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A special type of Streptococcus mutans expressing collagen-binding proteins (CBPs), Cnm, and Cbm, on the cell surface has been shown to be highly pathogenic. It is believed that S. mutans with CBPs that has entered the blood vessel attaches to collagen molecules exposed from the damaged blood vessel, inhibiting aggregation by platelets and increasing bleeding. Therefore, it is crucial to understand the molecular characteristic features of CBPs to protect against and cure S. mutans-related diseases. In this work, we highlighted the Cbm/collagen-binding domain (CBD) and examined its binding ability and thermal stability using its domain/region exchange variants in more detail. The CBD comprises the N1-domain, a linker, N2-domain, and a latch (N1–N2~), where the latch interacts with the N1-domain to form a β-sheet. The collagen-binding activity of the Cbm/CBD domain/region exchange variants was investigated using ELISA. Binding assays demonstrated that the N-domain_linker_N-domain composition is necessary for collagen binding as previously reported, newly that the latch is involved in binding through the β-sheet with the N1-domain when the N1-domain is present at the N-terminal position, and that the N2-domain is particularly important for collagen binding at both the N- and C-terminal positions. Thermal denaturation experiments newly revealed that the linker and latch bound to the N-domain contribute to N-domain stabilization but have no effect on the N-domain_linker_N-domain molecule, which contains two N-domains. It has also been shown that the N-terminal N2-domain destabilizes the N-domain_linker_N-domain structure. The results of this study will contribute to the rapid detection of CBP, development of CBP-targeted therapies, and application of CBPs to protein engineering using their collagen-binding ability.

A special type of Streptococcus mutans expressing collagen-binding proteins (CBPs), Cnm, and Cbm, on the cell surface has been shown to be highly pathogenic. It is believed that S. mutans with CBPs that has entered the blood vessel attaches to collagen molecules exposed from the damaged blood vessel, inhibiting aggregation by platelets and increasing bleeding. Therefore, it is crucial to understand the molecular characteristic features of CBPs to protect against and cure S. mutans-related diseases. In this work, we highlighted the Cbm/collagen-binding domain (CBD) and examined its binding ability and thermal stability using its domain/region exchange variants in more detail. The CBD comprises the N1-domain, a linker, N2-domain, and a latch (N1–N2~), where the latch interacts with the N1-domain to form a β-sheet. The collagen-binding activity of the Cbm/CBD domain/region exchange variants was investigated using ELISA. Binding assays demonstrated that the N-domain_linker_N-domain composition is necessary for collagen binding as previously reported, newly that the latch is involved in binding through the β-sheet with the N1-domain when the N1-domain is present at the N-terminal position, and that the N2-domain is particularly important for collagen binding at both the N- and C-terminal positions. Thermal denaturation experiments newly revealed that the linker and latch bound to the N-domain contribute to N-domain stabilization but have no effect on the N-domain_linker_N-domain molecule, which contains two N-domains. It has also been shown that the N-terminal N2-domain destabilizes the N-domain_linker_N-domain structure. The results of this study will contribute to the rapid detection of CBP, development of CBP-targeted therapies, and application of CBPs to protein engineering using their collagen-binding ability. [ABSTRACT FROM AUTHOR]

Introduction: Streptococcus mutans (SM) with the collagen-binding protein Cnm is a unique member of the oral resident flora because it causes hemorrhagic vascular disorders. In the multicenter study, we examined the relationship between Cnm-positive SM (CP-SM) and intracranial aneurysm (IA) rupture, which remains unknown. Methods: Between May 2013 and June 2018, we collected whole saliva samples from 431 patients with ruptured IAs (RIAs) and 470 patients with unruptured IAs (UIAs). Data were collected on age, sex, smoking and drinking habits, family history of subarachnoid hemorrhage, aneurysm size, number of teeth, and comorbidities of lifestyle disease. Results: There was no difference in the positivity rate of patients with CP-SM between the patients with RIAs (17.2%) and those with UIAs (19.4%). In subanalysis, the rate of positivity for CP-SM was significantly higher in all IAs <5 mm than in those ≥10 mm in diameter (p = 0.0304). In the entire cohort, the rate of positivity for CP-SM was lower in larger aneurysms than in smaller aneurysms (p = 0.0393). Conclusions: CP-SM was not involved in the rupture of UIAs. In the subanalysis, the possibility of its involvement in the formation of vulnerable aneurysms remained. [ABSTRACT FROM AUTHOR]

