Molecular and genetic analysis of multiple changes in the levels of production of virulence factors in a subcultured variant of Streptococcus mutans (original) (raw)
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The Biology of Streptococcus mutans
Microbiology spectrum, 2019
Gram-positive pathogens. In this chapter, we highlight some of the key studies that have led to our current understanding of S. mutans genetics, physiology and virulence. For a historical perspective and complete survey of the field, we direct the reader to the following review articles (2, 3, 6-9). Genetic ans phenotypic heterogeneity The first S. mutans genome sequenced (serotype c UA159 strain) was found to contain ~ 2.0 Mb of DNA and to encode approximately 2,000 genes (5). As the cost of Next Generation Sequencing (NGS) technologies has gone down, genomes from dozens of S. mutans strains have been sequenced, assembled, and are now available on public databases such as the NCBI (www.ncbi.nlm.nih.gov/assembly/?term=streptococcus+mutans). This influx of genome sequences has led to an increase in comparative genomic studies focused on S. mutans (10-13). One of the first such studies was based on the shotgun genome sequence from 57 geographically diverse S. mutans clinical isolates. This study concluded that the S. mutans pangenome contains a minimium of ~3,300 possible genes, and has a core genome (genes that are common to all strains) of 1,490 genes (14). This means that in any one S. mutans isolate, ~500 genes could be distinct from any other strain, perhaps significantly influencing virulence potential or fitness. The same group, using population demographic analysis based on single nucleotide polymorphisms of the core genes, determined that a large expansion took place in the S. mutans population between 3,000 and 10,000 years ago, which coincided with the advent of human agriculture and increased consumption of carbohydrates in the human host diet (14). This study also identified 73 unique core genes that are found only in S. mutans and not in its closest relatives, many of which are involved in carbohydrate metabolism and acid resistance (14). In a follow up study, Palmer et al. (15) characterized 15 of the most genetically diverse isolates of the 57 strains sequenced by Cornejo et al, and found great variation in the phenotypes directly related to virulence including the ability to form biofilm in the presence of sucrose and the ability to tolerate low pH and oxidative stresses, suggesting that not all strains of S. mutans are equally virulent and providing a rational explanation as to why attempts to correlate carriage of certain genotypes of S. mutans with incidence of dental caries has proven so difficult (10, 16-18). Work to characterize the unique core and non-core genes is ongoing and will likely lead to a greater understanding of the physiology and diversity of S. mutans as a species. For example one of the unique core hypothetical genes, SMu.1147, which encodes a small peptide, was found to regulate genetic competence and other traits of general importance to S. mutans virulence (19). In contrast, efforts to characterize the non-core genes indicate they likely provide a competitive advantage under particular circumstances. Such is the case for a galactose-specific PTS transporter found in several strains (20). Among the many non-core genes of S. mutans identified in recent years, those encoding the collagen-binding proteins (CBPs) Cnm and Cbm were shown to confer adhesion to collagen and laminin, invasion of endothelial and ephitelial cells, and virulence in the Galleria mellonella invertebrate model (21, 22) as well as in rabbit and rat models of infective endocarditis (IE) (23, 24). Epidemiological studies indicate that cnm is present in approximately 15% of S. mutans isolates, whereas cbm is rarely found (~ 2%) (21, 22). Genes encoding CBPs have an uneven distribution among the different serotypes, and are found at higher frequency among Lemos et al.
Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli
Journal of bacteriology, 1983
Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting pro...
Letters in Applied Microbiology, 2007
Aim-To assess potential function of each two-component signal transduction system in the expression of Streptococcus mutans virulence properties. Methods and Results-For each two-component system (TCS), the histidine kinase-encoding gene was inactivated by a polymerase chain reaction (PCR)-based deletion strategy and the effects of gene disruption on the cell's ability to form biofilms, become competent, and tolerate acid, osmotic, and oxidative stress conditions were tested. Our results demonstrated that none of the mutations were lethal for S. mutans. The TCS-2 (CiaRH) is involved in biofilm formation and tolerance to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal competence development. Conclusions-Our results confirmed the physiological role of the TCS in S. mutans cellular function, in particular the SncRK-like TCS and TCS-9 as they may represent new regulatory systems than can be involved in S. mutans pathogenesis. Significance and Impact of the Study-Multiple TCS govern important biological parameters of S. mutans enabling its survival and persistence in the biofilm community.
Journal of general microbiology, 1981
Five strains of Streptococcus mutans were grown in continuous culture with either a limited supply or an excess of glucose. Proteins secreted into the extracellular fluid by strains C67-1, 3209 and K1 rapidly catalysed the synthesis of insoluble glucan from sucrose (mutansucrase activity). The culture fluid from strains Ingbritt or C67-25 catalysed the synthesis of soluble glucan (dextransucrase activity) and fructan, but little or no mutansucrase activity was detected. The strains which secreted active mutansucrase readily colonized a smooth hard surface during growth in batch culture and were more cariogenic in pathogen-free rats than those which secreted little mutansucrase activity. There was no similar correlation between fructosyltransferase, dextransucrase or total glucosyltransferase activity and either adherence or cariogenicity. We conclude that the ability to catalyse insoluble glucan synthesis is a major determinant of the cariogenicity of S. mutans strains.
