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Biomedical subjects

H F Jenkinson

Publications and source records attributed to H F Jenkinson.

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

Cloning and expression of an endo-1,3-1,4-beta-glucanase gene from Bacillus macerans in Lactobacillus reuteri.

Strains of the gastrointestinal species Lactobacillus reuteri were electrotransformed with plasmid constructs containing the endo-1,3-1,4-beta-glucanase gene (bglM) of Bacillus macerans. The enzyme was expressed and secreted by the lactobacilli. A plasmid construct containing the bglM gene lacking its promoter was derived and was demonstrated to be useful as a promoter probe vector.

Bacillus↗
PubMed1997

Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides.

Cell surface proteins SspA and SspB in Streptococcus gordonii and SpaP in Streptococcus mutans are members of the antigen I/II family of polypeptides produced by oral streptococci. These proteins are adhesins and mediate species-specific binding of cells to a variety of host and bacterial receptors. Here we show that antigen I/II polypeptides are involved in the attachment of oral streptococci to collagen and that they also determine the ability of these bacteria to invade human root dentinal tubules. Wild-type S. gordonii DL1 (Challis) cells showed heavy invasion of tubules to a depth of approximately 200 microm, whereas the abilities of cells of isogenic mutant strains OB220 (sspA) and OB219 (sspA sspB) to invade were 50 and >90% reduced, respectively. Likewise, wild-type S. mutans NG8 cells invaded dentinal tubules, whereas cells of isogenic mutant strain 834 (spaP) did not. The invasive abilities of strains OB220 and OB219 were restored by heterologous expression of S. mutans SpaP polypeptide in these strains. The extents of tubule invasion by various wild-type and mutant strains correlated with their levels of adhesion to type I collagen, a major component of dentin. Furthermore, S. gordonii DL1 cells exhibited a growth response to collagen by forming long chains. This was not shown by ssp mutants but was restored by the expression of SpaP in these cells. The production of SspA polypeptide by S. gordonii DL1, but not production of SspB polypeptide by strain OB220 (sspA), was enhanced in the presence of collagen. These results are the first to demonstrate that antigen I/II family polypeptides bind collagen and mediate a morphological growth response of streptococci to collagen. These antigen I/II polypeptide activities are critical for intratubular growth of streptococci and thus for establishment of endodontic infections.

Adhesins, Bacterial↗
PubMed1997

Streptococcal adhesion and colonization.

Streptococci express arrays of adhesins on their cell surfaces that facilitate adherence to substrates present in their natural environment within the mammalian host. A consequence of such promiscuous binding ability is that streptococcal cells may adhere simultaneously to a spectrum of substrates, including salivary glycoproteins, extracellular matrix and serum components, host cells, and other microbial cells. The multiplicity of streptococcal adherence interactions accounts, at least in part, for their success in colonizing the oral and epithelial surfaces of humans. Adhesion facilitates colonization and may be a precursor to tissue invasion and immune modulation, events that presage the development of disease. Many of the streptococcal adhesins and virulence-related factors are cell-wall-associated proteins containing repeated sequence blocks of amino acids. Linear sequences, both within the blocks and within non-repetitive regions of the proteins, have been implicated in substrate binding. Sequences and functions of these proteins among the streptococci have become assorted through gene duplication and horizontal transfer between bacterial populations. Several adhesins identified and characterized through in vitro binding assays have been analyzed for in vivo expression and function by means of animal models used for colonization and virulence. Information on the molecular structure of adhesins as related to their in vivo function will allow for the rational design of novel acellular vaccines, recombinant antibodies, and adhesion agonists for the future control or prevention of streptococcal colonization and streptococcal diseases.

Adhesins, Bacterial↗
PubMed1996

Penetration of smeared or nonsmeared dentine by Streptococcus gordonii.

