The cytokine IL-23 activates the IL-23 receptor (IL-23R) and stimulates the differentiation of naïve T helper (Th) cells into a Th17 cell population that secretes inflammatory cytokines and chemokines. This IL-23/Th17 proinflammatory axis drives inflammation in Crohn's disease and ulcerative colitis and represents a therapeutic target of monoclonal antibodies. Non-immunoglobulin binding proteins based on the Streptococcus albumin-binding domain (ABD) provide a small protein alternative to monoclonal antibodies. They can be readily expressed in bacteria. Lactococcus lactis is a safe lactic acid bacterium that has previously been engineered as a vector for the delivery of recombinant therapeutic proteins to mucosal surfaces. Here, L. lactis was engineered to display or secrete ABD-variants against the IL-17 receptor (IL-17R). Its expression and functionality were confirmed with flow cytometry using specific antibody and recombinant IL-17R, respectively. In addition, L. lactis were engineered into multifunctional bacteria that simultaneously express two binders from pNBBX plasmid. First, binders of IL-17R were combined with binder of IL-17. Second, binders of IL-23R were combined with binders of IL-23. The dual functionality of the bacteria was confirmed by flow cytometry using corresponding targets, namely the recombinant receptors IL-17R and IL-23R or the p19 subunit of IL-23. Binding of IL-17 was confirmed by ELISA. With the latter, 97% of IL-17 was removed from solution by 2 × 109 recombinant bacteria. Moreover, multifunctional bacteria targeting IL-17/IL-17R prevented IL-17A-mediated activation of downstream signaling pathways in HEK-Blue IL-17 cell model. Thus, we have developed several multifunctional L. lactis capable of targeting multiple factors of the IL-23/Th17 proinflammatory axis. This represents a novel therapeutic strategy with synergistic potential for the treatment of intestinal inflammations.

• MeSH
• albuminy metabolismus   MeSH
• cytokiny * metabolismus   MeSH
• imunologické faktory MeSH
• interleukin-17 metabolismus   MeSH
• interleukin-23 chemie   metabolismus   MeSH
• Lactococcus lactis * genetika   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• rekombinantní proteiny metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cryptosporidium parvum infects enterocytes in diverse vertebrates, including humans, and causes diarrheal illness. However, no effective drugs are available for this protozoan infection. The P23 protein of C. parvum is a protective antigen, considered a potential candidate for developing an effective vaccine against cryptosporidiosis. In this study, the complementary DNA (cDNA) of the p23 gene was subcloned to Escherichia coli DH5α, with one nucleotide difference. The constructed plasmid pNZ8149-P23 was transferred by electroporation to Lactococcus lactis NZ3900, and the recombinant L. lactis NZ3900/pNZ8149-P23 strain was screened in Elliker-medium by adding bromocresolpurple indicator. A 23-kDa protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after nisin induction in LM17 broth medium, suggesting that P23 protein was in the form of glycosylation. Simultaneously, an optimal induction time of 9 h was determined, and the density of OD600 = 2.7 was tested. Through western blot and indirect immunofluorescence (IIF) analysis, the immunocompetence of expressed P23 antigen was identified, and its location of release to the cell interior of recombinant L. lactis was manifested. The first report of a food-grade genetically engineered L. lactis strain expressing a P23 antigen of C. parvum is herein presented. This result provides a novel and safe utilization method of P23 against C. parvum infection.

• MeSH
• Cryptosporidium parvum * genetika   metabolismus   MeSH
• Cryptosporidium * metabolismus   MeSH
• kryptosporidióza * prevence a kontrola   MeSH
• Lactococcus lactis * genetika   metabolismus   MeSH
• lidé MeSH
• pyridinolkarbamát MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Tyramine is one of the most toxic biogenic amines and it is produced commonly by lactic acid bacteria in fermented food products. In present study, we investigated the influence of selected nisin-producing Lactococcus lactis subsp. lactis strains and their cell-free supernatants (CFSs) on tyramine production by four Lactobacillus and two Lactiplantibacillus strains isolated from cheese and beer. Firstly, we examined the antimicrobial effect of the CFSs from twelve Lactococcus strains against tested tyramine producers by agar-well diffusion assay. Six Lactococcus strains whose CFSs showed the highest antimicrobial effect on tyramine producers were further studied. Secondly, we investigated the influence of the selected six Lactococcus strains and their respective CFSs on tyramine production by tested Lactobacillus and Lactiplantibacillus strains in MRS broth supplemented with 2 g.L-1 of l-tyrosine. Tyramine production was monitored by HPLC-UV. The tyramine formation of all tested Lactobacillus and Lactiplantibacillus strains was not detected in the presence of Lc. lactis subsp. lactis CCDM 71 and CCDM 702, and their CFSs. Moreover, the remainder of the investigated Lactococcus strains (CCDM 670, CCDM 686, CCDM 689 and CCDM 731) and their CFSs decreased tyramine production significantly (P < 0.05) - even suppressing it completely in some cases - in four of the six tested tyramine producing strains.

