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C A Genco

Publications and source records attributed to C A Genco.

At least 37 records · Page 2Linked to original sources
PubMed1997

A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.

Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells.

Antibodies, Blocking↗
PubMed1997

Identification of a second endogenous Porphyromonas gingivalis insertion element.

In this study a second endogenous Porphyromonas gingivalis insertion element (IS element) that is capable of transposition within P. gingivalis was identified. Nucleotide sequence analysis of the Tn4351 insertion site in a P. gingivalis Tn4351-generated transconjugant showed that a complete copy of the previously unidentified IS element, designated PGIS2, had inserted into IS4351R in Tn4351. PGIS2 is 1,207 bp in length with 19-bp imperfect terminal inverted repeats, and insertion resulted in a duplicated 10-bp target sequence. Results of Southern hybridization of chromosomal DNA isolated from several P. gingivalis strains with a PGIS2-specific probe demonstrated that the number of copies of PGIS2 per genome varies among different P. gingivalis strains. Computer analysis of the putative polypeptide encoded by PGIS2 revealed strong homologies to the products encoded by IS1358 from Vibrio cholerae, ISAS1 from Aeromonas salmonicida, and H-rpt in Escherichia coli K-12.

Amino Acid Sequence↗
PubMed1997

Promoter mapping and transcriptional regulation of the iron-regulated Neisseria gonorrhoeae fbpA gene.

In this study, we have mapped the promoter region of the Neisseria gonorrhoeae ferric iron binding protein-encoding gene fbpA, determined the start point of transcription, and examined the accumulation of fbpA mRNA Primer extension analysis of the fbpA promoter region indicated a single transcriptional start site located 51 bp upstream of the ATG translational start site. Northern blot analysis with a 200-bp fbpA structural gene probe detected one transcript of 1.0 kb in RNAs extracted from gonococcal cultures grown under iron-restricted conditions; the 1.0-kb transcript was observed to accumulate at a steady rate throughout the growth cycle. In comparison, in cultures grown under iron-sufficient conditions, the intensity of the 1.0-kb transcript was reduced considerably. Isolation of total RNA from rifampin-treated cells indicated that the half-life of the 1.0-kb fbpA transcript in cells grown under iron-restricted conditions was 1.2 +/- 0.2 min, while that of the 1.0-kb fbpA transcript obtained from cultures grown under iron-sufficient conditions was 0.5 +/- 0.1 min. Taken together, our results indicate that the fbpA promoter is regulated by iron and that transcription and translation of FbpA are closely linked.

Bacterial Outer Membrane Proteins↗
PubMed1996

Iron acquisition in the pathogenic Neisseria.

Pathogenic Neisseria species need to obtain iron from the host to grow in vivo. Several iron-transport systems are known, and regulation of Neisseria iron-transport genes occurs via the transcriptional regulator Fur. There is evidence that the ability to transport iron is crucial to the survival of these organisms in vivo.

Animals↗
PubMed1996

Hemin-induced modifications of the antigenicity and hemin-binding capacity of Porphyromonas gingivalis lipopolysaccharide.

Previous studies have shown that the physical, biochemical, and antigenic properties of the bacterial outer membrane are profoundly influenced by the growth environment. In the present study, the effects of growth in hemin-replete (H+) and hemin-depleted (H-) media on the lipopolysaccharide (LPS) of the oral pathogen Porphyromonas gingivalis were investigated. Our studies show that LPS from P. gingivalis cultured in H+ media (H+LPS) expressed additional low-molecular-mass antigens, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis. Particularly evident was a 26-kDa antigen (26 LPSC) that was lost from the LPS upon transfer of P. gingivalis into H- media. The loss of the 26 LPSC was accompanied by a marked reduction in the hemin-binding capacity of the LPS. The 26 LPSC was refractory to Coomassie blue staining and proteinase K digestion. H+LPS from strain W50/BE1, a nonpigmented pleiotropic strain, lacked the 26 LPSC and did not bind hemin. Polyclonal antiserum raised to whole-cell antigens of P. gingivalis A7436, W83, and HG405 grown in H+ media, but not in H- media, recognized the 26 LPSC in the purified H+LPS from any of the three strains. The immunoreactivities of sera from humans with (n = 24) or without (n = 25) periodontitis to the 26 LPSC and other H+LPS determinants were analyzed by Western blot. Overall, 75% of adult periodontitis patient sera reacted with multiple bands in the H+LPS stepladder, particularly in the range of 14 to 27 kDa. In contrast, only 20% of control sera reacted faintly with H+LPS bands in the range 27 to 34 kDa. The 26 LPSC was recognized by over 40% of sera from adult patients with periodontitis and none of the healthy control sera. Taken together, these results suggest that the antigenicity and hemin-binding properties of P. gingivalis LPS can be modified by growth in H+ media.

