RESUMO
Designer microbial consortia are an emerging frontier in synthetic biology that enable versatile microbiome engineering. However, the utilization of such consortia is hindered by our limited capacity in rapidly creating ecosystems with desired dynamics. Here we present the development of synthetic communities through social interaction engineering that combines modular pathway reconfiguration with model creation. Specifically, we created six two-strain consortia, each possessing a unique mode of interaction, including commensalism, amensalism, neutralism, cooperation, competition and predation. These consortia follow distinct population dynamics with characteristics determined by the underlying interaction modes. We showed that models derived from two-strain consortia can be used to design three- and four-strain ecosystems with predictable behaviors and further extended to provide insights into community dynamics in space. This work sheds light on the organization of interacting microbial species and provides a systematic framework-social interaction programming-to guide the development of synthetic ecosystems for diverse purposes.
Assuntos
Lactococcus lactis/metabolismo , Consórcios Microbianos , Interações Microbianas , Biologia Sintética , Lactococcus lactis/citologia , Lactococcus lactis/genética , Consórcios Microbianos/genética , Interações Microbianas/genética , Microbiota/genéticaRESUMO
The aim of the study was to neutralize zearalenone by lactic acid bacteria (LAB) such as Lactococcus lactis and Bifidobacterium sp. and investigate the mechanism of zearalenone (ZEA) binding. Neutralization of ZEA by LAB was confirmed by identification of binding kinetics and spectroscopic studies such as Fourier transform infrared spectroscopy (FT-IR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The obtained results showed that the kinetic process of zearalenone binding to L. lactis is not homogeneous but is expressed with an initial rapid stage with about 90% of ZEA biosorption and with a much slower second step. In case of Bifidobacterium sp., the neutralization process is homogeneous; the main stage can be described with about 88% of ZEA biosorption. MALDI-TOF-MS measurements and FTIR analysis confirmed the uptake of zearalenone molecules by bacterial species. Moreover, the assessment of dead and live lactic acid bacteria cells after zearalenone treatment was performed using fluorescence microscopy. Graphical abstract Microbiology neutralization of zearalenone using Lactococcus lactis and Bifidobacterium sp. was confirmed by identification of binding kinetics and spectroscopic studies such as FT-IR spectroscopy and MALDI-TOF-MS spectrometry. The mechanism of ZEA binding was also investigated.
Assuntos
Bifidobacterium/metabolismo , Lactococcus lactis/metabolismo , Zearalenona/metabolismo , Adsorção , Bifidobacterium/citologia , Lactococcus lactis/citologia , Viabilidade Microbiana , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Recombinante/metabolismo , Sistemas de Liberação de Medicamentos , Lactococcus lactis/fisiologia , Microrganismos Geneticamente Modificados/fisiologia , Nódulos Linfáticos Agregados/metabolismo , Vacinas de DNA/metabolismo , Administração Oral , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Translocação Bacteriana , Transporte Biológico , Células CACO-2 , Linhagem Celular , Polaridade Celular , DNA Recombinante/administração & dosagem , Feminino , Microbiologia de Alimentos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Lactococcus lactis/citologia , Lactococcus lactis/genética , Listeria monocytogenes/citologia , Listeria monocytogenes/genética , Listeria monocytogenes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microrganismos Geneticamente Modificados/citologia , Microrganismos Geneticamente Modificados/genética , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/microbiologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Vacinas de DNA/administração & dosagemRESUMO
In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.
Assuntos
Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/metabolismo , Ácidos/metabolismo , Animais , Bovinos , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Citometria de Fluxo , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Lactococcus lactis/citologia , Viabilidade Microbiana , Leite/química , Leite/metabolismoRESUMO
This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30 °C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3 months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization.
Assuntos
Membrana Celular/química , Ácidos Graxos Insaturados/química , Liofilização/métodos , Lactococcus lactis/citologia , Temperatura Baixa , Dessecação , Fermentação , Fluidez de Membrana , Transição de Fase , Espectroscopia de Infravermelho com Transformada de Fourier , Água/químicaRESUMO
The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1-0.9 h⻹) and initial glucose concentrations (20-60 g l⻹). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h⻹ dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml⻹ and 9,450 IU ml⻹ h⻹, respectively) with the combination of a 0.9-h⻹ dilution rate and a 40-g l⻹ initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.
Assuntos
Microbiologia Industrial , Lactococcus lactis/citologia , Lactococcus lactis/metabolismo , Nisina/metabolismo , Parede Celular/metabolismo , Quitina/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Octoxinol/metabolismoRESUMO
BACKGROUND: Controlled restriction of cellular movement using microfluidics allows one to study individual cells to gain insight into aspects of their physiology and behaviour. For example, the use of micron-sized growth channels that confine individual Escherichia coli has yielded novel insights into cell growth and death. To extend this approach to other species of bacteria, many of whom have dimensions in the sub-micron range, or to a larger range of growth conditions, a readily-fabricated device containing sub-micron features is required. RESULTS: Here we detail the fabrication of a versatile device with growth channels whose widths range from 0.3 µm to 0.8 µm. The device is fabricated using electron beam lithography, which provides excellent control over the shape and size of different growth channels and facilitates the rapid-prototyping of new designs. Features are successfully transferred first into silicon, and subsequently into the polydimethylsiloxane that forms the basis of the working microfluidic device. We demonstrate that the growth of sub-micron scale bacteria such as Lactococcus lactis or Escherichia coli cultured in minimal medium can be followed in such a device over several generations. CONCLUSIONS: We have presented a detailed protocol based on electron beam fabrication together with specific dry etching procedures for the fabrication of a microfluidic device suited to study submicron-sized bacteria. We have demonstrated that both Gram-positive and Gram-negative bacteria can be successfully loaded and imaged over a number of generations in this device. Similar devices could potentially be used to study other submicron-sized organisms under conditions in which the height and shape of the growth channels are crucial to the experimental design.
Assuntos
Elétrons , Escherichia coli/citologia , Lactococcus lactis/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Microtecnologia/instrumentação , Dimetilpolisiloxanos , Escherichia coli/crescimento & desenvolvimento , Corantes Fluorescentes/metabolismo , Ouro , Quimografia , Lactococcus lactis/crescimento & desenvolvimento , Microscopia Eletrônica de Varredura , Silício , Fatores de TempoRESUMO
Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum L-AI were used for production of D-tagatose from D-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of D-galactose to D-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L⻹ substrate and at 37.5 °C after 5 days. The D-tagatose production rate of 185 g L⻹ day ⻹ was obtained at 300 g L⻹ galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial D-tagatose production rate was 290 g L⻹ day⻹ under these conditions.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Bifidobacterium/enzimologia , Hexoses/biossíntese , Lactococcus lactis/metabolismo , Aldose-Cetose Isomerases/genética , Bifidobacterium/genética , Reatores Biológicos/microbiologia , Biotecnologia , Boratos , Soluções Tampão , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Galactose/metabolismo , Genes Bacterianos , Cinética , Lactococcus lactis/citologia , Lactococcus lactis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase de Repouso do Ciclo CelularRESUMO
The nisin leader is believed to be crucial for nisin biosynthesis. Here, by using a construct completely lacking the leader peptide, we show that an up to fivefold-dehydrated leaderless prenisin can be obtained, as judged by MALDI-TOF MS, and that some of these species are biologically active, thus suggesting that at least three lanthionine rings are present. Notably, by expressing the leader peptide in trans together with the leaderless prenisin, we were able to increase the dehydration/cyclization efficiency of both NisB and NisC, but still with limited efficiency until the fifth dehydratable residue (Thr13) was processed, thereby enabling three rings to form. This, for the first time, demonstrates that 1) the leader is not absolutely necessary for the dehydration reaction of class I lantibiotics to occur in vivo; 2) the leader acts in trans in vivo; 3) the leader increases the efficiency of modification. Based on previous work and our current study, a model for the interactions of NisB and NisC with prenisin is proposed, in which the leader induces a more active conformation and/or productive complex formation of the biosynthetic machinery, and, when covalently bound, is involved in increasing the efficiency of dehydration to the C-terminal end of the prenisin substrate molecule.