• MeSH
• antibakteriální látky analýza   metabolismus   farmakologie   MeSH
• kultivační média chemie   metabolismus   farmakologie   MeSH
• Lactobacillaceae účinky léků   růst a vývoj   izolace a purifikace   MeSH
• Lactobacillus účinky léků   růst a vývoj   izolace a purifikace   MeSH
• Lactococcus lactis chemie   metabolismus   MeSH
• pivo mikrobiologie   MeSH
• sýr mikrobiologie   MeSH
• tyramin analýza   metabolismus   farmakologie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH

The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

• MeSH
• bakteriální RNA chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• hořčík metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• Lactococcus lactis genetika   metabolismus   MeSH
• ligandy MeSH
• mangan metabolismus   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• mutace MeSH
• regulace genové exprese u bakterií MeSH
• riboswitch fyziologie   MeSH
• signální transdukce * MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• Xanthomonas metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis.

• MeSH
• buňky Th17 účinky léků   MeSH
• ELISA MeSH
• interleukin-23 metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• lidé MeSH
• proteiny genetika   metabolismus   farmakologie   MeSH
• průtoková cytometrie MeSH
• rekombinantní proteiny genetika   metabolismus   farmakologie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.

• MeSH
• antibakteriální látky farmakologie   MeSH
• CRISPR-Cas systémy MeSH
• editace genu metody   MeSH
• fermentace MeSH
• genetické inženýrství metody   MeSH
• genom bakteriální * MeSH
• guide RNA, Kinetoplastida genetika   metabolismus   MeSH
• imunoglobulin G genetika   metabolismus   MeSH
• klonování DNA MeSH
• Lactococcus lactis genetika   metabolismus   MeSH
• lidé MeSH
• methyltransferasy genetika   metabolismus   MeSH
• nisin farmakologie   MeSH
• plazmidy chemie   metabolismus   MeSH
• promotorové oblasti (genetika) účinky léků   MeSH
• proteiny teplotního šoku genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• transgeny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.

• MeSH
• albuminy metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• HeLa buňky MeSH
• imobilizované proteiny metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• lidé MeSH
• metody zobrazení buněčného povrchu MeSH
• podjednotky proteinů metabolismus   MeSH
• povrchová plasmonová rezonance MeSH
• proteinové domény MeSH
• průtoková cytometrie MeSH
• rekombinace genetická genetika   MeSH
• rekombinantní proteiny metabolismus   MeSH
• ribozomy metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• shiga toxin 1 chemie   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• biologické modely MeSH
• fruktosadifosfáty metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• metabolismus sacharidů * MeSH
• metoda Monte Carlo MeSH
• počítačová simulace MeSH
• statistické modely MeSH
• zpětná vazba fyziologická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lactococcus lactis subsp. lactis has been isolated from the intestines of marine fish and is a candidate probiotic for aquaculture. In order to use the bacterium as a probiotic, properties such as bile tolerance need to be assessed. Here, we compared bile tolerance in L. lactis strains derived from different sources. Three L. lactis subsp. lactis strains from marine fish (MFL), freshwater fish (FFL), and cheese starter (CSL) were used along with an Lactococcus lactis subsp. cremoris strain from cheese starter (CSC). The four strains were grown under various culture conditions: deMan-Rogosa-Sharpe (MRS) broth containing bile salts/acids, MRS agar containing oxgall, and phosphate-buffered saline (PBS) containing fish bile. Survival/growth of the strains in the presence of sodium cholate and sodium deoxycholate varied in the order MFL, CSL > CSC > FFL; in the presence of sodium taurocholate, the order was MFL > CSL > CSC > FFL. In liquid media containing various concentrations of oxgall, survival of the strains was observed in the order MFL > CSL > FFL and CSC. The survival of MFL was not affected by bile collected from the goldfish (Carassius auratus subsp. auratus) or the puffer fish (Takifugu niphobles), although the other strains showed significant inhibition of growth. It is a novel and beneficial finding that MFL has the highest resistance to bile acid.

• MeSH
• Lactococcus lactis metabolismus   MeSH
• počet mikrobiálních kolonií MeSH
• probiotika metabolismus   terapeutické užití   MeSH
• ryby mikrobiologie   MeSH
• vodní hospodářství MeSH
• žlučové kyseliny a soli farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1°C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.

• MeSH
• aerobióza MeSH
• anaerobióza MeSH
• chlorid sodný metabolismus   MeSH
• Lactococcus lactis klasifikace   enzymologie   růst a vývoj   metabolismus   MeSH
• laktosa metabolismus   MeSH
• sýr mikrobiologie   MeSH
• teplota MeSH
• tyramin biosyntéza   MeSH
• tyrosindekarboxylasa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH