The molecular architecture of Lactobacillus S-layer: Assembly and attachment to teichoic acids.

S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer's pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.

Mutualistic interactions of lactate-producing lactobacilli and lactate-utilizing Veillonella dispar: Lactate and glutamate cross-feeding for the enhanced growth and short-chain fatty acid production.

The human gut hosts numerous ecological niches for microbe-microbe and host-microbe interactions. Gut lactate homeostasis in humans is crucial and relies on various bacteria. Veillonella spp., gut lactate-utilizing bacteria, and lactate-producing bacteria were frequently co-isolated. A recent clinical trial has revealed that lactate-producing bacteria in humans cross-feed lactate to Veillonella spp.; however, their interspecies interaction mechanisms remain unclear. Veillonella dispar, an obligate anaerobe commonly found in the human gut and oral cavity, ferments lactate into acetate and propionate. In our study, we investigated the interaction between V. dispar ATCC 17748T and three representative phylogenetically distant strains of lactic acid bacteria, Lactobacillus acidophilus ATCC 4356T, Lacticaseibacillus paracasei subsp. paracasei ATCC 27216T, and Lactiplantibacillus plantarum ATCC 10241. Bacterial growth, viability, metabolism and gene level adaptations during bacterial interaction were examined. V. dispar exhibited the highest degree of mutualism with L. acidophilus. During co-culture of V. dispar with L. acidophilus, both bacteria exhibited enhanced growth and increased viability. V. dispar demonstrated an upregulation of amino acid biosynthesis pathways and the aspartate catabolic pathway. L. acidophilus also showed a considerable number of upregulated genes related to growth and lactate fermentation. Our results support that V. dispar is able to enhance the fermentative capability of L. acidophilus by presumably consuming the produced lactate, and that L. acidophilus cross-feed not only lactate, but also glutamate, to V. dispar during co-culture. The cross-fed glutamate enters the central carbon metabolism in V. dispar. These findings highlight an intricate metabolic relationship characterized by cross-feeding of lactate and glutamate in parallel with considerable gene regulation within both L. acidophilus (lactate-producing) and V. dispar (lactate-utilizing). The mechanisms of mutualistic interactions between a traditional probiotic bacterium and a potential next-generation probiotic bacterium were elucidated in the production of short-chain fatty acids.

Analyzing of colorectal cancerrelated genes and microRNAs expression profiles in response to probiotics Lactobacillus acidophilus and Saccharomyces cerevisiae in colon cancer cell lines.

BACKGROUND: Colorectal cancer is the world's third most frequent cancer and the fourth cause of mortality. Probiotics play an important function in preventing metastasis as well as the growth and proliferation of malignant cancer cells. METHODS AND RESULTS: The study investigated the anticancer effect of Lactobacillus acidophilus supernatant and Saccharomyces cerevisiae yeast on colorectal cell lines, including HT29 and SW480 as a colorectal cancer model. The extract from the Lactobacillus acidophilus and Saccharomyces cerevisiae standard probiotics were prepared, and probiotics characterization was confirmed by morphological and Biochemical tests. The viability of HT29 and SW480 colon cancer cell lines on effecting probiotic supernatant was evaluated by measuring the MTT colorimetric method. Comparison of the expression profile of several genes involved in apoptosis, cell cycle, and metastatic pathway in HT29 and SW480 cell lines with the treatment of probiotics extract showed an upregulation in the BAX, CASP3, and CASP9 and down regulation BCl-2, MMP2, and MMP9 genes. Also, a comparison of microRNA expression profiles indicated an increase of miR 34, 135, 25, 16, 195, 27, 98, let7 and a decrease of miR 9, 106b, 17, 21, 155, 221. CONCLUSIONS AND DISCUSSION: The findings of this study indicate that probiotics can effectively suppress the proliferation of colorectal cancer cells and even reverse their development. Additionally, the study of cellular genes and miRNA profiles associated with colorectal cancer have demonstrated that our probiotics play a crucial role in CRC prevention by increasing the expression of tumor suppressor microRNAs and their target genes while decreasing oncogenes.

Safety assessment of potential probiotic Lactobacillus acidophilus AM13-1 with high cholesterol-lowering capability isolated from human gut.

An important risk factor for cardiovascular disease is dyslipidemia, especially abnormal cholesterol levels. The relation between probiotics and cholesterol-lowering capability has been extensively studied. Lactobacillus acidophilus plays a significant role in affecting host health, and produces multitudinous metabolites, which have prohibitory functions against pathogenic microorganisms. In this study, we identified a cholesterol-lowering strain AM13-1, isolated from a fecal sample obtained from a healthy adult male, and performed comprehensive function analysis by whole-genome analysis and in vitro experiments. Genome analyses of L. acidophilus AM13-1 revealed that carbohydrate and amino acid transport, metabolism, translation, ribosomal structure, and biogenesis are abundant categories of functional genes. No virulence factors or toxin genes with experimentally verified were found in the genome of strain AM13-1. Besides, plenty of probiotic-related genes were predicted from the L. acidophilus AM13-1 genome, such as cbh, atpA-D, and dltD, with functions related to cholesterol-lowering and acid resistance. And strain AM13-1 showed high-efficiency of bile salt hydrolase activity and the capacity for removing cholesterol with efficiency rates of 70%. These function properties indicate that strain AM13-1 can be considered as a probiotic candidate for use in food and health care products.

Comparative genomic analysis reveals niche adaption of Lactobacillus acidophilus.

AIMS: Lactobacillus acidophilus has been extensively applied in plentiful probiotic products. Although several studies have been performed to investigate the beneficial characteristics and genome function of L. acidophilus, comparative genomic analysis remains scarce. In this study, we collected 74 L. acidophilus genomes from our gut bacterial genome collection and the public database and conducted a comprehensive comparative genomic analysis. METHODS AND RESULTS: This study revealed the potential correlation of the genomic diversity and niche adaptation of L. acidophilus from different perspectives. The pan-genome of L. acidophilus was found to be open, with metabolism, information storage, and processing genes mainly distributed in the core genome. Phage- and peptidase-associated genes were found in the genome of the specificity of animal-derived strains, which were related to the adaptation of the animal gut. SNP analysis showed the differences of the utilization of vitamin B12 in cellular of L. acidophilus strains from animal gut and others. CONCLUSIONS: This work provides new insights for the genomic diversity analysis of L. acidophilus and uncovers the ecological adaptation of the specific strains.

Functional Roles of N-Terminal Domains in Pullulanase from Human Gut Lactobacillus acidophilus.

Pullulanases are multidomain α-glucan debranching enzymes with one or more N-terminal domains (NTDs) including carbohydrate-binding modules (CBMs) and domains of unknown function (DUFs). To elucidate the roles of NTDs in Lactobacillus acidophilus NCFM pullulanase (LaPul), two truncated variants, Δ41-LaPul (lacking CBM41) and Δ(41+DUFs)-LaPul (lacking CBM41 and two DUFs), were produced recombinantly. LaPul recognized 1.3- and 2.2-fold more enzyme attack-sites on starch granules compared to Δ41-LaPul and Δ(41+DUFs)-LaPul, respectively, as measured by interfacial kinetics. Δ41-LaPul displayed markedly lower affinity for starch granules and ß-cyclodextrin (10- and >21-fold, respectively) in comparison to LaPul, showing substrate binding mainly stems from CBM41. Δ(41+DUFs)-LaPul exhibited a 12 °C lower melting temperature than LaPul and Δ41-LaPul, indicating that the DUFs are critical for LaPul stability. Notably, Δ41-LaPul exhibited a 14-fold higher turnover number (kcat) and 9-fold higher Michaelis constant (KM) compared to LaPul, while Δ(41+DUFs)-LaPul's values were close to those of LaPul, possibly due to the exposure of aromatic by truncation.

In silico prospection of Lactobacillus acidophilus strains with potential probiotic activity.

