Jovanka Lukić

Different Roles for Lactococcal Aggregation Factor and Mucin Binding Protein in Adhesion to Gastrointestinal Mucosa

ABSTRACT Adhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation (aggL) and possible interaction with mucin (mbpL), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL. In vivo and in vitro experiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated the in vitro and in vivo aggregation of the BGKP1-20 carrying plasmid with aggL to binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the other hand, the presence of AggL in the strain reduced its ability to adhere to the ileum. Moreover, MbpL protein showed an affinity to bind gastric type mucin proteins such as MUC5AC. This protein did not contribute to the binding of the strain to the ileal or colonic part of the intestine. Different potential functions of lactococcal AggL and MbpL proteins in the process of adhesion to the gastrointestinal tract are proposed.

Interaction of Lactobacillus fermentum BGHI14 with Rat Colonic Mucosa: Implications for Colitis Induction

ABSTRACT The present study was carried out to test the colonic mucosal response of rats to oral supplementation with Lactobacillus fermentum BGHI14 and to correlate the tissue reaction to trinitrobenzenesulfonate (TNBS)-induced colitis with mucosal barrier alterations caused by bacterial ingestion. An immune cell-mediated reaction of healthy colonic tissue was noticed after bacterial feeding. After prolonged bacterial treatment, the observed reaction had retreated to normality, but the mRNA levels of proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) remained elevated. These data point to the chronic low-grade inflammation that could be caused by long-term probiotic consumption. Although no detrimental effects of bacterial pretreatment were noticed in colitic rats, at least in the acute state of disease, the results obtained in our study point to the necessity of reassessment of existing data on the safety of probiotic preparations. Additionally, probiotic effects in experimental colitis models might depend on time coordination of disease induction with treatment duration.

Correlation of Gut Microbiota Composition with Resistance to Experimental Autoimmune Encephalomyelitis in Rats.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system (CNS). It is widely accepted that autoimmune response against the antigens of the CNS is the essential pathogenic force in the disease. It has recently become increasingly appreciated that activated encephalitogenic cells tend to migrate toward gut associated lymphoid tissues (GALTs) and that interrupted balance between regulatory and inflammatory immunity within the GALT might have decisive role in the initiation and propagation of the CNS autoimmunity. Gut microbiota composition and function has the major impact on the balance in the GALT. Thus, our aim was to perform analyses of gut microbiota in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Albino Oxford (AO) rats that are highly resistant to EAE induction and Dark Agouti (DA) rats that develop EAE after mild immunization were compared for gut microbiota composition in different phases after EAE induction. Microbial analyses of the genus Lactobacillus and related lactic acid bacteria showed higher diversity of Lactobacillus spp. in EAE-resistant AO rats, while some members of Firmicutes and Proteobacteria (Undibacterium oligocarboniphilum) were detected only in feces of DA rats at the peak of the disease (between 13 and 16 days after induction). Interestingly, in contrast to our previous study where Turicibacter sp. was found exclusively in non-immunized AO, but not in DA rats, in this study it was detected in DA rats that remained healthy 16 days after induction, as well as in four of 12 DA rats at the peak of the disease. Similar observation was obtained for the members of Lachnospiraceae. Further, production of a typical regulatory cytokine interleukin-10 was compared in GALT cells of AO and DA rats, and higher production was observed in DA rats. Our data contribute to the idea that gut microbiota and GALT considerably influence multiple sclerosis pathogenesis.

Genotypic diversity and virulent factors of Staphylococcus epidermidis isolated from human breast milk.

Staphylococcus epidermidis strains were isolated from the expressed human breast milk (EHM) of 14 healthy donor mothers. Genetic diversity was evaluated using RAPD-PCR REP-PCR and pulse-field gel electrophoresis (PFGE). PFGE allowed the best discrimination of the isolates, since it provided for the greatest diversity of the analyzed genomes. Among the S. epidermidis strains, resistance to gentamicin, tetracycline, erythromycin, clindamycin or vancomycin was detected, whilst four isolates were multiresistant. The results from our study demonstrate that staphylococci from EHM could be reservoirs of resistance genes, since we showed that tetK could be transferred from EHM staphylococci to Gram-negative Escherichia coli. Most of the staphylococcal strains displayed excellent proteolytic and lipolytic activities. Additionally, the presence of ica genes, which was related to their ability to form a biofilm on tissue culture plates, and the presence of virulence factors including autolysin/adhesin AtLE, point to their pathogenic potential.

Gut-associated lymphoid tissue, gut microbes and susceptibility to experimental autoimmune encephalomyelitis

Gut microbiota and gut-associated lymphoid tissue have been increasingly appreciated as important players in pathogenesis of various autoimmune diseases, including multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that can be induced with an injection of spinal cord homogenate emulsified in complete Freunds adjuvant in Dark Agouti (DA) rats, but not in Albino Oxford (AO) rats. In this study, mesenteric lymph nodes (MLN), Peyers patches (PP) and gut microbiota were analysed in these two rat strains. There was higher proportion of CD4(+) T cells and regulatory T cells in non-immunised DA rats in comparison to AO rats. Also, DA rat MLN and PP cells were higher producers of pro-inflammatory cytokines interferon-γ and interleukin-17. Finally, microbial analyses showed that uncultivated species of Turicibacter and Atopostipes genus were exclusively present in AO rats, in faeces and intestinal tissue, respectively. Thus, it is clear that in comparison of an EAE-susceptible with an EAE-resistant strain of rats, various discrepancies at the level of gut associated lymphoid tissue, as well as at the level of gut microbiota can be observed. Future studies should determine if the differences have functional significance for EAE pathogenesis.

Aggregation Factor as an Inhibitor of Bacterial Binding to Gut Mucosa

Modern research in the area of probiotics is largely devoted to discovering factors that promote the adherence of probiotic candidates to host mucosal surfaces. The aim of the present study was to test the role of aggregation factor (AggL) and mucin-binding protein (MbpL) from Lactococcus sp. in adhesion to gastrointestinal mucosa. In vitro, ex vivo, and in vivo experiments in rats were used to assess the adhesive potential of these two proteins expressed in heterologous host Lactobacillus salivarius BGHO1. Although there was no influence of MbpL protein expression on BGHO1 adhesion to gut mucosa, expression of AggL had a negative effect on BGHO1 binding to ileal and colonic rat mucosa, as well as to human HT29-MTX cells and porcine gastric mucin in vitro. Because AggL did not decrease the adhesion of bacteria to intestinal fragments in ex vivo tests, where peristaltic simulation conditions were missing, we propose that intestinal motility could be a crucial force for eliminating aggregation-factor-bearing bacteria. Bacterial strains expressing aggregation factor could facilitate the removal of pathogens through the coaggregation mechanism, thus balancing gut microbial ecosystems in people affected by intestinal bacteria overgrowth.

Lactobacillus fermentum Postbiotic-induced Autophagy as Potential Approach for Treatment of Acetaminophen Hepatotoxicity

The aim of this study was to investigate the potential of postbiotics originated from Lactobacillus fermentum BGHV110 strain (HV110) to counteract acetaminophen (APAP)-induced hepatotoxicity in HepG2 cells. This strain was selected according to its autophagy inducing potential, based on previous studies reporting protective role of autophagy in APAP caused cellular damage. Cell viability was assessed using MTT and LDH assays, while autophagy was monitored by qPCR analysis of BECN1, Atg5, p62/SQSTM1, and PINK1 mRNA expression and by Western blot analysis of p62/SQSTM1 and lipidated LC3 accumulation. Our results showed that detrimental effect of APAP on cell viability was suppressed in the presence of HV110 which was linked with increased conversion of LC3 protein and p62/SQSTM1 protein degradation. Additionally, higher p62/SQSTM1 and PINK1 mRNA transcription were noticed in cells co-treated with APAP/HV110, simultaneously. In conclusion, this study suggests that HV110 enhances activation of PINK1-dependent autophagy in HepG2 cells and its eventual co-supplementation with APAP could be potentially used for alleviation of hepatotoxic side effects caused by APAP overdose.