Collagen-binding proteins (CBP), Cnm and Cbm, from Streptococcus mutans are involved in infective endocarditis caused by S. mutans because of their collagen-binding ability. In this study, we focused on the collagen-binding domain (CBD), which is responsible for the collagen-binding ability of CBP, and analyzed its structure, binding activity, and stability using CBD domain variants. The CBD consists of the N1 domain, linker, N2 domain, and latch (N1-N2~) as predicted from the amino acid sequences. The crystal structure of the Cnm/CBD was determined at a 1.81 Å resolution. N1_linker_N2 forms a ring structure that can enfold collagen molecules, and the latch interacts with N1 to form a ring clasp. N1 and N2 have similar immunoglobulin folds. The collagen-binding activities of Cbm/CBD and its domain variants were examined using ELISA. N1-N2~ bound to collagen with KD = 2.8 μM, and the latch-deleted variant (N1-N2) showed weaker binding (KD = 28 μM). The linker-deleted variant (N1N2~) and single-domain variants (N1 and N2) showed no binding activity, whereas the domain-swapped variant (N2-N1~) showed binding ability, indicating that the two N-domains and the linker are important for collagen binding. Thermal denaturation experiments showed that N1-N2 was slightly less stable than N1-N2~, and that N2 was more stable than N1. The results of this study provide a basis for the development of CBD inhibitors and applied research utilizing their collagen-binding ability.

Enolase proteins play a significant role as moonlighting proteins. In their role as surface-associated enolase, they have multiple functions as they interact with extracellular matrix proteins. Type I and III collagens are the major constituents of this extracellular matrix, and collagen is one of the targets of interaction with the enolase of many pathogens, thereby helping the colonization process and promoting the subsequent invasion of the host. This work aimed to determine the participation of non-typeable H. influenzae enolase as a collagen-binding protein. In this study, through the use of in vitro tests it was demonstrated that recombinant enolase of non-typeable H. influenzae (rNTHiENO) strongly binds to type I collagen. Using molecular docking, the residues that could take part in the interaction of non-typeable H. influenzae enolase-type I collagen (NTHiENO-Cln I) and non-typeable H. influenzae enolase-type III collagen (NTHiENO-Cln III) were identified. However, in vitro assays show that NTHiENO has a better affinity to interact with Cln I, concerning type Cln III. The interaction of NTHiENO with collagen could play a significant role in the colonization process; this would allow H. influenzae to increase its virulence factors and strengthen its pathogenesis. [ABSTRACT FROM AUTHOR]

Collagen, the most abundant protein in mammal, is widely expressed in tissues and organs, as well as tumor extracellular matrix. Tumor collagen mainly accumulates in tumor stroma or beneath tumor blood vessel endothelium, and is exposed due to the fragmentary structure of tumor blood vessels. Through the blood vessels with enhanced permeability and retention (EPR) effect, collagen-binding macromolecules could easily bind to tumor collagen and accumulate within tumor, supporting tumor collagen to be a potential tumor-specific target. Recently, numerous studies have verified that targeting collagen within tumor extracellular matrix (TEM) would enhance the accumulation and retention of immunotherapy drugs at tumor, significantly improving their anti-tumor efficacy, as well as avoiding severe adverse effects. In this review, we would summarize the known collagen-binding domains (CBD) or proteins (CBP), their mechanism and application in tumor-targeting immunotherapy, and look forward to future development. [ABSTRACT FROM AUTHOR]

Collagen-binding proteins (CBP), Cnm and Cbm, from Streptococcus mutans are involved in infective endocarditis caused by S. mutans because of their collagen-binding ability. In this study, we focused on the collagen-binding domain (CBD), which is responsible for the collagen-binding ability of CBP, and analyzed its structure, binding activity, and stability using CBD domain variants. The CBD consists of the N1 domain, linker, N2 domain, and latch (N1-N2~) as predicted from the amino acid sequences. The crystal structure of the Cnm/CBD was determined at a 1.81 Å resolution. N1_linker_N2 forms a ring structure that can enfold collagen molecules, and the latch interacts with N1 to form a ring clasp. N1 and N2 have similar immunoglobulin folds. The collagen-binding activities of Cbm/CBD and its domain variants were examined using ELISA. N1-N2~ bound to collagen with KD = 2.8 μM, and the latch-deleted variant (N1-N2) showed weaker binding (KD = 28 μM). The linker-deleted variant (N1N2~) and single-domain variants (N1 and N2) showed no binding activity, whereas the domain-swapped variant (N2-N1~) showed binding ability, indicating that the two N-domains and the linker are important for collagen binding. Thermal denaturation experiments showed that N1-N2 was slightly less stable than N1-N2~, and that N2 was more stable than N1. The results of this study provide a basis for the development of CBD inhibitors and applied research utilizing their collagen-binding ability. [ABSTRACT FROM AUTHOR]

Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is a major pathogen of dental caries. The protein Cnm of S. mutans is involved in collagen binding, but its other biological functions are unknown. In this study, a Cnm-deficient isogenic mutant and a complementation strain were generated from a Cnm-positive S. mutans strain to help determine the properties of Cnm. Initially, comparison of the cell surface structure was performed by electron microscopy, which demonstrated that Cnm appears to be localized on the cell surface and associated with a protruding cell surface structure. Deep RNA sequencing of the strains revealed that the defect in Cnm caused upregulated expression of many genes related to ABC transporters and cell-surface proteins, while a few genes were downregulated. The amount of biofilm formed by the Cnm-defective strain increased compared with the parental and complemented strains, but the biofilm structure was thinner because of elevated expression of genes encoding glucan synthesis enzymes, leading to increased production of extracellular polysaccharides. Particular antibiotics, including bacitracin and chloramphenicol, had a lower minimum inhibitory concentration for the Cnm-defective strain than particular antibiotics, including bacitracin and chloramphenicol, compared with the parental and complemented strains. Our results suggest that S. mutans Cnm is located on the cell surface, gives rise to the observed protruding cell surface, and is associated with several biological properties related to membrane permeability. [ABSTRACT FROM AUTHOR]

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 micro-titer 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. [ABSTRACT FROM AUTHOR]

Streptococcus mutans, a cause of dental caries, is an opportunistic pathogen leading to subacute infective endocarditis and bacteremia. Bacterial attachment to the heart valves, requires the presence of collagen-binding proteins (CBP), such as Cnm and Cbm; encoded by cnm and cbm genes, respectively. Herein, the prevalent S. mutans serotypes and genes for CBPs among diabetics and non-diabetic controls from Northern Jordan were investigated. Teeth swabs were cultured on TYCSB agar for S. mutans isolation. PCR was used to confirm the isolates' identity, and to identify the isolates' serotypes and CBP genes. The most prevalent serotype among the diabetics was c (71.6 %), followed by k (43.2 %), f (32.1 %), and e (13.6 %). Among the diabetics, 44.4 % harbored two or more serotypes. The most prevalent serotype among the non-diabetics was k (92.1 %), followed by c (69.8 %), f (25.4 %), and e (15.9 %). Among the non-diabetics, 92.1 % harbored two or more serotypes. The prevalence rates for cnm and cbm were 75.3 % and 11.1 %, respectively among the diabetics' isolates, and 38.1 % each among the non-diabetics' isolates. Due to the high prevalence of serotype k and the isolates harboring CBP genes, the Jordanian population maybe at the risk of developing S. mutans-related complications, such as infective endocarditis. [ABSTRACT FROM AUTHOR]

InS. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in theGalleria mellonellainvertebrate model. To further characterize Cnm as a virulence factor, thecnmgene fromS. mutansstrain OMZ175 was expressed in the non-pathogenicLactococcus lactisNZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall ofL. lactis. Similar toS. mutans, expression of Cnm inL. lactisenabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence inG. mellonella. Using anex vivohuman heart tissue colonization model, we showed that Cnm-positive strains of eitherS. mutansorL. lactisoutcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitatedL. lactisadhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute ofS. mutansand confirm the usefulness of theL. lactisheterologous system for further characterization of bacterial virulence factors. [ABSTRACT FROM AUTHOR]

Collagen, the most abundant protein in mammal, is widely expressed in tissues and organs, as well as tumor extracellular matrix. Tumor collagen mainly accumulates in tumor stroma or beneath tumor blood vessel endothelium, and is exposed due to the fragmentary structure of tumor blood vessels. Through the blood vessels with enhanced permeability and retention (EPR) effect, collagen-binding macromolecules could easily bind to tumor collagen and accumulate within tumor, supporting tumor collagen to be a potential tumor-specific target. Recently, numerous studies have verified that targeting collagen within tumor extracellular matrix (TEM) would enhance the accumulation and retention of immunotherapy drugs at tumor, significantly improving their anti-tumor efficacy, as well as avoiding severe adverse effects. In this review, we would summarize the known collagen-binding domains (CBD) or proteins (CBP), their mechanism and application in tumor-targeting immunotherapy, and look forward to future development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Pan, Huang, Wang, Chen, Zhu and Ye.)

