The unwillingness of some researchers to share bacterial strains and reagents.
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Bacteria↗
Biomedical subjects
H Kuramitsu
Publications and source records attributed to H Kuramitsu.
Expression and role of superoxide dismutases (SOD) in pathogenic bacteria.
This review will be limited to the expression and roles of the family of metalloenzymes superoxide dismutases in pathogenic bacteria. Only animal pathogens will be described, with particular emphasis on those causing disease in man.
Animals↗
Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells.
Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a "zipping" mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.
Bacterial Adhesion↗
Development of shuttle vectors for spirochetes.
Constructions of Escherichia coli-spirochete shuttle vectors are based on naturally occurring plasmids, broad host range plasmids or bacteriophages. This review primarily focuses on genetic tools for Treponema denticola which is associated with periodontal diseases. The T. pallidum FlaA protein, E. coli beta-galactosidase, and the green fluorescent protein were successfully expressed in T. denticola from a shuttle vector system.
Flagellin↗
Characterization of a novel methyl-accepting chemotaxis gene, dmcB, from the oral spirochete Treponema denticola.
Immediately downstream from the previously isolated Treponema denticola ATCC 35405 prtB gene coding for a chymotrypsinlike protease activity, an open reading frame, ORF3, was identified which shared significant homology with the highly conserved domains (HCDs) of bacterial methyl-accepting chemotaxis proteins (MCPs). Nucleotide sequencing of this ORF revealed that the gene would code for a protein with a size of approximately 41 kDa. In addition, this sequence contained a domain which was virtually identical to the HCD of a recently characterized MCP, DmcA, of strain 35405. Therefore, this ORF was named dmcB. Northern blot analysis suggested that dmcB was part of an operon structure containing prtB. Insertional inactivation of dmcB utilizing an ermF-ermAM cassette resulted in a mutant with decreased chemoattraction toward nutrient supplements. In addition, the mutant displayed an altered pattern of methylated proteins under conditions of chemotaxis. Inactivation of the dmcB gene also attenuated the methylation of the DmcA protein. These results suggest that the dmcB gene codes for an MCP in T. denticola which may interact with other MCPs in these organisms.
Amino Acid Sequence↗
Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum flaA gene.
The present communication describes the construction of a new Escherichia coli-Treponema denticola shuttle vector based on the naturally occurring spirochete plasmid pTS1 and the expression of the heterologous T. pallidum flaA gene from the plasmid in T. denticola. This new shuttle vector system should prove useful in characterizing virulence factors from unculturable pathogenic spirochetes.
Flagellin↗
The sgp gene modulates stress responses of Streptococcus mutans: utilization of an antisense RNA strategy to investigate essential gene functions.
The sgp gene from Streptococcus mutans has been previously isolated, characterized, and demonstrated to encode a G-protein. In order to investigate the function of this gene, a novel antisense RNA strategy was developed. Expression of sgp antisense RNA in Escherichia coli led to transient inhibition of growth. In addition, sgp antisense RNA expression in S. mutans resulted in decreased growth under environmental stress conditions (44 degrees C, acidic pH, and high osmolarity). Therefore, these results suggest that the sgp gene plays a role in modulating the stress responses of S. mutans. This approach could be applicable for investigating the function of essential genes in other organisms for which mutants are not available.
Bacterial Proteins↗
Plasmid maintenance renders bacteria more susceptible to heat stress.
The presence of shuttle plasmid pTS749 in the oral bacterium Streptococcus mutans resulted in growth inhibition under heat stress conditions. This effect was dependent upon the growth stage of the inoculum used to initiate growth. Likewise, the introduction of plasmids containing distinct ori sequences into Escherichia coli also resulted in growth inhibition at elevated temperatures and could be correlated with the plasmid levels within the cells. These results suggest that these plasmids in bacteria render the cells to be more susceptible to heat stress conditions.
Culture Media↗
Role of Arg-gingipain A in virulence of Porphyromonas gingivalis.
In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37 degrees C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism.
Adhesins, Bacterial↗
Regulation of sucrose-6-phosphate hydrolase activity in Streptococcus mutans: characterization of the scrR gene.
Previous results have implicated an important role for the enzyme IIScr, the sucrose-specific permease, in the transport of sucrose by cariogenic Streptococcus mutans. The product of the scrB gene, sucrose-6-phosphate hydrolase (Suc-6PH), is required for the metabolism of phosphorylated sucrose. The results from the utilization of scrB::lacZ fusions in S. mutans GS-5 have suggested that sucrose-grown cells have higher levels of scrB gene expression than do cells grown with glucose or fructose. Northern blot analysis of scrB transcripts has also confirmed the relative strengths of expression as sucrose>glucose>fructose. Immediately downstream from the scrB gene, an open reading frame with homology to regulatory proteins of the GalR-LacI family as well as to ScrR proteins from several other bacteria has been identified. In addition, this gene appears to be transcribed in the same operon as scrB. Inactivation of this gene, scrR, did not alter the relative expression of the scrB gene in the presence of sucrose or fructose but did increase SUC-6PH levels in the presence of glucose to that observed with sucrose. Furthermore, the S. mutans ScrR homolog appears to bind to the scrB promoter region as determined from the results of gel shift assays. These results suggest that the scrR gene is involved in the regulation of scrB, and likely scrA, expression. However, it is not clear whether sucrose acts as an inducer of expression of these genes or, alternatively, whether glucose and fructose act as repressors.