Assuntos
Nisina/biossíntese , Lactococcus lactis/química , Lactococcus lactis/citologia , Lactococcus lactis/metabolismo , Nisina/química , Nisina/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In this work we focused on quantifying adhesion between Lactococcus lactis, the model for lactic acid bacteria (LAB) and mucins. Interactions between two strains of L. lactis (IBB477 and MG1820 as control) and pig gastric mucin-based coating were measured and compared with the use of atomic force microscopy. Analysis of retraction force-distance curves shed light on the differential contributions of nonspecific and specific forces. An increased proportion of specific adhesive events was obtained for IBB477 (20% vs. 5% for the control). Blocking assays with free pig gastric mucin and its O-glycan moiety showed that oligosaccharides play a major (but not exclusive) role in L. lactis-mucins interactions. Specific interactions were analyzed in terms of kinetic constants. An increase in the loading rate of atomic force microscope tip led to a higher force between interacting biological entities, which was directly linked to the kinetic dissociation constant (K(off)). Enhancing the contact time between the tip and the sample allowed an increase in the interaction probability, which can be related to the kinetic association constant (K(on)). Variations in the loading rate and contact time enabled us to determine K(on) (3.3 × 10(2) M(-1)·s(-1)) and K(off) (0.46 s(-1)), and the latter was consistent with values given in the literature for sugar-protein interactions.
Assuntos
Lactococcus lactis/metabolismo , Sondas Moleculares/metabolismo , Animais , Aderência Bacteriana , Sobrevivência Celular , Humanos , Cinética , Lactococcus lactis/citologia , Lactococcus lactis/ultraestrutura , Microscopia de Força Atômica , Microscopia de Fluorescência , Mucinas , Oligossacarídeos/metabolismo , Sus scrofa , Fatores de TempoRESUMO
Batch cultures of Lactococcus lactis NCDO 2118 and IL 1403 were performed in a Couette bioreactor operated in the modulated wavy vortex flow and the turbulent regimes. This study provides an overall analysis taking into account both mechanical stress and mixing in a Couette bioreactor. A unique phenotypic aspect has been proved to occur only in the modulated wavy vortex flow regime for the two studied strains, namely that the cells become entrapped in a filamentous form. No change in the metabolic behavior of the cells has been observed. The polymeric matrix has been microscopically observed through FISH and fluorescent lectin binding, showing cells entrapped in a glycoconjugate matrix. All hypotheses regarding insufficient mixing as a cause of this phenotype have been discarded, leading to the conclusion that this particular phenotypic feature is essentially due a combined effect of mechanical stress and flow structure. Particle size measurement during the fermentation course indicates that formation of filamentous form results from a continuous aggregation started in the early stages of the cultivation. According to our results a minimum shear is required to induce the ability for cells to aggregate. Then, it appears that both flow structure and mechanical stress (shear) are responsible for the appearance of such a filamentous form. As far as the authors know, this is the first experimental evidence of a bio polymerization induced by the flow structure.
Assuntos
Biopolímeros/metabolismo , Reatores Biológicos/microbiologia , Lactococcus lactis/citologia , Lactococcus lactis/crescimento & desenvolvimento , Aderência Bacteriana , Lactococcus lactis/metabolismo , Microscopia , Material ParticuladoRESUMO
Auxotrophy, the inability to produce an organic compound essential for growth, is widespread among bacteria. Auxotrophic bacteria rely on transporters to acquire these compounds from their environment. Here, we study the expression of both low- and high-affinity transporters of the costly amino acid methionine in an auxotrophic lactic acid bacterium, Lactococcus lactis. We show that the high-affinity transporter (Met-transporter) is heterogeneously expressed at low methionine concentrations, resulting in two isogenic subpopulations that sequester methionine in different ways: one subpopulation primarily relies on the high-affinity transporter (high expression of the Met-transporter) and the other subpopulation primarily relies on the low-affinity transporter (low expression of the Met-transporter). The phenotypic heterogeneity is remarkably stable, inherited for tens of generations, and apparent at the colony level. This heterogeneity results from a T-box riboswitch in the promoter region of the met operon encoding the high-affinity Met-transporter. We hypothesize that T-box riboswitches, which are commonly found in the Lactobacillales, may play as-yet unexplored roles in the predominantly auxotrophic lifestyle of these bacteria.