Lactic acid bacteria (LAB) are fermentative microorganisms and perform different roles in biotechnological processes, mainly in the food and pharmaceutical industries. Among the LAB, Lactobacillus acidophilus is a species that deserves to be highlighted for being used both in prophylaxis and in the treatment of pathologies. Most of the metabolites produced by this species are linked to the inhibition of pathogens. In this study, we utilized a pangenomic and metabolic annotation analysis using Roary and BlastKOALA, ML-based probiotic activity prediction with iProbiotic and whole-genome similarity using ANI to identify strains of L. acidophilus with potential probiotic activity. According to the results in BlastKOALA and iProbiotics, L. acidophilus NCTC 13721 had the greatest potential among the 64 strains tested, both in terms of its ability to be a Lactobacillus spp. probiotic, when in the amount of genes involved in the metabolism of organic acids and quorum sensing. In addition, DSM 20079 proved to be promising for prospecting new probiotic Lactobacillus from BlastKOALA analyses, as they presented similar results in the number of genes involved in the production of lactic acid, acetic acid, hydrogen peroxide, except for quorum sensing where the NCTC 13721 strain had 14 more genes. L. acidophilus NCTC 13721 and L. acidophilus La-5 strains showed greater ability to be Lactobacillus spp. probiotic capacity, showing 84.8% and 51.9% capacity in the iProbiotics tool, respectively. When analyzed in ANI, none of the evaluated strains showed genomic similarity with NCTC 13721. In contrast, the DSM 20079 strain showed genomic similarity with all evaluated strains except NCTC 13721. Furthermore, eight strains with characteristics with approximately 100% genomic similarity to La-5 were listed: S20_1, LA-5, FSI4, APC2845, LA-G80-111, DS1_1A, LA1, and BCRC 14065. Therefore, according to the findings in iProbiotics and BlastKoala, among the 64 strains evaluated, NCTC 13721 is the most promising strain to be used for future in vitro studies.

Induced Expression of the Acinetobacter sp. Oxa Gene in Lactobacillus acidophilus and Its Increased ZEN Degradation Stability by Immobilization.

Zearalenone (ZEN, ZEA) contamination in various foods and feeds is a significant global problem. Similar to deoxynivalenol (DON) and other mycotoxins, ZEN in feed mainly enters the body of animals through absorption in the small intestine, resulting in estrogen-like toxicity. In this study, the gene encoding Oxa, a ZEN-degrading enzyme isolated from Acinetobacter SM04, was cloned into Lactobacillus acidophilus ATCC4356, a parthenogenic anaerobic gut probiotic, and the 38 kDa sized Oxa protein was expressed to detoxify ZEN intestinally. The transformed strain L. acidophilus pMG-Oxa acquired the capacity to degrade ZEN, with a degradation rate of 42.95% at 12 h (initial amount: 20 µg/mL). The probiotic properties of L. acidophilus pMG-Oxa (e.g., acid tolerance, bile salt tolerance, and adhesion properties) were not affected by the insertion and intracellular expression of Oxa. Considering the low amount of Oxa expressed by L. acidophilus pMG-Oxa and the damage to enzyme activity by digestive juices, Oxa was immobilized with 3.5% sodium alginate, 3.0% chitosan, and 0.2 M CaCl2 to improve the ZEN degradation efficiency (from 42.95% to 48.65%) and protect it from digestive juices. The activity of immobilized Oxa was 32-41% higher than that of the free crude enzyme at different temperatures (20-80 °C), pH values (2.0-12.0), storage conditions (4 °C and 25 °C), and gastrointestinal simulated digestion conditions. Accordingly, immobilized Oxa could be resistant to adverse environmental conditions. Owing to the colonization, efficient degradation performance, and probiotic functionality of L. acidophilus, it is an ideal host for detoxifying residual ZEN in vivo, demonstrating great potential for application in the feed industry.

Insights from metagenome-assembled genomes on the genetic stability and safety of over-the-counter probiotic products.

The demand for and acceptance of probiotics is determined by their quality and safety. Illumina NGS sequencing and analytics were used to examine eight marketed probiotics. Up to the species level, sequenced DNA was taxonomically identified, and relative abundances were determined using Kaiju. The genomes were constructed using GTDB and validated through PATRICK and TYGS. A FastTree 2 phylogenetic tree was constructed using several type strain sequences from relevant species. Bacteriocin and ribosomally synthesized polypeptide (RiPP) genes were discovered, and a safety check was performed to test for toxins, antibiotic resistance, and genetic drift genes. Except for two products with unclaimed species, the labeling was taxonomically correct. In three product formulations, Lactobacillus acidophilus, Limosilactobacillus reuteri, Lacticaseibacillus paracasei, and Bifidobacterium animalis exhibited two to three genomic alterations, while Streptococcus equinus was found in one. TYGS and GDTB discovered E. faecium and L. paracasei in distinctly different ways. All the bacteria tested had the genetic repertoire to tolerate GIT transit, although some exhibited antibiotic resistance, and one strain had two virulence genes. Except for Bifidobacterium strains, the others revealed a variety of bacteriocins and ribosomally synthesized polypeptides (RiPP), 92% of which were unique and non-homologous to known ones. Plasmids and mobile genetic elements are present in strains of L. reuteri (NPLps01.et_L.r and NPLps02.uf_L.r), Lactobacillus delbrueckii (NPLps01.et_L.d), Streptococcus thermophilus (NPLps06.ab_S.t), and E. faecium (NPLps07.nf_E.f). Our findings support the use of metagenomics to build better and efficient production and post-production practices for probiotic quality and safety assessment.

