ABSTRACT
A shortcoming of currently available oral cholera vaccines is their induction of relatively short-term protection against cholera compared to that afforded by wild-type disease. We were interested in whether transcutaneous or subcutaneous boosting using a neoglycoconjugate vaccine made from a synthetic terminal hexasaccharide of the O-specific polysaccharide of Vibrio cholerae O1 (Ogawa) coupled to bovine serum albumin as a carrier (CHO-BSA) could boost lipopolysaccharide (LPS)-specific and vibriocidal antibody responses and result in protective immunity following oral priming immunization with whole-cell cholera vaccine. We found that boosting with CHO-BSA with immunoadjuvantative cholera toxin (CT) or Escherichia coli heat-labile toxin (LT) following oral priming with attenuated V. cholerae O1 vaccine strain O395-NT resulted in significant increases in serum anti-V. cholerae LPS IgG, IgM, and IgA (P < 0.01) responses as well as in anti-Ogawa (P < 0.01) and anti-Inaba (P < 0.05) vibriocidal titers in mice. The LPS-specific IgA responses in stool were induced by transcutaneous (P < 0.01) but not subcutaneous immunization. Immune responses following use of CT or LT as an adjuvant were comparable. In a neonatal mouse challenge assay, immune serum from boosted mice was associated with 79% protective efficacy against death. Our results suggest that transcutaneous and subcutaneous boosting with a neoglycoconjugate following oral cholera vaccination may be an effective strategy to prolong protective immune responses against V. cholerae.
Subject(s)
Antigens, Bacterial/immunology , Cholera Vaccines/immunology , Cholera/prevention & control , Oligosaccharides/immunology , Vibrio cholerae O1/immunology , Adjuvants, Immunologic/administration & dosage , Administration, Cutaneous , Administration, Oral , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/administration & dosage , Bacterial Toxins/administration & dosage , Blood Bactericidal Activity , Cholera/immunology , Cholera Toxin/administration & dosage , Cholera Vaccines/administration & dosage , Disease Models, Animal , Enterotoxins/administration & dosage , Escherichia coli Proteins/administration & dosage , Feces/chemistry , Female , Immunization, Secondary/methods , Immunoglobulin A/analysis , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Injections, Subcutaneous , Mice , Oligosaccharides/administration & dosage , Survival Analysis , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/immunologyABSTRACT
Transcriptional regulation of the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria is mediated by a conserved transcription antitermination system, in which readthrough of a termination site in the leader region of the mRNA is directed by a specific interaction with the cognate uncharged tRNA. The specificity of this interaction is determined in part by pairing of the anticodon of the tRNA with a "specifier sequence" in the leader, a codon representing the appropriate amino acid, as well as by pairing of the acceptor end of the tRNA with an unpaired region of the antiterminator. Previous studies have indicated that although these interactions are necessary for antitermination, they are unlikely to be sufficient. In the current study, the effect of multiple mutations in tRNA(Tyr) on readthrough of the tyrS leader region terminator, independent of other tRNA functions, was assessed using a system for in vivo expression of pools of tRNA variants; this system may be generally useful for in vivo expression of RNAs with defined end points. Although alterations in helical regions of tRNA(Tyr) that did not perturb base pairing were generally permitted, substitutions affecting conserved features of tRNAs were not. The long variable arm of tRNA(Tyr) could be replaced by either a short variable arm or a long insertion of a stable stem-loop structure. These results indicate that the tRNA-leader RNA interaction is highly constrained, and is likely to involve recognition of the overall tertiary structure of the tRNA.
Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Bacillus subtilis/genetics , Escherichia coli Proteins , RNA, Transfer/genetics , Transcription, Genetic , Base Sequence , Blotting, Northern , Endoribonucleases/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , HIV Long Terminal Repeat/genetics , Lac Operon , Models, Genetic , Molecular Sequence Data , Mutagenesis, Insertional , Mutation , Peptide Chain Termination, Translational , Plasmids/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Catalytic/metabolism , RNA, Transfer, Amino Acyl/metabolism , Ribonuclease P , beta-Galactosidase/metabolismABSTRACT
The Bacillus subtilis tyrS gene belongs to the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes, which are regulated by a common mechanism of transcriptional antitermination. Each gene is induced by specific amino acid limitation; the uncharged cognate tRNA is the effector inducing transcription of the full-length message. The leader regions of the genes in this family share a number of conserved primary sequence and secondary structural elements, the functions of which are unknown. In this study, we examine these regions and report the effects of mutations in several of these elements. In addition, two alternative basepairings in the F box region were found to be necessary for tyrS antitermination.
Subject(s)
Bacillus subtilis/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Tyrosine-tRNA Ligase/genetics , Base Sequence , Molecular Sequence Data , TATA BoxABSTRACT
The Bacillus subtilis tyrS gene, encoding tyrosyl-tRNA synthetase, is a member of the T-box family of genes, which are regulated by control of readthrough of a leader region transcriptional terminator. Readthrough is induced by interaction of the cognate uncharged tRNA with the leader; the system responds to decreased tRNA charging, caused by amino acid limitation or insufficient levels of the aminoacyl-tRNA synthetase. Recognition of the cognate tRNA is mediated by pairing of the anticodon of the tRNA with the specifier sequence of the leader, a codon specifying the appropriate amino acid; a second interaction between the acceptor end of the tRNA and an antiterminator structure is also important. Certain switches of the specifier sequence to a new codon result in a switch in the specificity of the amino acid response, while other switches do not. These effects may reflect additional sequence or structural requirements for the mRNA-tRNA interaction. This study includes investigation of the effects of a large number of specifier sequence switches in tyrS and analysis of structural differences between tRNA(Tyr) and tRNA species which interact inefficiently with the tyrS leader to promote antitermination.