The purpose of this study was to investigate the penetration of smeared and nonsmeared dentine by Streptococcus gordonii. Prepared human roots, grouped as either nonsmeared or smeared, were immersed in a suspension of S. gordonii cells for 3 weeks. The roots were then prepared for scanning electron microscopy and histological analysis. Dentine discs prepared from coronal dentine were grouped similarly. Using a fluid filtration apparatus, the hydraulic conductance (Lp) of each disc was determined before and after incubation with bacterial suspension. Scanning electron microscopy evaluation of the roots following infection with bacteria showed no change in the smear layer (P < 0.0001). Histological sections revealed that bacterial penetration of all the nonsmeared samples had occurred, while nine out of 10 smeared samples showed no bacterial penetration (P < 0.0001). The Lp of the nonsmeared discs was significantly reduced by 42% (P < 0.0001) after bacterial penetration. However, the smeared samples revealed a 1% reduction in Lp which was not significant (P > 0.05). The results suggest that dentinal smear layers are an effective barrier to dentinal tubule invasion by S. gordonii.

Dental Pulp Cavity↗
PubMed1996

Tandem genes encode cell-surface polypeptides SspA and SspB which mediate adhesion of the oral bacterium Streptococcus gordonii to human and bacterial receptors.

The highly conserved antigen I/II family of polypeptides produced by oral streptococci are believed to be colonization determinants and may mediate adhesion of bacterial cells to salivary glycoproteins absorbed to cells and tissues in the human oral cavity. Streptococcus gordonii is shown to express, on the cell surface, two antigen I/II polypeptides designated SspA and SspB (formerly Ssp-5) that are the products of tandemly arranged chromosomal genes. The structure and arrangement of these genes is similar in two independently isolated strains, DL1 and M5, of S. gordonii. The mature polypeptide sequences of M5 SspA (1539 amino acid (aa) residues) and SspB (1462 aa residues) are almost wholly conserved (98% identical) in the C-terminal regions (from residues 796 in SspA and 719 in SspB, to the respective C-termini), well-conserved (84%) at the N-terminal regions (residues 1-429), and divergent (only 27% identical residues) within the intervening central regions. Insertional inactivation of the sspA gene in S. gordonii DL1 resulted in reduced binding of cells to salivary agglutinin glycoprotein (SAG), human erythrocytes, and to the oral bacterium Actinomyces naeslundii. Further reductions in streptococcal cell adhesion to SAG and to two strains of A. naeslundii were observed when both sspA and sspB genes were inactivated. The results suggest that both SspA and SspB polypeptides are involved in adhesion of S. gordonii cells to human and bacterial receptors.

Actinomyces↗
PubMed1996

Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance.

Fluconazole-susceptible Candida albicans strains accumulated [3H]fluconazole at a rate of approximately 2 pmol/min per 10(9) cells. Fluconazole accumulation was not affected by the pretreatment of cells with sodium azide or with 2-deoxyglucose. The rate of fluconazole accumulation became saturated at high fluconazole concentrations and was not affected by the addition of ketoconazole, and there was no fluconazole accumulation in cells incubated at 4 degrees C. A fluconazole-resistant mutant of C. albicans SGY-243 was isolated following growth enrichment in fluconazole-containing medium. Cells of the mutant strain, designated FR2, showed a reduced rate of fluconazole accumulation compared with SGY-243 and were not resistant to other azole antifungal agents. The rates of fluconazole accumulation by C. albicans FR2 and the other azole-resistant strains, B59630, AD, and KB, were increased in the presence of sodium azide, suggesting that fluconazole resistance in these strains may be associated with an energy-dependent drug efflux. Fluconazole-resistant C. albicans strains all contained elevated amounts (2- to 17-fold) of mRNA encoding Cdr1, and an ATP-binding cassette-type transporter. In addition, C. albicans FR2 also contained increased amounts of mRNA encoding Benr, a major facilitator superfamily transporter. These results suggest that fluconazole enters C. albicans cells by facilitated diffusion and that fluconazole resistance may involve energy-dependent drug efflux associated with increased expression of Benr and/or Cdr1.

Antifungal Agents↗
PubMed1996

Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.

