-literature
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.
| S-EPMC6222205 | biostudies-literature
Project description:Cerebral microbleeds (CMBs) are an important risk factor for stroke and dementia. We have shown that the collagen binding surface Cnm protein expressed on cnm-positive Streptococcus mutans is involved in the development of CMBs. However, whether the collagen binding activity of cnm-positive S. mutans is related to the nature of the CMBs or to cognitive impairment is unclear. Two-hundred seventy nine community residents (70.0 years) were examined for the presence or absence of cnm-positive S. mutans in the saliva by PCR and collagen binding activity, CMBs, and cognitive function were evaluated. Cnm-positive S. mutans was detected more often among subjects with CMBs (p < 0.01) than those without. The risk of CMBs was significantly higher (odds ratio = 14.3) in the group with S. mutans expressing collagen binding activity, as compared to the group without that finding. Deep CMBs were more frequent (67%) and cognitive function was lower among subjects with cnm-positive S. mutans expressing collagen binding activity. This work supports the role of oral health in stroke and dementia and proposes a molecular mechanism for the interaction.
| S-EPMC5146923 | biostudies-literature
Project description: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.
| S-EPMC7192136 | biostudies-literature
Project description:Expression of the surface protein Cnm has been directly implicated in the ability of certain strains of Streptococcus mutans to bind to collagen and to invade human coronary artery endothelial cells (HCAEC) and in the killing of Galleria mellonella. Sequencing analysis of Cnm(+) strains revealed that cnm is located between the core genes SMU.2067 and SMU.2069. Reverse transcription-PCR (RT-PCR) analysis showed that cnm is cotranscribed with SMU.2067, encoding a putative glycosyltransferase referred to here as PgfS (protein glycosyltransferase of streptococci). Notably, Cnm contains a threonine-rich domain predicted to undergo O-linked glycosylation. The previously shown abnormal migration pattern of Cnm, the presence of the threonine-rich domain, and the molecular linkage of cnm with pgfS lead us to hypothesize that PgfS modifies Cnm. A ΔpgfS strain showed defects in several traits associated with Cnm expression, including collagen binding, HCAEC invasion, and killing of G. mellonella. Western blot analysis revealed that Cnm from the ΔpgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ΔpgfS was highly susceptible to proteinase K degradation, in contrast to the high-molecular-weight Cnm version found in the parent strain. Lectin-binding analyses confirmed the glycosylated nature of Cnm and strongly suggested the presence of N-acetylglucosamine residues attached to Cnm. Based on these findings, the phenotypes observed in ΔpgfS are most likely associated with defects in Cnm glycosylation that affects protein function, stability, or both. In conclusion, this study demonstrates that Cnm is a glycoprotein and that posttranslational modification mediated by PgfS contributes to the virulence-associated phenotypes linked to Cnm.
| S-EPMC4135665 | biostudies-literature
Project description:Streptococcus mutans is a major pathogen of dental caries. Collagen-binding proteins (CBPs) (approximately 120 kDa), termed Cnm and Cbm, are regarded as important cell surface antigens related to the adherence of S. mutans to collagenous tissue. Furthermore, CBP-positive S. mutans strains are associated with various systemic diseases involving bacteremia, such as infective endocarditis. Endodontic infection is considered to be an important cause of bacteremia, but little is known regarding the presence of S. mutans in dental pulp tissue. In the present study, the distribution and virulence of S. mutans in dental pulp tissues were investigated by focusing on CBPs. Adhesion and invasion properties of various S. mutans strains were analyzed using human dental pulp fibroblasts (HDPFs). CBP-positive strains had a significantly higher rate of adhesion to HDPFs compared with CBP-defective isogenic mutant strains (P<0.001). In addition, CBP-positive strains induced HDPF proliferation, which is a possible mechanism related to development of hyperplastic pulpitis. The distribution of S. mutans strains isolated from infected root canal specimens was then analyzed by PCR. We found that approximately 50% of the root canal specimens were positive for S. mutans. Approximately 20% of these strains were Cnm-positive, while no Cbm-positive strains were isolated. The Cnm-positive strains isolated from the specimens showed adhesion to HDPFs. Our results suggest that CBP-positive S. mutans strains exhibit high colonization in dental pulp. This could be a possible virulence factor for various systemic diseases.
| S-EPMC4956251 | biostudies-literature
Project description:Although several risk factors for stroke have been identified, one-third remain unexplained. Here we show that infection with Streptococcus mutans expressing collagen-binding protein (CBP) is a potential risk factor for haemorrhagic stroke. Infection with serotype k S. mutans, but not a standard strain, aggravates cerebral haemorrhage in mice. Serotype k S. mutans accumulates in the damaged, but not the contralateral hemisphere, indicating an interaction of bacteria with injured blood vessels. The most important factor for high-virulence is expression of CBP, which is a common property of most serotype k strains. The detection frequency of CBP-expressing S. mutans in haemorrhagic stroke patients is significantly higher than in control subjects. Strains isolated from haemorrhagic stroke patients aggravate haemorrhage in a mouse model, indicating that they are haemorrhagic stroke-associated. Administration of recombinant CBP causes aggravation of haemorrhage. Our data suggest that CBP of S. mutans is directly involved in haemorrhagic stroke.
