Protective Properties of S-layer Protein 2 from Lactobacillus crispatus 2029 against Candida albicans Infections.

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.

Ligilactobacillus salivarius 7247 Strain: Probiotic Properties and Anti-Salmonella Effect with Prebiotics.

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.

Limosilactobacillus fermentum 3872 That Produces Class III Bacteriocin Forms Co-Aggregates with the Antibiotic-Resistant Staphylococcus aureus Strains and Induces Their Lethal Damage.

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.

Anti-Salmonella Defence and Intestinal Homeostatic Maintenance In Vitro of a Consortium Containing Limosilactobacillus fermentum 3872 and Ligilactobacillus salivarius 7247 Strains in Human, Porcine, and Chicken Enterocytes.

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.

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-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.

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-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.

Binding of LcrV protein from Yersinia pestis to human T-cells induces apoptosis, which is completely blocked by specific antibodies.

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.

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens.

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacillus crispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 ± 0.7) × 10(-10) M). Lactobacillus crispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacillus crispatus induced NF-κB activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1ß, IL-1α and TNF-α. Lactobacillus crispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacillus crispatus 2029 produced H2O2 and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents.

Properties of the Probiotic Strain Lactobacillus plantarum 8-RA-3 Grown in a Biofilm by Solid Substrate Cultivation Method.

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.