Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium.

Bifidobacterium is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in B. animalis subsp. lactis, demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.

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.

Portable CRISPR-Cas9N System for Flexible Genome Engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei.

Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplement industries, and specific strains, such as Lactobacillus acidophilus NCFM, have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR (clustered regularly interspaced palindromic repeats)-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for a customized single guide RNA (sgRNA) and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS, and ltaS), yielding 35 to 100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted an mCherry fluorescent-protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate a concurrent single-base substitution and gene deletion in Lactobacillus gasseri ATCC 33323 and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered with by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli and open new avenues for the engineering of health-promoting lactic acid bacteria.IMPORTANCE This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three Lactobacillus species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.

S-layer associated proteins contribute to the adhesive and immunomodulatory properties of Lactobacillus acidophilus NCFM.

BACKGROUND: Surface layers (S-layers) are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost layer of many bacterial cell envelopes. Within the Lactobacillus genus, S-layer presence is frequently associated with probiotic-relevant properties such as improved adherence to host epithelial cells and modulation of the immune response. However, recent studies have demonstrated that certain S-layer functions may be supplemented by a novel subset of proteins embedded within its lattice, termed S-layer associated proteins (SLAPs). In the following study, four Lactobacillus acidophilus NCFM SLAPs (LBA0046, LBA0864, LBA1426, and LBA1539) were selected for in silico and phenotypic assessment. RESULTS: Despite lacking any sequence similarity or catalytic domains that may indicate function, the genes encoding the four proteins of interest were shown to be unique to S-layer-forming, host-adapted lactobacilli species. Likewise, their corresponding deletion mutants exhibited broad, host-relevant phenotypes including decreased inflammatory profiles and reduced adherence to Caco-2 intestinal cells, extracellular matrices, and mucin in vitro. CONCLUSIONS: Overall, the data presented in this study collectively links several previously uncharacterized extracellular proteins to roles in the underlying host adaptive mechanisms of L. acidophilus.

An 1,4-α-Glucosyltransferase Defines a New Maltodextrin Catabolism Scheme in Lactobacillus acidophilus.

The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic α-amylase of family 13, subfamily 20 (LaGH13_20), a maltose phosphorylase of GH65 (LaGH65), and a family 13, subfamily 31, member (LaGH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that LaGH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS with a degree of polymerization of ≥2, with a preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus revealed that the substrate preference of LaGH13_31B toward maltotriose complements the ~40-fold lower kcat of LaGH13_20 toward this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose. The concerted action of LaGH13_20 and LaGH13_31B confers the efficient conversion of MOS to maltose that is phosphorolyzed by LaGH65. Structural analyses revealed the presence of a flexible elongated loop that is unique for a previously unexplored clade of GH13_31, represented by LaGH13_31B. The identified loop insertion harbors a conserved aromatic residue that modulates the activity and substrate affinity of the enzyme, thereby offering a functional signature of this clade, which segregates from 1,6-α-glucosidases and sucrose isomerases previously described within GH13_31. Genomic analyses revealed that the LaGH13_31B gene is conserved in the MOS utilization loci of lactobacilli, including acidophilus cluster members that dominate the human small intestine.IMPORTANCE The degradation of starch in the small intestine generates short linear and branched α-glucans. The latter are poorly digestible by humans, rendering them available to the gut microbiota, e.g., lactobacilli adapted to the small intestine and considered beneficial to health. This study unveils a previously unknown scheme of maltooligosaccharide (MOS) catabolism via the concerted activity of an 1,4-α-glucosyltransferase together with a classical hydrolase and a phosphorylase. The intriguing involvement of a glucosyltransferase likely allows the fine-tuning of the regulation of MOS catabolism for optimal harnessing of this key metabolic resource in the human small intestine. The study extends the suite of specificities that have been identified in GH13_31 and highlights amino acid signatures underpinning the evolution of 1,4-α-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli.

Deletion of S-Layer Associated Ig-Like Domain Protein Disrupts the Lactobacillus acidophilus Cell Surface.

Bacterial surface-layers (S-layers) are crystalline arrays of repeating proteinaceous subunits that coat the exterior of many cell envelopes. S-layers have demonstrated diverse functions in growth and survival, maintenance of cell integrity, and mediation of host interactions. Additionally, S-layers can act as scaffolds for the outward display of auxiliary proteins and glycoproteins. These non-covalently bound S-layer associated proteins (SLAPs) have characterized roles in cell division, adherence to intestinal cells, and modulation of the host immune response. Recently, IgdA (LBA0695), a Lactobacillus acidophilus SLAP that possesses a Group 3 immunoglobulin (Ig)-like domain and GW (Gly-Tryp) dipeptide surface anchor, was recognized for its high conservation among S-layer-forming lactobacilli, constitutive expression, and surface localization. These findings prompted its selection for examination within the present study. Although IgdA and corresponding orthologs were shown to be unique to host-adapted lactobacilli, the Ig domain itself was specific to vertebrate-adapted species suggesting a role in vertebrate adaptation. Using a counterselective gene replacement system, igdA was deleted from the L. acidophilus NCFM chromosome. The resultant mutant, NCK2532, exhibited a visibly disrupted cell surface which likely contributed to its higher salt sensitivity, severely reduced adhesive capacity, and altered immunogenicity profile. Transcriptomic analyses revealed the induction of several stress response genes and secondary surface proteins. Due to the broad impact of IgdA on the cellular physiology and probiotic attributes of L. acidophilus, identification of similar proteins in alternative bacterial species may help pinpoint next-generation host-adapted probiotic candidates.

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus.

CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5'-AAA-3' protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.

Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus.

CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems.

Comparative Analysis of Lactobacillus gasseri and Lactobacillus crispatus Isolated From Human Urogenital and Gastrointestinal Tracts.

Lactobacillus crispatus and Lactobacillus gasseri are two of the main Lactobacillus species found in the healthy vaginal microbiome and have also previously been identified and isolated from the human gastrointestinal (GI) tract. These two ecological niches are fundamentally different, notably with regards to the epithelial cell type, nutrient availability, environmental conditions, pH, and microbiome composition. Given the dramatic differences between these two environments, we characterized strains within the same Lactobacillus species isolated from either the vaginal or intestinal tract to assess whether they are phenotypically and genetically different. We compared the genomes of the Lactobacillus strains selected in this study for genetic features of interest, and performed a series of comparative phenotypic assays including small intestinal juice and acid resistance, carbohydrate fermentation profiles, lactic acid production, and host interaction with intestinal Caco-2 and vaginal VK2 cell lines. We also developed a simulated vaginal fluid (SVF) to study bacterial growth in a proxy vaginal environment and conducted differential transcriptomic analysis between SVF and standard laboratory MRS medium. Overall, our results show that although strain-specific variation is observed, some phenotypic differences seem associated with the isolation source. We encourage future probiotic formulation to include isolation source and take into consideration genetic and phenotypic features for use at various body sites.

Characterization and Repurposing of Type I and Type II CRISPR-Cas Systems in Bacteria.

CRISPR-Cas systems constitute the adaptive immune system of bacteria and archaea, as a sequence-specific nucleic acid targeting defense mechanism. The sequence-specific recognition and cleavage of Cas effector complexes has been harnessed to developed CRISPR-based technologies and drive the genome editing revolution underway, due to their efficacy, efficiency, and ease of implementation in a broad range of organisms. CRISPR-based technologies offer a wide variety of opportunities in genome remodeling and transcriptional regulation, opening new avenues for therapeutic and biotechnological applications. To repurpose CRISPR-Cas systems for these applications, the various elements of the system need to be first identified and functionally characterized in their native host. Bioinformatic tools are first used to identify putative CRISPR arrays and their associated genes, followed by a comprehensive characterization of the CRISPR-Cas system, encompassing predictions for guide and target sequences. Subsequently, interference assays and transcriptomic analyses should be performed to probe the functionality of the CRISPR-Cas system. Once an endogenous CRISPR-Cas system is characterized as functional, they can be readily repurposed by delivering an engineered synthetic CRISPR array or a small RNA guide for targeted gene manipulation. Alternatively, developing a plasmid-based system for heterologous expression of the necessary CRISPR components can enable exploitation in other organisms. Altogether, there is a wide diversity of native CRISPR-Cas systems in many bacteria and most archaea that await functional characterization and repurposing for genome editing applications in prokaryotes.

Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli.

Our evolving understanding on the mechanisms underlying the health-promoting attributes of probiotic lactobacilli, together with an expanding genome editing toolbox have made this genus an ideal chassis for the development of living therapeutics. The rising adoption of CRISPR-based technologies for prokaryotic engineering has demonstrated precise, efficient and scalable genome editing and tunable transcriptional regulation that can be translated into next-generation development of probiotic lactobacilli with enhanced robustness and designer functionalities. Here, we discuss how these tools in conjunction with the naturally abundant and diverse native CRISPR-Cas systems can be harnessed for Lactobacillus cell surface engineering and the delivery of biotherapeutics.

Deletion-based escape of CRISPR-Cas9 targeting in Lactobacillus gasseri.

Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system. Although the primary outcome of the assay was efficient interference, a small proportion of the transformed population overcame targeting. Mutants containing plasmids that had escaped were recovered to investigate the genetic routes of escape and their relative frequencies. Deletion of the targeting spacer in the native CRISPR array was the dominant pattern of escape, accounting for 52-70 % of the mutants from two L. gasseri strains. We repeatedly observed internal deletions in the chromosomal CRISPR array, characterized by polarized excisions from the leader end that spanned 1-15 spacers, and systematically included the leader-proximal targeting spacer. This study shows that deletions of spacers within CRISPR arrays constitute a key escape mechanism to evade CRISPR targeting, while preserving the functionality of the CRISPR-Cas system. This mechanism enables cells to maintain an active immune system, but allows the uptake of potentially beneficial plasmids. Our study revealed the co-occurrence of other genomic mutations associated with various phenotypes, showing how this selection process uncovers population diversification.

Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals.

Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-ß-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host.IMPORTANCE Thousands of therapeutically active plant-derived compounds are widely present in berries, fruits, nuts, and beverages like tea and wine. The bioactivity and bioavailability of these compounds, which are typically glycosylated, are altered by microbial bioconversions in the human gut. Remarkably, little is known about the bioconversion of PGs by the gut microbial community, despite the significance of this metabolic facet to human health. Our work provides the first molecular insights into the metabolic routes of diet relevant and therapeutically active PGs by Lactobacillus acidophilus and related human gut lactobacilli. This taxonomic group is adept at metabolizing the glucoside moieties of select PG and externalizes their aglycones. The study highlights an important role of lactobacilli in the bioconversion of dietary PG and presents a framework from which to derive molecular insights into their metabolism by members of the human gut microbiota.

Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of Lactobacillus acidophilus Using Quantitative Proteomics.

Bacterial surface-layers (S-layers) are semi-porous crystalline arrays that self-assemble to form the outermost layer of some cell envelopes. S-layers have been shown to act as scaffolding structures for the display of auxiliary proteins externally. These S-layer associated proteins have recently gained attention in probiotics due to their direct physical contact with the intestinal mucosa and potential role in cell proliferation, adhesion, and immunomodulation. A number of studies have attempted to catalog the S-layer associated proteome of Lactobacillus acidophilus NCFM under a single condition. However, due to the versatility of the cell surface, we chose to employ a multiplexing-based approach with the intention of accurately contrasting multiple conditions. In this study, a previously described lithium chloride isolation protocol was used to release proteins bound to the L. acidophilus S-layer during logarithmic and early stationary growth phases. Protein quantification values were obtained via TMT (tandem mass tag) labeling combined with a triple-stage mass spectrometry (MS3) method. Results showed significant growth stage-dependent alterations to the surface-associated proteome while simultaneously highlighting the sensitivity and reproducibility of the technology. Thus, this study establishes a framework for quantifying condition-dependent changes to cell surface proteins that can easily be applied to other S-layer forming bacteria.

The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM.

Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease homolog, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (ΔprtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ΔprtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ΔprtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ΔprtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms' immunomodulatory properties and interactions with some intestinal epithelial cell components.

An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus.

Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 (LaPul13_14), and its role in branched α-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for α-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of α-glucans but was repressed by glucose. The debranching activity is conferred exclusively by LaPul13_14 and is abolished in a mutant strain lacking a functional LaPul13_14 gene. Hydrolysis kinetics of recombinant LaPul13_14 confirmed the preference for short-branched α-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest Km reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by ß-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut.IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched α-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of health-beneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched α-glucan oligomers (α-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for α-glucans with short α-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.

Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose - an emerging prebiotic.

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; ß-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.

Prebiotics: why definitions matter.

The prebiotic concept was introduced twenty years ago, and despite several revisions to the original definition, the scientific community has continued to debate what it means to be a prebiotic. How prebiotics are defined is important not only for the scientific community, but also for regulatory agencies, the food industry, consumers and healthcare professionals. Recent developments in community-wide sequencing and glycomics have revealed that more complex interactions occur between putative prebiotic substrates and the gut microbiota than previously considered. A consensus among scientists on the most appropriate definition of a prebiotic is necessary to enable continued use of the term.