Amino Acid Sequence↗
The role of the Streptococcus mutans glucosyltransferases in the sucrose-dependent attachment to smooth surfaces: essential role of the GtfC enzyme.
Previous results have indicated that the glucosyltransferase activities of mutans streptococci are required for sucrose-dependent colonization of tooth surfaces. We have constructed mutants of Streptococcus mutans GS5 that are altered in varying combinations of the three gtf genes present in this organism. A quantitative in vitro sucrose-dependent attachment system was used to demonstrate that the inactivation of the gtfC gene drastically reduced adherence to smooth surfaces. By contrast, inactivation of the gtfB gene resulted in a smaller, but significant, reduction in attachment while the gtfD mutant was only marginally affected. Furthermore, production of only the glucosyltransferase C enzyme allowed for attachment although at reduced levels compared to the wild-type organism. The results from reintroduction of single copies of each of the gtf genes into a mutant of strain GS5 lacking glucosyltransferase activity also demonstrated the crucial role of the glucosyltransferase C enzyme in colonization. These results suggest a unique role for the glucosyltransferase C enzyme in the sucrose-dependent colonization of tooth surfaces by S. mutans strains.
Bacterial Adhesion↗
Multiple colonization defects in a cysteine protease mutant of Porphyromonas gingivalis.
A cysteine protease mutant, G-102, of Porphyromonas gingivalis 381 defective in the rgp-1 gene has been recently constructed in this laboratory. In order to evaluate the role of the protease in the virulence properties of P. gingivalis, a number of putative periodontopathic properties of the mutant were evaluated. Relative to the parental strain, mutant G-102 was demonstrated to be defective in interacting with Gram-positive bacteria as well as cultured epithelial cells. In addition, the mutant was altered in attaching to the protein components of extracellular matrix as well as to type I collagen. Some of these alterations could result from the decreased autoaggregation displayed by mutant G-102 relative to strain 381. However, since the epithelial cell attachment assays were carried out at very low bacterial densities, it is unlikely that reduced autoaggregation of the mutant is responsible for its decreased ability to attach to these eucaryotic cells. Electron microscopic examination of the cells also revealed that mutant G-102 was altered in normal fimbrae expression. In addition, reduced expression of the 43 kDa fimbrial subunit in the mutant was detected with both Western and Northern blotting. These results indicated that the rgp-1 gene product can play either a direct or indirect role in the colonization properties of P. gingivalis.
Adhesins, Bacterial↗
Characterization of a methyl-accepting chemotaxis protein gene, dmcA, from the oral spirochete Treponema denticola.
A gene, dmcA, expressing a methyl-accepting chemotaxis protein (MCP) from the oral spirochete Treponema denticola has been characterized. The gene was initially identified as an open reading frame immediately upstream from the previously characterized prtB protease gene of strain ATCC 35405. Nucleotide sequencing of the dmcA gene revealed a potential 57-kDa protein product with extensive homology with the signaling regions of MCPs from a variety of bacteria. The protein expressed in Escherichia coli cross-reacted with anti-Trg (E. coli MCP) serum, confirming its homology with MCPs. Northern blot and primer extension analyses identified the transcription start site of the monocistronic dmcA mRNA. By utilizing a T. denticola gene inactivation system recently developed in this laboratory, a mutant defective in the dmcA gene, HL0501, was constructed. The mutant was demonstrated to be defective in chemotaxis toward nutrients. In addition, the methylation profiles of cellular proteins indicated altered MCPs in the mutant relative to those of the parental strain. These results indicate that we have identified an MCP gene in the oral spirochete which plays a significant role in the chemotactic response of the organism.
Amino Acid Sequence↗
Relationship of Treponema denticola periplasmic flagella to irregular cell morphology.
Treponema denticola is an anaerobic, motile, oral spirochete associated with periodontal disease. We found that the periplasmic flagella (PFs), which are located between the outer membrane sheath and cell cylinder, influence its morphology in a unique manner. In addition, the protein composition of the PFs was found to be quite complex and similar to those of other spirochetes. Dark-field microscopy revealed that most wild-type cells had an irregular twisted morphology, with both planar and helical regions, and a minority of cells had a regular right-handed helical shape. High-voltage electron microscopy indicated that the PFs, especially in those regions of the cell which were planar, wrapped around the cell body axis in a right-handed sense. In those regions of the cell which were helical or irregular, the PFs tended to lie along the cell axis. The PFs caused the cell to form the irregular shape, as two nonmotile, PF-deficient mutants (JR1 and HL51) were no longer irregular but were right-handed helices. JR1 was isolated as a spontaneously occurring nonmotile mutant, and HL51 was isolated as a site-directed mutant in the flagellar hook gene flgE. Consistent with these results is the finding that wild-type cells with their outer membrane sheath removed were also right-handed helices similar in shape to JR1 and HL51. Purified PFs were analyzed by two-dimensional gel electrophoresis, and several protein species were identified. Western blot analysis using antisera to Treponema pallidum PF proteins along with N-terminal amino acid sequence analysis indicated T. denticola PFs are composed of one class A sheath protein of 38 kDa (FlaA) and three class B proteins of 35 kDa (FlaB1 and FlaB2) and one of 34 kDa (FlaB3). The N-terminal amino acid sequences of the FlaA and FlaB proteins of T. denticola were most similar to those of T. pallidum and Treponema phagedenis. Because these proteins were present in markedly reduced amounts or were absent in HL51, PF synthesis is likely to be regulated in a hierarchy similar to that found for flagellar. synthesis in other bacteria.
