RESUMEN

The Vi antigen in Salmonella typhi is stably expressed and may act to protect the strain against the defensive system of the host. Citrobacter freundii, not usually a common human pathogen, also expresses the Vi antigen but expresses it unstably, exhibiting a reversible transition between the Vi+ and Vi- states. Two widely separated chromosomal regions, ViaA and ViaB, are needed for Vi synthesis. Escherichia coli K-12 harboring a functional ViaB plasmid can also express Vi antigen, but the cloned ViaB sequence can only be stably maintained and expressed in recA hosts. Vi- derivatives arise either through IS1-like insertional events occurring in ViaB sequences or by chromosomal mutations at the ViaA region. P1vir mapping indicates that the ViaA mutations are located at min 47.75 on the E. coli chromosome. All the spontaneous viaA mutants isolated from E. coli and S. typhi were identified as rcsB mutants by complementation tests using plasmid pJB100. Introduction of rcsA::Tn10 into E. coli harboring functional ViaB sequences eliminates the expression of Vi antigen. These results indicate that Vi antigen synthesis is regulated by the same regulatory proteins involved in colanic acid synthesis in E. coli.

Asunto(s)

Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Citrobacter freundii/genética , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Polisacáridos Bacterianos , Factores de Transcripción , Alelos , Antígenos Bacterianos/biosíntesis , Cápsulas Bacterianas/biosíntesis , Proteínas Bacterianas/biosíntesis , Clonación Molecular , Escherichia coli/genética , Genes Reguladores , Prueba de Complementación Genética , Mutagénesis Insercional , Rec A Recombinasas/genética , Mapeo Restrictivo , Virulencia/genética

RESUMEN

When Escherichia coli HB101 harbors pWR127, a plasmid comprising the viaB gene from Citrobacter freundii WR7004 and the ColE1-derived pACKC1, the strain produces the virulence (Vi) antigen. Vi antigen expression is abolished (Vi- phenotype), however, when an IS1 or IS1-like DNA element inserts into the viaB region. To determine the sites of IS1 insertion, pWR127 DNAs extracted from 95 independently isolated Vi- strains were analyzed by digestion with the restriction endonuclease PstI and agarose gel electrophoresis. Ten insertion sites were found distributed non-randomly in an area of about 1.3 kb. Nine Vi+ strains (two Citrobacter, two E. coli, and five Salmonella strains), four of which contain pWR127, were then tested for the presence of IS1 by DNA-DNA hybridization. Of the nine strains, five were stable Vi+ and did not contain IS1. The other four which generated Vi- strains, contained IS1. When pRR134, a plasmid that contains IS1 was transferred into a stable Vi+ Salmonella typhimurium strain carrying pWR127 (OU5140), Vi- strains were produced from which pWR127 derivatives carrying IS1 inserts could be isolated. It appears, therefore, that the presence of an IS1 or IS1-like element in a strain is required for conversion of the Vi+ expression state to the Vi- expression state.

Asunto(s)

Antígenos Bacterianos/genética , Elementos Transponibles de ADN/genética , Enterobacteriaceae/genética , Regulación Bacteriana de la Expresión Génica/genética , Polisacáridos Bacterianos , Virulencia/genética , Hibridación de Ácido Nucleico , Plásmidos/genética

RESUMEN

Colonial variation of Shigella flexneri serotype 2a from the translucent (2457T) to the opaque form (2457O) occurs spontaneously once in 10(4) cell divisions, with concomitant loss of ipa gene expression and virulence. The appearance of 2457O was associated with the insertional inactivation of virF, an invasion plasmid-encoded positive regulator of ipa gene expression. Plasmid pWR110, a Tn5-tagged invasion plasmid that restores the invasive phenotype to plasmid-cured Shigella derivatives, was conjugally transferred into 2457O. Synthesis of the invasion-associated IpaB and IpaC polypeptides, normally present on the surface of virulent shigellae, and the invasive phenotype were restored in 2457O(pWR110) transconjugants. Plasmid DNA restriction endonuclease patterns of 2457T and 2457O, along with hybridization analysis, showed that a SalI fragment carrying the virF gene in 2457O had increased in size relative to its counterpart in 2457T. Analysis of virF DNA sequences amplified by the polymerase chain reaction revealed that the virF sequence from 2457O was 780 bp larger than that amplified from 2457T. Moreover, the virF sequence amplified from 2457O hybridized to an IS1 DNA probe whereas the amplified 2457T virF sequence did not. DNA sequence analysis mapped the insertion element, designated IS1SFO, within an A.T-rich region of the virF open reading frame and identified a 9-bp virF target sequence that was duplicated at the insertion site of IS1SFO. The DNA sequence of IS1SFO was greater than 99% homologous to IS1F. Plasmid pWR600, carrying a 1,260-bp HpaII fragment encoding a wild-type virF gene, was able to restore the virulent phenotype and translucent colonial morphology to nine independently isolated 2457O hosts.

