RESUMEN
BACKGROUND: Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition. RESULTS: A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype. CONCLUSIONS: This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.
Asunto(s)
Bacteriófagos/genética , ADN Bacteriano/genética , Familia de Multigenes/genética , Serogrupo , Shigella flexneri/genética , Shigella flexneri/virología , Integración Viral/genética , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Southern Blotting , Disentería Bacilar/microbiología , Evolución Molecular , Genoma Viral , Glucosiltransferasas/genética , Antígenos O/genética , Reacción en Cadena de la Polimerasa , Profagos/genética , ARN de Transferencia , Análisis de Secuencia , Serotipificación , Shigella flexneri/inmunologíaRESUMEN
Coli surface antigen 6 (CS6) is one of the most prevalent non-fimbrial colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) bacteria, which are the most common cause of diarrhea among infants and children in developing countries. Since immune protection against ETEC is mainly mediated by locally produced IgA antibodies in the gut, much effort is focused on the development of an oral CF-based vaccine. Previous work has described the preparation of candidate E. coli vaccine strains expressing immunogenic amounts of fimbrial CF antigens such as CFA/I and CS2, which are retained after formalin treatment. However, attempts to generate E. coli expressing immunogenic amounts of CS6 and to preserve the immunological activity of the CS6 protein in a killed whole-cell vaccine have failed until now. Here we describe the construction of a recombinant non-toxigenic E. coli strain, with thyA as a non-antibiotic-based selection, which expresses large amounts of CS6 antigen on the bacterial surface, and show that phenol inactivation of the bacteria does not destroy the CS6 antigen properties. Oral immunization of mice with such phenol-killed CS6 over-expressing E. coli bacteria induced strong fecal and intestinal IgA and serum IgG+IgM antibody responses to CS6 that exceeded the responses induced by an ETEC reference strain naturally expressing CS6 and previously used as a vaccine strain. Our data indicate that the described phenol-inactivated non-toxigenic and CS6 over-expressing E. coli strain may be a useful component in an oral ETEC vaccine.
Asunto(s)
Formación de Anticuerpos , Antígenos Bacterianos/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Escherichia coli/inmunología , Administración Oral , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Femenino , Formaldehído , Vectores Genéticos , Inmunidad Mucosa , Inmunoglobulina A/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Intestinos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FenolRESUMEN
The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.
Asunto(s)
Glucosiltransferasas/metabolismo , Antígenos O/metabolismo , Shigella flexneri/enzimología , Mapeo Cromosómico , Cromosomas Bacterianos , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Familia de Multigenes , Filogenia , Shigella flexneri/clasificación , Shigella flexneri/genéticaRESUMEN
The nptA gene of Vibrio cholerae has significant protein sequence homology with type II sodium-dependent phosphate (P(i)) cotransporters found in animals but not previously identified in prokaryotes. The phylogeny of known type II cotransporter sequences indicates that nptA may be either an ancestral gene or a gene acquired from a higher eukaryotic source. The gene was cloned into an expression vector under the control of an inducible promoter and expressed in Escherichia coli. The results demonstrate that nptA encodes a functional protein with activity similar to that of the animal enzyme, catalyzing high-affinity, sodium-dependent P(i) uptake with comparable affinities for both sodium and phosphate ions. Furthermore, the activity of NptA is influenced by pH, again in a manner similar to that of the NaPi-2a subtype of the animal enzyme, although it lacks the corresponding REK motif thought to be responsible for this phenomenon. P(i) uptake activity, a component of which appeared to be sodium dependent, was increased in V. cholerae by phosphate starvation. However, it appears from the use of a reporter gene expressed from the nptA promoter that none of this activity is attributable to the induction of expression from nptA. It is thus proposed that the physiological function of NptA protein may be the rapid uptake of P(i) in preparation for rapid growth in nutrient-rich environments and that it may therefore play a role in establishing infection.