Transcriptome analysis of the Clostridioides difficile response to a specific lactobacilli probiotic formulation: explanations for its mechanisms of action.

AIMS: Clostridioides difficile infections (CDI) are a major cause of morbidity and mortality in hospitalized patients. A probiotic formulation (Bio-K+) comprised of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacti. rhamnosus CLR2 strains have been shown to reduce the incidence of CDI and antibiotic-associated diarrhea (AAD). This research aims to therefore elucidate the mechanism of action of the three probiotic strained against C. difficile R20291, independently of the acidification of the environment. . METHODS AND RESULTS: Antitoxin activity was evaluated using ELISA method and the expression of C. difficile genes was evaluated using transcriptomic analysis in co-culture assays conducted in a bioreactor allowing precise control of the pH. The fermentation results demonstrated a decrease for toxin A and many genes directly related to C. difficile virulence were underexpressed in the co-cultures. CONCLUSIONS: The lactobacilli tested could have a role in the motility, the quorum sensing (QS), the survival of the spores, and the germination potential of the spores, which are essential elements for the virulence of C. difficile. .

Novel targets in rectal cancer by considering lncRNA-miRNA-mRNA network in response to Lactobacillus acidophilus consumption: a randomized clinical trial.

We aimed to explore the lncRNA-miR-mRNA network in response to Lactobacillus acidophilus (L. acidophilus) consumption in rectal cancer patients. The candidate miRs were first taken from the GEO and TCGA databases. We constructed the lncRNA-miR-mRNA network using the high-throughput sequencing data. At last, we created a heatmap based on the experimental data to show the possible correlation of the selected targets. The expression levels of selected targets were measured in the samples of 107 rectal cancer patients undergoing placebo and probiotic consumption and 10 noncancerous subjects using Real-Time PCR. Our analysis revealed a group of differentially expressed 12 miRs and 11 lncRNAs, and 12 genes in rectal cancer patients. A significant expression increase of the selected tumor suppressor miRs, lncRNAs, and genes and a substantial expression decrease of the selected oncomiRs, onco-lncRNAs, and oncogenes were obtained after the probiotic consumption compared to the placebo group. There is a strong correlation between some network components, including miR-133b and IGF1 gene, miR-548ac and MSH2 gene, and miR-21 and SMAD4 gene. In rectal cancer patients, L. acidophilus consumption was associated with improved expression of the lncRNA-miR-mRNA network, which may provide novel monitoring and therapeutic approaches.

Computational design of Lactobacillus Acidophilus α-L-rhamnosidase to increase its structural stability.

α-L-rhamnosidase catalyzes hydrolysis of the terminal α-L-rhamnose from various natural rhamnoglycosides, including naringin and hesperidin, and has various applications such as debittering of citrus juices in the food industry and flavonoid derhamnosylation in the pharmaceutical industry. However, its activity is lost at high temperatures, limiting its usage. To improve Lactobacillus acidophilus α-L-rhamnosidase stability, we employed molecular dynamics (MD) to identify a highly flexible region, as evaluated by its root mean square fluctuation (RMSF) value, and computational protein design (Rosetta) to increase rigidity and favorable interactions of residues in highly flexible regions. MD results show that five regions have the highest flexibilities and were selected for design by Rosetta. Twenty-one designed mutants with the best ΔΔG at each position and ΔΔG < 0 REU were simulated at high temperature. Eight designed mutants with ΔRMSF of highly flexible regions lower than -10.0% were further simulated at the optimum temperature of the wild type. N88Q, N202V, G207D, Q209M, N211T and Y213K mutants were predicted to be more stable and could maintain their native structures better than the wild type due to increased hydrogen bond interactions of designed residues and their neighboring residues. These designed mutants are promising enzymes with high potential for stability improvement.