Amino Acid Sequence↗
Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis.
An hemR (hemin-regulated) gene from Porphyromonas gingivalis ATCC 53977 has been isolated and characterized. This gene is present downstream from the prtT gene, previously cloned in this laboratory. In addition, another putative gene, ORF1, was identified between hemR and prtT. The complete nucleotide sequences of ORF1 and hemR were determined, and the deduced amino acid sequence of ORF1 and HemR proteins corresponded to 16- and 48-kDa proteins, respectively. The amino termini of the HemR protein exhibited significant homology with iron-regulated, TonB-dependent outer membrane receptor proteins from various bacteria, while the carboxyl terminus of the HemR protein displayed almost complete identity with a P. gingivalis PrtT protease domain. PCR analyses confirmed the existence of such extensive homology between the carboxyl termini of both the prtT and hemR genes on the P. gingivalis chromosome. Northern blots indicated that ORF1 was part of a 1.0-kb mRNA and was positively regulated by hemin levels. On the other hand, the hemR gene was apparently a part of a 3.0-kb polycistronic message and was negatively regulated at the transcriptional level by hemin. Primer extension analysis of the hemR gene revealed that the transcription start site was at a C residue located within ORF1. An examination of HemR::lacZ constructs in both Escherichia coli and P. gingivalis confirmed hemin repression of hemR expression in both organisms. Moreover, the HemR protein expressed in E. coli was detected by an antiserum from a periodontitis patient heavily colonized with P. gingivalis but not by serum from a periodontally healthy patient or by antisera against hemin-grown P. gingivalis cells. Therefore, it is likely that the 48-kDa HemR protein can be expressed only under hemin-restricted conditions. These results suggest that we have isolated a hemin-regulated gene, hemR, which encodes a 48-kDa protein that may be a TonB-dependent outer membrane protein.
Amino Acid Sequence↗
Gene inactivation in the oral spirochete Treponema denticola: construction of an flgE mutant.
Treponema denticola is implicated in the etiology of periodontal diseases. We now report the construction of a specific flgE mutant of T. denticola ATCC 35405 following electroporation utilizing an erythromycin resistance cassette inserted into an flgE DNA fragment. The resulting mutant displays no visible motility and lacks periplasmic flagella as would be predicted from inactivation of the gene for the flagellar hook protein.
Bacterial Proteins↗
Cariogenicity of Streptococcus mutans strains with defects in fructan metabolism assessed in a program-fed specific-pathogen-free rat model.
To define the role of dental plaque fructans and the enzymes involved in their metabolism in the initiation and progression of dental caries, we constructed otherwise-isogenic mutants of Streptococcus mutans defective in the ability to synthesize fructans, to degrade fructans, or to do both. The cariogenic potential of these organisms was evaluated in a specific-pathogen-free rat model in which the feeding patterns of the animals were controlled by means of a König-Hofer programed feeder. Specifically, rats were infected with wild-type S. mutans UA159 or derivatives of this strain which contained an insertionally-inactivated fructanase (fruA) gene, fructosyltransferase (ftf) gene, or which had both genes inactivated. The animals were fed 17 meals per day of Diet 2000 containing 56% sucrose at 70-minute intervals for five weeks, and caries experience was evaluated. Animals infected with S. mutans with a mutated fruA gene only had statistically significant decreases in sulcal caries severity. Such a decrease was not observed in previous studies with ad libitum-fed animals (Wexler et al., 1992). The manifestation of diminished virulence in the programmed feeding model, but not in ad libitum-fed animals, supports the concept that the primary contribution of FruA to virulence is through the utilization of fructans storage polysaccharides. Animals infected with strains carrying the ftf mutation or simultaneous mutations in ftf and fruA did not display decreased virulence, perhaps indicating that sucrose utilization pathways may compete for substrate in vivo, or that accumulation of fructans may affect the ecology or the physicochemical characteristics of dental plaque in such a way as to reduce its cariogenic potential. The results of this study also emphasize that the contribution of a particular virulence determinant to the caries process may be highly dependent on the experimental design, feeding regimen and diet, and the presence or absence of other enzymatic activities.
Animals↗
Simple and rapid method for isolation of RNA from gram-negative bacteria.
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Biotechnology↗