Subject(s)
Bacillus subtilis/genetics , Gene Expression Regulation, Bacterial/genetics , RNA, Messenger/genetics , RNA, Transfer/genetics , Tyrosine-tRNA Ligase/genetics , Base Sequence , Codon/genetics , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Messenger/chemistry , RNA, Transfer, Thr/genetics , RNA, Transfer, Tyr/geneticsABSTRACT
The Bacillus subtilis tyrS gene is a member of a group of gram-positive aminoacyl-tRNA synthetase and amino acid biosynthesis genes which are regulated by transcription antitermination. Each gene in the group is specifically induced by limitation for the appropriate amino acid. This response is mediated by interaction of the cognate tRNA with the mRNA leader region to promote formation of an antiterminator structure. The tRNA interacts with the leader by codon-anticodon pairing at a position designated the specifier sequence which is upstream of the antiterminator. In this study, an additional site of possible contact between the tRNA and the leader was identified through covariation of leader mRNA and tRNA sequences. Mutations in the acceptor end of tRNA(Tyr) could suppress mutations in the side bulge of the antiterminator, in a pattern consistent with base pairing. This base pairing may thereby directly affect the formation and/or function of the antiterminator. The discriminator position of the tRNA, an important identity determinant for a number of tRNAs, including tRNA(Tyr), was shown to act as a second specificity determinant for assuring response to the appropriate tRNA. Furthermore, overproduction of an unchargeable variant of tRNA(Tyr) resulted in antitermination in the absence of limitation for tyrosine, supporting the proposal that uncharged tRNA is the effector in this system.
Subject(s)
Bacillus subtilis/genetics , Gene Expression Regulation, Bacterial , RNA, Transfer, Tyr/genetics , Regulatory Sequences, Nucleic Acid/genetics , Tyrosine-tRNA Ligase/genetics , Base Sequence , Models, Genetic , Molecular Sequence Data , Mutation , RNA, Transfer, Tyr/metabolism , Suppression, Genetic , Terminator Regions, Genetic/genetics , Transcription, Genetic , Tyrosine-tRNA Ligase/metabolismABSTRACT
These experiments were conducted to study the possible involvement of macrophage-derived gelatinases in the bleomycin-induced model of pulmonary fibrosis. Normal rat alveolar macrophages and the rat alveolar macrophage cell line NR8383 were stimulated in vitro with 0-1.0 microgram/ml bleomycin for 18 h. Gelatinase activity in the medium was assayed on zymograms in which gelatin or collagen were used as substrates. Macrophages stimulated with 0.01-1.0 microgram/ml of bleomycin secreted significantly more of a 92-kDa gelatinase than did unstimulated controls. Addition of cycloheximide during stimulation decreased gelatinase activity by 86 +/- 4%, and activity was completely inhibited by the addition of EDTA to zymograms. This gelatinase degraded denatured type I collagen and native type IV collagen. Western blot analysis using a monoclonal mouse anti-rat antibody demonstrated that this enzyme was the same as a metalloproteinase secreted by rat mammary carcinoma cells. Gelatinase secreted by macrophages in fibrotic lungs may enhance macrophage migration through the lung and may also be active in the remodeling process.
Subject(s)
Bleomycin/pharmacology , Gelatinases/metabolism , Macrophages/enzymology , Animals , Cell Line , Cells, Cultured , Culture Media, Conditioned , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Gelatinases/isolation & purification , Hydrogen-Ion Concentration , Kinetics , Macrophages/drug effects , Male , Molecular Weight , Rats , Rats, Inbred F344ABSTRACT
Bleomycin-induced fibrosis in rodents has been used extensively as a model of human pulmonary fibrosis. The influx of monocytes observed during the early stages of fibrosis is at least partially regulated by the elaboration of chemotactic factors in the lung. Exposure of alveolar macrophages (AM phi) to bleomycin either in vivo or in vitro stimulated secretion of monocyte chemotactic activity (MCA). This MCA has been previously characterized as being primarily due to fibronectin fragments. The present experiments revealed that bleomycin also induced AM phi to secrete a second chemotactic factor, transforming growth factor-beta (TGF-beta). However, the TGF-beta secreted by macrophages was in latent form, since no TGF-beta activity was detected unless AM phi conditioned medium (CM) was acid-activated. After acidification, chemotactic activity in CM from AM phi stimulated with bleomycin in vitro was increased by 3.6, whereas activity in AM phi CM from fibrotic rats increased by 2 and that of a bleomycin-stimulated AM phi cell line increased by 1.6. This acid-activatable chemotactic activity was inhibited by antibody to TGF-beta. Bleomycin-stimulated AM phi s secreted significantly more TGF-beta than did unstimulated controls. Further, in vitro exposure of AM phi to bleomycin induced TGF-beta mRNA expression in a time- and concentration-dependent manner, with maximal mRNA being detected following a 16-h incubation with 1 microgram/ml bleomycin.
