RESUMO
The strong immunogenicity of bacterial fimbriae results from their polymeric and proteinaceous nature, and the protective role of these immunogens in experimental or commercial vaccines is associated with their capacity to induce antiadhesive antibodies. Fimbria-mediated intestinal colonization by enteropathogens typically leads to similar antibody responses. The possibility of taking advantage of these properties was investigated by determining whether enteroadhesive fimbriae, like the 987P fimbriae of enterotoxigenic Escherichia coli, can serve as carriers for foreign antigens without losing their adhesive characteristics. Random linker insertion mutagenesis of the fasA gene encoding the major 987P subunit identified five different mutants expressing wild-type levels of fimbriation. The linker insertion sites of these mutants were used to introduce three continuous segments of viral surface glycoproteins known to be accessible to antibodies. These segments encode residues 11 to 19 or 272 to 279 of herpes simplex virus type 1 (HSV-1) glycoprotein D [gD(11-19) and gD(272-279), respectively] or residues 379 to 388 of the transmissible gastroenteritis virus (TGEV) spike protein [S(379-388)]. Studies of bacteria expressing fimbriae incorporating mutated FasA subunits alone or together with wild-type FasA subunits (hybrid fimbriae) indicated that foreign epitopes were best exported and displayed on assembled fimbriae when they were inserted near the amino terminus of FasA. Fimbriated bacteria expressing FasA subunits carrying the HSV gD(11-19) or the TGEV S(379-388) epitope inserted between the second and third residues of mature FasA elicited high levels of foreign epitope antibodies in all rabbits immunized parenterally. Antibodies against the HSV epitope were also shown to recognize the epitope in the context of the whole gD protein. Because the 987P adhesive subunit FasG was shown to be present on mutated fimbriae and to mediate bacterial attachment to porcine intestinal receptors, polymeric display of foreign epitopes on 987P offers new opportunities to test the potential beneficial effect of enteroadhesion for mucosal immunization and protection against various enteric pathogens.
Assuntos
Antígenos Virais/genética , Escherichia coli/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/imunologia , Adesinas de Escherichia coli/genética , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , Epitopos/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Fímbrias Bacterianas/imunologia , Fímbrias Bacterianas/virologia , Expressão Gênica , Genes Virais , Humanos , Imunidade nas Mucosas , Imunização , Microscopia Imunoeletrônica , CoelhosRESUMO
The mercury resistance determinant of marine Pseudomonas sp. strain MR1 plasmid pMR1 was cloned into a narrow-host-range vector pUC18. A direct selection of mercury resistant clones was successful and 12 clones were evolved; 9 from direct selection on mercury agar plates and 3 from ampicillin-resistant white colonies. All the predicted clones efficiently volatilized mercury. One of the hybrid plasmids pMRD5, containing a 15.5-kb insert, conferred inducible resistance to both HgCl2 and phenyl mercury acetate with over a 40-fold increase in mer resistance in Escherichia coli HB101. No DNA homology existed between the mer operon of Pseudomonas sp. strain MR1 and the characterized determinants of Tn501 mer DNA.
Assuntos
Escherichia coli/efeitos dos fármacos , Cloreto de Mercúrio/farmacologia , Compostos de Fenilmercúrio/farmacologia , Plasmídeos/isolamento & purificação , Pseudomonas/efeitos dos fármacos , Autorradiografia , Clonagem Molecular , DNA Bacteriano/análise , Resistência Microbiana a Medicamentos , Eletroforese em Gel de Ágar , Escherichia coli/genética , Técnicas In Vitro , Pseudomonas/genéticaRESUMO
Pseudomonas strain MR1 isolated from the coastal waters of Bay of Bengal was found to resist Hg, As, Cd, Cu, Zn and Pb. It efficiently detoxified both organic and inorganic mercuric compounds to non toxic metallic mercury by an inducible enzyme, mercuric reductase. Its resistance to arsenic might be due to energy-dependent arsenate efflux system. Cadmium was detected intracellularly and in the surrounding medium. The bacterium accumulated copper and lead intracellularly.
Assuntos
Metais/farmacologia , Pseudomonas/efeitos dos fármacos , Poluentes Químicos da Água/farmacologia , Relação Dose-Resposta a Droga , Resistência Microbiana a Medicamentos , Pseudomonas/crescimento & desenvolvimentoRESUMO
Pseudomonas sp isolated from the Bay of Bengal (Madras coast) contained a single large plasmid (pMR1) of 146 kb. Plasmid curing was not successful with mitomycin C, sodium dodecyl sulfate, acridine orange, nalidixic acid or heat. Transfer of mercury resistance from marine Pseudomonas to Escherichia coli occurred during mixed culture incubation in liquid broth at 10(-4) to 10(-5) ml(-1). However, transconjugants lacked the plasmid pMR1 and lost their ability to resist mercury. Transformation of pMR1 into E. coli competent cells was successful; however, the efficiency of transformation (1.49 x 10(2)Hgr transformants microsgm-1 pMR1 DNA) was low. E. coli transformants containing the plasmid pMR1 conferred inducible resistance to mercury, arsenic and cadmium compounds similar to the parental strain, but with increased expression. The mercury resistant transformants exhibited mercury volatilization activity. A correlation existed between metal and antibiotic resistance in the plasmid pMR1.
