RESUMEN
Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection.
Asunto(s)
Antígenos Bacterianos/metabolismo , Membrana Externa Bacteriana/metabolismo , Bacteroides thetaiotaomicron/metabolismo , Vacuna contra la Peste/inmunología , Peste/inmunología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Vesículas Transportadoras/inmunología , Yersinia pestis/fisiología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Bacteroides thetaiotaomicron/genética , Bioingeniería , Muerte Celular , Células Cultivadas , Microbioma Gastrointestinal/genética , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunoglobulina A/metabolismo , Inmunoglobulina G/sangre , Macaca , Peste/prevención & control , Vacuna contra la Peste/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Vesículas Transportadoras/metabolismoRESUMEN
Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed. The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator. Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype. Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L. sakei. A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L. sakei by two successive crossovers. A strain deleted of the lacLM operon encoding the beta-galactosidase of L. sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation of atkYB. Results show that the atkYB operon is induced by small concentrations of CuSO(4) (30 to 40 microM) but not when CuSO(4) is omitted or added at higher concentrations.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Lactobacillus/enzimología , Lactobacillus/genética , Operón , Proteínas Represoras/genética , beta-Galactosidasa/genética , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Clonación Molecular , Intercambio Genético , Escherichia coli/genética , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Proteínas Represoras/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A 7-kb DNA fragment of Lactobacillus sakei, containing the rbsD, rbsK and rbsR genes was sequenced. The genes responsible for ribose utilization are organized differently from what was previously described for model organisms such as Escherichia coli and Bacillus subtilis. No gene encoding RbsA, RbsB and RbsC, the subunits of the ribose ABC-transporter, were present in the rbs gene cluster. Instead, we found an open reading frame coding for RbsU, a protein similar to GltA, the glucose transporter of Staphylococcus xylosus. The disruption of rbsK, encoding the ribokinase, impaired growth on ribose. The disruption of rbsR, encoding the repressor, had no effect on the ability to grow on ribose, but led to overexpression of a large transcript corresponding to rbsU, rbsD and rbsK, suggesting that RbsU might be involved in ribose utilization. Ribose uptake and phosphorylation assays on the wild type strain and various mutants showed that, in ptsI mutants, both ribose uptake and phosphorylation are increased. These increased activities can explain the faster growth rate on ribose that was observed in ptsI mutants. The phosphotransferase system is thus involved in the negative regulation of ribose utilization. This regulation might not act at the transcriptional level since the overexpression of the rbs genes in the rbsR mutant did not lead to the same phenotype. A gene sharing high similarity scores with ackA genes, encoding the acetate kinase, was found upstream from the rbs gene cluster. The unusual location of this gene is maybe not fortuitous since acetate kinase is involved in ribose catabolism.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Lactobacillus/genética , Operón , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Represoras/genética , Ribosa/metabolismo , Secuencia de Bases , Paseo de Cromosoma , Cromosomas Bacterianos , Clonación Molecular , ADN Bacteriano , Genes Bacterianos , Glucosa/metabolismo , Lactobacillus/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosforilación , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/genética , Reacción en Cadena de la Polimerasa/métodos , Transcripción GenéticaRESUMEN
The ptsH and ptsI genes of Lactobacillus sake, encoding the general enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), were cloned and sequenced. HPr (88 amino acids), encoded by ptsH, and enzyme I (574 amino acids), encoded by ptsI, are homologous to the corresponding known enzymes of other bacteria. Nucleotide sequence and mRNA analysis showed that the two genes are cotranscribed in a large transcript encoding both HPr and enzyme I. The transcription of ptsHI was shown to be independent of the carbon source. Four ptsI mutants were constructed by single-crossover recombination. For all mutants, growth on PTS carbohydrates was abolished. Surprisingly, the growth rates of mutants on ribose and arabinose, two carbohydrates which are not transported by the PTS, were accelerated. This unexpected phenotype suggests that the PTS negatively controls ribose and arabinose utilization in L. sake by a mechanism different from the regulation involving HPr described for other gram-positive bacteria.