RESUMO
The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.
Assuntos
Animais , Anticorpos Monoclonais/química , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Índia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes/química , Virulência , Fatores de Virulência , Yersinia/metabolismo , Yersinia enterocolitica/metabolismo , Yersinia pestis/metabolismo , Yersinia pseudotuberculosis/metabolismoRESUMO
BACKGROUND & OBJECTIVES: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences of the major subunit protein of the toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between the classical and El Tor biotypes. However, the presence of classical biotype specific tcpA amplicon with the El Tor strains often complicates the interpretation. The effect of PCR variables on the amplification of biotype specific tcpA in the multiplex PCR has been investigated. METHODS: Reference strains of toxigenic V. cholerae O1 belonging to classical and El Tor biotypes were selected to optimize the PCR variables for the unambiguous biotype determination by multiplex PCR. RESULTS: In the multiplex PCR assay, a reduction in the reaction volume from 100 microliters to 25 microliters and the annealing temperature of 64 degrees C, the El Tor strain produced ctxA amplicon (302 bp) along with tcpA amplicons of 618 bp and 472 bp which are specific for classical and El Tor tcpA respectively. The simplex PCR with biotype specific tcpA primer pairs showed the amplification of either 472 bp or 618 bp tcpA amplicon with El Tor template. With the classical biotype strain, the specific primer pair yielded tcpA amplicon of the expected size. Lowering of PCR annealing temperature from 64 to 60 degrees C resulted in the elimination of the amplification of the nonspecific tcpA amplicon with El Tor strain. INTERPRETATION & CONCLUSION: A comparison of the theoretical melting temperature (Tm) values of the reacting primers, and their alignment to the biotype specific tcpA revealed the basis of unambiguous biotyping of V. cholerae O1 at a PCR annealing temperature of 60 degrees C.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Técnicas de Tipagem Bacteriana , Sequência de Bases , Cólera/microbiologia , Proteínas de Fímbrias , Fímbrias Bacterianas/metabolismo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Subunidades Proteicas , Temperatura , Vibrio cholerae/classificaçãoRESUMO
The outer membrane protein (OMP) and lipopolysaccharide (LPS) patterns of 12 strains of serogroups of enterotoxigenic E. coli frequntly isolated in Säo Paulo city werte determined by fractionation techniques and by sodium dodecyl sulfate-plyacrylamide gel electrophoresis (SDS-PAGE). Five O6, three O78 and four O128 serogroup isolates of different serotypes (flagellar antigens) and virulence factors (toxins and colonization factor antigens) showed a high degree of variability in their OMP pattern and at least nine groups could be identified. The analysis of LPS aptterns by SDS-PAGE showed a homogenous profile for the O6 strains and some minor differences for the O128 and 078 strains. The oresented data indicate that analysis of OMP and LPS by SDS-PAGE may further improve the discriminating ability of extensively used serological techniques or the detection of virulence factors and could be a useful tool in epidemiological studies of enterotoxigenic E. coli (ETEC) strains from this area