Asunto(s)

Proteínas Bacterianas/genética , Elementos Transponibles de ADN , Mutagénesis Insercional , Shigella flexneri/patogenicidad , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , ADN/análisis , Genes Bacterianos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , Plásmidos/inmunología , Reacción en Cadena de la Polimerasa , Shigella flexneri/genética , Transducción Genética , Virulencia/genética

RESUMEN

New typhoid vaccines have been sought to replace the reactogenic parenteral whole cell vaccines. Both Ty21a, a live oral attenuated Vi-negative Salmonella typhi vaccine strain, and parenteral Vi polysaccharide vaccine are safe and efficacious in field trials. To achieve potentially greater protective efficacy, a derivative of Ty21a that expresses Vi polysaccharide was prepared and its safety and immunogenicity assessed in 27 adult volunteers. The volunteers received either one dose of 5 x 10(5), 5 x 10(7), or 5 x 10(9) cfu or three doses of 5 x 10(9) cfu of lyophilized vaccine and were observed for adverse effects on a research isolation ward. The vaccine was well tolerated; however, at the highest dose, 13% of volunteers had mild diarrhea. Serial blood cultures were negative for the vaccine strain. Vaccine was shed in the stool of most volunteers at the higher doses for 1-4 days. No serum antibodies to Vi, circulating cells secreting antibody to Vi, or jejunal fluid antibodies to Vi were detected in any volunteer.

Asunto(s)

Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Salmonella typhi/inmunología , Vacunas Tifoides-Paratifoides/inmunología , Adulto , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Polisacáridos Bacterianos/inmunología , Vacunas Tifoides-Paratifoides/efectos adversos , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología

RESUMEN

A number of plasmidless strains were obtained by curing the 90 kilobase pair (kb) virulence plasmid from six strains, C5, TML, W118, SR11, LT2 and Fisher, of Salmonella serovar Typhimurium. A number of transposon (Tn5) tagged 90 kb plasmids, also derived from these Typhimurium strains, were then transferred back into these plasmidless strains. Plasmid-cured strains, reconstituted strains, and the parental strains were tested for their virulence in BALB/c mice. There were two groups of Typhimurium strains: one required the 90 kb plasmid to express high virulence (LD50 less than 50 bacteria), and the other, regardless of the presence or absence of the 90 kb plasmid, maintained the same level of virulence at LD50 = 10 to 7 x 10(5) bacteria. Among the plasmidless strains, there were strains with a virulence level as low as LD50 = 10(7) bacteria, which was unaffected by the presence of the 90 kb plasmid.

Asunto(s)

Plásmidos , Salmonella typhimurium/patogenicidad , Animales , Composición de Base , Elementos Transponibles de ADN , Femenino , Dosificación Letal Mediana , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Salmonella typhimurium/clasificación , Salmonella typhimurium/genética , Especificidad de la Especie , Virulencia

RESUMEN

Vaccine development and understanding of cellular immune stimulatory mechanisms have been impeded by the paucity of data on microbial antigens that stimulate protective immunity. We describe here a general method for identifying and isolating peptide antigens that specifically stimulate sensitized lymphocytes. First, Salmonella typhimurium C5 genomic DNA fragments were subcloned into Escherichia coli by use of the lambda gt11 expression vector. Next, antigens expressed by recombinant phage from this genomic library were tested for their capacity to stimulate proliferative responses in pooled lymphocytes obtained from BALB/c mice infected 14 days earlier with S. typhimurium. Of 2000 recombinant phages tested, 5 stimulated a polypeptide-antigen-specific proliferative response. Physical analyses of these 5 recombinant phages revealed cloned inserts of 0.5-2.4 kilobase pairs representing nonoverlapping regions of the C5 chromosome. Four of the five insert DNAs hybridized at high stringency to both S. typhimurium and Salmonella typhi total chromosomal DNA, suggesting that these pathogens of different host specificity share several antigenic determinants. Use of sensitized primary polyclonal lymphocytes provides a rapid and simple method for screening recombinant DNA libraries for clones that stimulate specific immune responses and avoids the use of cloned lymphocyte cell lines. This approach should be generally applicable to similar studies in different hosts of many other microbial pathogens.

Asunto(s)

Antígenos Bacterianos/inmunología , Activación de Linfocitos , Salmonella typhimurium/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Bacteriófago lambda/genética , Clonación Molecular , Sondas de ADN , Replicación del ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Femenino , Biblioteca de Genes , Genes Bacterianos , Ratones , Ratones Endogámicos BALB C , Mapeo Restrictivo , Infecciones por Salmonella/inmunología , Salmonella typhimurium/genética

RESUMEN

The chromosomally encoded galactose utilization (gal) operons of Salmonella typhimurium and S. typhi were each cloned on similar 5.5-kilobase HindIII fragments into pBR322 and were identified by complementation of Gal- Escherichia coli strains. Restriction endonuclease analyses indicated that these Salmonellae operons share considerable homology, but some heterogeneities in restriction sites were observed. Subcloning and exonuclease mapping experiments showed that both operons have the same genetic organization as that established for the E. coli gal operon (i.e., 5' end, promoter, epimerase, transferase, kinase, and 3' end). Two gal operator regions (oE and oI) of S. typhimurium, identified by repressor titration in an E. coli superrepressor [galR(Sup)] mutant, were sequenced and found to flank the promoter region. This promoter region is identical to the -10 and -35 regions of the E. coli gal operon. Minicell studies demonstrated that the three gal structural genes of S. typhimurium encode separate polypeptides of 39 kilodaltons (kDa) (epimerase, 337 amino acids [aa's]), 41 kDa (transferase, 348 aa's), and 43 kDa (kinase, 380 aa's). Despite functional and organizational similarities, DNA sequence analysis revealed that the S. typhimurium gal genes show less than 70% homology to the E. coli gal operon. Because of codon degeneracy, the deduced amino acid sequences of these polypeptides are highly conserved (greater than 90% homology) as compared with those of the E. coli gal enzymes. These studies have defined basic genetic parameters of the gal genes of two medically important Salmonella species, and our findings support the hypothesized divergent evolution of E. coli and Salmonella spp. from a common ancestral parent bacterium.

Asunto(s)

Galactosa/metabolismo , Operón , Salmonella typhi/genética , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Exodesoxirribonucleasas , Biblioteca de Genes , Prueba de Complementación Genética , Metionina/metabolismo , Datos de Secuencia Molecular , Mapeo Nucleótido , Conformación Proteica , Mapeo Restrictivo

RESUMEN

We examined the compatibility of the Salmonella virulence plasmids of serovars choleraesuis, dublin, enteritidis, gallinarum and pullorum and the cryptic Salmonella plasmids of serovars copenhagen, durban, give, infantis and sendai, with the 90 kilobase pair (kb) virulence plasmid of S. typhimurium. The 90 kb virulence plasmid of S. typhimurium in the form of pWR33, a cointegrate of F'::Tn10lac+ts and the 90 kb virulence plasmid, was transferred by bacterial conjugation into the Salmonella strains (except for S. sendai). The compatibility of their plasmids with the 90 kb virulence plasmid of S. typhimurium was then tested. Separately, a 90 kb virulence plasmid tagged with Tn5 was transformed into the S. sendai strain. The 90 kb virulence plasmid of S. typhimurium was found to be incompatible with the plasmids of serovars choleraesuis, copenhagen, dublin, enteritidis, and sendai, but compatible with the plasmids of serovars durban, gallinarum, give, infantis, and pullorum.