Objective Cerebral hemorrhage has been shown to occur in animals experimentally infected with Streptococcus mutans carrying the collagen-binding Cnm gene. However, the relationship between cerebral microbleeds and oral hygiene, with a focus on Cnm gene-positive S. mutans infection, remains unclear. Material and Methods One hundred and thirty-nine subjects participated. The presence or absence of Cnm-positive S. mutans and its collagen-binding activity were investigated using saliva samples, and relationship with cerebral microbleeds detected on MRI investigated, including clinical information and oral parameters. Results Fifty-one subjects were identified as Cnm-positive S. mutans carriers (36.7%), with cerebral microbleeds being detected in 43 (30.9%). A significantly larger number of subjects carried Cnm-positive S. mutans in the cerebral microbleeds (+) group. S. mutans with Cnm collagen-binding ability was detected in 39 (28.1%) of all subjects, and the adjusted odds ratio for cerebral microbleeds in the Cnm-positive group was 14.4. Regarding the presence of cerebral microbleeds, no significant differences were noted in the number of remaining teeth, dental caries, or in classic arteriosclerosis risk factors. Conclusions The occurrence of cerebral microbleeds was higher in subjects carrying Cnm-positive S. mutans, indicating that the presence of Cnm-positive S. mutans increases cerebral microbleeds, and is an independent risk for the development of cerebrovascular disorders. [ABSTRACT FROM AUTHOR]

Background: IgA nephropathy (IgAN) is the most common primary chronic glomerulonephritis; however, its precise initiating pathogenesis remains unclear. Streptococcus mutans is a major pathogen of human dental caries. S. mutans strains with the cnm gene encoding Cnm, a collagen-binding protein, have been reported to contribute to the development of systemic diseases. However, the contribution of S. mutans with Cnm in the development of IgAN has not been reported. The aim of this study was to investigate the prevalence of cnm-positive S. mutans in IgAN patients and clarify the effects of cnm-positive S. mutans on the histological pathology of IgAN. Methods: We identified the cnm gene in S. mutans isolated in saliva specimens, which were collected from IgAN patients ( n = 53) and control subjects ( n = 50). We evaluated the collagen-binding properties of S. mutans in IgAN patients and controls. The clinical parameters and histological scores were also assessed in IgAN patients. Results: The rates of S. mutans isolation in IgAN and control groups were 84.0 and 84.9 %, respectively, not significantly dfferent. cnm-positive strains were significantly more prevalent in the IgAN group than in controls (32.1 vs. 14.0 %, p < 0.05). With regard to collagen-binding assays, the binding rates of cnm-positive strains were significantly higher in the IgAN group than in controls (96.6 vs. 30.0, p < 0.05). In addition, the segmental glomerulosclerosis scores were significantly higher in cnm-positive patients with IgAN than in cnm-negative patients with IgAN (0.94 vs. 0.57, p < 0.05). Conclusion: cnm-positive S. mutans strains are potentially associated with the pathogenesis of IgAN. [ABSTRACT FROM AUTHOR]

Cnm, a collagen- and laminin-binding protein present in a subset of Streptococcus mutans strains, mediates binding to extracellular matrices (ECM), intracellular invasion and virulence in the Galleria mellonella model. Antibodies raised against Cnm were used to confirm expression and the cell surface localization of Cnm in the highly invasive OMZ175 strain. Sequence analysis identified two additional genes (cnaB and cbpA) encoding putative surface proteins immediately upstream of cnm. Inactivation of cnaB and cbpA in OMZ175, individually or in combination, did not decrease the ability of this highly invasive and virulent strain to bind to different ECM proteins, invade human coronary artery endothelial cells (HCAEC), or kill G. mellonella. Similarly, expression of cnaB and cbpA in the cnm(-) strain UA159 revealed that these genes did not enhance Cnm-related phenotypes. However, integration of cnm in the chromosome of UA159 significantly increased its ability to bind to collagen and laminin, invade HCAEC, and kill G. mellonella. Moreover, the presence of antibodies against Cnm nearly abolished the ability of OMZ175 to bind to collagen and laminin and invade HCAEC, and significantly protected G. mellonella against OMZ175 infection. We concluded that neither CnaB nor CbpA is necessary for the expression of Cnm-related traits. We also provided definitive evidence that Cnm is an important virulence factor and a suitable target for the development of novel preventive and therapeutic strategies to combat invasive S. mutans strains. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Streptococcus mutans, an aetiologic agent of dental caries, is a pathogen for infective endocarditis (IE). We investigated strains that express collagen-binding proteins (CBPs) with further classification based on expression of the 190-kDa protein antigen (PA). Method: Zeta-potential values of strains TW871 (CBP+/PA+) and MT8148 (CBP−/PA+), and their respective PA-defective mutant strains TW871PD (CBP+/PA−) and MT8148PD (CBP−/PA−), were analysed, as were their adhesion to and invasion of human umbilical vein endothelial cells (HUVECs). The distribution of strains from the oral cavities of 200 healthy individuals was analysed for CBP and/or PA expression and the strains were characterised for their adhesion and invasion properties. Results: TW871PD and MT8148PD showed significantly lower zeta-potential values than TW871 and MT8148, respectively. Collagen-binding rates were significantly higher for TW871PD than for TW871 but nearly negligible for MT8148 and MT8148PD. The adhesion and invasion rates of HUVECs were significantly higher for TW871PD than for TW871 and significantly higher for TW871 than for MT8148 and MT8148PD. The prevalence of CBP+ strains was ∼10% and ∼3% in the case of CBP+/PA− strains. Analyses of 200 clinical strains showed the CBP+/PA− group to have higher adhesion and invasion rates than other groups. Conclusions: CBP+/PA− S. mutans strains, despite their low distribution frequency, may be highly virulent for infective endocarditis. [Copyright &y& Elsevier]

Abstract: Objective: Streptococcus mutans, an oral pathogen associated with infective endocarditis (IE), possesses two genes encoding collagen-binding proteins, namely cnm and cbm. In this study, we used multilocus sequence typing (MLST) of S. mutans with the cbm gene. Design: Forty-five S. mutans strains including 15 strains with the cnm gene, 15 strains with the cbm gene, and 15 strains without these two genes were analysed by MLST. In addition, the collagen-binding properties as well as the abilities to adhere to and invade human umbilical vein endothelial cells (HUVEC) were also evaluated for all strains. Results: In the groups of cnm-positive and cbm-positive strains, all properties, including collagen binding, adhesion, and invasion were significantly greater than those of the cnm–cbm-negative group. Moreover, MLST revealed three clonal complexes of S. mutans possessing the cbm gene. These three clones showed no close relatedness with clones of strains containing the cnm gene. Among three clones harbouring the cbm gene, two clones belong to serotype k, and appeared to be associated with the pathogenesis of IE due to their strong collagen binding and relatively enhanced abilities to adhere to and invade endothelial cells. However, such properties were relatively weak in the other non-serotype k clone possessing the cbm gene. Conclusions: MLST indicated a difference in evolution between S. mutans strains with the cbm gene and those with the cnm gene. In addition, this technique also suggested the importance of cbm-positive S. mutans clones relative to the pathogenesis of IE. [Copyright &y& Elsevier]

Streptococcus mutans , a Gram-positive facultative anaerobic bacterium, is a major pathogen of dental caries. The protein Cnm of S. mutans is involved in collagen binding, but its other biological functions are unknown. In this study, a Cnm-deficient isogenic mutant and a complementation strain were generated from a Cnm-positive S. mutans strain to help determine the properties of Cnm. Initially, comparison of the cell surface structure was performed by electron microscopy, which demonstrated that Cnm appears to be localized on the cell surface and associated with a protruding cell surface structure. Deep RNA sequencing of the strains revealed that the defect in Cnm caused upregulated expression of many genes related to ABC transporters and cell-surface proteins, while a few genes were downregulated. The amount of biofilm formed by the Cnm-defective strain increased compared with the parental and complemented strains, but the biofilm structure was thinner because of elevated expression of genes encoding glucan synthesis enzymes, leading to increased production of extracellular polysaccharides. Particular antibiotics, including bacitracin and chloramphenicol, had a lower minimum inhibitory concentration for the Cnm-defective strain than particular antibiotics, including bacitracin and chloramphenicol, compared with the parental and complemented strains. Our results suggest that S. mutans Cnm is located on the cell surface, gives rise to the observed protruding cell surface, and is associated with several biological properties related to membrane permeability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Naka, Matsuoka, Goto, Misaki, Nagasawa, Ito, Nomura, Nakano and Matsumoto-Nakano.)

