Expression and immunogenicity of a recombinant diphtheria toxin fragment A in Streptococcus gordonii

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA(2)) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA(2) was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.     

Expression of a pertussis toxin S1 fragment by inducible promoters in oral Streptococcus and the induction of immune responses during oral colonization in mice

Previous work aimed at developing a live oral vaccine expressing pertussis toxin S1 fragment on the surface of the bacterium Streptococcus gordonii elicited a lower than expected antibody response, perhaps because of low antigen expression. In this study, in-frame promoter fusions were constructed to investigate whether an increase in antigen production by the streptococcal vaccine strain results in a better antibody response. The promoters tested were (i) the Streptococcus mutans sucrose-inducible fructosyltransferase (ftf) promoter and (ii) the Bacillus subtilis/Escherichia coli chimeric tetracycline-inducible xyl/tetO promoter. Each of these two promoters was placed upstream of the spaP/s1 fusion gene to drive its expression. The constructs were introduced into S. gordonii DL1 and S. mutans 834. The inducibility of the promoters was confirmed through the determination of SpaP/S1 production via Western blottings. Induced production of SpaP/S1 was observed in S. gordonii and S. mutans with each of the promoters, but the level of expression was the highest in S. mutans, using the xyl/tetO promoter. Thus, S. mutans carrying the xyl/tetO/spaP/s1 construct (S. mutans PM14) was used in oral colonization studies in BALB/c mice. Streptococccus mutans PM14 was able to colonize the animals for the 14-week duration of experimentation. A mucosal IgA response was observed in all the treatment groups but was highest in mice receiving tetracycline induction. In the mouse model of Bordetella pertussis respiratory infection, animals colonized with S. mutans PM14 showed a decreased in B. pertussis lung colony count (P = 0.03) on day 3 compared with control mice colonized by the parent S. mutans 834.     

Expression and Immunogenicity of a Recombinant Diphtheria Toxin Fragment A in Streptococcus gordonii

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA₂) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA₂ was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.     

Oral immunization of BALB/c mice by intragastric delivery of Streptococcus gordonii-expressing Giardia cyst wall protein 2 decreases cyst shedding in challenged mice

Giardia lamblia (Giardia duodenalis or Giardia intestinalis) is a protozoan parasite of vertebrates with broad host specificity. Specific antibodies directed against cyst antigens can interfere with the cyst wall-building process. In this study, we engineered Streptococcus gordonii to express a 26 kDa fragment of cyst wall protein 2 (CWP2), containing a relevant B cell epitope, on the cell surface. This is the first report of S. gordonii expressing a protein of parasite origin. As S. gordonii was intended for intestinal delivery of CWP2, it was determined that this oral commensal bacterium is able to persist in the murine intestine for 30 days. Immunization with recombinant streptococci expressing the 26 kDa fragment resulted in higher antibody levels. Specific anti-CWP2 IgA antibodies were detected in fecal samples and anti-CWP2 IgG antibodies were detected in serum demonstrating the efficacy of S. gordonii for intragastric antigen delivery. In a pilot challenge experiment, immunized mice demonstrated a significant 70% reduction in cyst output.     

Expression of saliva-binding epitopes of the Porphyromonas gingivalis FimA protein on the surface of Streptococcus gordonii

Porphyromonas gingivalis, a gram-negative oral anaerobic bacterium, has been implicated in the onset and development of periodontitis. The P. gingivalis fimbriae which mediate bacterial adherence to host oral sites and induce host inflammatory responses have been suggested as a potential antigen candidate. for vaccine development. This study was undertaken to generate Streptococcus gordonii vectors expressing the major subunit protein (FimA) of P. gingivalis fimbriae for testing as a potential live vaccine against periodontitis. We report here the expression of the C-terminal saliva-binding epitopes of P. gingivalis FimA on the surface of S. gordonii and demonstrate that domains containing free cysteine residues are poorly expressed on the surface of S. gordonii.     

Expression of measles virus antigens in Streptococcus gordonii

The measles virus proteins haemagglutinin (HA) and fusion protein (F), which together mediate attachment and penetration of the virus in the host cell and can elicit production of neutralising antibodies in the course of natural infection were expressed in the vaccine vector Streptococcus gordonii, a Gram-positive bacterium normally present in the human oral cavity. HA and F were expressed as fusion proteins attached to the bacterial surface, and were both found to be immunogenic when the recombinant S. gordonii were inoculated subcutaneously in mice.     

Role of the cell wall microenvironment in expression of a heterologous SpaP-S1 fusion protein by Streptococcus gordonii

The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of Streptococcus mutans and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium Streptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the dlt mutant strain, which has a mutation in the dlt operon preventing d-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the dlt mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca(2+), Mg(2+), and K(+), presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolyl cis/trans isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression in S. gordonii and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.     

