RESUMO
Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains-Lacticaseibacillus paracasei BL23 and Lacticaseibacillus rhamnosus GG-in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.
RESUMO
Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.
Assuntos
Ácido Láctico/análise , Sequência de Bases , Produtos Fermentados do Leite , Fermentação , Técnicas Genéticas , Variação Genética , Técnicas In Vitro , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Leite , Reação em Cadeia da Polimerase , Amostras de Alimentos , Genótipo , Cabras , Métodos , MétodosRESUMO
Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.
RESUMO
Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.
RESUMO
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
Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.
Assuntos
Proteínas do Capsídeo/metabolismo , Lacticaseibacillus casei/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Recombinação Genética , Vacinas Virais , Vírion/metabolismo , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Células Cultivadas , Papillomavirus Humano 16/metabolismo , Humanos , Imunização , Lacticaseibacillus casei/genética , Lactose/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Oncogênicas Virais/administração & dosagem , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Óperon , Regiões Promotoras Genéticas , Conformação Proteica , Spodoptera , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.
Assuntos
Feminino , Humanos , /classificação , VacinasRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Lacticaseibacillus casei/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras , Adesinas Bacterianas , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Expressão Gênica , Lacticaseibacillus casei/genética , Lipoproteínas/genética , Lipoproteínas/imunologia , Vacinas Pneumocócicas/genética , Vacinas Pneumocócicas/imunologia , Vacinas Pneumocócicas/farmacologia , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/genéticaRESUMO
Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source. However, ptsHI genes encoding HPr and EI proteins belonging to a PTS were cloned, sequenced and characterized. Both HPr and EI proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins. HPr(His(6)) was purified from wild-type and a Ser46/Gln mutant of B. sphaericus, and used for in vitro phosphorylation experiments using extracts from either B. sphaericus or Bacillus subtilis as kinase source. The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained. The findings also proved indirectly the existence of an HPr kinase activity in B. sphaericus. The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orf2). In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic. Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an EII(Nag) element of the PTS, as yet uncharacterized. In addition, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc. These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B. sphaericus. They also open new possibilities for genetic improvements in industrial applications.
Assuntos
Acetilglucosamina/metabolismo , Bacillus/metabolismo , Proteínas de Bactérias , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Trifosfato de Adenosina/metabolismo , Bacillus/genética , Sequência de Bases , Transporte Biológico Ativo , Clonagem Molecular , DNA Bacteriano/genética , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Fosfoenolpiruvato/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosfotransferases (Aceptor do Grupo Nitrogenado)/genética , Fosfotransferases (Aceptor do Grupo Nitrogenado)/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Transcrição GênicaRESUMO
Some strains of Bacillus sphaericus are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the pfk gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes.