- Klíčová slova
- granadaene,
- MeSH
- bakteriální zoonózy etiologie MeSH
- brouci mikrobiologie patogenita MeSH
- feces mikrobiologie MeSH
- grampozitivní koky * chemie izolace a purifikace patogenita MeSH
- jedlý hmyz * mikrobiologie patogenita MeSH
- Lactococcus chemie izolace a purifikace patogenita MeSH
- Leontopithecus mikrobiologie MeSH
- riziko MeSH
- střevní mikroflóra MeSH
- Publikační typ
- přehledy MeSH
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
Out of six samples of wastewater produced in the dairy industry, taken in 2017 at various places of dairy operations, 86 bacterial strains showing decarboxylase activity were isolated. From the wastewater samples, the species of genera Staphylococcus, Lactococcus, Enterococcus, Microbacterium, Kocuria, Acinetobacter, Pseudomonas, Aeromonas, Klebsiella and Enterobacter were identified by the MALDI-TOF MS and biochemical methods. The in vitro produced quantity of eight biogenic amines (BAs) was detected by the HPLC/UV-Vis method. All the isolated bacteria were able to produce four to eight BAs. Tyramine, putrescine and cadaverine belonged to the most frequently produced BAs. Of the isolated bacteria, 41% were able to produce BAs in amounts >100 mg L-1. Therefore, wastewater embodies a potential vector of transmission of decarboxylase positive microorganisms, which should be taken into consideration in hazard analyses within foodstuff safety control. The parameters of this wastewater (contents of nitrites, nitrates, phosphates, and proteins) were also monitored.
- MeSH
- Acinetobacter MeSH
- Aeromonas MeSH
- biogenní aminy chemie MeSH
- chemické látky znečišťující vodu chemie izolace a purifikace MeSH
- Enterobacter MeSH
- Enterococcus MeSH
- karboxylyasy chemie MeSH
- Klebsiella MeSH
- Lactobacillus MeSH
- Lactococcus MeSH
- Microbacterium MeSH
- mikrobiologie vody MeSH
- mlékárenství * MeSH
- odpadní voda analýza mikrobiologie MeSH
- Pediococcus MeSH
- Pseudomonas MeSH
- spektrofotometrie ultrafialová MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Staphylococcus MeSH
- Streptococcus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Evropa 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
Lactococcus garvieae has emerged as an important zoonotic pathogen. However, information regarding mechanisms and factors related to its pathogenicity is lacking. In the present study, we investigated the distribution and functionality of genes related to virulence factors in L. garvieae strains isolated from different niches (diseased fish, humans, meat and dairy products, vegetables), using both post-genomic and genotypic analysis. Putative genes encoding hemolysin, fibronectin-binding protein, and penicillin acylase were detected in all analyzed genomes/strains. Their expression was significantly induced by bile salt stress. Putative genes encoding bile salt hydrolase were found in a few strains from dairy and human sources, as well as the mobilizable tet genes. Finally, all genomes possessed a folate gene cluster, in which mutations in the dihydropteroate synthase gene (folP) could be related to sulfonamide resistance. To the best of our knowledge, this is the first study aimed to explore the pathogenic potential of L. garvieae through the analysis of numerous L. garvieae genomes/strains, coming from different sources. This approach allowed the detection of virulence-related genes not yet investigated in the species and the study of their expression after exposure to different environmental stresses. The results obtained suggest a virulence potential in some L. garvieae strains that can be exploited for survival in the human gastrointestinal tract.
- MeSH
- DNA bakterií genetika MeSH
- faktory virulence genetika MeSH
- genomika * MeSH
- Lactococcus genetika růst a vývoj patogenita MeSH
- lidé MeSH
- regulace genové exprese u bakterií * MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie 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
