Biomedical subjects

D W Dyer

Publications and source records attributed to D W Dyer.

At least 37 records · Page 2Linked to original sources

PubMed1994

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.

Alveolar Bone Loss↗

PubMed1993

Genetic structure of populations of Porphyromonas gingivalis associated with periodontitis and other oral infections.

One hundred isolates of the oral pathogenic bacterium Porphyromonas gingivalis were genetically characterized by determining the electrophoretic mobilities of 16 metabolic enzymes and the presence or absence of catalase activity. A total of 78 distinct electrophoretic types (ETs), representing multilocus genotypes, were identified, and cluster analysis placed them in three major phylogenetic divisions. Division I (71 ETs) included all 88 human isolates examined, most of which had been recovered from patients with periodontitis, together with 4 monkey isolates. The strains in division II (four ETs) and division III (three ETs) are strongly differentiated from those in division I and apparently represent two previously unclassified (cryptic) species. The mean genetic diversity per enzyme locus among the 92 isolates of division I (P. gingivalis, strict sense) was 0.321, and the strains were distributed among 14 phylogenetic clusters and single-ET lineages. The population structure is basically clonal, with some clonal genotypes being widespread, and even global, in distribution. There was no evidence of association between specific genetic lineages or clusters of ETs and the type of disease (periodontitis or root canal infections), invasive potential, serogroup, or fimbrial restriction fragment length polymorphism group. The finding that dental patients are infected by strains of a wide variety of chromosomal genotypes suggests that interstrain variation in pathogenicity is small. On the basis of the observed genetic structure of natural populations of P. gingivalis, we hypothesize that the role of this microorganism in the pathogenesis of periodontitis and other dental infections is largely opportunistic.

Alleles↗

PubMed1993

A siderophore production mutant of Bordetella bronchiseptica cannot use lactoferrin as an iron source.

Bordetella bronchiseptica secreted a hydroxamate siderophore when grown in Fe-depleted medium. A Tn5lac insertion mutant of B. bronchiseptica, DBB22, did not produce this hydroxamate siderophore and was incapable of using lactoferrin as an Fe source. Our data suggest that B. bronchiseptica uses a siderophore for removal of Fe from lactoferrin and transferrin rather than relying upon a receptor for these host Fe-binding proteins.

Animals↗

PubMed1993

Membrane protein expression by Actinobacillus actinomycetemcomitans in response to iron availability.

Iron-limited growth conditions under anaerobiosis were established for Actinobacillus actinomycetemcomitans strains Y4, JP2, and 75 by use of the ferrous ion chelator 2,2'-dipyridyl. Growth inhibition was reversible with both ferrous and ferric iron sources. Sarcosyl-insoluble membrane fractions of iron-stressed anaerobic A. actinomycetemcomitans cultures revealed a similar iron-repressible protein of approximately 70 kDa in A. actinomycetemcomitans strains Y4, JP2, and 75. This 70-kDa protein was recognized by serum from localized juvenile periodontitis patients and a periodontally healthy subject. This suggested that the 70-kDa iron-repressible protein may be expressed in vivo. When A. actinomycetemcomitans was grown under aerobic conditions, the ferric iron chelator, ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) was utilized for growth limitation. EDDA inhibition was reversible in strain Y4 with ferrous and ferric iron sources. An iron-repressible protein of approximately 70 kDa was also noted in iron-stressed aerobic cultures. The 70-kDa protein may be involved in iron transport by A. actinomycetemcomitans. Preliminary experiments were performed to examine potential iron transport systems for A. actinomycetemcomitans. Production of the two most common chemical types of siderophore was not detected in A. actinomycetemcomitans culture supernatants. Iron-starved A. actinomycetemcomitans cells did not bind transferrin or lactoferrin in a dot blot assay.

2,2'-Dipyridyl↗

PubMed1992

Conjugal transfer of plasmid and transposon DNA from Escherichia coli into Porphyromonas gingivalis.

