Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains.

A simple and versatile system has been developed using a new cloning vector which can serve as a vehicle for integrating DNA fragments, which direct the expression of heterologous antigens, into the aroC gene on the Salmonella chromosome. The system is based on Escherichia coli plasmid vectors which contain the DNA fragment, cloned from the chromosome of S. typhimurium C5, which encodes the aroC gene. The aroC gene was modified using synthetic oligodeoxyribonucleotides so that it contained several unique restriction sites into which DNA, directing the expression of heterologous antigens, could be cloned. DNA was integrated into the S. typhimurium chromosome at aroC by transferring the vectors into S. typhimurium polA mutants and allowing homologous recombination to occur between the cloned and chromosomal aroC genes. The vectors were used to integrate nucleotide sequences into the S. typhimurium chromosome which directed the expression of tetanus toxin fragment C and the Treponema pallidum lipoprotein. The expression of both antigens was detected by Western blotting.

A novel flow cytometric assay for quantitating adherence of Helicobacter pylori to gastric epithelial cells.

Adherence may be an important virulence factor for Helicobacter pylori. Current methods available for quantitation of adherence are time consuming and liable to observer error. A new direct technique for fluorescent labelling of bacteria has been developed to quantitate adherence of H. pylori to epithelial cells by fluorescence activated cell sorting (FACS). Type strains of H. pylori, H. mustelae, H. cinaedi and H. fennelliae were grown microaerobically in broth culture for 24 h and fluorescently labelled by incubation with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37 degrees C. After washing to remove excess CFDA-SE, bacteria were co-incubated (ratio 10:1) with gastric epithelial cells at 37 degrees C for up to 24 h. After washing to remove non-adherent bacteria, epithelial cells were detached with EDTA (2 mM) and fixed with formaldehyde for flow cytometry. Adherence was quantitated both in terms of the proportion of cells with adherent H. pylori and as the mean number of adherent bacteria per cell. All H. pylori strains adhered to gastric-type epithelial cells. The proportion of cells with bound bacteria varied from 40-99% and the number of bacteria per cell from 1-50, both of which correlated with microscopy (r = 0.6, and r = 0.8 respectively, n = 35). Time course studies demonstrated saturation of binding by H. pylori within 90 min. For H. mustelae, H. cinaedi and H. fennelliae the proportion of cells with bound bacteria varied from 5-15% and the mean number of bacteria per cell was < 4. Binding of H. pylori to epithelial cells could be partly blocked by pre-incubation with polyclonal anti-sera or using oligosaccharides against potential binding epitopes of gastric mucus. Fluorescent labelling of H. pylori with CFDA-SE in combination with flow cytometry provides a quick, specific, and sensitive method to quantitate in vitro the adherence of H. pylori.

Immune response to Treponema pertenue and Treponema pallidum Nichols in patients with yaws.

Human sera from African patients with acute yaws were analysed by Western blot (WB) against antigens of Treponema pallidum Nichols and two Treponema pertenue isolates. The Western blot patterns were remarkably similar from one patient to another, and strains of both subspecies exhibited exactly the same banding pattern. Sera from yaws patients failed to detect at least one antigen in T. pertenue which was absent from T. pallidum.

Identification of flagellar and associated polypeptides of Helicobacter (formerly Campylobacter) pylori.

Flagella of Helicobacter pylori were isolated from intact organisms by shearing and differential centrifugation. Treatment of the flagella with the detergent Triton X-100 removed the flagellar sheath, which was confirmed by electron microscopy, and the remaining naked flagella were shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to consist primarily of a single 54 kilodalton (kDa) polypeptide. This was confirmed by immunogold labelling and electron microscopy of detergent treated whole organisms, using a mouse antiserum specific for the 54 kDa polypeptide. Polypeptides solubilised from crude flagellar preparations by detergent treatment were found to have molecular weights of 26, 30, 58, 62, 66 and 80 kDa. These polypeptides are possible components of the flagellar sheath and they may represent outer membrane proteins, based on the assumption that the flagellar sheath is related in composition to the outer membrane of the organism. Analysis and definition of these components of the surface structures of the organism are important in understanding the interaction between the organism and its host in pathogenesis.

Investigation of microbial growth in vivo: evaluation of a novel in vivo chamber implant system.