Asunto(s)

Salmonella/patogenicidad , Conjugación Genética , Electroforesis en Gel de Agar , Plásmidos/genética , Salmonella/clasificación , Salmonella/genética , Serotipificación , Virulencia/genética

RESUMEN

The viaB locus coding for the Vi antigen of Salmonella typhi Ty2 was cloned on a 40.6-kilobase fragment into the cosmid vector pHC79. The live, oral, attenuated Vi-negative S. typhi Ty21a vaccine strain was transformed with the recombinant cosmid encoding the viaB locus. Homologous recombination of the viaB locus into the chromosome of S. typhi Ty21a was induced by UV irradiation, and Vi-positive recombinants were selected in the presence of D-cycloserine. One such isolate, termed WR4103, contained no plasmids or the attendant antibiotic resistance markers and expressed the Vi antigen stably. Vi antigen extracted from WR4103 was immunologically indistinguishable from Vi antigen purified from S. typhi Ty2. The only detectable difference between Ty21a and WR4103 was in the production of Vi antigen. The mean lethal doses of Ty21a and WR4103 for mice were nearly identical. Immunization of mice with WR4103 engendered a Vi antibody response and afforded complete protection against fatal infection with virulent S. typhi Ty2. Thus, S. typhi WR4103 may serve as an improved oral vaccine for protection against typhoid fever.

Asunto(s)

Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Polisacáridos Bacterianos , Salmonella typhi/inmunología , Vacunas Sintéticas/inmunología , Vacunas/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Proteínas Bacterianas/análisis , Vacunas Bacterianas/genética , Clonación Molecular , Galactosa/metabolismo , Hígado/microbiología , Ratones , Fenotipo , Salmonella typhi/genética , Salmonella typhi/patogenicidad , Bazo/microbiología , Fiebre Tifoidea/microbiología , Fiebre Tifoidea/prevención & control

RESUMEN

Salmonella typhi, the causative agent of typhoid fever, must invade the human gastrointestinal tract and multiply within the host to cause disease. We have cloned from S. typhi Ty2 a chromosomal region that confers upon Escherichia coli HB101 the ability to invade cultured human intestinal epithelial cells. Three invasion-positive recombinant cosmids were isolated and restriction endonuclease analyses of the inserts showed a 33-kilobase region of identity. Transmission electron microscopy of epithelial cells invaded by S. typhi Ty2 or E. coli HB101 carrying an invasion cosmid showed intracellular bacteria contained within endocytic vacuoles. One of the invasion cosmids was mutagenized with transposon Tn5 to identify the cloned sequences that are required for the invasive phenotype. Seven of 92 independent Tn5 insertions within the common 33-kilobase region eliminated invasive ability and revealed at least four separate loci that are required for invasion. Penetration of epithelial cells by Ty2 and HB101 carrying the cloned invasion determinants was inhibited by cytochalasin B and D, indicating that epithelial cell endocytosis of S. typhi is a microfilament-dependent event. The invasion cosmids were found to carry the recA and srlC genes indicating that the cloned invasion determinants are located at about 58 minutes on the S. typhi chromosome. With a segment of the cloned S. typhi invasion region used as a probe, homologous sequences were isolated from Salmonella typhimurium. Two independent S. typhimurium recombinant cosmids containing the entire 33-kilobase common region identified in S. typhi were isolated, but these cosmids did not confer upon HB101 the ability to invade epithelial cells.

Asunto(s)

Clonación Molecular , Escherichia coli/genética , Intestinos/microbiología , Salmonella typhi/patogenicidad , Línea Celular , Cósmidos , Citocalasina B/farmacología , Epitelio/microbiología , Epitelio/ultraestructura , Humanos , Microscopía Electrónica , Mutación , Mapeo Restrictivo , Salmonella typhi/efectos de los fármacos , Salmonella typhi/genética

RESUMEN

Mice infected subcutaneously with wild-type Salmonella typhimurium, SR11, developed a significant splenomegaly when compared with mice infected with an equal number of a plasmid-cured strain. Further, the bacterial load in the spleen at 14 days after infection, measured as colony-forming units per gram tissue, was significantly higher in mice infected with the parent strain than in mice infected with the plasmid-cured strain. These data confirm the previously reported plasmid-associated ability of Salmonella to multiply within the spleen. In addition, lymph node cells (LNC) from mice infected with the parent strain had a significantly reduced ability to proliferate in response to concanavalin A, a T-cell mitogen, and to heat-killed S. typhimurium cells when compared with LNC isolated from mice infected with the plasmid-cured strain. Finally, reintroduction of a functional Tn5-tagged 90-kb plasmid into a plasmid-free strain restored its capacity to cause a marked splenomegaly and to suppress lymph node cell proliferation in BALB/c mice. These data demonstrate that the 90-kb plasmid of highly virulent S. typhimurium strains mediates several novel pathogenic properties in infected mice: (1) enhancement of the ability of Salmonella to multiply within the spleen; (2) stimulation of a splenic inflammatory response as displayed by marked splenomegaly; and (3) a general suppression of lymphocyte responsiveness to both T-cell mitogens and specific Salmonella antigens.

