Enteropathogenic Escherichia coli (EPEC) express a plasmid-encoded type IV pilus termed bundle-forming pilus, which is associated with the formation of bacterial microcolonies on cultured epithelial cells. Bacterial attachment and effacement of the enterocyte brush border membrane is attributed to a surface outer membrane protein adhesin termed intimin and EPEC-secreted proteins EspA, EspB, and EspD. Except for intimin, production in vivo or antibody response against these virulence determinants during natural EPEC infections in young children has not been demonstrated. Antibody responses against BfpA, intimin, EspA, and EspB were investigated in Brazilian children naturally infected with EPEC. Generally, IgG antibodies against BfpA and EspB were the most commonly found, followed by anti-EspA and intimin antibodies. Thus, bundle-forming pilus and locus of enterocyte attachment-encoded products are produced in vivo during natural EPEC infections and elicit an immune response against heterologous EPEC virulence determinants. These findings have important implications in the immunoprophylaxis against EPEC infections.

Human enterotoxigenic Escherichia coli (ETEC) produces a type IV pilus termed tongus which is encoded on large plasmids in association with colonization factor antigens (CFAs) and enterotoxins. A plasmid-derived 7 kbp BamHl DNA fragment hybridizing with an oligonucleotide probe designed from the aminoterminal amino acid sequence of the denatured 22 kDa structural longus pilin subunit was subcloned and sequenced. DNA sequencing analysis revealed an open reading frame, designated lngA, whose predicted amino acid sequence matched perfectly the N-terminal sequence of LngA obtained by Edman degradation. h g A is the first gene described of the longus gene cluster. Cloned h g A encoded and expressed a prepilin protein of 236 residues with a calculated mass of 25-17 kDa. The prepilin is apparently processed into a mature pilin of 206 residues with a calculated mass of 21.5 kDa. The predicted peptide sequence of lngA showed 7 8 8 and 37% identity to CFAllll pilin (CofA) of ETEC and the toxin-coregulated pilus (TcpA) of Vibrio cholerae. Peptide sequence homology between lngA and cofA was more prominent towards the amino terminus than within the carboxy region. Like other type IV pilins, LngA contains two cysteine residues towards the carboxy-terminal region.Transmission electron microscopy and immunoblot analysis of ETEC strains expressing either longus or CFNIIi detected antigenic differences between native and denatured epitopes of these pili. In addition, differential regulation of pilus expression was identified when ETEC strains were grown in different media. Our data indicate that longus and CFAllll are two distinct but yet highly related type IV pili of ETEC.

Enterotoxigenic Escherichia coli produces a long type 4 pilus called Longus. The regulatory elements and the environmental signals controlling the expression of Longus-encoding genes are unknown. We identified two genes lngR and lngS in the Longus operon, whose predicted products share homology with transcriptional regulators. Isogenic lngR and lngS mutants were considerably affected in transcription of lngA pilin gene. The expression of lngA, lngR and lngS genes was optimally expressed at 37°C at pH 7.5. The presence of glucose and sodium chloride had a positive effect on Longus expression. The presence of divalent ions, particularly calcium, appears to be an important stimulus for Longus production. In addition, we studied H-NS, CpxR and CRP global regulators, on Longus expression. The response regulator CpxR appears to function as a positive regulator of lng genes as the cpxR mutant showed reduced levels of lngRSA expression. In contrast, H-NS and CRP function as negative regulators since expression of lngA was up-regulated in isogenic hns and crp mutants. H-NS and CRP were required for salt- and glucose-mediated regulation of Longus. Our data suggest the existence of a complex regulatory network controlling Longus expression, involving both local and global regulators in response to different environmental signals.

La vaginosis bacteriana (VB) es una alteración frecuente de la microbiota vaginal en mujeres en edad reproductiva. El diagnóstico puede ser efectuado aplicando criterios clínicos o por la evaluación de los morfotipos bacterianos presentes en la tinción de Gram realizada a la secreción vaginal o mediante procedimientos microbiológicos, los cuales se desarrollaron como una alternativa al diagnóstico clínico, reemplazándolo paulatinamente. El objetivo del presente trabajo fue determinar la efectividad de los métodos de Amsel e Ison-Hay, para el diagnóstico de vaginosis bacteriana, empleando el método de Nugent como estándar. En este estudio se analizaron 305 muestras de secreción vaginal de pacientes del Hospital Regional ISSSTE Puebla. Las muestras se procesaron y analizaron para el diagnóstico de VB, siguiendo las recomendaciones de los métodos de Amsel, Nugent e Ison-Hay. El análisis de los resultados indicó un 12.8 % por Nugent, 31.1 % de VB por el método de Amsel y 36.7 % por Ison-Hay; sugiriendo que ambas técnicas muestran una alta tasa de falsos positivos. La sensibilidad para el método de Amsel e Ison-Hay fue de 97.44 %, la especificidad fue de 78.57 % y 72.18 % para Amsel e Ison-Hay, respectivamente. En conclusión, dado a los resultados obtenidos y a las mínimas diferencias entre los métodos analizados, se recomienda realizar los criterios de Amsel, seguidos de la observación de la tinción de Gram del flujo vaginal para la valoración de la microbiota por el método de Ison-Hay, para tener un mejor diagnóstico de VB, cuando no se emplea el método de Nugent.

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.