Using the broad host-range vector R751 to provide transfer functions, plasmid pVAL-1 and transposon Tn4351 were conjugally mobilized from Escherichia coli into Porphyromonas gingivalis. Transfer frequencies for both elements varied between 10(-6) and 10(-11), depending upon the recipient. The behavior of pVAL-1 and Tn4351 in P. gingivalis was essentially as described previously in Bacteroides spp. These data indicate that plasmid and transposon DNA can be conjugally transferred into P. gingivalis and that these elements can be used to genetically manipulate the organism in examining putative virulence determinants that may participate in the induction or exacerbation of periodontal disease.

Autoradiography↗

PubMed1992

Siderophore production and membrane alterations by Bordetella pertussis in response to iron starvation.

Bordetella pertussis was grown in iron (Fe)-free defined medium to limit the growth of the organism. Doubling times of the Fe-starved organism increased by approximately 1 h, and a 40% reduction in the final extent of growth in Fe-depleted medium was observed. Under these conditions, a hydroxamate siderophore named bordetellin was secreted by B. pertussis. Lactoferrin and transferrin supported growth of B. pertussis even when the protein was sequestered inside dialysis tubing. This suggested that binding of lactoferrin and transferrin to B. pertussis was not essential and that bordetellin production plays a major role in Fe uptake. Solid-phase dot blot assays indicated weak binding of lactoferrin to the cell surface, consistent with previous reports of a lactoferrin receptor. Three new proteins of 97, 77, and 63 kDa were synthesized in response to Fe starvation. Fe-inducible proteins of 103, 72, 24, 21, and 18 kDa were also observed. The synthesis of lipopolysaccharide was also altered by Fe availability.

Blotting, Western↗

PubMed1992

A statistical approach to the ecology of Porphyromonas gingivalis.

Previous studies have suggested that infections with Porphyromonas gingivalis, associated with periodontal disease, may consist of one clonal type. It has also been shown that each individual patient carries a unique clonal type of P. gingivalis, as assessed by DNA fingerprinting. This issue was further examined by random collection of multiple isolates of P. gingivalis from multiple sites in several patients, and characterization of these isolates by DNA fingerprinting. Although most patients appeared to be infected exclusively by one clonal type of P. gingivalis, at least one patient was found to harbor two distinct clonal types. This indicates that the simultaneous presence of different clonal types of P. gingivalis can occur. A statistical method was developed for retrospective analysis of these data for estimation of whether the remainder of these patients were actually infected with single or multiple clonal types of P. gingivalis. With this statistical method, a confidence interval was calculated for estimation of the true proportion of a single observed clonal type in the potential population of P. gingivalis that might be recovered from an infected patient. Statistically, the sampling of small numbers of sites per patient or isolates per site leads to a wide confidence interval for the estimated true proportion of the observed clonal type in the infecting P. gingivalis population. For example, when five sites in an oral cavity yield indistinguishable P. gingivalis isolates, then the true proportion of this clonal type in the total P. gingivalis population in the infected oral cavity is estimated to be in the interval between 55% and 100% (at a 95% confidence level).(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Fingerprinting↗

PubMed1992

Restriction fragment length polymorphism analysis of the fimbrillin locus, fimA, of Porphyromonas gingivalis.

With hybridization probes derived from the fimbrial locus of Porphyromonas gingivalis strain 381, fimA381, restriction fragment length polymorphisms (RFLPs) were examined at the fimbrillin locus in 39 human and animal strains of this species. The 39 strains were subdivided into nine RFLP groups (I-IX) after genomic digests were probed with the internal coding sequence of the fimA381 gene. Thirty-three strains showed one or more AluI fragments of moderate-to-high homology (greater than or equal to 77%) with the internal coding sequence of fimA381. These strains were distributed into the first seven RFLP groups, based solely on the size of the major hybridizing AluI fragment. Five human strains (RFLP Group VIII) had only one AluI fragment that hybridized very poorly with this probe. One animal strain did not have homology at all (RFLP Group IX). When all AluI fragments that hybridized with fimA381 were analyzed, RFLP groups I-VIII were further differentiated into 25 distinct RFLP patterns. Hybridizations were also performed with the internal coding sequence of fimA381 to probe PstI genomic digests of selected strains that appeared to have lesser homology with fimA381. These hybridizations were performed to determine the level and location of the region of poor homology within the fimA genes of these strains. The results suggested that fimbrial coding sequences are more commonly conserved between these strains in the 5'-region of the fimA locus (greater than or equal to 92% sequence homology).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Human immunoglobulin G antibody response to iron-repressible and other membrane proteins of Porphyromonas (Bacteroides) gingivalis.