| S-EPMC3220351 | biostudies-literature
Project description:BACKGROUND AND PURPOSE:Cerebral microbleeds (CMB) are associated with stroke and cognitive impairment. We previously reported a high prevalence of CMB in people with Streptococcus mutans expressing Cnm, a collagen-binding protein in the oral cavity. S. mutans is a major pathogen responsible for dental caries. Repeated challenge with S. mutans harboring the cnm gene encoding Cnm induced cerebral bleeding in stroke-prone spontaneously hypertensive rats. The purpose of this longitudinal study is to examine the relationship of cnm-positive S. mutans to the development of CMB. METHODS:We retrospectively investigated patients with stroke receiving oral microbiological examination and head 3T magnetic resonance imaging evaluations twice in the period 2014 to 2019, allowing >180-day interval. Patients with cnm-positive S. mutans were compared with those without. Quasi-Poisson regression models were used to explore associations between cnm-positive S. mutans and the increase in number of CMB between the 2 magnetic resonance imaging scans. RESULTS:A total of 111 patients were identified; 21 (19%) with cnm-positive S. mutans and 90 (81%) without. Clinical history, including blood pressure and the use of antithrombotic agents, were comparable between the 2 groups. New CMB were more commonly observed in patients with cnm-positive S. mutans (52% versus 23%; P=0.008). The incidence of CMB was significantly higher in the group with cnm-positive S. mutans, especially in deep areas, (incidence rate ratios [95% CI], 5.1 [1.9-13.6] for CMB in any brain region; 15.0 [5.4-42.0] for deep CMB), which persisted after adjusting for age, sex, hypertension, and renal impairment (4.7 [1.8-11.9] for CMB in any brain region; 13.9 [4.3-44.5] for deep CMB). CONCLUSIONS:This study demonstrates that cnm-positive S. mutans is associated with an increased incidence of CMB. Treatment for cnm-positive S. mutans infection may be a novel microbiota-based therapeutic approach for stroke and cognitive impairment.
| S-EPMC7678651 | biostudies-literature
Project description:A collection of 113 Streptococcus strains from supragingival dental plaque of caries-free individuals were recently tested in vitro for direct antagonism of the dental caries pathogen Streptococcus mutans, and for their capacity for arginine catabolism via the arginine deiminase system (ADS). To advance their evaluation as potential probiotics, twelve strains of commensal oral streptococci with various antagonistic and ADS potentials were assessed in a mouse model for oral (i.e., oral mucosal pellicles and saliva) and dental colonization under four diets (healthy or high-sucrose, with or without prebiotic arginine). Colonization by autochthonous bacteria was also monitored. One strain failed to colonize, whereas oral colonization by the other eleven strains varied by 3 log units. Dental colonization was high for five strains regardless of diet, six strains increased colonization with at least one high-sucrose diet, and added dietary arginine decreased dental colonization of two strains. Streptococcus sp. A12 (high in vitro ADS activity and antagonism) and two engineered mutants lacking the ADS (ΔarcADS) or pyruvate oxidase-mediated H2O2 production (ΔspxB) were tested for competition against S. mutans UA159. A12 wild type and ΔarcADS colonized only transiently, whereas ΔspxB persisted, but without altering oral or dental colonization by S. mutans In testing four additional candidates, S. sanguinis BCC23 markedly attenuated S. mutans' oral and dental colonization, enhanced colonization of autochthonous bacteria, and decreased severity of smooth surface caries under highly cariogenic conditions. Results demonstrate the utility of the mouse model to evaluate potential probiotics, revealing little correlation between in vitro antagonism and competitiveness against S. mutans in vivoIMPORTANCE Our results demonstrate in vivo testing of potential oral probiotics can be accomplished and can yield information to facilitate the ultimate design and optimization of novel anti-caries probiotics. We show human oral commensals associated with dental health are an important source of potential probiotics that may be used to colonize patients under dietary conditions of highly varying cariogenicity. Assessment of competitiveness against dental caries pathogen Streptococcus mutans and impact on caries identified strains or genetic elements for further study. Results also uncovered strains that enhanced oral and dental colonization by autochthonous bacteria when challenged with S. mutans, suggesting cooperative interactions for future elucidation. Distinguishing a rare strain that effectively compete with S. mutans under conditions that promote caries further validates our systematic approach to more critically evaluate probiotics for use in humans.
| S-EPMC7851695 | biostudies-literature