Asunto(s)

Terapia de Inmunosupresión , Plásmidos , Infecciones por Salmonella/inmunología , Salmonella typhimurium/patogenicidad , Esplenomegalia/microbiología , Animales , Células Cultivadas , Elementos Transponibles de ADN , Femenino , Activación de Linfocitos , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Salmonella/patología , Salmonella typhimurium/genética , Esplenomegalia/inmunología , Virulencia

RESUMEN

Citrobacter freundii strain WR7004 reversibly expresses the virulence capsular antigen Vi, whose production is controlled by two distinct chromosomal loci, viaA and viaB. The rate of oscillation between the Vi-producing (Vi+) state and the Vi-nonproducing (Vi-) state in strain WR7004 ranges from 2 X 10(-4) to 7 X 10(-3) transitions per bacterium per generation. A similarly high conversion rate from Vi+ to Vi- occurs in Escherichia coli HB101 harboring pWR127, a plasmid that contains the 18-kilobase-pair (kb) viaB region cloned from WR7004. However, the Vi- state in HB101 harboring pWR127 was so stable that transition to the Vi+ state was not detected (less than 10(-10) per bacterium per generation). When pWR127 DNA derived from Vi- strains of HB101 harboring pWR127 was transformed in HB101 recipient, a small number of Vi+ transformants were seen among the transformants, which were predominantly Vi-. The viaB region consists of two EcoRI digestion fragments, A (8.6 kb) and B (9.4 kb). A discrete 700- to 800-base-pair DNA element was found to be inserted in the EcoRI B fragment of the viaB region of pWR127 when derived from Vi- strains. However, no such DNA element was found in the pWR127 DNA isolated from Vi+ strains. This discrete DNA element inserts into a recombinational hot spot 1.1 kb from the end of the EcoRI B fragment and behaves as an insertion sequence 1 (IS1)-like element. The insertion of this IS1-like element in the viaB region thus disrupts expression of the Vi antigen. Restoration of Vi expression results when this element is excised.

Asunto(s)

Antígenos Bacterianos/genética , Deleción Cromosómica , Citrobacter/genética , Elementos Transponibles de ADN , Escherichia coli/genética , Genes Bacterianos , Genes , Polisacáridos Bacterianos , Enzimas de Restricción del ADN , Plásmidos , Transformación Bacteriana

RESUMEN

Immunization with radiation-attenuated malaria sporozoites induces potent cellular immune responses, but the target antigens are unknown and have not previously been elicited by subunit vaccines prepared from the circumsporozoite (CS) protein. A method is described here for inducing protective cell-mediated immunity to sporozoites by immunization with attenuated Salmonella typhimurium transformed with the Plasmodium berghei CS gene. These transformants constitutively express CS antigens and, when used to immunize mice orally, colonize the liver, induce antigen-specific cell-mediated immunity, and protect mice against sporozoite challenge in the absence of antisporozoite antibodies. These data indicate that the CS protein contains T cell epitopes capable of inducing protective cell-mediated immunity, and emphasize the importance of proper antigen presentation in generating this response. Analogous, orally administered vaccines against human malaria might be feasible.

Asunto(s)

Antígenos de Superficie/inmunología , Vacunas Bacterianas/inmunología , Malaria/inmunología , Plasmodium berghei/inmunología , Proteínas Protozoarias , Salmonella typhimurium/inmunología , Animales , Femenino , Hígado/microbiología , Malaria/prevención & control , Ratones , Ratones Endogámicos BALB C , Plásmidos , Salmonella typhimurium/genética

RESUMEN

For protection against dysentery caused by Shigella flexneri 2a, an in vivo-constructed recombinant plasmid with genes specifying the S. flexneri type and group antigens located near the pro (min 6) and his (min 44) chromosomal markers, respectively, was made and transferred to the galE Salmonella typhi strain Ty21a. Strain Ty21a carrying this recombinant plasmid was shown by immunological and biochemical analyses to express the S. flexneri 2a type and group antigens. Mice immunized with this vaccine strain were found to be protected against challenge with virulent S. flexneri 2a, but not significantly against S. typhi challenge, presumably because synthesis of the Shigella antigens interfered with expression of the typhoid antigens. Elimination of the recombinant plasmid from Ty21a allowed this strain to again express typical S. typhi O antigens. Mouse protection against both S. typhi and S. flexneri 2a challenges was achieved with a whole-cell vaccine mixture composed of equal parts of Ty21a and the Ty21a-S. flexneri 2a hybrid strain.

