RESUMO
The probiotic Lactobacillus casei catabolizes galacto-N-biose (GNB) and lacto-N-biose (LNB) by using a transport system and metabolic routes different from those of Bifidobacterium. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Inactivation of gnbC (EIIC) or ptsI (Enzyme I) of the phosphoenolpyruvate : sugar phosphotransferase system (PTS) prevented the growth on those three carbohydrates, indicating that they are transported and phosphorylated by the same PTS(Gnb) . Enzyme activities and growth analysis with knockout mutants showed that GnbG (phospho-ß-galactosidase) hydrolyses both disaccharides. However, GnbF (N-acetylgalactosamine-6P deacetylase) and GnbE (galactosamine-6P isomerase/deaminase) are involved in GNB but not in LNB fermentation. The utilization of LNB depends on nagA (N-acetylglucosamine-6P deacetylase), showing that the N-acetylhexosamine moieties of GNB and LNB follow different catabolic routes. A lacAB mutant (galactose-6P isomerase) was impaired in GNB and LNB utilization, indicating that their galactose moiety is channelled through the tagatose-6P pathway. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR, which binds to a 26 bp DNA region containing inverted repeats exhibiting a 2T/2A conserved core. The data represent the first characterization of novel metabolic pathways for human milk oligosaccharides and glycoconjugate structures in Firmicutes.
Assuntos
Acetilglucosamina/análogos & derivados , Dissacarídeos/metabolismo , Lacticaseibacillus casei/genética , Lacticaseibacillus casei/metabolismo , Leite Humano/química , Mucosa/química , Família Multigênica , Fosfoenolpiruvato/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Galactose/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Humanos , Mutação , Óperon , Polissacarídeos , Reação em Cadeia da Polimerase em Tempo Real , beta-Galactosidase/genéticaRESUMO
Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Hemaglutinação/imunologia , Lacticaseibacillus casei/genética , Fatores de Virulência de Bordetella/imunologia , Coqueluche/prevenção & controle , Adesinas Bacterianas/genética , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/genética , Feminino , Imunidade nas Mucosas , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Vacina contra Coqueluche/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia , Transformação Bacteriana , Vacinas Sintéticas/imunologia , Fatores de Virulência de Bordetella/genéticaRESUMO
Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 10(9) CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/análise , Aderência Bacteriana/imunologia , Lipoproteínas/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Mucosa Respiratória/imunologia , Streptococcus pneumoniae/imunologia , Vacinação , Vacinas de DNA/administração & dosagem , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/genética , Administração Intranasal , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Feminino , Imunoglobulina A/análise , Imunoglobulina G/sangue , Lactobacillus/genética , Lactobacillus/metabolismo , Lipoproteínas/biossíntese , Lipoproteínas/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Saliva/imunologia , Especificidade da EspécieRESUMO
A number of recent research works in lactic acid bacteria aim towards the design of new strains that could be used as live vectors for the delivery of antigens for oral vaccination, or other therapeutic molecules. In this work, an inducible expression system based on the Lactobacillus casei lactose operon promoter was used to express three important surface antigens of Streptococcus pneumoniae in this lactic acid bacterium: a virulence-related pneumococcal surface antigen (PsaA) and two variants of the virulence factor PspA (pneumococcal surface protein A). Expression of the three proteins was induced upon growth on lactose and strongly repressed by glucose. These proteins were produced intracellularly. Also, secretion to the growth medium was achieved by means of a fusion to the secreting and processing signals from the L. casei surface proteinase. Interestingly, while secreted PspA proteins were found in the culture supernatants, PsaA remained trapped in the cell wall. Expression of pneumococcal antigens in a food-grade organism opens an alternative for mucosal vaccination against this important pathogen.