Multiplex SYBR Green real-time PCR for Lactobacillus acidophilus group species targeting biomarker genes revealed by a pangenome approach.

The Lactobacillus acidophilus group consists of seven closely related species. Among these, Lb. acidophilus, Lb. gallinarum, and Lb. helveticus help maintain gut health and are used as a starter for fermented food. However, these species are difficult to differentiate using conventional methods due to the high similarity between the 16S rRNA and housekeeping genes. Thus, in this study, we selected biomarker genes to identify and discriminate the three species via pangenome analysis, and a multiplex SYBR Green real-time PCR that can be detected simultaneously in a single tube was developed. Pangenome analysis revealed three specific target genes: mucus-binding protein precursor to detect Lb. acidophilus, an amino acid ABC superfamily ATP binding cassette transporter carrier protein to detect Lb. gallinarum, and selenocysteine lyase to detect Lb. helveticus. The specificity was robustly verified using 26 Lb. acidophilus group strains and 62 other strains. The detection limits were 101 colony-forming units (CFU)/ml in pure culture. As per our findings, the developed method satisfactorily monitored Lb. acidophilus group species in probiotic and dairy products. This result suggests that real-time PCR based on specific targets provides a promising approach for the rapid, accurate, and sensitive identification of these three species.

Heterologous expression and biological characteristics of UGPases from Lactobacillus acidophilus.

Herein, two genes (LBA0625 and LBA1719) encoding UGPases (UDP-glucose pyrophosphorylase) in Lactobacillus acidophilus (L. acidophilus) were successfully transformed into Escherichia coli BL21 (DE3) to construct recombinant overexpressing strains (E-0625, E-1719) to investigate the biological characteristics of UGPase-0625 and UGPase-1719. The active sites, polysaccharide yield, and anti-freeze-drying stress of L. acidophilus ATCC4356 were also detected. UGPase-0625 and UGPase-1719 belong to the nucleotidyltransferase of stable hydrophilic proteins; contain 300 and 294 amino acids, respectively; and have 20 conserved active sites by prediction. Αlpha-helixes and random coils were the main secondary structures, which constituted the main skeleton of UGPases. The optimal mixture for the high catalytic activity of the two UGPases included 0.5 mM UDP-Glu (uridine diphosphate glucose) and Mg2+ at 37 °C, pH 10.0. By comparing the UGPase activities of the mutant strains with the original recombinant strains, A10, L130, and L263 were determined as the active sites of UGPase-0625 (P < 0.01) and A11, L130, and L263 were determined as the active sites of UGPase-1719 (P < 0.01). In addition, UGPase overexpression could increase the production of polysaccharides and the survival rates of recombinant bacteria after freeze-drying. This is the first study to determine the enzymatic properties, active sites, and structural simulation of UGPases from L. acidophilus, providing in-depth understanding of the biological characteristics of UGPases in lactic acid bacteria.Key points• We detected the biological characteristics of UGPases encoded by LBA0625 and LBA1719.• We identified UGPase-0625 and UGPase-1719 active sites.• UGPase overexpression elevates polysaccharide levels and post-freeze-drying survival.

Probiotic activity traits in vitro and production of antimicrobial peptides by Lactobacillaceae isolates from pulque using Lactobacillus acidophilus NCFM as control.

The objective of this work was to determine in vitro probiotic activity traits of 11 lactic acid bacteria (LAB) strains isolated from pulque obtained from three different locations in the Mexican states of Oaxaca and Puebla using the probiotic strain Lactobacillus acidophilus NCFM as a positive control, and to detect their production of antimicrobial peptides, including bacteriocins and peptidoglycan hydrolases (PGH). The LAB isolates were identified by sequencing of their 16S rRNA as belonging to four different genera of the Lactobacillaceae family: Lactiplantibacillus, Levilactobacillus, Lacticaseibacillus and Liquorilactobacillus, corresponding to the species plantarum, brevis, paracasei and ghanensis, respectively. Most of the strains showed resistance to high acidity (pH 2) and bile salts (0.5%), with survival rates up to 87 and 92%, respectively. In addition, most of the strains presented good antimicrobial activity against the foodborne pathogens Listeria monocytogenes, ECEC and Salmonella Typhi. The strain Liquorilactobacillus ghanensis RVG6, newly reported in pulque, presented an outstanding overall performance on the probiotic activity tests. In terms of their probiotic activity traits assessed in this work, the strains compared positively with the control L. acidophilus NCFM, which is a very-well documented probiotic strain. For the antimicrobial peptide studies, four strains presented bacteriocin-like mediated antibiosis and six had significant PGH activity, with two strains presenting outstanding overall antimicrobial peptide production: Lacticaseibacillus paracasei RVG3 and Levilactobacillus brevis UTMB2. The probiotic performance of the isolates was mainly dependent on strain specificity. The results obtained in this work can foster the revalorization of pulque as a functional natural product.

Positive Effect of Lactobacillus acidophilus EG004 on Cognitive Ability of Healthy Mice by Fecal Microbiome Analysis Using Full-Length 16S-23S rRNA Metagenome Sequencing.

Evidence for the concept of the "gut-brain axis" (GBA) has risen. Many types of research demonstrated the mechanism of the GBA and the effect of probiotic intake. Although many studies have been reported, most were focused on neurodegenerative disease and, it is still not clear what type of bacterial strains have positive effects. We designed an experiment to discover a strain that positively affects brain function, which can be recognized through changes in cognitive processes using healthy mice. The experimental group consisted of a control group and three probiotic consumption groups, namely, Lactobacillus acidophilus, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus. Three experimental groups fed probiotics showed an improved cognitive ability by cognitive-behavioral tests, and the group fed on L. acidophilus showed the highest score. To provide an understanding of the altered microbial composition effect on the brain, we performed full 16S-23S rRNA sequencing using Nanopore, and operational taxonomic units (OTUs) were identified at species level. In the group fed on L. acidophilus, the intestinal bacterial ratio of Firmicutes and Proteobacteria phyla increased, and the bacterial proportions of 16 species were significantly different from those of the control group. We estimated that the positive results on the cognitive behavioral tests were due to the increased proportion of the L. acidophilus EG004 strain in the subjects' intestines since the strain can produce butyrate and therefore modulate neurotransmitters and neurotrophic factors. We expect that this strain expands the industrial field of L. acidophilus and helps understand the mechanism of the gut-brain axis. IMPORTANCE Recently, the concept of the "gut-brain axis" has risen and suggested that microbes in the GI tract affect the brain by modulating signal molecules. Although many pieces of research were reported in a short period, a signaling mechanism and the effects of a specific bacterial strain are still unclear. Besides, since most of the research was focused on neurodegenerative disease, the study with a healthy animal model is still insufficient. In this study, we show using a healthy animal model that a bacterial strain (Lactobacillus acidophilus EG004) has a positive effect on mouse cognitive ability. We experimentally verified an improved cognitive ability by cognitive behavioral tests. We performed full 16S-23S rRNA sequencing using a Nanopore MinION instrument and provided the gut microbiome composition at the species level. This microbiome composition consisted of candidate microbial groups as a biomarker that shows positive effects on cognitive ability. Therefore, our study suggests a new perspective for probiotic strain use applicable for various industrialization processes.

A Method of Direct Quantitation of Lactobacillus spp. in Intestinal Contents Based on Real-Time PCR.

The method of direct quantitation of Lactobacillus spp. and L. acidophilus in intestinal contents based on real-time PCR was developed. It does not require culturing and allows estimating the number of living lactobacilli cells (measured in lg CFU) in absolute quantitative PCR format.

A genome guided evaluation of the Lab4 probiotic consortium.

Draft genome sequences of the Lab4 probiotic consortium were deposited in Genbank: Bifidobacterium animalis subsp lactis CUL34 (PRJNA482550), Bifidobacterium bifidum CUL20 (PRJNA559984), Lactobacillus acidophilus CUL60 (PRJNA482335), Lactobacillus acidophilus CUL21 (PRJNA482434). Probiogenomic analyses confirmed existing taxonomies and identified putative gene sequences that were functionally related to the performance of each organism during in vitro assessments of bile and acid tolerability, adherence to enterocytes and susceptibility to antibiotics. Genomic stability predictions identified no significant risk of gene acquisition of both antibiotic resistance and virulence genes. These observations were supported by acute phase and repeat dose tolerability studies in Wistar rats. High doses of Lab4 did not result in mortalities, clinical/histopathological abnormalities nor systemic toxicity. Increased faecal numbers of Lab4 in supplemented rats implied survival through the gastrointestinal tract and/or impact the intestinal microbiota composition. In summary, this study provides multifaceted support for probiotic functionality and the safety of the Lab4 consortium.

