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F L Macrina

Publications and source records attributed to F L Macrina.

At least 37 records · Page 2Linked to original sources
PubMed1994

Genetic regulation of fructosyltransferase in Streptococcus mutans.

Streptococcus mutans possesses several extracellular sucrose-metabolizing enzymes which have been implicated as important virulence factors in dental caries. This study was initiated to investigate the genetic regulation of one of these enzymes, the extracellular fructosyltransferase (Ftf). Fusions were constructed with the region upstream of the S. mutans GS5 Ftf gene (ftf) and a promoterless chloramphenicol acetyltransferase (CAT) gene. The fusions were integrated at a remote site in the chromosome, and transcriptional activity in response to the addition of various carbohydrates to the growth medium was measured. A significant increase in CAT activity was observed when glucose-grown cells were shifted to sucrose-containing medium. Sucrose-induced expression was repressed immediately upon addition of phosphoenolpyruvate phosphotransferase system sugars to the growth media. Deletion analysis of the ftf upstream region revealed that an inverted repeat structure was involved in the control of ftf expression in response to carbohydrate. However, the control of the level of ftf transcription appeared to involve a region distinct from that mediating carbohydrate regulation. CAT gene fusions also were constructed with the ftf upstream region from S. mutans V403, a fructan-hyperproducing strain which synthesizes increased levels of Ftf. Sequence analysis of the upstream ftf region in this strain revealed several nucleotide sequence changes which were associated with high-level ftf expression. Comparison of the GS5 and V403 ftf expression patterns suggested the presence of a trans-acting factor(s) involved in modulation of ftf expression in response to carbohydrate. This factor(s) was either absent or altered in V403, resulting in the inability of this organism to respond to the presence of carbohydrate. The sequences of the ftf regions from three additional fructan-hyperproducing strains were determined and compared with that of V403. Only one strain displayed nucleotide changes similar to those of V403. Two additional strains did not have these changes, suggesting that several mechanisms for up-regulation of ftf expression exist.

Base Sequence↗
PubMed1994

Genetic analysis of fructan-hyperproducing strains of Streptococcus mutans.

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries. It may serve as an extracellular storage polysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulation of bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of increased production of fructan have been discovered. By using eight independent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA were examined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared to correlate with the serotype of the organism and the geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produced by these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutants deficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strain which does not hyperproduce fructan. All of the fruA mutant strains were devoid of fructan hydrolase activity when levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability to hydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.

Animals↗
PubMed1993

Infectivity of a glucan synthesis-defective mutant of Streptococcus gordonii (Challis) in a rat endocarditis model.

Streptococcus gordonii, a member of the human indigenous oral microflora, colonizes smooth tooth surfaces and contributes to dental plaque formation. Although it is not recognized as being a cariogenic pathogen, it may cause endocarditis following invasion of the bloodstream. Using allelic exchange mutagenesis, we have constructed a mutant of S. gordonii (Challis) which is defective in its single functional glucosyltransferase gene and, hence, is unable to synthesize glucan exopolymers from sucrose. When examined in a rat endocarditis model, the sucrose-grown mutant did not differ significantly from S. gordonii wild-type, suggesting that glucan polymers did not contribute to infectivity. This result was in striking contrast to that previously observed with a polymer-defective S. mutans mutant.

Animals↗
PubMed1993

Graduate teaching in principles of scientific integrity.

Training in the responsible conduct of research in the biomedical sciences is on its way to being a standard component in the graduate biomedical curriculum. Emphasis in this area has been catalyzed by certain requirements for such training imposed by the National Institutes of Health and by an increased awareness of relevant issues created by the publicity and discussion of incidents of scientific misconduct. The authors describe the development and formalization of a course about the responsible conduct of science for students in the biomedical disciplines at Virginia Commonwealth University. In addition, they discuss general considerations of course content important in teaching scientific integrity; examine the use of published resource materials and documents from professional societies, institutions, states, and the federal government as teaching tools; and describe the importance of case studies as vehicles for teaching scientific integrity.

Biological Science Disciplines↗
PubMed1993

Sucrose-derived exopolysaccharides of Streptococcus mutans V403 contribute to infectivity in endocarditis.

We used an isogenic mutant of Streptococcus mutans V403, which differs from the wild-type V403 in genes involved in glucan and fructan production, to examine the importance of these exopolysaccharides as factors affecting infectivity in endocarditis. Rats inoculated with V403 developed endocarditis more frequently than animals inoculated with the mutant strain which produced neither glucan nor fructan (58% versus 12%, P < 0.01). In phagocytosis assays, both strains were found to be associated with the human granulocytes but a greater number of live V403 than of mutant organisms could be recovered. Colony counts recovered from fibrin plates incubated with the mutant were lower than those incubated with V403. These experiments indicate that exopolysaccharides produced by Streptococcus mutans contribute to its infectivity in endocarditis.

Alleles↗
PubMed1992

Liposomes and conjugate vaccines for antigen delivery and induction of mucosal immune responses.

In this brief review, emphasis was placed on the effectiveness of liposomes as carriers/vehicles of soluble antigens and as adjuvants for use in oral vaccine development. Evidence was provided that oral administration of antigen in liposomes resulted in a mucosal response which was higher than that obtained when the oral vaccine consisted of antigen alone. Specific mucosal responses were enhanced by incorporating lipophilic MDP into the antigen/liposome vaccines. Antigens shown to be effective in inducing a protective mucosal response when given in an oral liposome vaccine were anti-idiotypic antibodies, purified S. mutans GTF, CHO and Ag I/II. Evidence is also provided that CTB may be an effective oral adjuvant when coupled to proteins or peptides by either chemical or genetic methods. Further studies, however, will be required to characterize the effectiveness and safety of CTB in conjugate vaccines for inducing specific mucosal responses and to develop practical means to prepare oral liposome vaccines for use in humans.

Animals↗
PubMed1991

Identification of a region that influences host range of the streptococcal conjugative plasmid pIP501.

pIP501 is a member of a group of conjugative plasmids that are self-transmissible to a wide variety of streptococci as well as to other gram-positive bacteria. Several pIP501 restriction fragment deletion derivatives have been isolated and characterized. In this paper we describe one such derivative (pVA1702) which was conjugally proficient but had a limited host range. The loss of host range ability was seen as decreased conjugal transfer from Enterococcus faecalis to Streptococcus sanguis and was coincident with the deletion of a 4.5-kb DNA fragment. Transformation of pVA1702 into S. sanguis also was dramatically reduced as compared to its progenitor, suggesting the 4.5-kb fragment encoded a factor(s) necessary for stable maintenance in this host but not in E. faecalis. These observations suggest that pIP501 employs specific mechanisms enabling its maintenance in certain gram-positive bacteria.

Chromosome Deletion↗
PubMed1991

Molecular survey of clindamycin and tetracycline resistance determinants in Bacteroides species.

We have examined 13 clinical isolates of the intestinal Bacteroides group using DNA probes representing Bacteroides macrolide-lincosamide-streptogramin B (MLS) (ermF) and tetracycline resistance (tetQ) determinants as well as an insertion sequence (IS4351) previously seen in association with erm genes. tetQ-hybridizing sequences were detected in 11 of 13 tetracycline-resistant clinical isolates. On the other hand, ermF-like sequences were detected in only three of eight clindamycin-resistant strains. One isolate displayed low-level, inducible resistance to clindamycin and was sensitive to erythromycin. This same isolate had IS4351-like sequences but was missing ermF-like sequences, in contrast to previous reports which demonstrated the common association of IS4351 and erm genes. Our results suggest the occurrence of unclassified MLS genes in the Bacteroides group and furthermore suggest that IS4351-like sequences are not always linked to Bacteroides ermF-like sequences. Finally, 4 of 13 isolates conjugally transferred tetracycline resistance or linked tetracycline-clindamycin (MLS) resistances, but this process did not involve plasmids.

Autoradiography↗
PubMed1991

Repeated DNA sequence involved in mutations affecting transport of sucrose into Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase system.

Mutants of Streptococcus mutans V403 defective in the intracellular sucrose-6-phosphate hydrolase (product of the scrB gene) are sensitive to sucrose because of the intracellular accumulation of the phosphorylated sugar. Using a scrB mutant prepared by allelic exchange, we have isolated and characterized a number of sucrose-resistant revertants. One such mutant was found to lack the ability to transport sucrose into the cell via the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS). Genetic analysis of this strain revealed this lesion to be linked to the scrB gene. This was corroborated by the physical demonstration of an insertion mutation very near scrB. Taken together with DNA sequence information (Y. Sato, F. Poy, G. R. Jacobson, and H. K. Kuramitsu, J. Bacteriol. 171:263-271, 1989), our results indicated that all of the mutations characterized were located in the adjoining scrA gene which encodes the membrane-associated, sugar-specific enzyme II (EIIsucrose) component of the sucrose PTS in S. mutans. Biochemically, such a genetic lesion disables the sucrose PTS and prevents sucrose from entering the cell by this system. In this paper, we detail the nature of two independent insertion mutations and conclude them to be the result of duplicative transposition events into the scrA gene. This region of the chromosome was amplified and purified in large quantities by using the polymerase chain reaction. Examination of the amplified DNA revealed that the two independent insertion mutations were composed of sequences that were indistinguishable by size and by restriction site endonuclease maps. Their insertion points in the scrA gene were approximately 200 bp apart. The amplified DNA fragment was also used as a probe to demonstrate the presence of five copies of this element on the S. mutans V403 chromosome. A second strain, S. mutans V310, also was found to carry similarly arranged, multiple copies of this sequence on its chromosome, suggesting a clonal origin of V403 and V310. The small size of this sequence, its presence in multiple copies on the V403 chromosome, and its ability to duplicate itself semiconservatively into remote sites argue compellingly that it is an insertion sequence element. One such insertion mutant, with a defective sucrose PTS, was tested for virulence in rats and was found to cause caries at levels similar to those of the wild-type strain.

Animals↗
PubMed1991

Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange.

Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.

Alleles↗
PubMed1990

Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein.

A synthetic peptide, encoding amino acid residues 345 to 359 of the glucosyltransferase B enzyme of Streptococcus mutans GS-5, was genetically fused to the N-terminal end of the B-subunit gene of cholera toxin. The protein was overexpressed in Escherichia coli and retained the antigenicity associated with cholera toxin B subunit (CTB) as well as that associated with glucosyltransferase B. The addition of 15 amino acids to the N-terminal end of CTB did not appear to affect the gross structure of the protein significantly. The chimeric protein monomers assembled into a functional oligomer which exhibited only minor conformational differences from native CTB as measured by circular dichroism. The chimera bound to GM1 ganglioside and thus retained the biological activity of CTB. These results demonstrate that genetic fusion of small peptides to the N terminus of CTB has only a minimal effect on the structure and function of the protein. Furthermore, the chimera was shown to be immunogenic when fed to mice. This work has important implications in the construction of CTB chimeras for use as oral vaccines.

Amino Acid Sequence↗
PubMed1990

Inhibition of Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide.

An antigenic 15-amino-acid peptide sequence (gtfB.1) from the glucosyltransferase B enzyme of the cariogenic bacterium Streptococcus mutans GS-5 was identified previously from the genetic fusion of this sequence to the B subunit of cholera toxin. The resulting chimeric protein was used to raise antiserum in rabbits. This antiserum was shown to recognize the native glucosyltransferase enzyme and to inhibit its activity. The antiserum inhibited the synthesis of water-soluble glucan by approximately 40% and the synthesis of water-insoluble glucan by greater than 90%. The antiserum was shown to partially inhibit fructosyltransferase activity as well. The ability of this antipeptide antiserum to inhibit several enzymes from S. mutans suggests that these enzymes share an epitope related to enzymatic activity.

Amino Acid Sequence↗
PubMed1990

Genetic approaches to the study of oral microflora: a review.

As the study of oral microorganisms intensified almost 2 decades ago, the application of genetic techniques resulted in important contributions to the understanding of this clinically and ecologically important group of bacteria. The isolation and characterization of mutants of cariogenic streptococci helped to focus attention on traits that were important in colonization and virulence. Such classic genetic approaches gave way to molecular genetic techniques, including recombinant DNA methodology in the late 1970s. Gene cloning systems and methods to move DNA into cells have been developed for oral streptococci. Many streptococcal genes thought to be important in colonization and virulence have since been cloned and their nucleotide sequence determined. Mutant strains have been constructed using defective copies of cloned genes in order to create specific genetic lesions on the bacterial chromosome. By testing such mutants in animal models, a picture of the cellular and molecular basis of dental caries is beginning to emerge. These modern genetic methodologies also are being employed to develop novel and efficacious cell-free or whole cell vaccines against this infection. Genetic approaches and analyses are now being used to dissect microorganisms important in periodontal disease as well. Such systems should be able to exploit advances made in genetically manipulating related anaerobes, such as the intestinal Bacteroides. Gene cloning techniques in oral anaerobes, Actinomyces and Actinobacillus, are already beginning to pay dividends in helping understand gene structure and expression. Additional effort is needed to develop facile systems for genetic manipulation of these important groups of microorganisms.

Bacteria↗
PubMed1989

Plasmid vectors for constructing translational fusions to the B subunit of cholera toxin.

A family of plasmid cloning vectors has been developed for creating translational fusions to the ctxB gene encoding the B subunit of cholera toxin (CTB) in Escherichia coli. These vectors permit insertion of transcriptionally and translationally competent gene sequences upstream from ctxB. To test the utility of the system, a portion of the glucosyltransferase B (GTF) gene (gtfB) from the cariogenic bacterium Streptococcus mutans GS-5 (Bratthall serotype c), encoding the N-terminal one-third of the protein, was inserted into each vector. E. coli lysates containing the constructs were partially purified by passage over a GM1 ganglioside affinity column. Western blotting analysis of the column retentate from one of the lysates revealed the presence of a novel 58-kDa protein which cross-reacted with antisera to GTF and CTB. These vectors are of general use for making other translational fusions to ctxB. The high binding affinity of CTB can be exploited in purifying large polypeptides fused to this relatively small protein. Moreover, these vectors can be used to create neoantigens with altered immunogenicity for use in polypeptide-based vaccines.

Cholera Toxin↗
PubMed1989

Biochemical characterization and evaluation of virulence of a fructosyltransferase-deficient mutant of Streptococcus mutans V403.

The Streptococcus mutans extracellular fructosyltransferase (FTF) enzyme may play a role in the formation of dental caries by synthesizing a fructan polymer that serves as an extracellular storage polysaccharide. We sought to determine if an FTF-deficient strain of S. mutans was less virulent than wild-type cells in a rat animal model system. Cloned ftf gene sequences from S. mutans GS5 were used to generate a defective copy of the ftf gene by inserting into the ftf coding region a DNA fragment which encoded erythromycin resistance. The plasmid which carried the defective ftf construct was introduced into S. mutans V403 by using genetic transformation. This defective construct replaced, by allelic exchange, the wild-type copy of the ftf gene carried on the V403 chromosome. FTF activity assays indicated that the recombinant strain, V1741, was deficient in fructan synthesis. However, extracellular protein preparations from this strain displayed an increased ability to generate glucose polymers (glucans) compared with V403 preparations. Levels of adherence to glass and rat tooth surfaces by strain V1741 were similar to those of the V403 strain. Both strains caused moderate decay on rat tooth surfaces; however, the FTF-deficient strain was less pathogenic compared with the wild-type strain. These results suggest that FTF activity contributes to the pathogenicity of S. mutans V403, possibly by generating extracellular fructans which serve as storage compounds.

Animals↗
PubMed1989

Genetic analysis of the conjugal transfer determinants encoded by the streptococcal broad-host-range plasmid pIP501.

pIP501 is a 30.2-kilobase (kb) broad-host-range conjugative streptococcal plasmid which encodes chloramphenicol and erythromycin resistance. A smaller conjugation-proficient derivative of pIP501 has been constructed and designated pVA1702. pVA1702 is 25.2 kb, encodes only kanamycin resistance, and confers conjugative ability in an Enterococcus faecalis mating system. The temperature-sensitive transposon delivery vectors pTV1ts and pTV32 carrying Tn917 and Tn917lac, respectively, were used to create insertion mutants of pVA1702 in E. faecalis. These insertions enabled us to localize the determinants conferring conjugation to two separate regions of pVA1702: the 7.5-kb region A and the 8.8-kb region B. A nested set of deletions of the 7.5-kb region was subcloned in Escherichia coli, and the expression of these deletions was examined in an E. coli minicell system. The genes for three conjugal proteins have been located and designated cnjA, cnjB, and cnjC (for conjugation). Two other peptides that are transcribed in a divergent direction from the cnj genes were encoded in the A region, but it is not known whether they are involved in conjugation. These studies indicate that the conjugation genes of pIP501 are arranged in multiple transcriptional units.

Chromosome Deletion↗