Application of <i>In Vitro</i> Mutagenesis to Identify the Gene Responsible for Cold Agglutination Phenotype of <i>Streptococcus mutans</i>

Satō, Yutaka; Okamoto, Kazuko; Kagami, Akiyoshi; Yamamoto, Yoh; Ohta, Kaoru; Igarashi, Takeshi; Kizaki, Harutoshi

doi:10.1111/j.1348-0421.2004.tb03535.x

Cited by 13 publications

(14 citation statements)

References 25 publications

(18 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This pattern of glycosylation was predicted for all Cnm variants available in the sequence repository, although the number of TTTEAP repeats may vary between strains (data not shown). In addition, we and others showed that Cnm migrates at ϳ120 kDa on SDS-PAGE despite a predicted molecular mass of 51 kDa (15,33). Collectively, these observations suggest that Cnm undergoes O-linked glycosylation and that sugar side chains attached to Cnm by SM.2067 may be responsible, at least in part, for the unexpected migration pattern.…”

Section: Resultsmentioning

confidence: 48%

“…Cnm is composed of an N-terminal conserved collagen-binding domain (A domain), a unique threoninerich B-repeat domain (B domain), and a C-terminal sortase signal LPXTG motif for cell wall anchoring (14). Initially, Cnm was identified as the factor responsible for the cold agglutination phenotype with a predicted molecular mass of 54 kDa (33). However, Cnm has been shown to migrate at ϳ120 kDa in SDS-PAGE, and such a discrepancy in size was, until now, poorly understood (15,33).…”

Section: Discussionmentioning

confidence: 99%

“…Initially, Cnm was identified as the factor responsible for the cold agglutination phenotype with a predicted molecular mass of 54 kDa (33). However, Cnm has been shown to migrate at ϳ120 kDa in SDS-PAGE, and such a discrepancy in size was, until now, poorly understood (15,33).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence

et al. 2014

View full text Add to dashboard Cite

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 ⌬pgfS 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 ⌬pgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ⌬pgfS 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 ⌬pgfS 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.

show abstract

Section: Resultsmentioning

confidence: 48%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Some S. mutans surface structures, such as the P1 protein (also known as antigen I/II or SpaP), the wall-anchored protein A (WapA), the biofilm regulatory protein A (BrpA), the autolysin AtlA, the glucosyltransferases (GtfB, GtfC, and GtfD), and the serotype-specific RGP, have been implicated in the pathogenesis of IE by promoting adherence to endothelial tissues and triggering inflammatory responses (17,55,61). More recently, a new surface protein with collagen-and laminin-binding activity, Cnm, which has an uneven distribution among the different serotypes of S. mutans, was identified (47,51,52). Interestingly, Cnm is found frequently in strains belonging to the uncommon serotype f but rarely in serotype c strains.…”

mentioning

confidence: 99%

The Collagen-Binding Protein Cnm Is Required for Streptococcus mutans Adherence to and Intracellular Invasion of Human Coronary Artery Endothelial Cells

et al. 2011

View full text Add to dashboard Cite

Streptococcus mutans is considered the primary etiologic agent of dental caries, a global health problem that affects 60 to 90% of the population, and a leading causative agent of infective endocarditis. It can be divided into four different serotypes (c, e, f, and k), with serotype c strains being the most common in the oral cavity. In this study, we demonstrate that in addition to OMZ175 and B14, three other strains (NCTC11060, LM7, and OM50E) of the less prevalent serotypes e and f are able to invade primary human coronary artery endothelial cells (HCAEC). Invasive strains were also significantly more virulent than noninvasive strains in the Galleria mellonella (greater wax worm) model of systemic disease. Interestingly, the invasive strains carried an additional gene, cnm, which was previously shown to bind to collagen and laminin in vitro. Inactivation of cnm rendered the organisms unable to invade HCAEC and attenuated their virulence in G. mellonella. Notably, the cnm knockout strains did not adhere to HCAEC as efficiently as the parental strains did, indicating that the loss of the invasion phenotype observed for the mutants was linked to an adhesion defect. Comparisons of the invasive strains and their respective cnm mutants did not support a correlation between biofilm formation and invasion. Thus, Cnm is required for S. mutans invasion of endothelial cells and possibly represents an important virulence factor of S. mutans that may contribute to cardiovascular infections and pathologies.

show abstract

“…The S. mutans strains used were Z1 (6), which is serotype f , gbpC -negative, and cnm -positive strain previously isolated from a saliva sample at Tokyo Dental College (6, 11), MT8148 (12), and UA159 (13). The streptococci were maintained on Todd-Hewitt (TH) broth/agar plates.…”

Section: Methodsmentioning

confidence: 99%

A mechanism for extremely weak SpaP-expression in Streptococcus mutans strain Z1

Satō

Okamoto-Shibayama

Azuma

2011

Journal of Oral Microbiology

Self Cite

View full text Add to dashboard Cite

Background Streptococcus mutans surface-protein antigen (SpaP, PAc, or antigen I/II) has been well known to play an important role in initial attachment to tooth surfaces. However, strains with weak SpaP-expression were recently reported to be found in natural populations of S. mutans. The S. mutans gbpC-negative strain Z1, which we previously isolated from saliva and plaque samples, apparently expresses relatively low levels of SpaP protein compared to S. mutans strains MT8148 or UA159.ObjectiveTo elucidate the mechanism for weak SpaP-expression in this strain, the spaP gene region in strain Z1 was amplified by polymerase chain reaction (PCR) and analyzed.MethodsAllelic exchange mutants between strains Z1 and UA159 involving the spaP gene region were constructed. The SpaP protein expressed in the mutants was detected with Coomasie Brilliant Blue (CBB)-staining and Western blot analysis following SDS-PAGE.ResultsThe 4689 bp spaP gene coding sequence for Z1 appeared to be intact. In contrast, a 20 bp nucleotide sequence appeared to be deleted from the region immediately upstream from the Z1 spaP gene when compared to the same region in UA159. The 216 bp and 237 bp intergenic fragments upstream from the spaP gene, respectively, from Z1 and UA159 were isolated, modified, and transformed into the other strain by allelic replacement. The resultant UA159-promoter region-mutant exhibited extremely weak SpaP-expression similar to that of strain Z1 and the Z1 complemented mutant expressed Spa protein levels like that of strain UA159.ConclusionThese results suggest that weak SpaP-expression in strain Z1 resulted from a 20 bp-deletion in the spaP gene promoter region.

show abstract

Application of In Vitro Mutagenesis to Identify the Gene Responsible for Cold Agglutination Phenotype of Streptococcus mutans

Cited by 13 publications

References 25 publications

Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence

Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence

The Collagen-Binding Protein Cnm Is Required for Streptococcus mutans Adherence to and Intracellular Invasion of Human Coronary Artery Endothelial Cells

A mechanism for extremely weak SpaP-expression in Streptococcus mutans strain Z1

Contact Info

Product

Resources

About