The N-terminus of Lactobacillus amylovorus feruloyl esterase plays an important role in its secretion by Lactobacillus plantarum and Escherichia coli.

BACKGROUND: Feruloyl esterase is a multifunctional esterase with potential industrial applications. In the present study, we found the Lactobacillus amylovorus feruloyl esterase (FaeLam) could be secreted by L. plantarum and Escherichia coli. However, no signal peptide was detected in this protein as predicted by SignalP-5.0. Therefore, experiments were carried out to propose an explanation for the extracellular release of FaeLam. RESULTS: Here, we identified that the FaeLam could be secreted to the culture medium of L. plantarum CGMCC6888 and E. coli DH5α, respectively. To exclude the possibility that FaeLam secretion was caused by its hydrolytic activity on the cell membrane, the inactive FaeLamS106A was constructed and it could still be secreted out of L. plantarum and E. coli cells. Furthermore, the truncated version of the FaeLam without the N-terminal residues was constructed and demonstrated the importance of the 20 amino acids of N-terminus (N20) on FaeLam secretion. In addition, fusion of heterologous proteins with N20 or FaeLam could carry the target protein out of the cells. These results indicated the N-terminus of FaeLam played the key role in the export process. CONCLUSIONS: We proved the N-terminus of L. amylovorus FaeLam plays an important role in its secretion by L. plantarum and E. coli. To our best knowledge, this is the first reported protein which can be secreted out of the cells of both Gram-positive and Gram-negative bacteria. Furthermore, the results of this study may provide a new method for protein secretion in L. plantarum and E. coli through fusion the target protein to N20 of FaeLam.

In Vivo Transcriptome of Lactobacillus acidophilus and Colonization Impact on Murine Host Intestinal Gene Expression.

Lactobacillus acidophilus NCFM is a probiotic strain commonly used in dairy products and dietary supplements. Postgenome in vitro studies of NCFM thus far have linked potential key genotypes to its probiotic-relevant attributes, including gut survival, prebiotic utilization, host interactions, and immunomodulatory activities. To corroborate and extend beyond previous in vivo and in vitro functional studies, we employed a dual RNA sequencing (RNA-seq) transcriptomic approach to identify genes potentially driving the gut fitness and activities of L. acidophilus NCFM in vivo, and in parallel, examine the ileal transcriptional response of its murine hosts during monocolonization. Spatial expression profiling of NCFM from the ileum through the colon revealed a set of 134 core genes that were consistently overexpressed during gut transit. These in vivo core genes are predominantly involved in the metabolism of carbohydrates, amino acids, and nucleotides, along with mucus-binding proteins and adhesion factors, confirming their functionally important roles in nutrient acquisition and gut retention. Functional characterization of the highly expressed major S-layer-encoding gene established its indispensable role as a cell shape determinant and maintenance of cell surface integrity, essential for viability and probiotic attributes. Host colonization by L. acidophilus resulted in significant downregulation of several proinflammatory cytokines and tight junction proteins. Genes related to redox signaling, mucin glycosylation, and circadian rhythm modulation were induced, suggesting impacts on intestinal development and immune functions. Metagenomic analysis of NCFM populations postcolonization demonstrated the genomic stability of L. acidophilus as a gut transient and further established its safety as a probiotic and biotherapeutic delivery platform.IMPORTANCE To date, our basis for comprehending the probiotic mechanisms of Lactobacillus acidophilus, one of the most widely consumed probiotic microbes, was largely limited to in vitro functional genomic studies. Using a germfree murine colonization model, in vivo-based transcriptional studies provided the first view of how L. acidophilus survives in the mammalian gut environment, including gene expression patterns linked to survival, efficient nutrient acquisition, stress adaptation, and host interactions. Examination of the host ileal transcriptional response, the primary effector site of L. acidophilus, has also shed light into the mechanistic roles of this probiotic microbe in promoting anti-inflammatory responses, maintaining intestinal epithelial homeostasis and modulation of the circadian-metabolic axis in its host.