Interactions between oral bacteria: inhibition of Streptococcus mutans bacteriocin production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

Purification and immunogenicity of a recombinant Bordetella pertussis S1S3FHA fusion protein expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model

BACKGROUND: Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. MATERIALS AND METHODS: KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. RESULTS: KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. CONCLUSIONS: Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo.     

Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.     

戈登链球菌的生物学特性及在黏膜疫苗的应用

戈登氏链球菌是一种非致病性革兰氏阳性黏膜共生菌, 参与组成人类口腔正常菌群。它具有特殊的生物学特性, 非常适合作为黏膜疫苗的载体。了解戈登氏链球菌的生物学特性, 常用表达体系及该菌在黏膜疫苗的应用情况, 将为其黏膜疫苗的进一步研制提供重要参考。     

戈登链球菌的生物学特性及在黏膜疫苗的应用

戈登氏链球菌是一种非致病性革兰氏阳性黏膜共生菌,参与组成人类口腔正常菌群.它具有特殊的生物学特性,非常适合作为黏膜疫苗的载体.了解戈登氏链球菌的生物学特性,常用表达体系及该菌在黏膜疫苗的应用情况,将为其黏膜疫苗的进一步研制提供重要参考.     

Expression of functional Porphyromonas gingivalis fimbrillin polypeptide domains on the surface of Streptococcus gordonii.

Genetically engineering bacteria to express surface proteins which can antagonize the colonization of other microorganisms is a promising strategy for altering bacterial environments. The fimbriae of Porphyromonas gingivalis play an important role in the pathogenesis of periodontal diseases. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to be an important virulence factor, which likely promotes adherence of the bacterium to saliva-coated oral surfaces and induces host responses. Immunization of gnotobiotic rats with synthetic peptides based on the predicted amino acid sequence of fimbrillin has also been shown to elicit a specific immune response and protection against P. gingivalis-associated periodontal destruction. In this study we engineered the human oral commensal organism Streptococcus gordonii to surface express subdomains of the fimbrillin polypeptide fused to the anchor region of streptococcal M6 protein. The resulting recombinant S. gordonii strains expressing P. gingivalis fimbrillin bound saliva-coated hydroxyapatite in a concentration-dependent manner and inhibited binding of P. gingivalis to saliva-coated hydroxyapatite. Moreover, the recombinant S. gordonii strains were capable of eliciting a P. gingivalis fimbrillin-specific immune response in rabbits. These results show that functional and immunologically reactive P. gingivalis fimbrillin polypeptides can be expressed on the surface of S. gordonii. The recombinant fimbrillin-expressing S. gordonii strains may provide an effective vaccine or a vehicle for replacement therapy against P. gingivalis. These experiments demonstrated the feasibility of expressing biologically active agents (antigens or adhesin molecules) by genetically engineered streptococci. Such genetically engineered organisms can be utilized to modulate the microenvironment of the oral cavity.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

Characterization of diphtheria toxin's catalytic domain interaction with lipid membranes

In response to a low environmental pH and with the help of the B fragment (DTB) the catalytic domain of diphtheria toxin (DTA) crosses the endosomal membrane to inhibit protein synthesis. In this study, we investigated the interaction of DTA with lipid membranes by biochemical and biophysical approaches. Data obtained from proteinase K and trypsin digestion experiments of membrane-inserted DTA suggested that residues 134-157 may adopt a transmembrane orientation and residues 77-100 could be membrane-associated, adopting either a surface or a transmembrane orientation. Fourier transform infrared spectroscopy analysis (FTIR) was used to characterize the secondary and tertiary structure of DTA along its pathway, from the native secreted form at pH 7.2 to the refolded structure at neutral pH after interaction with and desorption from a lipid membrane. We found that the association of DTA with lipid membranes at low pH was characterized by an increase of β-sheet structures and that the refolded structure at neutral pH after interaction with the membrane was identical to the native structure at the same pH. We also investigated the desorption of DTA from the membrane at neutral pH as a function of temperature. Although a complete desorption was observed at 37 °C, no desorption took place at 4 °C. A model of translocation involving the possibility that DTA might insert one or several transient transmembrane domains during translocation is discussed.     

Cloning of the saliva-interacting protein gene from Streptococcus mutans.