The human immunoglobulin G (IgG) immune response against Porphyromonas (Bacteroides) gingivalis A7A1-28 iron-repressible membrane proteins (IRMPs) and other membrane proteins was examined by immunoblot analysis. Thirty sera from patients with adult periodontitis and 30 sera from periodontally healthy subjects were included. Iron limitation of P. gingivalis was achieved by growing bacteria in brain heart infusion broth supplemented with protoporphyrin IX and 250 microM alpha, alpha'-dypyridyl, a ferrous iron chelator. Iron-sufficient growth was achieved by growing bacteria in the same medium without alpha, alpha'-dypyridyl. Human sera, in particular those from patients with periodontitis who exhibited high levels of IgG against whole cells of P. gingivalis A7A1-28 in serum in an enzyme-linked immunosorbent assay (ELISA), commonly reacted with five membrane proteins with apparent molecular masses of 80, 67.5, 51, 40.5, and 28 kDa and four IRMPs of 46, 43, 37.5, and 22 kDa. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with the 46-, 43-, and 37.5-kDa IRMPs, and 40% of these subjects expressed immunoreactivity against the 22-kDa IRMP. Sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects exhibited less immunoreactivity against IRMPs and the five membrane proteins of P. gingivalis. The present study indicates that P. gingivalis IRMPs are immunogenic and that these proteins are expressed in vivo.

Antibodies, Bacterial↗

PubMed1990

Effect of iron limitation on Bacteroides gingivalis.

This study was undertaken to describe the effects of iron limitation on Bacteroides gingivalis. Four strains of B. gingivalis were grown in brain heart infusion broth, substituting protoporphyrin IX for hemin. Culture with protoporphyrin IX resulted in a loss of a 28 kDa membrane protein, but no decrease in growth. Iron-restricted cultural conditions for the growth of B. gingivalis were achieved using alpha/alpha'-dipyridyl, a ferrous iron chelator, at concentrations from 12.5 microM to 300 microM. Total suppression of bacterial growth for strain A7A1-28 and strain 381 was achieved at 200 microM alpha/alpha'-dipyridyl. At 300 microM alpha/alpha'-dipyridyl, strain W50 and Bowden 18/10 showed 100% and 80% suppression of growth, respectively. The ferric iron chelator Desferal did not show suppression of growth in concentrations up to 500 microM. The dipyridyl inhibition of cell growth for strain A7A1-28 could be reversed by adding excess ferrous ammonium sulphate but not by ferric nitrate. Iron regulation of proteolytic enzymes could not be demonstrated. Two new membrane proteins 42 kDa and 24 kDa are expressed with iron limitation, and the 45 kDa membrane protein was decreased with iron limitation.

Bacteroides↗

PubMed1990

Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin.

Transferrin (TF) and lactoferrin (LF) are probably the major sources of iron (Fe) for Neisseria gonorrhoeae in vivo. We isolated mutants of N. gonorrhoeae FA19 that were unable to grow with Fe bound to either TF (TF-) or LF (LF-) or to both TF and LF ([TF LF]-). The amount of Fe internalized by each of the mutants was reduced to background levels from the relevant iron source(s). The wild-type parent strain exhibited saturable specific binding of TF and LF; receptor activity was induced by Fe starvation. The TF(-)-specific or LF(-)-specific mutants were almost completely lacking in receptor activity for TF or LF, respectively, whereas the [TF LF]- mutants bound both TF and LF as well as the wild-type strain. All mutants utilized citrate and heme normally as Fe sources. These results demonstrate that ability to bind TF or LF is essential for gonococci to scavenge appreciable amounts of Fe from these sources in vitro. In addition, the TF and LF Fe acquisition pathways are linked by the mutual use of a nonreceptor gene product that is essential to Fe scavenging from both of these sources; this gene product is not required for Fe acquisition from other sources.