Streptococcus mutans, a major pathogen of dental caries, is occasionally isolated from the blood of patients with infective endocarditis. Bacterial attachment of exposed collagen tissue in the impaired endothelium is an important step in the onset of infective endocarditis. In our previous studies, some S. mutans strains were shown to possess collagen-binding activities and most of them had an approximately 120-kDa cell-surface collagen-binding protein called Cnm. However, several strains without Cnm proteins show collagen-binding properties. In the present study, another collagen-binding protein, Cbm, was characterized and its coding gene cbm was sequenced in these strains. The amino acid alignment in the putative collagen-binding domain of Cbm was shown to have approximately 80% identity and 90% similarity to the comparable region of Cnm. Cbm-deficient isogenic mutant strains constructed by insertional inactivation of the cbm gene, lacked collagen-binding properties, which were recovered in the complemented mutant. Analyses of a large number of clinical isolates from Japan, Thailand and Finland revealed that cbm-positive strains were present in all of these countries and that cnm-positive and cbm-positive strains were detected in the oral cavity of approximately 10 and 2% of systemically healthy subjects, respectively. In addition, cnm-positive strains were predominantly identified in the serotype f group, whereas cbm-positive strains were frequently detected in serotype k. These results suggest that Cbm as well as Cnm are major cell surface proteins of S. mutans associated with binding to type I collagen and predominantly identified in serotype k strains. [ABSTRACT FROM AUTHOR]

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase ( bsh) and collagen-binding protein ( cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles. [ABSTRACT FROM AUTHOR]

Abstract: HPLC–MS analysis of tryptic protein digests in combination with fluorescence detection is presented as a convenient and quantitative method to gain insight into the relative reactivity of lysine side chains. In this scheme (tandem) mass spectrometry was used for identification of the modified residue, whereas fluorescence detection allowed determination of their relative abundance. Our method identified ‘labeling hot-spots’ at two flexible parts of the collagen-binding protein CNA35, positions that were consistent with all available structural and biochemical data on the collagen-binding properties of CNA35. [Copyright &y& Elsevier]

Abstract: Collagen-binding protein (CNA) is the major Staphylococcus aureus adhesin responsible for high affinity binding to collagen and is assumed to be a major virulence factor in infection and disease. Mutants lacking the cna gene are less virulent than the parent strain in models of septic arthritis, osteomyelitis, keratinitis, and endocarditis. In order to investigate the immunological and protective properties of a CNA-based DNA vaccine, a eukaryotic expression vector pCNA was constructed which expressed the collagen-binding domain of this adhesin in transfected cells. Three groups of 11 Balb/c mice received three injection of either pCNA, the empty expression vector (pCI) or PBS. Those injected with pCNA showed hi titre (64000) antibody and evidence of a cell-mediated immune response (CMI). The anti-CNA antibodies recognized the intact bacteria and prevented binding to collagen in vitro. However, the vaccination did not protect against bacterial challenge using the intra-peritoneal route of infection. Moreover, S. aureus that had been treated with sera from vaccinated mice caused a more severe infection than bacteria treated with sera from non-vaccinated mice. In summary, DNA vaccination against CNA produced a strong antibody and cellular response in mice but failed to protect from i.p. infection by S. aureus. [Copyright &y& Elsevier]

A cDNA expression library of Entamoeba histolytica was screened with antiserum to native amoebic collagen binding proteins (CBPs), and two clones C13 and C7 which partially encode for the 30kDa CBP were obtained. The sequenced clones were 90% homologous. C7 had a 69bp deletion at the 5′ end that is present in C13 and encodes for a Glu-Cys-Lys rich region and a four amino acids repeat (Glu-Lys-Glu-Cys). Purified fusion proteins from these cDNA clones were able to bind native type I collagen gels in a pH, calcium, ionic strength, and temperature dependent way. The binding of pgtC13 to collagen gel was time and temperature stable, while pgtC7 binding was not, suggesting that the deleted region in C7 is important for the binding. The clones reported here partially encode a 30kDa CBP that also belong to an antioxidant molecule family. We demonstrated that the fusion protein pgtC13 is immunogenic and partially protective as a subunit vaccine in the hamster model of amoebic liver abscess. [Copyright &y& Elsevier]

Presents a letter to the editor commenting on resonance assignments of the vonWillebrand factor, plays an essential role in platelet adhesion, A3 domain.