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1.
Mol Plant Microbe Interact ; 14(7): 823-31, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11437255

ABSTRACT

We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs. In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.


Subject(s)
Quaternary Ammonium Compounds/metabolism , Repressor Proteins/metabolism , Rhizobium leguminosarum/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , DNA, Bacterial/genetics , Fabaceae/microbiology , Genes, Bacterial , Glutamate-Ammonia Ligase/metabolism , Mutagenesis, Site-Directed , Mutation , Nitrogen/metabolism , Nitrogen Fixation/genetics , Promoter Regions, Genetic , Repressor Proteins/genetics , Rhizobium leguminosarum/genetics , Rhizobium leguminosarum/growth & development , Symbiosis
2.
Vaccine ; 19(15-16): 1931-9, 2001 Feb 28.
Article in English | MEDLINE | ID: mdl-11228363

ABSTRACT

Tetanus toxin fragment C (TTFC) was expressed on the surface of the vaccine vector Streptococcus gordonii, a Gram-positive commensal bacterium of the human oral cavity. The immunogenicity of recombinant S. gordonii expressing TTFC was assayed in mice immunized by the parenteral and mucosal routes. High serum TTFC-specific IgG responses were induced in both BALB/c and C57BL/6 mice immunized subcutaneously. A total of 82% of vaccinated BALB/c mice were protected from the lethal challenge with 50 LD(50) of tetanus toxin (TT) and a direct correlation between the serum TTFC-specific IgG concentration and survival time of unprotected animals was observed. Intranasal immunization of BALB/c mice was also effective in inducing TTFC-specific serum IgG and local IgA in lung washes. Furthermore, 38% of animals immunized intranasally were protected from the lethal challenge with 10 LD(50) of TT while all control animals died within 24 h. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after parenteral immunization in BALB/c mice (IgG1/IgG2a ratio congruent with6) while following mucosal immunization a mixed IgG1 and IgG2a pattern (IgG1/IgG2a ratio congruent with1) was observed. These data show that TTFC expressed on the surface of S. gordonii is immunogenic by the subcutaneous and mucosal routes and the immune response induced is capable of conferring protection from the lethal challenge with TT.


Subject(s)
Bacterial Vaccines/immunology , Peptide Fragments/immunology , Streptococcus/immunology , Tetanus Toxin/immunology , Administration, Intranasal , Animals , Antibodies, Bacterial/biosynthesis , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Base Sequence , Clostridium tetani/genetics , Clostridium tetani/immunology , Clostridium tetani/pathogenicity , DNA Primers/genetics , Female , Humans , Immunity, Mucosal , Immunization , Immunoglobulin G/biosynthesis , Immunoglobulin G/classification , Injections, Subcutaneous , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptide Fragments/genetics , Recombination, Genetic , Streptococcus/genetics , Tetanus/immunology , Tetanus/prevention & control , Tetanus Toxin/genetics , Tetanus Toxin/toxicity , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
3.
Int Rev Immunol ; 20(2): 275-87, 2001.
Article in English | MEDLINE | ID: mdl-11878770

ABSTRACT

The gram-positive bacterium Streptococcus gordonii was engineered to express the microbicidal molecule H6, which is an antiidiotypic single chain antibody mimicking a yeast killer toxin. S. gordonii is a human commensal which we developed as a model system for mucosal delivery of heterologous proteins. The in vivo candidacidal activity of both H6-secreting and H6-surface-displaying streptococcal strains were assayed in a well-established rat model of vaginal candidiasis. At day 21 full clearance of Candida albicans infection was observed in 75% of animals treated with the H6-secreting strain, and in 37.5% of animals treated with the strain expressing H6 on the surface, while all animals treated with the control strain were still infected. The observed candidacidal effect was comparable with that observed with the antimycotic drug fluconazole. These data confirm the potential of H6 as a candidacidal agent and show how promising is the approach of using recombinant bacteria for mucosal delivery of biologically active molecules.


Subject(s)
Antifungal Agents/administration & dosage , Immunity, Mucosal , Immunoglobulin Variable Region/genetics , Streptococcus/genetics , Streptococcus/immunology , Animals , Candidiasis, Vulvovaginal/immunology , Candidiasis, Vulvovaginal/therapy , Female , Fungal Proteins/administration & dosage , Fungal Proteins/genetics , Fungal Proteins/immunology , Humans , Immunoglobulin Variable Region/administration & dosage , Immunotherapy , In Vitro Techniques , Mice , Molecular Mimicry , Mycotoxins/administration & dosage , Mycotoxins/genetics , Rats , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunology
4.
Nat Biotechnol ; 18(10): 1060-4, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11017043

ABSTRACT

Two recombinant strains of Streptococcus gordonii, secreting or displaying a microbicidal single-chain antibody (H6), and stably colonizing rat vagina, were used to treat an experimental vaginitis caused by Candida albicans. A post-challenge intravaginal delivery of the H6-secreting strain was as efficacious as fluconazole in rapidly abating the fungal burden. Three weeks after challenge, 75% and 37.5% of the rats treated with the H6-secreting or displaying bacteria, respectively, were cured of the infection, which persisted in 100% of the animals treated with a S. gordonii strain expressing an irrelevant single-chain antibody. Thus, a human commensal bacterium can be suitably engineered to locally release a therapeutic antibody fragment.


Subject(s)
Candida albicans/immunology , Candidiasis/therapy , Immunoglobulin Idiotypes/immunology , Immunoglobulin Idiotypes/therapeutic use , Streptococcus/genetics , Vaginitis/therapy , Administration, Intravaginal , Animals , Anti-Bacterial Agents , Anti-Infective Agents/administration & dosage , Anti-Infective Agents/immunology , Anti-Infective Agents/therapeutic use , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Candida albicans/drug effects , Candida albicans/physiology , Candidiasis/immunology , Candidiasis/microbiology , Colony Count, Microbial , Disease Models, Animal , Female , Fluconazole/pharmacology , Fluconazole/therapeutic use , Humans , Immunization, Passive , Immunoglobulin Idiotypes/administration & dosage , Immunoglobulin Idiotypes/genetics , Mycotoxins/administration & dosage , Mycotoxins/chemistry , Mycotoxins/immunology , Mycotoxins/therapeutic use , Protein Engineering , Rats , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Streptococcus/cytology , Streptococcus/physiology , Vaginitis/immunology , Vaginitis/microbiology
5.
Res Microbiol ; 151(5): 361-8, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10919516

ABSTRACT

Spores of various Bacillus species, including B. subtilis, B. cereus and B. clausii, are used as probiotics, although they are generally absent from the normal microflora of man. We used two nonpathogenic Bacillus species, B. subtilis and B. clausii, to follow the fate of spores inoculated intragastrically in mice. We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species. Both spores and cells were detected in the lymph nodes and spleen of one mouse. Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B. anthracis germination in macrophages. These results indicate that any claimed probiotic effect of B. subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine.


Subject(s)
Bacillus/physiology , Intestines/microbiology , Probiotics , Spores, Bacterial/physiology , Administration, Oral , Animals , Bacillus/drug effects , Bacillus subtilis/drug effects , Bacillus subtilis/physiology , Bile Acids and Salts/pharmacology , Deoxycholic Acid/pharmacology , Female , Lymph Nodes/microbiology , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Spores, Bacterial/drug effects , Taurodeoxycholic Acid/pharmacology
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