Genomic libraries from Streptococcus mutans OMZ175 were constructed in bacteriophage vectors. DNA fragments 1 to 2 kilobases in length were cloned in expression vector lambda gt11. S. mutans DNA fragments 15 to 20 kilobases in length were inserted in the BamHI site of phage EMBL3. Rabbit antiserum raised against an S. mutans saliva-interacting protein with a molecular weight of 74,000, designated 74K SR, was used to screen the lambda gt11 library. A recombinant phage carrying an S. mutans DNA sequence of 1.45 kilobases, lambda SmAD2, was detected and isolated. This fragment, named SmAD2, was used to construct the recombinant expression plasmid pSAD2-4 which encoded for the expression of a 60,000-molecular-weight protein controlled by the beta-galactosidase promoter from plasmid pUC8. The SmAD2 fragment and polyclonal anti-74K SR antibodies were used to screen the EMBL3 library. A total coincidence between the screening with antibodies and the DNA probe was observed, and two phages, lambda SmAD9 and lambda SmAD10, were isolated. They contained a common S. mutans DNA sequence of about 11.8 kilobases and coded for a protein with a molecular weight of about 195,000, which comigrated with a protein of an S. mutans cell wall extract. The expressed protein was purified, and a very strong relationship with the S. mutans 74K SR protein was found by competitive enzyme-linked immunosorbent assay. Thus, cloning of the 74K SR gene allowed us to demonstrate that the saliva receptor appears to be a part of an S. mutans precursor molecule with a molecular mass of 195,000 daltons.     

Research

Gram-positive bacterium Streptococcus gordonii, a human oral commensal, was engineered to display a single-chain Fv (scFv) antibody fragment at the cell surface. The previously developed host-vector system allowed expression of the Guy’s 13 scFv as a fusion with the streptococcal surface protein M6. Surface expression of the 515-amino acid M6/scFv fusion protein was confirmed by Western blot analysis on cellular fractions and flow cytometric analysis. Guy’s 13 scFv was derived from the Guy’s 13 monoclonal antibody, which was raised against streptococcal antigen I/II (SA I/II), the major adhesin of the caries-producing bacterium Streptococcus mutans. Surface plasmon resonance was used to test binding of scFv-expressing S. gordonii to SA I/II. Whole cells of recombinant S. gordonii were found to specifically bind to immobilised SA I/II and binding was inhibited by fluid-phase SA I/II in a dose-dependent manner. Production of a functional scFv in S. gordonii is the first step towards the development of genetically engineered commensal bacteria that, by colonizing mucosal surfaces, may provide the host with sustained delivery of recombinant antibodies.     

Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli

Pertussis toxoid, diphtheria toxoid, and tetanus toxoid are key components of diphtheria-tetanus-acellular pertussis vaccines. The efficacy of the vaccines is well documented, however, the vaccines are expensive partly because the antigens are derived from three different bacteria. In this study, a fusion protein (PDT) composed of the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin, and fragment C of tetanus toxin was constructed via genetic means. The correct fusion was verified by restriction endonuclease analysis and Western immunoblotting. Escherichia coli carrying the recombinant plasmid (pCoPDT) produced a 161kDa protein that was recognized by antibodies specific to the three toxins. The expression of the PDT protein was inducible by isopropyl-beta-d-thio-galactoside but the total amount of protein produced was relatively low. Attempts to improve the protein yield by expression in an E. coli strain (Rosetta-gami 2) that could alleviate rare-codon usage bias and by supplementation of the growth media with amino acids deemed to be a limiting factor in translation were not successful. The PDT protein remained in the insoluble fraction when the recombinant E. coli was grown at 37 degrees C but the protein became soluble when the bacteria were grown at 22 degrees C. The PDT protein was isolated via affinity chromatography on a NiCAM column. The protein was associated with five other proteins via disulfide bonds and non-covalent interactions. Following treatment with beta-mercaptoethanol, the PDT fusion was purified to homogeneity by preparative polyacrylamide gel electrophoresis with a yield of 45 microg/L of culture. Antisera generated against the purified PDT protein recognized the native toxins indicating that some, if not all, of the native epitopes were conserved.     

Characterization of diphtheria toxin's catalytic domain interaction with lipid membranes

In response to a low environmental pH and with the help of the B fragment (DTB) the catalytic domain of diphtheria toxin (DTA) crosses the endosomal membrane to inhibit protein synthesis. In this study, we investigated the interaction of DTA with lipid membranes by biochemical and biophysical approaches. Data obtained from proteinase K and trypsin digestion experiments of membrane-inserted DTA suggested that residues 134-157 may adopt a transmembrane orientation and residues 77-100 could be membrane-associated, adopting either a surface or a transmembrane orientation. Fourier transform infrared spectroscopy analysis (FTIR) was used to characterize the secondary and tertiary structure of DTA along its pathway, from the native secreted form at pH 7.2 to the refolded structure at neutral pH after interaction with and desorption from a lipid membrane. We found that the association of DTA with lipid membranes at low pH was characterized by an increase of beta-sheet structures and that the refolded structure at neutral pH after interaction with the membrane was identical to the native structure at the same pH. We also investigated the desorption of DTA from the membrane at neutral pH as a function of temperature. Although a complete desorption was observed at 37 degrees C, no desorption took place at 4 degrees C. A model of translocation involving the possibility that DTA might insert one or several transient transmembrane domains during translocation is discussed.