Adult↗
PubMed1996

Analysis of Fur binding to operator sequences within the Neisseria gonorrhoeae fbpA promoter.

The gene encoding Neisseria gonorrhoeae periplasmic binding protein FbpA contains two regions whose sequences exhibit homology with the Escherichia coli ferric uptake regulator protein (Fur) consensus binding sequence. In this study, DNase I footprinting experiments were employed to characterize the operator sequences within the fbpA promoter region to which E. coli Fur binds. A 160-bp fragment encompassing the promotor region and the putative iron boxes of the fbpA promoter was incubated with Fur, DNaseI was added, and the products of these reactions were sequenced to identify nucleotide peaks that were protected. At 50 nM Fur, a protected region that spanned 33 bp and extended 19 bp upstream and 8 bp downstream of the -35 region of the fbpA promoter was observed. At higher concentrations of Fur (75 and 100 nM), an extension of this protected region upstream of the -35 region was observed. Introduction of a plasmid carrying an fbpA-cat transcriptional fusion in E. coli H1717 (Fur+) resulted in an 88% induction of chloramphenicol acetyltransferase expression under conditions of iron restriction; however, chloramphenicol acetyltransferase expression was not responsive to iron in E. coli H1745 (Fur-), indicating that transcriptional regulation of fbpA in response to iron occurs via the negative regulator Fur. The extent of the fbpA operator sequence (42 bp), as defined by our footprinting analysis, would suggest the binding of two Fur repressor dimers.

Bacterial Outer Membrane Proteins↗
PubMed1995

Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis.

Adult periodontitis is a chronic inflammatory disease that affects over 49 million people in the USA alone. Porphyromonas (formerly Bacteroides) gingivalis, a Gram-negative anaerobe, has a diverse repertoire of virulence factors that may be involved in the induction or progression of periodontitis.

Bacteroidaceae Infections↗
PubMed1995

Binding and accumulation of hemin in Neisseria gonorrhoeae.

The ability to utilize hemin and hemin-containing compounds for nutritional iron (Fe) uptake has been documented for several pathogenic bacteria. Neisseria gonorrhoeae can utilize free hemin as a source of Fe for growth; however, little is known concerning the mechanisms involved in hemin transport. In this study we have characterized the binding and accumulation of hemin by N. gonorrhoeae and defined the specificity of the gonococcal hemin receptor. N. gonorrhoeae F62 was grown in a chemically defined medium containing the iron chelator Desferal, and hemin transport was initiated by the addition of [59Fe]hemin (4.0 or 8.0 microM; specific activity, 7.0 Ci/mol). 59Fe uptake from radiolabeled hemin by N. gonorrhoeae was energy dependent, and 59Fe was shown to accumulate in the cell at a constant rate during logarithmic growth. However, we observed a decrease in the uptake of 59Fe from radiolabeled hemin when inorganic iron was present in the growth medium. Binding of 59Fe from radiolabeled hemin was inhibited by the addition of either cold hemin, hematoporphyrin, or hemoglobin, but not by ferric citrate. Although [14C]hemin was found to support the growth of N. gonorrhoeae, we did not detect the uptake of 14C from radiolabeled hemin. Extraction of the gonococcal periplasmic ferric binding protein (Fbp) from cultures grown with [59Fe]hemin indicated that a majority of the 59Fe was associated with the Fbp. Taken together, the results presented here indicate that hemin binds to a gonococcal outer membrane receptor through the protoporphyrin portion of the molecule and that following binding, iron is removed and transported into the cell, where it is associated with the gonococcal periplasmic ferric binding protein, Fbp.

Binding, Competitive↗
PubMed1995

Resistance of a Tn4351-generated polysaccharide mutant of Porphyromonas gingivalis to polymorphonuclear leukocyte killing.