Asunto(s)

Antígenos Bacterianos/genética , Vacunas Bacterianas/genética , Salmonella typhi/inmunología , Shigella flexneri/inmunología , Administración Oral , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Disentería/prevención & control , Regulación de la Expresión Génica , Lipopolisacáridos/inmunología , Ratones , Plásmidos , Shigella flexneri/genética , Fiebre Tifoidea/prevención & control , Vacunas Sintéticas/genética

RESUMEN

Coliphage lambda gene expression is regulated temporally by systems of termination and antitermination of transcription. The lambda-encoded N protein (pN) acting with host factors (Nus) at sites (nut) located downstream from early promoters is the first of these systems to operate during phage development. We report observations on some of the components of this complex system that, in part, address the way in which these elements interact to render RNA polymerase termination-resistant. (1) The isolation of a conditionally lethal cold-sensitive nusA mutation demonstrates that NusA is essential for bacterial growth. (2) The effect on lambda growth in a host in which the Salmonella NusA protein is overproduced suggests that NusA is essential for N-mediated antitermination in phage lambda. (3) A truncated NusA product, representing only the amino two-thirds of the native protein, is active for both bacterial growth and pN action, indicating that the carboxy end of the molecule may not be a functionally important region. (4) lambda pN can function with the heterologous nut region from Salmonella typhimurium phage P22 when lambda pN is overproduced, demonstrating that lambda pN can function with the nut regions of other lambdoid phages. (5) A single base-pair change in the lambda nutR boxA sequence that was selected to permit a lambda derivative to utilize the Salmonella NusA protein restores lambda growth in the Escherichia coli nusA1 host.

Asunto(s)

Proteínas Bacterianas/genética , Bacteriófago lambda/genética , Genes Reguladores , Regiones Terminadoras Genéticas , Factores de Transcripción/genética , Bacteriófago lambda/crecimiento & desarrollo , Secuencia de Bases , ADN Viral , Regulación de la Expresión Génica , Mutación , Transcripción Genética

RESUMEN

Mutations in two Escherichia coli genes, sipA and sipB, result in a specific inhibition of the growth of certain hybrid lambdoid bacteriophages, lambda immP22, that have the early regulatory regions and adjacent genes from bacteriophage P22. The sipB391 mutation maps near minute 56 and exerts the strongest inhibitory effect on the growth of the hybrid phages. The sipA1 mutation maps near minute 72 and plays an auxiliary role: enhancing the action of sipB391. Such a role is not limited to sipA1, since there is a similar enhancement by the nusA1 and nusE71 mutations. The Sip-imposed restriction on the growth of lambda immP22 phages is not observed if the phage carries a mutation in the c1 gene. Perhaps this reflects the fact that the c1 product regulates phage DNA replication and is a major determinant in the decision governing whether the phage takes the lytic or lysogenic pathway. Consistent with this idea is the observation that lambda immP22 DNA replication is severely inhibited in bacteria carrying the sipB391 mutation. It is suggested that sip mutations exaggerate the normal role of c1 in limiting lytic growth. This causes a failure in the expression of sufficient amounts of some or all of the lytic gene products required for phage growth.

Asunto(s)

Bacteriófago lambda/crecimiento & desarrollo , Escherichia coli/genética , Genes Bacterianos , Fagos de Salmonella/inmunología , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Clonación Molecular , Replicación del ADN , ADN Viral/biosíntesis , Genes Virales , Prueba de Complementación Genética , Ligamiento Genético , Mutación , Fenotipo , Replicación Viral

RESUMEN

This study was undertaken to identify a DNA sequence that could be used to facilitate the diagnostic identification of Salmonella typhi, the causative agent of typhoid fever. All virulent S. typhi strains encode a relatively unique capsular antigen termed the virulence (Vi) antigen. Two distinct genetic loci, viaA and viaB, are involved in the synthesis of this antigen. The structural genes, located at viaB, were considered as a possible specific DNA probe. The viaB locus, contained in a recombinant cosmid, was subcloned to various plasmid vectors for this purpose. Selected viaB-region DNA fragments were then analyzed for specificity in DNA colony hybridization reactions with more than 170 strains representing a variety of enteric bacteria. An 8.6-kilobase EcoRI fragment was highly specific for the viaB gene region and was considered a good hybridization probe. This DNA probe should prove useful in rapid diagnostic assays set up to detect S. typhi in mixed bacterial samples (e.g., stools) within a few hours of specimen collection.