Isogenic mutants of Streptococcus gordonii DL1 (Challis) in which the genes encoding high-molecular-mass cell surface polypeptides CshA and/or CshB were inactivated were deficient in binding to four strains of Actinomyces naeslundii and two strains of Streptococcus oralis. Lactose-sensitive interactions of S. gordonii with A. naeslundii ATCC 12104 and PK606 were associated with expression of cshA but not of cshB. Lactose-insensitive interactions of S. gordonii with A. naeslundii T14V and WVU627, and with S. oralis C104 and 34, were dependent on expression of cshA and cshB. S. gordonii DL1 cells bound to immobilized human fibronectin (Fn), but not to soluble Fn, in a dose-dependent manner, and binding was noninhibitable by heparin. S. gordonii cshA and cshB mutants were also deficient in binding to immobilized human Fn. Antibodies to an NH2-terminal nonrepetitive region (amino acid residues 42 to 886) of recombinant CshA inhibited binding of S. gordonii DL1 cells to A. naeslundii T14V and PK606 and to immobilized Fn. Conversely, antibodies to an amino acid repeat block segment of the COOH-terminal domain (amino acid residues 2026 to 2508) were not inhibitory to adherence. Assays using CshA-specific antibodies revealed that surface expression of CshA was reduced in cshB mutants. The results suggest that CshA acts as a multifunctional adhesin in S. gordonii and that major adhesion-mediating sequences are specified within the nonrepetitive NH2-terminal region of the polypeptide.

Animals↗
PubMed1996

Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions.

Candida albicans binds to several species of oral streptococci, in particular Streptococcus gordonii, through recognition of a streptococcal cell wall polysaccharide receptor (A. R. Holmes, P. K. Gopal, and H. F. Jenkinson, Infect. Immun. 63:1827-1834, 1995). We now show that isogenic cell surface protein mutants of S. gordonii DL1, unaltered in expression of cell wall polysaccharide, are reduced in ability to support adherence of C. albicans cells in a solid-phase assay. Inactivation of the S. gordonii cshA and cshB genes, encoding high-molecular-mass cell surface polypeptides, and inactivation of the sspA and sspB genes, encoding antigen I/II salivary adhesins, resulted in 40 and 79% reductions, respectively, in adherence of C. albicans cells. Inactivation of the S. gordonii scaA gene encoding a cell surface lipoprotein had no effect on C. albicans adherence. Polyclonal antiserum to streptococcal antigen I/II protein SpaP and antibodies specific to the amino-terminal nonrepetitive (NR) domain of CshA both inhibited adherence of C. albicans to S. gordonii cells. Conversely antibodies to the amino acid repeat block repetitive (R) domain of CshA, or to ScaA, did not inhibit C. albicans adherence. Immobilized recombinant polypeptide fragments of CshA comprising NR domain or R domain sequences both supported adherence of C. albicans cells. Expression of S. gordonii SspB protein on the surface of Enterococcus faecalis conferred on the enterococcal cells the ability to bind C. albicans, and this was ablated by antigen I/II antiserum. Collectively the results suggest that interaction of C. albicans with S. gordonii is mediated by a complement of adhesin-receptor interactions that involves two families of streptococcal multifunctional polypeptide adhesins, bacterial cell wall polysaccharide, and as yet unidentified yeast cell surface components.

Adhesins, Bacterial↗
PubMed1996

A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides.

Cells of the oral bacterium Streptococcus gordonii express three cytoplasmic membrane-bound lipoproteins with apparent molecular masses of 76 to 78 kDa that are the products of three genes (designated hppA, hppG, and hppH). The lipoproteins are immunologically cross-reactive, contain 60% or more identical amino acid residues, and are highly similar to the AmiA, AliA (PlpA), and AliB substrate-binding protein components of an oligopeptide permease in Streptococcus pneumoniae. Insertional inactivation of the hppA or hppH gene resulted in loss of the ability of S. gordonii cells to utilize specific peptides of five to seven amino acid residues for growth. An insertion within the COOH-terminal coding region of hppG that caused apparent truncation of the HppG polypeptide had a similar effect; however, S. gordonii mutants in which HppG polypeptide production was abolished were still able to grow on all oligopeptides tested. Inactivation of hppA gene (but not inactivation of the hppG or hppH gene) caused reduced growth rate of cells in complex medium, slowed the rate of development of competence for transformation, reduced the efficiency of transformation, and increased the resistance of cells to aminopterin. These results suggest that the formation of a solute-binding-protein complex consisting of at least the HppA and the HppH lipopolypeptides is necessary for binding and subsequent uptake of primarily hexa- or heptapeptides by a Hpp (Hexa-heptapeptide permease) system in S. gordonii. In addition, Hpp may play a role in the control of metabolic functions associated with the growth of streptococcal cells on complex nitrogen sources and with the development of competence.

Amino Acid Sequence↗
PubMed1996

Ins and outs of antimicrobial resistance: era of the drug pumps.

Over the past five years, concerns have heightened over the escalating numbers of pathogenic micro-organisms isolated that are resistant to many antibiotics and drugs. This phenomenon poses major problems in the treatment of patients with hospital- or community-acquired infections caused by bacteria, fungi, or parasitic organisms. Microbial cells have acquired resistances to specific antibiotics and drugs by mechanisms that include antibiotic inactivation, target alteration, or drug exclusion. More recently, the importance of another mechanism, that of drug expulsion, has been recognized as contributing significantly to antibiotic and drug resistance in microbes. Drug expulsion, mediated by membrane-associated drug efflux pumps, can protect cells from a range of toxic compounds and therefore may confer single-step multidrug resistance. It is imperative that new drugs be designed or discovered that will poison the pumps or bypass the efflux mechanisms.

Bacterial Infections↗
PubMed1995

Genetic analysis of adherence by oral streptococci.

Streptococci are one of the most successful bacterial colonizers of the human body and are major components of oral biofilms. The bacterial cells express multiple cell-surface adhesins that are responsible for the ability of streptococci to adhere to a wide range of substrates which include salivary and serous proteins, epithelial cells and other bacterial cells. Analysis of adherence-defective mutants has indicated the importance of high molecular mass wall-associated polypeptides and of enzymes catalyzing extracellular glucan polysaccharide synthesis to the adherence and accumulation of oral streptococci. The analysis of isogenic mutants of streptococci, generated through insertional inactivation (or allelic exchange), has confirmed the essential roles of specific surface polypeptides both to adhesive processes and to correct assembly of the cell wall layers.

Bacterial Adhesion↗
PubMed1995

Interactions of Candida albicans with bacteria and salivary molecules in oral biofilms.

The yeast Candida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells. C. albicans and Candida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind to Streptococcus gordonii NCTC 7869 while two other Candida species (Candida krusei and Candida kefyr) do not. Adherence of C. albicans was greatest when the yeast had been grown at 30 degrees C to mid-exponential growth phase. For 21 strains of C. albicans there was a positive correlation between the ability to adhere to S. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence of C. albicans to S. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins of C. albicans are co-expressed.

Bacterial Adhesion↗
PubMed1995

Adherence of Candida albicans to human salivary components adsorbed to hydroxylapatite.

Colonization of the oral cavity by Candida albicans involves adherence of yeast cells to oral surfaces. An assay was developed to measure the attachment of C. albicans cells, metabolically labelled with [35S]methionine, to saliva-coated hydroxylapatite (SHA) beads--a model for the tooth surface. Using this assay approximately 1.26 x 10(6) C. albicans cells (50.5% of input cells) attached to the SHA beads (12 mg). Different strains of C. albicans adhered to varying degrees to SHA beads, but in general adherence was promoted by growth of cells at 28 degrees C and by starvation of cells for glucose. Proteins in human whole, or parotid, saliva samples were fractionated by gel filtration (Sephacryl S-200 column) and fractions were adsorbed to hydroxylapatite beads. Fractions that contained proline-rich proteins or statherin promoted attachment of C. albicans ATCC 10261 cells. Neuraminidase treatment of SHA beads, but not of cells, significantly increased yeast cell binding to the SHA beads. Neuraminidase activity in the oral cavity may unmask glycoprotein receptors involved in the adhesion of C. albicans to saliva-coated surfaces.

Adsorption↗
PubMed1995

Polypeptides associated with tufts of cell-surface fibrils in an oral Streptococcus.

Cells of the oral bacterium Streptococcus oralis CN3410 produce lateral tufts of cell-surface fibrils of two lengths. Treatment of cells with trypsin resulted in loss of the tufts and release of longer fibrils intact. SDS-PAGE analysis of trypsin extracts containing fibrils revealed two groups of high molecular mass polypeptides which were denoted group A (molecular mass 227-246 kDa) and group B (molecular mass 175-208 kDa). Antibodies were raised to these two groups of trypsin-extracted polypeptides (TEPs) and to purified fibrils, and the reactivities of the three different antisera were found to be similar both on nitrocellulose blots of cell-surface polypeptides and in ELISA with whole cells. Similar patterns of TEPs were obtained from cells of a spontaneously derived mutant strain, KP34V, which lacked the short fibril components of tufts. Cells of strain KP34V had similar cell-surface hydrophobicity to strain CN3410 cells, and adhered to the same extent to parotid salivary pellicle or human buccal epithelial cells (BECs) as the wild-type cells. Trypsin treatment of strain CN3410 cells abolished their surface hydrophobicity and ability to adhere to BECs, but did not affect streptococcal cell binding to experimental salivary pellicle. Antibodies to TEPs or fibrils had no effect on cell adhesion to BECs or salivary pellicle. The results imply that the short fibril components of tufts are not involved in the cell adhesion properties tested. It is suggested that the TEPs are components of long fibrils, but they are not determinants of streptococcal cell adhesion to pellicle or to epithelial cells.

Antigens, Bacterial↗
PubMed1995

Comparison of Lactobacillus strains with respect to bile salt hydrolase activity, colonization of the gastrointestinal tract, and growth rate of the murine host.

The significance of bile salt hydrolase production by lactobacilli in the microecology of the murine intestinal tract has not been extensively studied previously. Assays of bile salt hydrolase (sodium taurocholate as substrate) associated with cell extracts of five Lactobacillus strains of murine origin gave a range of activities (from 915 nmol of cholate released per mg of protein per 30 min to none detected). All of the strains tested colonized the murine gastrointestinal tract equally well. The growth rates of mice were not affected by colonization of their intestinal tracts by lactobacilli whether or not the bacteria produced bile salt hydrolase.

Amidohydrolases↗
PubMed1995

Adherence of Candida albicans to a cell surface polysaccharide receptor on Streptococcus gordonii.

Candida albicans ATCC 10261 and CA2 bound to cells of the oral bacteria Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguis when these bacteria were immobilized onto microtiter plate wells, but they did not bind to cells of Streptococcus mutans or Streptococcus salivarius. Cell wall polysaccharide was extracted with alkali from S. gordonii NCTC 7869, the streptococcal species to which C. albicans bound with highest affinity, and was effective in blocking the coaggregation of C. albicans and S. gordonii cells in the fluid phase. When fixed to microtiter plate wells, the S. gordonii polysaccharide was bound by all strains of C. albicans tested. The polysaccharide contained Rha, Glc, GalNAc, GlcNAc, and Gal and was related compositionally to previously characterized cell wall polysaccharides from strains of S. oralis and S. sanguis. The adherence of yeast cells to the immobilized polysaccharide was not inhibitable by a number of saccharides. Antiserum raised to the S. gordonii NCTC 7869 polysaccharide blocked adherence of C. albicans ATCC 10261 to the polysaccharide. The results identify a complex cell wall polysaccharide of S. gordonii as the coaggregation receptor for C. albicans. Adherent interactions of yeast cells with streptococci and other bacteria may be important for colonization of both hard and soft oral surfaces by C. albicans.

Candida albicans↗
PubMed1995

Lipoprotein receptors in oral streptococci.

Streptococcus gordonii produces cell-surface lipopolypeptides that have been implicated in the determination of cell adherence and aggregation properties. SarA lipopolypeptide produced by S. gordonii is highly similar to the oligopeptide-binding protein AmiA in Streptococcus pneumoniae and to the OppA and SpoOKA oligopeptide-binding proteins in Bacillus subtilis. Insertional mutagenesis was used to inactivate the genes encoding SarA (76kDa) lipoprotein and a related 78-kDa lipoprotein denoted SarG. SarA- mutants were defective in serum-induced aggregation, competence, growth on complex nitrogen sources, and ability to colonize the oral cavity. Conversely, SarG- mutants were unaltered in the above properties, but were deficient in growth on simple nitrogen sources. It is proposed that SarA plays a central role in environmental sensing of extracellular factors by streptococci leading to modulation of cell-surface composition and growth responses of cells.

Adhesins, Bacterial↗