Assuntos
Lactococcus lactis/genética , Riboswitch/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Lactococcus lactis/citologia , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Óperon/genética , Fenótipo , Análise de Célula Única , Transcrição GênicaRESUMO
Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. By combining results from conventional isolation methods and molecular investigation of 16S rRNA gene and lactococcal/enterococcal specific genes, we identify at species level catalase negative gram positive thermoresistant cocci isolated from traditional 'leben', a 24-h fermented milk in arid area of west Algeria. Forty strains phenotypically related to cocci LAB were identified as belonging to the species Lactococcus lactis ssp. lactis, Enterococcus faecalis, Enterococcus faecium, and other Enterococcus species. No Streptococcus thermophilus strain was isolated. Ten different phenotype groups were recognized, and the species content of these groups were in some cases different from the expected features usually given in genus and species descriptions. In particular, atypical lactococci, able to grow in 6.5% NaCl, at pH 9.5 and showing high resistance to thermal stresses were isolated. Lactococci, but also enterococci isolated from traditional 'leben' produced in the desert area, may be therefore of interest in milk fermentation. Further studies to assess this source of diversity within the wild microbial population should provide starter new strains for product innovation.
Assuntos
DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Microbiologia de Alimentos , Cocos Gram-Positivos/classificação , Cocos Gram-Positivos/isolamento & purificação , Argélia , Análise por Conglomerados , DNA Bacteriano/química , DNA Ribossômico/química , DNA Ribossômico/genética , Cocos Gram-Positivos/citologia , Cocos Gram-Positivos/genética , Lactococcus lactis/classificação , Lactococcus lactis/citologia , Lactococcus lactis/genética , Lactococcus lactis/isolamento & purificação , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild-type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress-resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.
Assuntos
Fermentação , Manipulação de Alimentos , Lactococcus lactis/citologia , Viabilidade Microbiana , Leite/microbiologia , Mutação/genética , Estresse Oxidativo , Ácidos , Animais , Contagem de Colônia Microbiana , Produtos Fermentados do Leite/microbiologia , TemperaturaRESUMO
With the aim of constructing an efficient protein display system for lactic acid bacteria (LABs), the effect of fusion direction on the cell-surface binding activity of the C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 was studied. CPH fused to the alpha-amylase (AMY) of Streptococcus bovis 148 either at its C-terminus (CPH-AMY) or at its N-terminus (AMY-CPH) was expressed intracellularly in Escherichia coli. This domain was able to direct binding of AMY to the surface of L. lactis ATCC 19435 in both constructs. However, the number of bound molecules per cell and the specific activity for starch digestion in the case of CPH-AMY were 3 and 14 times greater than those in the case of AMY-CPH, respectively. Of the LABs tested, L. lactis ATCC 19435 showed the highest binding capability for CPH-AMY, up to 6 x 10(4) molecules per cell, with a dissociation rate constant of 5.00 x 10(-5) s(-1). The binding of CPH-AMY to the surface of Lactobacillus delbrueckii ATCC 9649 cells was very stable with a dissociation rate constant of 6.96 x 10(-6) s(-1). The production of CPH-AMY in the soluble form increased 3-fold as a result of coexpression with a molecular chaperone, trigger factor. The results of this study suggest the usefulness of CPH as a bidirectional anchor protein for the production of cell-surface adhesive enzymes in E. coli. Furthermore, the importance of the fusion direction of CPH in determining cell-surface binding and enzymatic activities was shown.