Exopolysaccharide Produced by Probiotic Strain Lactobacillus paraplantarum BGCG11 Reduces Inflammatory Hyperalgesia in Rats

The aim of this study was to test the potential of high molecular weight exopolysaccharide (EPS) produced by the putative probiotic strain Lactobacillus paraplantarum BGCG11 (EPS CG11) to alleviate inflammatory pain in Wistar rats. The EPS CG11 was isolated from bacterial surface and was subjected to Fourier-transform infrared spectroscopy (FTIR) and thermal analysis. FTIR spectra confirmed the polysaccharide structure of isolated sample, while the thermal methods revealed good thermal properties of the polymer. The antihyperalgesic and antiedematous effects of the EPS CG11 were examined in the rat model of inflammation induced by carrageenan injection in hind paw. The results showed that the intraperitoneal administration of EPS CG11 produced a significant decrease in pain sensations (mechanical hyperalgesia) and a paw swelling in a dose-dependent manner as it was measured using Von Frey anesthesiometer and plethysmometer, respectively. These effects were followed by a decreased expression of IL-1β and iNOS mRNAs in rat’s paw tissue suggesting that the antihyperalgesic and antiedematous effects of the EPS CG11 are related to the suppression of inflammatory response. Additionally, we demonstrated that EPS CG11 exhibits immunosuppressive properties in the peritonitis model induced by carrageenan. Expression levels of pro-inflammatory mediators IL-1β, TNF-α and iNOS were decreased, together with the enhanced secretion of anti-inflammatory IL-10 and IL-6 cytokines, while neutrophil infiltration was not changed. To the best of our knowledge, this is the first study which reports an antihyperalgesic effect as the novel property of bacterial EPSs. Given the high demands of pharmaceutical industry for the replacement of commonly used analgesics due to numerous side effects, this study describes a promising natural compound for the future pharmacological testing in the area.

Supplementation of lactobacilli improves growth, regulates microbiota composition and suppresses skeletal anomalies in juvenile pike-perch ( Sander lucioperca ) reared in recirculating aquaculture system (RAS): A pilot study

This research aimed to test the effects of lactobacilli, applied to cultured pike-perch, either through hydrolyzed OTOHIME fish diet, or through Artemia nauplii, on fish growth, microbiota balance and skeletal development. On the 12th Day Post Hatching (DPH) fish were divided into following treatment groups: two groups received the combination of OTOHIME and nauplii enriched either with Lactobacillus paracasei BGHN14+Lactobacillus rhamnosus BGT10 or with Lactobacillus reuteri BGGO6-55+Lactobacillus salivarius BGHO1, and one group received OTOHIME hydrolyzed by BGHN14+BGT10 and non-enriched nauplii. Control group received non-enriched nauplii and non-hydrolyzed OTOHIME. The treatment lasted 14days and fish were sacrificed on the 26th DPH for the assessment of digestive enzyme activity and microbiota composition. Individual total lengths and individual body weights were recorded at the end of the treatments, on the 26th DPH, and also on the 45th DPH, in parallel with the evaluation of skeletal deformities and fish survival. Our results indicated positive effect of Artemia enriched with BGGO6-55+BGHO1 on fish growth, skeletal development and trypsin to chymotrypsin activity ratio (T/C), as an indicator of protein digestibility. Hydrolysis of OTOHIME was also associated with better skeletal development, higher T/C values and lower levels of Aeromonas and Mycobacterium spp., which are important fish pathogens. Though additional testing in larger cohort studies is needed, these observations are promising in terms of usage of probiotics for improved environmentally friendly production of pike-perch in Recirculating Aquaculture System (RAS).

Survey on proteolytic activity and diversity of proteinase genes in mesophilic lactobacilli

Lactocepins or CEPs are large cell wall bound extracellular proteinases of lactic acid bacteria, involved in protein breakdown and utilization. They are responsible for many health-promoting traits of food products fermented with these organisms, but also essential for probiotic effects of certain strains. Different mesophilic strains selected within the species Lactobacillus zeae, Lb. casei, Lb. rhamnosus, and Lb. plantarum were analyzed for their proteolytic activity towards main fractions of milk proteins—caseins and whey proteins. The strains showing excellent proteolytic features were further examined for presence of corresponding proteinase gene(s). It was found that Lb. zeae LMG17315 possessed catalytic domains of three distinct proteinase genes, unique feature in Lb. casei group, which are similar but not identical to previously characterized prtP and prtR genes. Lb. casei neotype strain ATCC393 was also analysed and based on obtained results its reclassification in taxon Lb. zeae is supported. In addition, we report catalytic domain of prtR-type gene in Lb. plantarum LMG9208, which is first such report in this species, and it is first time that this gene is reported outside Lb. casei group.