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1.
Polymers (Basel) ; 15(14)2023 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-37514425

RESUMO

Cationic copolymers based on 2-(N,N-dimethylamino)ethyl methacrylate and polyethylene glycol monomethyl ether (pDMAEMA-co-PEO) with different molecular weights have been synthesized. Their physicochemical properties were studied by NMR spectroscopy, sedimentation, and potentiometric titration. According to the data of potentiometric titration for the synthesized pegylated cationic copolymers, the apparent dissociation constants were determined in the pH range from 4.5 to 8.5. The physicochemical properties of interpolyelectrolyte complexes of these polycations with circular DNA (IPEC DNA) were also studied by dynamic light scattering, electrophoretic mobility, and TEM methods. It has been established that the diameter and electrokinetic potential (ζ-potential) of interpolyelectrolyte complexes can be varied over a wide range (from 200 nm to 1.5 µm and from -25 mV to +30 mV) by changing the ratio of oppositely charged ionizable groups in pegylated cationic copolymers and DNA, as well as by regulating medium pH. The resistance of the IPEC DNA/polycation complex to the action of nucleases was studied by electrophoresis in agarose gel; the cytotoxic effect of the polymers in vitro, and the efficiency of penetration (transfection) of IPEC DNA with PDMAEMA-co-PEO-polycations into eukaryotic cells of a cell line derived from human embryonic kidneys HEK 293 in vitro.

2.
Antibiotics (Basel) ; 12(3)2023 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-36978338

RESUMO

LF3872 was isolated from the milk of a healthy lactating and breastfeeding woman. Earlier, the genome of LF3872 was sequenced, and a gene encoding unique bacteriocin was discovered. We have shown here that the LF3872 strain produces a novel thermolabile class III bacteriolysin (BLF3872), exhibiting antimicrobial activity against antibiotic-resistant Staphylococcus aureus strains. Sequence analysis revealed the two-domain structural (lysozyme-like domain and peptidase M23 domain) organization of BLF3872. At least 25% residues of this protein are expected to be intrinsically disordered. Furthermore, BLF3872 is predicted to have a very high liquid-liquid phase separation. According to the electron microscopy data, the bacterial cells of LF3872 strain form co-aggregates with the S. aureus 8325-4 bacterial cells. LF3872 produced bacteriolysin BLF3872 that lyses the cells of the S. aureus 8325-4 mastitis-inducing strain. The sensitivity of the antibiotic-resistant S. aureus collection strains and freshly isolated antibiotic-resistant strains was tested using samples from women with lactation mastitis; the human nasopharynx and oral cavity; the oropharynx of pigs; and the cows with a diagnosis of clinical mastitis sensitive to the lytic action of the LF3872 strain producing BLF3872. The co-cultivation of LF3872 strain with various antibiotic-resistant S. aureus strains for 24 h reduced the level of living cells of these pathogens by six log. The LF3872 strain was found to be able to co-aggregate with all studied S. aureus strains. The cell-free culture supernatant of LF3872 (CSLF3872) induced S. aureus cell damage and ATP leakage. The effectiveness of the bacteriolytic action of LF3872 strain did not depend on the origin of the S. aureus strains. The results reported here are important for the creation of new effective drugs against antibiotic-resistant strains of S. aureus circulating in humans and animals.

3.
Antibiotics (Basel) ; 12(10)2023 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-37887236

RESUMO

The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman's intestines and reproductive system, has been studied for its probiotic potential, particularly against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti-Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti-Salmonella activity, with a partial reduction upon neutralization with NaOH (p < 0.05), suggesting the presence of anti-Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0-1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes (p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes (p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.

4.
Biomolecules ; 13(12)2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-38136611

RESUMO

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni, Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans, and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans-induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human ß-defensin 3 in various epithelial cells. These findings support the anti-Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.


Assuntos
Candidíase , Lactobacillus crispatus , Feminino , Humanos , Candida albicans , Células HeLa , Células Epiteliais/metabolismo
5.
Antibiotics (Basel) ; 13(1)2023 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-38247590

RESUMO

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti-Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor-intestinal alkaline phosphatase (IAP)-in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1ß, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

