S-layer protein 2 of 'Lactobacillus crispatus' 2029, its structural and immunomodulatory characteristics and roles in protective potential of the whole bacteria against foodborne pathogens

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-1β, 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

'Limosilactobacillus fermentum' Strain 3872 : antibacterial and immunoregulatory properties and synergy with prebiotics against socially significant antibiotic-resistant infections of animals and humans

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-1β, 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-β, 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

Effects of Medium Components on Isocitric Acid Production by Yarrowia lipolytica Yeast

The microbiological production of isocitric acid (ICA) is more preferable for its application in medicine and food, because the resulting product contains only the natural isomer—threo-DS. The aim of the present work was to study ICA production by yeast using sunflower oil as carbon source. 30 taxonomically different yeast strains were assessed for their capability for ICA production, and Y. lipolytica VKM Y-2373 was selected as a promising producer. It was found that ICA production required: the limitation of Y. lipolytica growth by nitrogen, phosphorus, sulfur or magnesium, and an addition of iron, activating aconitate hydratase, a key enzyme of isocitrate synthesis. Another regulatory approach capable to shift acid formation to a predominant ICA synthesis is the use of inhibitors (itaconic and oxalic acids), which blocks the conversion of isocitrate at the level of isocitrate lyase. It is recommended to cultivate Y. lipolytica VKM Y-2373 under nitrogen deficiency conditions with addition of 1.5 mg/L iron and 30 mM itaconic acid. Such optimized nutrition medium provides 70.6 g/L ICA with a ratio between ICA and citric acid (CA) equal 4:1, a mass yield (YICA) of 1.25 g/g and volume productivity (QICA) of 1.19 g/L·h

Effect of Nitrogen Concentration on the Biosynthesis of Citric Acid, Protein, and Lipids in the Yeast Yarrowia lipolytica

Yarrowia lipolytica yeast is well known to be able to synthesize citric acid (CA) in large amounts. This study deals with CA biosynthesis, the production of biomass, as well as the accumulation and composition of proteins and lipids in Y. lipolytica VKM Y-2373 grown in media with glucose at different concentrations of ammonium sulfate (from 2 to 10 g/L). It was found that these concentrations of nitrogen source are limiting for the growth of Y. lipolytica and that nitrogen deficiency is the main cause of CA excretion. At the high concentration of (NH4)2SO4 (10 g/L), the accumulation of cell biomass, biomass yield (YX/S), and protein concentration was higher than in the medium with 2 g/L ammonium sulfate by 4.3 times, 143%, and 5.1 times, respectively. CA was accumulated in meaningful quantities only in media containing 3–10 g/L (NH4)2SO4 with the maximum concentration of CA (99.9 g/L) at 4 g/L ammonium sulfate. Also of interest is the technological mode with 6 g/L (NH4)2SO4, which is characterized by high productivity (1.11 g/L × h). It should be noted that biomass contains large amounts of essential amino acids and unsaturated fatty acids and can be used in food biotechnologies and agriculture

Fermentation Conditions and Media Optimization for Isocitric Acid Production from Ethanol by Yarrowia lipolytica

Isocitric acid exists in the form of four stereoisomers, of which only the threo-Ds-form (ICA) is a natural active compound, an intermediate of Krebs cycle, and suitable for nutritional and pharmaceutical use. In this paper, we propose a method for ICA production from ethanol by yeast Yarrowia lipolytica. The effects of temperature, pH of the medium, and aeration on the growth of the producer Y. lipolytica VKM Y-2373 and synthesis of ICA were studied. An optimal fermentation regime, which ensures a good growth of the producer and directed synthesis of the target product, was determined. The producer is advised to carry out cultivation at 29°C and various pH of the medium and the oxygen concentration (pH 5 and pO2 20–25% (of saturation) during the growth period and pH 6 and pO2 50–55% (of saturation) during the acid formation) on a nutrient medium containing an increased content of zinc (0.6 mg/L), iron (1.2 mg/L), and 30 mM itaconic acid (inhibitor of isocitrate lyase—the key enzyme of ICA metabolism) should also be introduced into the nutrition medium. Such fermentation production mode provides 90.5 g/L ICA with process selectivity of 80%, mass yield (YICA) of 0.77 g/g, and energy yield (ηICA) of 0.278 g/g

S-белок пробиотического штамма Lactobacillus crispatus 2029 предотвращает рост проницаемости монослоя Caco-2 энтероцитов человека, индуцируемый возбудителями кишечных инфекций = S-protein of the probiotic strain lactobacillus crispatus 2029 prevents the growth of permeability of the Caco-2 monolayer of human enterocytes induced by intestinal infections

The use of a biomodel of the confluent monolayer Caco-2 of enterocytes allowed us to reveal the ability of the S-protein isolated from strain LC2029 to prevent permeability disorders resulting from intestinal infections induced by pathogens (E. coli O157: H7, C. jejuni ATCC 33291, S. enteritidis ATCC 25928). This is important for maintaining the efficiency of the intestinal barrier

Направленная регуляция микробиома человека : вклад в решение демографической проблемы в России = Directed regulation of the human microbiome : contribution to the solution of the demographic problem in Russia

In order to solve the demographic problem that has arisen in Russia, comprehensive multilateral approaches are required, aimed at increasing the birth rate and reducing child and adult mortality. One approach to solve this problem is directed regulation of the human microbiome

S-layer protein 2 of vaginal 'Lactobacillus crispatus' 2029 enhances growth, differentiation, VEGF production and barrier functions in intestinal epithelial cell line Caco-2

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