An intraperitoneal chamber implant system has been used to investigate the phenotype of Staphylococcus aureus growing in the rat and the effect of the antibiotic flucloxacillin on bacterial growth in vivo. Titanium chambers were implanted in the peritoneum: a period of 3-4 days equilibration allowed diffusion of host proteins into the chamber fluid prior to inoculation with bacteria. S. aureus inoculated into the chamber fluid, grew rapidly over a 72 h period, reaching counts of > 10(9) per ml. Organisms harvested from chambers were analysed by SDS-PAGE and showed significant differences in polypeptide profiles from the same strain grown in nutrient broth in vitro. Analysis of whole cell extracts by Western-blotting revealed that protein A expression was repressed in S. aureus grown in vivo. Following subcutaneous administration, flucloxacillin levels in serum peaked earlier and were higher than those detected in chamber fluid. The inhibitory effect of the antibiotic on the growth of S. aureus in chambers in treated animals could be monitored easily by sequential sampling of the chamber fluid. These results indicate the potential of the chamber implant model for investigation of microbial phenotype in vivo and development of alternative methods for assessment of antimicrobial efficacy in vivo.

Clinical and molecular aspects of the pathogenesis of Staphylococcus aureus bone and joint infections.

Staphylococcus aureus is an important cause of bone and joint infections. In recent years, significant changes in the incidence of septic arthritis and osteomyelitis have occurred. Haematogenous osteomyelitis is now less common during childhood, but secondary spread of infection to bone or joint from a contiguous site in adults is increasing in incidence. Infection introduced at the time of surgery or arising by the haematogenous route is a significant complication of prosthetic joint implantation, and the effect of bone cement on local immune function may be important in this setting. ALthough S. epidermis is a more common cause of prosthetic joint infection, S. aureus is more difficult to treat. S. aureus produces a number of extracellular and cell-associated factors, but it is unclear what role these have as virulence factors in vivo. Furthermore, it is difficult in animal models to simulate transient bacteraemia followed by non-fulminating septic arthritis or osteomyelitis, as occurs in the patient. Surface factors which may be important in pathogenesis include the cell wall (activates complement and stimulates cytokine release), capsular polysaccharide (promotes adhesion to host cell surfaces), collagen receptors and fibronectin-binding protein. Staphylococcal toxic shock syndrome toxin (TSST-1) and the enterotoxins are superantigens and have the potential to suppress plasma cell differentiation and antibody responsiveness. TSST-1-positive isolates have been shown to cause more severe joint infection in one animal model, but most other studies to date have focused on in-vitro rather than in-vivo effects. There is little evidence supporting a role for coagulase, lipase and the haemolysins in staphylococcal bone and joint infections. Despite the clinical importance of these infections, surprisingly little is known about pathogenesis at the cellular level. Future research should focus on the role of the host immune system in limiting spread of infection, and the expression of virulence factors in animal or other models incorporating isogenic mutant strains.

Responses to free lipopolysaccharide and Escherichia coli by normal human intestinal macrophages, following their migration out of the lamina propria.

Intestinal macrophage responses to luminal bacteria and their constituents are important in mucosal inflammatory responses. We investigated the responses of intestinal macrophages to free lipopolysaccharide (LPS) and Escherichia coli. Macrophages were isolated from normal terminal ileum and colon by allowing them to migrate out of the lamina propria of mucosal samples denuded of epithelial cells. Following exposure to free LPS or fluorescein-labelled E. coli, responsiveness was studied by intracellular expression of tumour necrosis factor-alpha (TNF-alpha). CD14, CD33, CD68, TLR2 and TLR4 expression was studied by fluorescence-activated cell sorter (FACS). TLR and NOD2 expression was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). CD14 was expressed by 36.5 +/- 4.0% of the macrophages obtained following migration out of the lamina propria. These cells also expressed TLR2, TLR4 and NOD2. Of cells exposed to free LPS or those that had taken up E. coli, a greater proportion of CD14(+) than CD14(-) macrophages expressed intracellular TNF-alpha. Moreover, a greater proportion of macrophages (CD14(+) and CD14(-)) demonstrated responses to E. coli than free LPS. In conclusion, a proportion of macrophages obtained following migration out of the lamina propria of normal terminal ileal and colonic mucosal samples express CD14, TLR2 and TLR4. These cells respond to free LPS and E. coli, as demonstrated by the expression of TNF-alpha.

Interactions of Candida albicans yeast cells, germ tubes and hyphae with human polymorphonuclear leucocytes in vitro.

Suspensions of Candida albicans yeast cells, germ tubes and hyphae with biomass standardized by ATP measurement were compared for their relative susceptibilities to phagocytosis and intracellular killing by human polymorphonuclear leucocytes. All three forms were ingested to a similar extent, but significantly fewer yeast cells were killed intracellularly after ingestion than were filamentous forms of the fungus. Ketoconazole pretreatment significantly enhanced the susceptibility of hyphae, but not of germ tubes, to phagocytosis and intracellular killing. The opsonic requirements of the yeasts and filamentous forms for efficient phagocytosis and killing differed.

Inhibitory effect of polyunsaturated fatty acids on the growth of Helicobacter pylori: a possible explanation of the effect of diet on peptic ulceration.