Assuntos
Adesão Celular/fisiologia , Membrana Celular/metabolismo , Lactococcus lactis/citologia , Lactococcus lactis/fisiologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos/fisiologiaRESUMO
Rotaviruses are the primary cause of acute gastroenteritis in children worldwide. Although the implementation of live attenuated vaccines has reduced the number of rotavirus-associated deaths, variance in their effectiveness has been reported in different countries. This fact, among other concerns, leads to continuous efforts for the development of new generation of vaccines against rotavirus.In this work, we describe the obtention of cell wall-derived particles from a recombinant Lactococcus lactis expressing a cell wall-anchored version of the rotavirus VP6 protein. After confirming by SDS-PAGE, Western blot, flow cytometry and electronic immunomicroscopy that these particles were carrying the VP6 protein, their immunogenic potential was evaluated in adult BALB/c mice. For that, mucosal immunizations (oral or intranasal), with or without the dmLT [(double mutant Escherichia coli heat labile toxin LT(R192G/L211A)] adjuvant were performed. The results showed that these cell wall-derived particles were able to generate anti-rotavirus IgG and IgA antibodies only when administered intranasally, whether the adjuvant was present or not. However, the presence of dmLT was necessary to confer protection against rotavirus infection, which was evidenced by a 79.5 percent viral shedding reduction.In summary, this work describes the production of cell wall-derived particles which were able to induce a protective immune response after intranasal immunization. Further studies are needed to characterize the immune response elicited by these particles as well as to determine their potential as an alternative to the use of live L. lactis for mucosal antigen delivery.
Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Parede Celular/metabolismo , Portadores de Fármacos/metabolismo , Lactococcus lactis/citologia , Mucosa/metabolismo , Infecções por Rotavirus/prevenção & controle , Rotavirus/fisiologia , Animais , Especificidade de Anticorpos , Antígenos Virais/metabolismo , Proteínas do Capsídeo/metabolismo , Modelos Animais de Doenças , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from ß-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle.
Assuntos
Lactococcus lactis/citologia , Lactococcus lactis/enzimologia , Proteínas de Ligação às Penicilinas/metabolismo , Divisão Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/crescimento & desenvolvimento , beta-Lactamas/farmacologiaRESUMO
BACKGROUND: A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria. RESULTS: Electrotransformation efficiencies of up to 105 transformants per mug DNA have been reported in the literature for L. lactis spp.lactis LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 +/- 52.5 x 107 transformants per mug DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 +/- 0.5 x 105 transformants per mug DNA. Results of the same trend were obtained with L. lactis ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 1010 cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient. CONCLUSION: This investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for L. lactis spp. lactis. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries.
Assuntos
Acetatos/farmacologia , Ditiotreitol/farmacologia , Eletroporação/métodos , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/genética , Transformação Bacteriana , DNA Bacteriano/metabolismo , Vetores Genéticos , Lactococcus lactis/citologiaRESUMO
Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.
Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Íleo/microbiologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Engenharia de Proteínas/métodos , Timidilato Sintase/deficiência , Sequência de Aminoácidos , Animais , Divisão Celular , Sobrevivência Celular , Colite/microbiologia , Colite/terapia , Sistemas de Liberação de Medicamentos/métodos , Engenharia Genética/métodos , Humanos , Interleucina-10/uso terapêutico , Lactococcus lactis/citologia , Lactococcus lactis/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Probióticos/uso terapêutico , Suínos , Timidilato Sintase/genética , Timidilato Sintase/metabolismoRESUMO
OBJECTIVE: To assess the relief effect of beta-galactosidase genetically engineered Lactococcus lactis on the cell toxicity caused by lactose in vitro. METHODS: An in vitro toxic Caco-2 cell model caused by lactose was established to evaluate the relief effect of beta-galactosidase genetically engineered Lactococcus lactis. Cell morphological parameters and proliferation activity parameter were used. RESULTS: The in vitro toxic Caco-2 cell model caused by lactose was successfully established; the genetically engineered Lactococcus lactis constructed in the authors' laboratory could enable the Caco-2 cell to have normal appearance with the presence of lactose and could improve the proliferation activity with the presence of high concentration of lactose (P < 0.01). CONCLUSION: The beta-galactosidase genetically engineered Lactococcus lactis has significant relief effect on the cell toxicity caused by lactose in vitro, which lays a foundation for food-grade alternation of this bacterium.