RESUMEN
A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some serogroup Icterohaemorrhagiae agglutinating epitopes. However, LaiMut displayed an increased reactivity to antisera against related serogroups, suggesting that the disruption of some lipopolysaccharide epitopes resulted in greater exposure to cross-reactive epitopes, not accessible to antibodies in the wild type (LaiWT). Comparison of the nucleotide sequences of the lipopolysaccharide loci of LaiMut and Lai wild type (LaiWT) strains showed an inframe stop mutation in the gene encoding undecaprenyl-galactosyltransferase, a protein that provides a fundamental and nonredundant function essential for lipopolysaccharide biosynthesis. Despite this, the biosynthesis of lipopolysaccharide agglutinating antigens was not abolished by the mutation. Based on the phenotype of LaiMut and analysis of the domain structure of the undecaprenyl-galactosyltransferase in relation to the mutation, we propose that the mutation results in the expression of two functional proteins in place of the undecaprenyl-galactosyltransferase. We hypothesize that the loss of coordination of the coupled function afforded by the intact dual function protein present in the parent strain results in an inefficient production of lipopolysaccharide in LaiMut.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Leptospira interrogans/genética , Lipopolisacáridos/biosíntesis , Mutación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Leptospira interrogans/efectos de los fármacos , Leptospira interrogans/crecimiento & desarrollo , Leptospira interrogans/metabolismo , Lipopolisacáridos/inmunología , Datos de Secuencia MolecularRESUMEN
Plasmid pGL4W15-96 was constructed from the pGL4W74 vector without promoter for kanamycin and a sequence of 480pb containing the supposed J15 promoter with the objective of confirming the role of J15 sequence as a promoter for Leptospira, which restored the transcription of the gene of resistance to kanamycin in Escherichia coli and Leptospira biflexa, corroborating this way the function of the insertion as a promoter for both organisms.
Asunto(s)
Proteínas Bacterianas , Farmacorresistencia Bacteriana/genética , Kanamicina/farmacología , Leptospira interrogans/efectos de los fármacos , Leptospira interrogans/genética , Lipopolisacáridos/biosíntesis , Plásmidos/genética , Transcripción Genética , Escherichia coli/efectos de los fármacos , Leptospira interrogans/metabolismo , Antígenos O , Regiones Promotoras Genéticas/genéticaRESUMEN
A fragment of 1.539 pb of the gene homologous to gspD of Hardjobovis was clonated in the pET28a vector and it was transformed into E. coli C43 and Rosetta strains. The sequence of GspD(L) showed 46 % of similitude with E. coli GspD secretin. The expression of recombinant GspD(L) was obtained in Rosetta strain.