Diets high in polyunsaturated fatty acids may protect against duodenal ulcer, possibly through inhibiting the growth of Helicobacter pylori. This hypothesis was tested in vitro by incubating H pylori microaerophilically with a range of polyunsaturated fatty acids. omega-3 Linolenic acid significantly, but reversibly, inhibited growth at 1.8, 2.5, and 5 x 10(-4) M (p < 0.01), while concentrations of 10(-3) M killed virtually all organisms, with cell lysis observed by electron microscopy. Similar inhibitory effects were seen with other polyunsaturated fatty acids, at concentrations of 2.5 x 10(-4) M the relative inhibitory potencies were oleic (C18:1) < linoleic (C18:2) < arachidonic (C20:4) < omega-3 linolenic (C18:3) = omega-6 linolenic (C18:3) = eicosapentanoic (C20:5) acid. Cell fractionation studies with 14C labelled linolenic acid showed that the linolenic acid was associated with the membrane fraction. Commonly ingested dietary polyunsaturated fatty acids inhibit the growth of H pylori in vitro, an effect which deserves further in vivo study.

Surface properties of Treponema pallidum in relation to phagocytosis by human polymorphonuclear leucocytes in vitro.

Surface charge and hydrophobicity of Treponema pallidum have been investigated in relation to phagocytosis by human polymorphonuclear leucocytes (PMNs) in vitro. The treponemal surface was relatively hydrophobic and negatively charged but despite these properties, phagocytosis, as assessed by luminol-enhanced chemiluminescence, was minimal in the absence of serum. Preopsonization of bacteria with serum reduced surface hydrophobicity but promoted phagocytosis, suggesting that specific immune mechanisms may be more important in controlling phagocytosis of T. pallidum in vitro than non-specific surface properties. T. pallidum evoked a much weaker chemiluminescence response from PMNs than the non-pathogenic treponeme Treponema phagedenis biotype Reiterii even though similar numbers of bacteria were phagocytosed, suggesting differences in the reactivity of the surface components of the two organisms toward PMNs. The reactivity of T. pallidum towards PMNs could be increased by removal of the bacterial outer membrane by Triton X-100 treatment. These observations reinforce the suggestion that the outer surface of T. pallidum is inherently inert.

The axial filament antigen of Treponema pallidum.

Axial filaments (flagella) of Treponema pallidum have been purified in large enough quantities to be analysed electrophoretically. They produced a characteristic linear precipitate in two-dimensional immunoelectrophoresis. Polyacrylamide gel electrophoresis showed three major polypeptides, the most prominent having an apparent molecular weight of 37,000, about 1500 less than the corresponding component of axial filaments of the Reiter treponeme. A doublet of less abundant polypeptides of 33,500-34,000 MW also differed slightly from those of the Reiter treponeme. Western blot analysis showed that the principal polypeptide of the T. pallidum axial filament was strongly antigenic, and antibody to it was prominent in sera from hyperimmunized, as well as acutely infected (orchitic), rabbits, and in soluble fractions from acutely infected rabbit testes from which large numbers of viable treponemes had been extracted. This indicated that antibody to this component was ineffective in eliminating treponemes from the tissue.

The outer membrane of Treponema pallidum: biological significance and biochemical properties.

Rabbits infected intravenously with Treponema pallidum were not markedly febrile, and the pyrogenicity of treponeme preparations administered intravenously to rabbits was negligible. The antibiotic polymyxin B did not induce any ultrastructural changes on the treponemal surface and was not lethal (immobilizing) for T. pallidum, which was, however, highly susceptible to detergents such as SDS. Extraction of treponemes with Triton X-100 removed the outer membrane (despite the presence of Mg2+) as shown by electron microscopy, and solubilized a limited number of proteins detectable by SDS-PAGE, including a dominant antigen (47 kDal) demonstrated by immunoblotting. None of the proteins were heat-modifiable. Periodic acid-silver staining of polyacrylamide gels for carbohydrate together with protease K digestion did not demonstrate major carbohydrate components in whole treponemes, or in the Triton-soluble fraction. Surface iodination of intact treponemes revealed very little surface exposure of treponemal proteins, although a protein which co-migrated with host albumin was labelled and appeared to be associated with the treponemal surface. Many treponemal proteins were, however, labelled when iodination was done in the presence of Triton. These observations, indicate that the outer membrane of T. pallidum differs significantly from those of many Gram-negative pathogens.

Analysis of sheath and core structures of the axial filament of Treponema pallidum.