Virulence of a spaP Mutant of Streptococcus mutans in a Gnotobiotic Rat Model
Infection and Immunity, 1999
Streptococcus mutans, the principal etiologic agent of dental caries in humans, possesses a variety of virulence traits that enable it to establish itself in the oral cavity and initiate disease. A 185-kDa cell surface-localized protein known variously as antigen I/II, antigen B, PAc, and P1 has been postulated to be a virulence factor in S. mutans. We showed previously that P1 expression is necessary for in vitro adherence of S. mutans to salivary agglutinin-coated hydroxyapatite as well as for fluid-phase aggregation. Since adherence of the organism is a necessary first step toward colonization of the tooth surface, we sought to determine what effect deletion of the gene for P1, spaP, has on the colonization and subsequent cariogenicity of this organism in vivo. Germ-free Fischer rats fed a diet containing 5% sucrose were infected with either S. mutans NG8 or an NG8-derived spaP mutant strain, PC3370, which had been constructed by allelic exchange mutagenesis. At 1-week intervals for 6 weeks after infection, total organisms recovered from mandibles were enumerated. At week 6, caries lesions also were scored. A significantly lower number of enamel and dentinal carious lesions was observed for the mutant-infected rats, although there was no difference between parent and mutant in the number of organisms recovered from teeth through 6 weeks postinfection. Coinfection of animals with both parent and mutant strains resulted in an increasing predominance of the mutant strain being recovered over time, suggesting that P1 is not a necessary prerequisite for colonization. These data do, however, suggest a role for P1 in the virulence of S. mutans, as reflected by a decrease in the cariogenicity of bacteria lacking this surface protein.
Nucleotide sequence of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme
Journal of general microbiology, 1990
The nucleotide sequence of the Streptococcus mutans GS-5 gtfD gene coding for the glucosyltransferase which synthesizes water-soluble glucan (GTF-S) has been determined. The complete gene contains 4293 base pairs and the unprocessed protein is composed of 1430 amino acids with a molecular mass of 159814 Da. The amino terminus of the unprocessed protein resembles the signal sequences of other extracellular proteins secreted by S. mutans and that of the GTF-I secreted by Streptococcus downei. In addition, the GTF-S protein exhibits high amino acid similarity with the strain GS-5 enzymes responsible for insoluble glucan synthesis (GTF-I, GTF-SI) previously isolated and sequenced in this laboratory. These results indicate that all three gtf genes evolved from a common ancestral gene.
Journal of Clinical Microbiology, 2003
We recently identified the genes responsible for the serotype c-specific glucose side chain formation of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. These genes were located downstream from the rgpA through rgpF locus that is involved in the synthesis of RGP. In the present study, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The rgpA through rgpF homologs were well conserved among the three serotypes. By contrast, the regions downstream from the rgpF homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotypespecific glucose side chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes. S. mutans was isolated from 198 of 432 preschool children (3 to 4 years old). The serotypes of all but one S. mutans isolate were identified by serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occured rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection by multiple serotypes of S. mutans was significantly higher than that in the group with a monoinfection by a single serotype.
Molecular analyses of glucosyltransferase genes among strains of Streptococcus mutans
FEMS Microbiology Letters, 1998
Three glucosyltransferase (GTase) genes (gtfB, gtfC and gtfD) were cloned and sequenced from clinically isolated strains of Streptococcus mutans MT8148 (serotype c), MT4239 (c), MT4245 (e), MT4467 (e) and MT4251 (f), respectively. Comparison of the gtf genes revealed that interstrain difference of gtfB and gtfD was limited, while gtfC showed significant interstrain variations. Similar to gtfB and gtfD, gtfC possessed five direct repeats composed of homologous unit in the carboxyl-terminal portion. The repeating unit consisted of 63^65 amino acid residues and is responsible for glucan binding. The gtfC gene from S. mutans MT4245 lacked the fourth unit. Multiple alignment with the gtf sequence of strain GS-5 (c) revealed several changes in these gtf genes due to frameshift mutations. The peptides encoded by the gtfB, gtfC and gtfD genes of GS-5 were 1, 80, and 32 amino acid residues shorter than those of the test strains except strain MT4245. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
Cloning and inactivation of the gene responsible for a major surface antigen on Streptococcus mutans
Archives of Oral Biology, 1990
Antigen P1, also called I/TI, is one of the most abundant cell wall proteins of the mutans streptococci. It has been suggested that P1 may be involved in cell adherence to tooth surfaces and in sucrose-induced cell aggregation. As a first step toward fully understanding its biological functions, the P1 gene, which has been designated spaPI, from Streptococcus mutans NG5 (serotype c) has been cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. The recombinant strain expressing P1 carries a 5.2-kilobase DNA insert whose restriction map has been determined. This map is completely different from that of spaA of Streptococcus sobrinus (serotype g), even though P1 and SpaA are antigenically related. Southern hybridization revealed that DNA sequences closely homologous to spaPI were present in serotypes c, e, and f, and similar sequences also existed in strains of serotypes a and d. The expression of the cloned spaPI was found to be independent of the lac inducer and the orientation of the DNA insert, suggesting that it carries its own promoter. Western blotting (immunoblotting) revealed at least 20 bands reacting with a mixture of three anti-Pl monoclonal antibodies. The highest-molecular-weight reactive band was comparable in size to the parent P1 (185 kilodaltons [kDa]); however, the major reactive bands were smaller (-160 kDa). Expression of cloned P1 in E. coli LC137 (htpR lonR9) resulted in the increased prominence of the 185-kDa protein reactive band. Ouchterlony immunodiffusion showed partial identity between the parent and cloned P1. In E. coli, P1 was detected primarily in the periplasm and extracellular fluid.