Asunto(s)

Antígenos Bacterianos/genética , Técnicas Bacteriológicas , ADN Bacteriano/genética , Hibridación de Ácido Nucleico , Polisacáridos Bacterianos , Salmonella typhi/aislamiento & purificación , Clonación Molecular , Enzimas de Restricción del ADN , ADN Recombinante , Desoxirribonucleasa EcoRI , Enterobacteriaceae/genética , Genes , Genes Bacterianos , Salmonella typhi/genética , Salmonella typhi/inmunología , Salmonella typhi/patogenicidad , Especificidad de la Especie , Virulencia

RESUMEN

phi 80immP22dis, a hybrid between phi 80 and P22, carries all the late genes of phi 80 and most of the P22 early region including the immC and immI bipartite immunity loci. The presence of the immI region allows this hybrid to grow on lysogens of phi 80immP22 hybrids which have the immC locus, but not the immI locus. In addition to these P22 immunity regions, phi 80immP22dis contains the P22 att marker so that the prophage can be inserted into the chromosomal P22 attachment site adjacent to the proA-proB region of the host. Unlike its phi 80 parent which performs specialized transduction of the trp region, phi 80immP22dis transduces markers located adjacent to its attachment site to Escherichia coli K12 recipients at high frequencies (0.3% for argF and 0.18% for proA). Induction of phi 80immP22dis lysogens yields new hybrid phage clones which have incorporated E. coli K12 chromosomal segments in place of the P22 immI to att segment. Having lost the immI region, the new hybrids no longer grow in phi 80immP22 lysogens. These new hybrids, termed phi 80immP22dis-, possess specialized transducing properties, transferring the argF and proA markers at higher frequencies (21% for argF and 12% for proA) than previously obtained with the phi 80immP22dis phage.

Asunto(s)

Colifagos/genética , Escherichia coli/genética , Fagos de Salmonella/genética , Salmonella/genética , Transducción Genética , Genes Virales , Lisogenia , Mutación , Hibridación de Ácido Nucleico , Plásmidos

Asunto(s)

Disentería Bacilar/microbiología , Disentería/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/patogenicidad , Shigella/patogenicidad , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/genética , Vacunas Bacterianas , Mapeo Cromosómico , Cromosomas Bacterianos , Citotoxicidad Inmunológica , ADN Bacteriano/genética , Disentería Bacilar/fisiopatología , Disentería Bacilar/prevención & control , Enterotoxinas/inmunología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Escherichia coli/fisiología , Genes Bacterianos , Humanos , Antígenos O , Plásmidos , Polisacáridos Bacterianos/inmunología , Toxinas Shiga , Shigella/genética , Shigella/crecimiento & desarrollo , Shigella/inmunología , Shigella/fisiología , Shigella flexneri/genética , Shigella flexneri/patogenicidad , Shigella sonnei/genética , Shigella sonnei/patogenicidad , Transformación Bacteriana , Virulencia

RESUMEN

A living oral vaccine, designed to protect against Shigella flexneri 2a infections, was constructed by using Escherichia coli K-12 as a carrier strain. The hybrid strain, designated EC104, contained both chromosomal and plasmid genes from S. flexneri donor strains. In addition to expressing the S. flexneri 2a somatic antigen, it had inherited the property of epithelial-cell invasion. After the oral administration to rhesus monkeys, EC104 was isolated from the feces for up to 3 days, but by day 4 all stool cultures were negative. The serum antibody response against S. flexneri 2a somatic antigen was variable, but the vaccine conferred significant protection against an oral challenge with virulent S. flexneri 2a.

Asunto(s)

Vacunas Bacterianas/inmunología , Disentería Bacilar/prevención & control , Escherichia coli/inmunología , Shigella flexneri/inmunología , Administración Oral , Animales , Vacunas Bacterianas/administración & dosificación , Escherichia coli/genética , Hibridación Genética , Macaca mulatta/inmunología , Plásmidos , Shigella flexneri/genética