In this study, we describe the development of an efficient transpositional mutagenesis system for Porphyromonas gingivalis using the Bacteroides fragilis transposon Tn4351. Using this system, we have isolated and characterized a Tn4351-generated mutant of P. gingivalis A7436, designated MSM-1, which exhibits enhanced resistance to polymorphonuclear leukocyte (PMN) phagocytosis and killing. P. gingivalis MSM-1 was initially selected based on its colony morphology; MSM-1 appeared as a mucoid, beige-pigmented colony. Analysis of P. gingivalis MSM-1 by electron microscopy and staining with ruthenium red revealed the presence of a thick ruthenium red-staining layer that was twice the thickness of this layer observed in the parent strain. P. gingivalis MSM-1 was found to be more hydrophilic than strain A7436 by hydrocarbon partitioning. Analysis of phenol-water extracts prepared from P. gingivalis A7436 and MSM-1 by Western (immunoblot) analysis and immunodiffusion with hyperimmune sera raised against A7436 and MSM-1 revealed the loss of a high-molecular-weight anionic polysaccharide component in extracts prepared from MSM-1. P. gingivalis MSM-1 was also found to be more resistant to PMN phagocytosis and intracellular killing than the parent strain, as assessed in a fluorochrome phagocytosis microassay. These differences were statistically significant (P < 0.05) when comparing PMN phagocytosis in nonimmune serum and intracellular killing in nonimmune and immune sera. P. gingivalis MSM-1 was also more resistant to killing by crude granule extracts from PMNs than was P. gingivalis A7436. These results indicate that the increased evasion of PMN phagocytosis and killing exhibited by P. gingivalis MSM-1 may result from alterations in polysaccharide-containing antigens.

Antigens, Bacterial↗
PubMed1995

Characterization of a Tn4351-generated hemin uptake mutant of Porphyromonas gingivalis: evidence for the coordinate regulation of virulence factors by hemin.

The ability of Porphyromonas gingivalis to acquire iron in the iron-limited environment of the host is crucial to the colonization of this organism. We report here on the isolation and characterization of a transpositional insertion mutant of P. gingivalis A7436 (designated MSM-3) which is defective in the utilization and transport of hemin. P. gingivalis MSM-3 was selected on the basis of its nonpigmented phenotype on anaerobic blood agar following mutagenesis with the Bacteroides fragilis transposon Tn4351. P. gingivalis MSM-3 grew poorly when supplied with hemin as a sole source of iron; however, growth was observed with hemoglobin or inorganic iron. P. gingivalis MSM-3 grown in either hemin-replete or hemin-depleted conditions bound and transported less [14C]hemin or [59Fe]hemin than did the parent strain. At 4 h, P. gingivalis MSM-3 grown in hemin-replete conditions transported only 10,000 pmol of hemin per mg of protein, or 14% of the amount transported by P. gingivalis A7436. Unlike P. gingivalis A7436, hemin binding and transport by P. gingivalis MSM-3 were not tightly regulated by hemin or iron. Examination of P. gingivalis MSM-3 cultures by electron microscopy revealed an overproduction of membrane vesicles, and determination of the dry weight of purified vesicles indicated that P. gingivalis MSM-3 produced twice as much membrane vesicles as did strain A7436. Extracellular vesicles isolated from P. gingivalis MSM-3 also were found to express increased hemolytic and trypsin-like protease activities compared with the parent strain. When inoculated into subcutaneous chambers implanted in mice, P. gingivalis MSM-3 was highly infectious and more invasive than the parent strain, as indicated by secondary lesion formation and death. Taken together, these results indicate that the decreased transport of hemin by P. gingivalis MSM-3 results in the increased expression of several virulence factors which may be coordinately regulated by hemin.

Animals↗
PubMed1995

Regulation of hemin and iron transport in Porphyromonas gingivalis.

As with other pathogens, a requirement for the in vivo growth of Porphyromonas gingivalis is that the organism must be capable of obtaining iron from the host. The ability to utilize hemin and hemin-containing compounds for nutritional iron has been documented for several pathogenic bacteria, including P. gingivalis; however, the mechanisms involved in hemin uptake are poorly defined. I have determined that P. gingivalis transports the entire hemin moiety into the cell by an energy-dependent mechanism and that the binding and accumulation of hemin are induced by growth of cultures in the presence of hemin. A model of hemin transport in P. gingivalis consistent with these results is presented. I have also found that, in P. gingivalis, hemin regulates the expression of several putative virulence factors; this in turn results in the increased virulence potential of P. gingivalis as assessed in an animal model. Regulation of hemin-responsive genes in P. gingivalis may occur by a negative regulator, as has been described in other pathogenic organisms.

Bacterial Outer Membrane Proteins↗
PubMed1995

Analysis of the insertion characteristics of Tn4351 during high frequency transposition of Porphyromonas gingivalis.

In this study, we characterized the high frequency transfer of the Bacteroides fragilis transposon Tn4351 into the chromosome of Porphyromonas gingivalis using a system developed by Genco, et al. Using this system, transconjugants were isolated and genomic sequences analyzed using both Ava-I and EcoR1 to determine the insertion characteristics of Tn4351 into the genome of P. gingivalis. Although previous evidence pointed to the possibility of random insertion, this study shows that, in fact, the insertion characteristics of Tn4351 into the genome of P. gingivalis are nonrandom and predictable.

Bacteroides fragilis↗
PubMed1994

Genetic diversity of the iron-binding protein (Fbp) gene of the pathogenic and commensal Neisseria.

The pathogenic Neisseria and most commensal Neisseria species produce an iron-binding protein (Fbp) when grown under iron-limited conditions. In the current study, we confirmed the presence of Fbp, as well as DNA sequences homologous to the gonococcal fbp, in strains of N. gonorrhoeae, N. meningitidis, N. cinerea, N. lactamica, N. subflava, N. kochii and N. polysaccharea. The fbp genes from these strains were amplified by the polymerase chain reaction, digested with StuI or RsaI, and the restriction patterns examined. The patterns for the gonococcal and meningococcal fbp were virtually identical; however, variations were observed in the fbp sequences of the commensal Neisseria species. N. flavescens, N. mucosa, N. sicca, N. ovis and Branhamella catarrhalis, did not produce Fbp as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with an Fbp specific monoclonal antibody, nor did they hybridize to an fbp-specific DNA probe.

Amino Acid Sequence↗
PubMed1994

Binding and accumulation of hemin in Porphyromonas gingivalis are induced by hemin.

Although hemin is an essential nutrient for the black-pigmented oral bacterium Porphyromonas gingivalis, the mechanisms involved in hemin binding and uptake are poorly defined. In this study, we have examined the binding of hemin and Congo red (CR) to P. gingivalis whole cells and have defined the conditions for maximal binding. Additionally, the accumulation of hemin by P. gingivalis under growing conditions has been characterized. P. gingivalis A7436 was grown under hemin- or iron-deplete conditions (basal medium [BM] or Schaedler broth with dipyridyl [SBD]) or under hemin- or iron-replete conditions (BM with hemin [BMH] or Schaedler broth [SB]), and hemin and CR binding were assessed spectrophotometrically. Binding of hemin by P. gingivalis whole cells was rapid and was observed in samples obtained from cells grown under hemin- and iron-replete and hemin-deplete conditions but was not observed in cells grown under iron limitation. We also found that P. gingivalis whole cells bound more hemin when grown in BMH or SB than cells grown in BM or SBD. Binding of CR by P. gingivalis A7436 was also enhanced when cells were grown in the presence of hemin or when cells were incubated with hemin prior to CR binding. Hemin binding and accumulation were also assessed using [14C]hemin and [59Fe]hemin under growing conditions. Both [14C]hemin and [59Fe]hemin were accumulated by P. gingivalis, indicating that iron and the porphyrin ring were taken into the cell. Binding and accumulation of hemin under growing conditions were also induced by growth of P. gingivalis in hemin-replete media. Hemin accumulation was inhibited by the addition of KCN to P. gingivalis cultures, indicating that active transport was required for hemin uptake. [14C]hemin binding and accumulation were also inhibited by the addition of either cold hemin or protoporphyrin IX. Taken together, these results indicate that P. gingivalis transports the entire hemin moiety into the cell and that the binding and accumulation of hemin are induced by growth of cultures in the presence of hemin.

Binding, Competitive↗
PubMed1992

Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model.

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.

Animals↗
PubMed1991

Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers.

Iron-uptake mutants of Neisseria gonorrhoeae strain 340 were obtained following treatment with streptonigrin, and one such mutant (Fud14) was characterized. N. gonorrhoeae strain Fud14 was unable to grow with human transferrin or haemoglobin as the sole source of iron, but grew normally with heat-inactivated normal human serum or haemin. Internalization of 55Fe from transferrin by strain Fud14 was only 25% of the parent level. Strain Fud14 (less than or equal to 1 x 10(8) c.f.u.) did not grow in subcutaneous chambers implanted in mice, whereas the parent strain was infective at an ID50 of 4.3 x 10(1) c.f.u. Supplementation of chambers with either normal human serum or haemin resulted in the establishment of strain Fud14 in vivo for at least 240 h post-inoculation. Electroporation of Fud14 with wild-type DNA and selection for growth on medium containing human transferrin resulted in a recombinant (Fud15) that was capable of utilizing haemoglobin, and was virulent in mice. These results suggest that a gonococcal strain defective in the ability to utilize in vivo iron sources is not capable of survival in vivo.

Animals↗