6.
Antibiotics (Basel) ; 11(10)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36290095

RESUMO

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. The high-quality genome sequencing of LF3872 was performed, and a gene encoding a unique bacteriocin was discovered. It was established that the bacteriocin produced by LF3872 (BLF3872) belongs to the family of cell-wall-degrading proteins that cause cell lysis. The antibacterial properties of LF3872 were studied using test cultures of antibiotic-resistant Gram-positive and Gram-negative pathogens. Gram-positive pathogens (Staphylococcus aureus strain 8325-4 and S. aureus strain IIE CI-SA 1246) were highly sensitive to the bacteriolytic action of LF3872. Gram-negative pathogens (Escherichia coli, Salmonella strains, and Campylobacter jejuni strains) were more resistant to the bacteriolytic action of LF3872 compared to Gram-positive pathogens. LF3872 is a strong co-aggregator of Gram-negative pathogens. The cell-free culture supernatant of LF3872 (CSLF3872) induced cell damage in the Gram-positive and Gram-negative test cultures and ATP leakage. In the in vitro experiments, it was found that LF3872 and Actigen prebiotic (Alltech Inc., Nicholasville, KY, USA) exhibited synergistic anti-adhesive activity against Gram-negative pathogens. LF3872 has immunoregulatory properties: it inhibited the lipopolysaccharide-induced production of proinflammatory cytokines IL-8, IL-1ß, and TNF-α in a monolayer of Caco-2 cells; inhibited the production of IL-12 and stimulated the production of IL-10 in immature human dendritic cells; and stimulated the production of TGF-ß, IFN-γ, and IgA in the immunocompetent cells of intestinal Peyer's patches (PPs) in mice. These results indicate the possibility of creating a synbiotic based on LF3872 and a prebiotic derived from Saccharomyces cerevisiae cell wall components. Such innovative drugs and biologically active additives are necessary for the implementation of a strategy to reduce the spread of antibiotic-resistant strains of socially significant animal and human infections.

7.
Int J Biol Macromol ; 189: 410-419, 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34437917

RESUMO

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes.


Assuntos
Diferenciação Celular , Enterócitos/metabolismo , Lactobacillus crispatus/química , Glicoproteínas de Membrana/farmacologia , Vagina/microbiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Impedância Elétrica , Enterócitos/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Lactase/genética , Lactase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sacarase/genética , Sacarase/metabolismo
8.
Int J Biol Macromol ; 150: 400-412, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32045605

RESUMO

We have previously demonstrated that human vaginal Lactobacillus crispatus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slp1 and slp2. Whilst we found no evidence for the expression of slp1 under the growth conditions used, a very high level of expression of the slp2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SlpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein of L. crispatus JCM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. crispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kB activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators Il-8, Il-1ß, and TNF-α. Slp2 and LC2029 inhibited Il-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine Il-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodborne pathogens Campylobacter jejuni, Salmonella enteritidis, and Escherichia coli O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and Slp2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. Slp2-positive LC2029 strain but not Slp2 alone provided bactericidal effect on foodborne pathogens. These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell-adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizing enterocolitis (NEC) in newborns and foodborne infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis.


Assuntos
Antibiose , Doenças Transmitidas por Alimentos/dietoterapia , Doenças Transmitidas por Alimentos/microbiologia , Imunomodulação , Lactobacillus crispatus/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Probióticos , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Ácidos e Sais Biliares , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Sobrevivência Celular , Células Epiteliais , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Estresse Fisiológico , Relação Estrutura-Atividade
9.
Int J Biol Macromol ; 122: 1062-1070, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30218736

RESUMO

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective protein that is considered as a vaccine component for humans. LcrV mediates the delivery of Yop toxins into host cells and upregulates TLR2-dependent IL-10 production. Although LcrV can interact with the receptor-bound human interferon-γ (hIFN-γ), the significance of these interactions in plague pathogenesis is not known. In this study, we determined the parameters of specific interactions of LcrV and LcrV68-326 with primary human thymocytes and Jurkat T-leukemia cells in the presence of receptor-bound hIFN-γ. Although the C-terminal region of hIFN-γ contains a GRRA138-141 site needed for high-affinity binding of LcrV and LcrV68-326, in the hIFN-γ homodimer, these GRRA138-141 target sites becomes accessible for targeting by LcrV or LcrV68-326 only after immobilization of the hIFN-γ homodimer on the hIFN-γ receptors of thymocytes or Jurkat T-cells. The interaction of LcrV or LcrV68-326 with receptor-bound hIFN-γ on the thymocytes or Jurkat T-cells caused apoptosis of both cell types, which can be completely blocked by the addition of monoclonal antibodies specific to the LEEL32-35 and DEEI203-206 sites of LcrV. The ability of LcrV to utilize hIFN-γ is insidious and may account in part for the severe symptoms of plague in humans.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Apoptose , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Humanos , Lactente , Células Jurkat , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/química , Ligação Proteica , Conformação Proteica
10.
Cell Immunol ; 249(1): 46-53, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18082675

RESUMO

Supernatant obtained from high dose hydrocortisone resistant thymocytes can induce migration of the bone marrow cell precursors to the periphery. This biological activity depends on the presence of the 18 kDa protein, whose amino acid sequence fits with the sequence of the secretory form of murine cyclophilin A (SP-18). Cyclophilin A isolated from the supernatant of the cortisone-resistant thymoma EL-4 shows its characteristic functional features as it demonstrates isomerase activity and binds with cyclosporine A. The cyclophilin A obtained manifests chemotactic activity that regulates migration of bone marrow cell precursors of neutrophils, T-, B- and dendritic cells.