Electron microscopy and SDS-PAGE have been used to analyse the polypeptide and antigenic composition of the sheath and core components of the axial filament of Treponema pallidum. The sheath contains a major 37 kDa polypeptide which was solubilized by a combination of trypsin and urea treatments with concurrent loss of binding of anti-37 kDa monoclonal antibody. These studies also indicated some antigenic heterogeneity within the axial filament population. Trypsin treatment alone removed a number of antigenic determinants from the axial filament but left others intact, suggesting differences in their location within the sheath structure. A second 31.5 kDa polypeptide may also be associated with the sheath. The axial filament core comprises at least two components, an antigenically dominant 33.5 kDa polypeptide and a second of 34 kDa. The structure of the axial filament in T. pallidum and Treponema phagedenis biotype Reiterii was similar, but antigenic cross-reactivity of sheath and core components was incomplete.

Production of murine monoclonal antibodies to the major axial filament polypeptide of Treponema pallidum.

A suitable immunization protocol for the stimulation of a murine antibody response to the axial filament polypeptides of Treponema pallidum was established. A range of monoclonal antibodies (Mabs) specific for different epitopes of the major axial filament polypeptide (37 kDa) was generated which demonstrated diversity in their ability to react with other treponemal species. Immunogold electron microscopy located the 37 kDa antigen on the surface of the axial filament structure. The early appearance of specific antibody to this polypeptide in infected man and rabbit indicates that such Mabs are potentially useful for the diagnosis of early syphilis.

Molecular and antigenic analysis of treponemes.

The treponemes comprise the essentially non-cultivable Treponema pallidum subspecies (agents of syphilis, yaws and other human trepanematoses), the gut pathogen of pigs, T. hydysenteriae, and a group of antigenically related, cultivable species, some of which are strongly implicated in human periodontal or gastrointestinal disease. Technical developments during the last decade have made possible the molecular analysis of components of this diverse group of organisms. Polypeptides and other macromolecular components have been characterized by techniques including electron microscopy, gel electrophoresis and immunoblotting. Antigenic analysis has been greatly enhanced by the use of monoclonal antibodies. Finally, DNA cloning and genetic manipulation have enabled the detailed investigation of important antigens at a genetic, structural and functional level. We examine these developments and provide a current overview of the data now available, which is an important foundation for applications in diagnosis, therapy, and, potentially, immunization against disease.

Monoclonal antibodies directed against surface-associated polypeptides of Treponema pallidum define a biologically active antigen.

Murine monoclonal antibodies (Mabs) were raised against two outer-membrane-associated polypeptides of Treponema pallidum (47 and 44 kDa). Three Mabs against each polypeptide were investigated further and only those directed against the 44 kDa polypeptide were demonstrated to have immobilizing activity. The specificity of the Mabs for T. pallidum was determined by Western blotting procedures and the surface association of the antigens was inferred by immunogold electron microscopy. The clear distinction between these two polypeptides in their biological activity could help to explain the pathobiology of syphilis infections as the 47 kDa antigen has been shown to be associated with the outer membrane of this organism. Inactivity of such a surface-located protein in antibody-mediated anti-treponemal mechanisms could account for the observed ability of this organism to survive in the face of strong antibody responses in infection.

Immunochemical characterization and purification of Treponema pallidum antigen TpD expressed by Escherichia coli K12.

The immunochemical properties of the Treponema pallidum antigen TpD, as expressed by Escherichia coli K12, was investigated by crossed immunoelectrophoresis in which an affinity-purified antibody to this antigen was used. Two immunologically cross-reacting components of TpD with different mobility were demonstrated. Affinity-purified antibodies were used in obtaining purified TpD and in determining the cellular localization of TpD in T. pallidum by immunoelectron microscopy. TpD was localized on the surface of methanol-fixed T. pallidum. Twenty sera from patients with secondary syphilis and 20 sera from nonsyphilitics were examined in crossed immunoelectrophoresis. All sera from patients with secondary syphilis and none from nonsyphilitics contained antibodies to the TpD components. Because TpD seems to be surface associated and a major immunogen during infection with T. pallidum, this antigen might be useful for development of a vaccine against syphilis and for development of improved methods for serodiagnosis of syphilis.

Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae.

Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5-fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10(-8) M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10(-7) M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10(-5) M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth.

Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter.

Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC transporters with proven or putative metal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it may be involved in either siderophore- or transferrin-mediated iron uptake in S. epidermidis.

Comparative antigenic analysis of Treponema pallidum laboratory and street strains.

The polypeptide and antigenic profiles of Treponema pallidum Nichols strain and two other more recently isolated 'street' strains of T. pallidum have been compared. PAGE and immunoblotting identified a 34.5 kDa polypeptide present in the Nichols strain which was absent from one of the other street strains. This polypeptide was shown to be associated with the axial filament in T. pallidum. Three other axial-filament-associated polypeptides of 37, 33 and 30 kDa were present in all strains examined. Axial filaments of all three strains were morphologically identical and all three strains were equally motile.