Binding, Competitive↗

PubMed1988

Preferential uptake of restriction fragments from a gonococcal cryptic plasmid by competent Neisseria gonorrhoeae.

Factors involved in the specificity of DNA uptake by competent Neisseria gonorrhoeae were examined. Host-controlled modification did not affect uptake. Certain restriction fragments of the 4.2 kb gonococcal cryptic plasmid pFA1 and of the replicative form of the bacteriophage M13 were taken up in preference to others, independent of differences in fragment size. A 600 bp fragment from the 4.2 kb plasmid was cloned into pLES2, a gonococcal-Escherichia coli shuttle vector; the 600 bp fragment was taken up into a DNAase-I-resistant state in preference to the vector fragment. A second 370 bp fragment in pFA1 was also taken up preferentially. The 600 bp and 370 bp fragments share a 10 bp sequence, which is found in pFA1 only on fragments that were taken up readily. However, a fragment from M13 which was efficiently taken up did not contain this 10 bp sequence. In addition, this sequence was not sufficient to direct preferential DNA uptake by gonococci, since a recombinant plasmid containing this 10 bp sequence was not taken up appreciably better than the vector plasmid or another recombinant plasmid containing an unrelated 10 bp sequence. Sequence comparisons of the three restriction fragments which were preferentially taken up did not yield any consensus sequences greater than 7 bp. Although it is likely that efficient uptake of DNA by gonococci is determined by DNA structure, a single short sequence could not be found that accounted for specific uptake.

Base Sequence↗

PubMed1988

Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source.

Although Neisseria meningitidis does not produce siderophores, it is able to obtain iron from human transferrin. We observed saturable specific binding of 125I-labeled human transferrin to meningococci. Human lactoferrin and mouse transferrin did not compete with human transferrin for binding, whereas human apotransferrin and 100% iron-saturated transferrin competed equally well. Meningococci thus have a specific receptor for human transferrin. Scatchard analysis yielded a relatively low Kd of 0.7 microM and an apparent copy number of 2,900 receptors per CFU. Receptor activity was iron-regulated. A meningococcal transformant specifically unable to utilize transferrin as an iron source had decreased transferrin receptor activity. These data are consistent with the hypothesis that receptor-mediated binding of transferrin is a rate-limiting step in meningococcal iron uptake from transferrin.

Blotting, Western↗

PubMed1988

Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae.

The major iron (Fe) sources available to Neisseria gonorrhoeae in the human host are probably transferrin (TF) and lactoferrin (LF). Although a number of studies have examined Fe uptake by Neisseria meningitidis, no comparable studies have been done on Fe uptake by the gonococcus from TF and LF. We found that, like meningococci, gonococci removed Fe from TF and LF in an energy-dependent manner; uptake was repressed by Fe and did not proceed by a siderophore-mediated uptake system. Unlike published studies examining meningococcal Fe uptake from TF, our study showed that gonococcal Fe uptake from both TF and LF was highly efficient; uptake saturated at 1 microM protein, and growth with 5% saturated TF and LF occurred at maximal rates when the protein was present in appreciable concentrations. We conclude that the availability of protein-bound Fe probably does not limit growth of N. gonorrhoeae in the human body.

Biological Transport↗

PubMed1988

A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-kilodalton iron-regulated protein.

We isolated an iron-uptake mutant of Neisseria meningitidis M986-NCV-1 that was severely limited in the ability to use several sources of iron in the form of Fe3+. This mutant, FAM11, grew poorly or not at all with human transferrin (TF) or lactoferrin (LF) as the sole iron source in a defined medium but grew as well as wild-type meningococci with hemin or hemoglobin. Uptake of 55Fe bound to TF, LF, dicitrate complexes, aerobactin, or nitrilotriacetate was reduced to 0 to 4% of the wild-type level. FAM11 did not produce an iron-repressible outer membrane protein (FeRP) of 70 kilodaltons (kDa) found in membranes of iron-stressed M986-NCV-1. Western blot (immunoblot) analysis using rabbit antiserum against this protein revealed that at least 17 of 18 meningococcal and 10 of 14 gonococcal strains produced an FeRP of ca. 70 kDa. The 70-kDa FeRP was shown to be surface exposed by radioimmunoprecipitation with human immune sera. These data suggest that the 70-kDa FeRP is somehow involved in Fe uptake from TF and LF. However, we were unable to transform the iron-uptake phenotype from FAM11 into wild-type meningococci to confirm this. Revertants of FAM11 that grew with TF and LF did not regain the ability to make the 70-kDa FeRP but also did not completely regain the Fe-uptake phenotype of M986-NCV-1.

Antigens, Bacterial↗

PubMed1987

Isolation by streptonigrin enrichment and characterization of a transferrin-specific iron uptake mutant of Neisseria meningitidis.

The bactericidal action of the antibiotic streptonigrin is enhanced by large intracellular iron pools. Using this observation, we have utilized a simple enrichment protocol to aid in the isolation of iron uptake mutants of N. meningitidis, based on the relative resistance of iron-starved meningococci to streptonigrin. One such mutant, FAM29, was impaired in its use of transferrin-bound iron; transferrin is the principal iron-binding protein in human plasma. FAM29 retained wild-type ability to utilize iron bound to lactoferrin, heme, or ferric citrate. FAM29 did not produce two iron-repressible outer membrane proteins, of 85,000 and 95,000 daltons, made by the parent strain. However, genetic transformation experiments indicated that the outer membrane protein alterations were not necessary for the transferrin-deficient phenotype.

Bacterial Outer Membrane Proteins↗

PubMed1987

Effects of serum carrier proteins on the growth of pathogenic neisseriae with heme-bound iron.

The pathogenic neisseriae can use free heme and hemoglobin as an essential source of iron (Fe) for growth in vitro, but it is unknown whether they can utilize heme bound to human hemopexin or to human serum albumin, or hemoglobin bound to haptoglobin. We found that neither Neisseria meningitidis nor Neisseria gonorrhoeae used bound heme, but bound hemoglobin was used as an Fe source by two meningococcal strains and one gonococcal strain. A second gonococcal strain, previously shown to use free hemoglobin poorly or not at all, also did not grow with hemoglobin-haptoglobin complex as an Fe source. These observations suggest that hemoglobin might act as an Fe source in vivo for many pathogenic neisseriae even when in complexed (bound) form, but heme probably would not support growth in vivo if bound to serum carrier proteins.

Blood Proteins↗

PubMed1986

Human immune response to iron-repressible outer membrane proteins of Neisseria meningitidis.

Neisseria meningitidis grown under iron-limiting conditions in vitro expresses additional iron-repressible outer membrane proteins (FeRPs). To see which FeRPs were expressed and immunogenic in human infection, we examined purified membranes from four meningococcal disease isolates with Western blotting of patient sera. Convalescent serum from each patient contained immunoglobulin G (IgG) and IgM antibodies against the homologous 70-kilodalton (kDa) FeRP and IgG antibody to the homologous 94-kDa FeRPs. Three other immunoreactive FeRPs were identified in two or more strains. Neither acute-phase sera nor pooled normal human sera contained appreciable levels of these antibodies. Antigenic cross-reactivity among FeRPs was suggested by the observation that the convalescent sera of two patients contained IgG antibodies reactive with the 70- and 94-kDa FeRPs and IgM antibodies reactive with the 70-kDa FeRPs from all four strains. Additionally, rabbit antiserum against the 70-kDa FeRP from one of these disease isolates contained IgG and IgM antibodies that reacted in Western blots with the 70-kDa FeRPs in all four strains. These results demonstrate that meningococcal FeRPs are expressed and immunogenic in vivo and that certain of these proteins are immunologically cross-reactive.

Antibodies, Bacterial↗