Assuntos
Células da Medula Óssea/fisiologia , Ciclofilina A , Animais , Antígeno CD11b , Antígeno CD11c , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Quimiotaxia , Cortisona/farmacologia , Meios de Cultivo Condicionados/farmacologia , Ciclofilina A/biossíntese , Células Dendríticas/fisiologia , Resistência a Medicamentos , Feminino , Granulócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Molécula-1 de Adesão Celular Endotelial a Plaquetas , Células Precursoras de Linfócitos B , Células Precursoras de Linfócitos T/fisiologia , Timo/citologia , Timo/efeitos dos fármacos
11.
Genome Announc ; 4(1)2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26744375

RESUMO

A draft genome sequence of Lactobacillus plantarum 2025 was derived using Ion Torrent sequencing technology. The total size of the assembly (3.33 Mb) was in agreement with the genome sizes of other strains of this species. The data will assist in revealing the genes responsible for the specific properties of this strain.

12.
Genome Announc ; 2(1)2014 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-24407651

RESUMO

This report describes a draft genome sequence of Lactobacillus plantarum 2165. The data demonstrate the presence of a large number of genes responsible for sugar metabolism and the fermentation activity of this bacterium. Different cell surface proteins, including fibronectin and mucus-binding adhesins, may contribute to the beneficial probiotic properties of this strain.

13.
Genome Announc ; 2(1)2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24558253

RESUMO

This report describes a draft genome sequence of Lactobacillus crispatus 2029. The reads generated by the Ion Torrent PGM were assembled into contigs with a total size of 2.2 Mb. The data were annotated using the NCBI GenBank and RAST servers. A comparison with the reference strain revealed specific features of the genome.

14.
Genome Announc ; 2(1)2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24558254

RESUMO

In this report, we present a draft sequence of the genome of Lactobacillus rhamnosus strain 2166, a potential novel probiotic. Genome annotation and read mapping onto a reference genome of L. rhamnosus strain GG allowed for the identification of the differences and similarities in the genomic contents and gene arrangements of these strains.

15.
Genome Announc ; 1(6)2013 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-24285651

RESUMO

A draft genome sequence of Lactobacillus jensenii strain MD IIE-70(2) was determined using Ion PGM technology. The reads were mapped to a reference strain and assembled using a combination of tools. The genetic features revealed in this study will assist in understanding the probiotic properties of Lactobacillus bacteria.

16.
Genome Announc ; 1(6)2013 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-24285652

RESUMO

This report describes a draft genome sequence of Lactobacillus fermentum strain 3872. The data revealed remarkable similarity to and dissimilarity with the published genome sequences of other strains of the species. The absence of and variation in structures of some adhesins and the presence of an additional adhesin may reflect adaptation of the bacterium to different host systems and may contribute to specific properties of this strain as a new probiotic.

17.
Genome Announc ; 1(4)2013 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-23969051

RESUMO

Different common factors contribute to the antagonistic properties of Lactobacillus gasseri toward various pathogens. However, there is strain-to-strain variation in the probiotic properties of this bacterium. The draft genome sequence of L. gasseri strain 2016 determined in this study will assist in understanding the genetic basis for such variation.

18.
Probiotics Antimicrob Proteins ; 4(3): 180-6, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26782044

RESUMO

The biofilm formation took place in 48 h within the solid substrate cultivation of Lactobacillus plantarum 8-RA-3 strain on the wheat bran saturated with the MRS medium. The drying of the bran fermented by lactobacilli resulted in a decrease in the number of colony-forming units (CFU) from 23.0 × 10(8) to 6.9 × 10(5) CFU/g in daily samples and to less than 10(4) CFU/g in 2- and 3-day samples. However, according to the fluorescence-based live/dead assay data, more than 40 % of the non-cultured bacteria were viable. As a result of mice kept on a diet with the introduction of bran fermented by Lact. plantarum 8-RA-3 for 72 h into the fodder, a recovery of normal level of intestinal lactobacilli, inhibited by administration of antibiotic was noted. The strain genetically identical to the Lact. plantarum 8-RA-3 was isolated from the feces of these mice. The results indicate that solid substrate cultivated Lact. plantarum 8-RA-3 strain formed a biofilm. Once dried and transferred into a non-cultured state, biofilm cells retained its viability and biological activity.

19.
J Proteome Res ; 6(6): 2222-31, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17441749

RESUMO

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.


Assuntos
Antígenos de Bactérias/imunologia , Interferon gama/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Receptores de Interferon/imunologia , Receptor 2 Toll-Like/imunologia , Yersinia pestis/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Sítios de Ligação , Linhagem Celular , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Proteínas Citotóxicas Formadoras de Poros/química , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia
20.
J Proteome Res ; 1(4): 307-15, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12645886

RESUMO

Yersinia pestis capsular antigen Caf1 is shown to be a beta-structural protein that in polymeric form possesses very high conformational stability. Different approaches show that a dimer is the minimal cooperative block of Caf1 adhesin. Caf1 dimer interacts effectively with IL-1 receptors of human macrophage and epithelial cells. The specificity of such interaction is confirmed by the inhibition of IL-1alpha binding by Caf1. The Caf1 role in pneumonic plague pathogenesis is discussed.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peste/microbiologia , Yersinia pestis/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Cápsulas Bacterianas/química , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Linhagem Celular , Dicroísmo Circular , Dimerização , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Guanidina/química , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/citologia , Macrófagos/metabolismo , Modelos Biológicos , Peste/imunologia , Peste/metabolismo , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Receptores de Interleucina-1/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Yersinia pestis/química
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