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

NCFM is a probiotic strain commonly used in dairy products and dietary supplements. Postgenome 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 and functional studies, we employed a dual RNA sequencing (RNA-seq) transcriptomic approach to identify genes potentially driving the gut fitness and activities of NCFM , and in parallel, examine the ileal transcriptional response of its murine hosts during monocolonization.

Get Cultured: Eat Bacteria.

The Klaenhammer group at North Carolina State University pioneered genomic applications in food microbiology and beneficial lactic acid bacteria used as starter cultures and probiotics. Dr. Todd Klaenhammer was honored to be the first food scientist elected to the National Academy of Sciences (2001).

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.

Phenotypic and genotypic diversity of Lactobacillus buchneri strains isolated from spoiled, fermented cucumber.

Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol.

Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner.

Identification of probiotic effector molecules: present state and future perspectives.

Comprehension of underlying mechanisms of probiotic action will support rationale selection of probiotic strains and targeted clinical study design with a higher likelihood of success. This will consequently contribute to better substantiation of health claims. Here, we aim to provide a perspective from a microbiology point of view that such comprehensive understanding is not straightforward.

Shared mechanisms among probiotic taxa: implications for general probiotic claims.

Strain-specificity of probiotic effects has been a cornerstone principle of probiotic science for decades. Certainly, some important mechanisms are present in only a few probiotic strains. But scientific advances now reveal commonalities among members of certain taxonomic groups of probiotic microbes.

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in .

NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown.

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, Pul13_14 (Pul13_14), and its role in branched α-glucan metabolism in the well-characterized NCFM, which is widely used as a probiotic.

CRISPR-Cas Technologies and Applications in Food Bacteria.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms.

Impact of short-chain galactooligosaccharides on the gut microbiome of lactose-intolerant individuals.

Directed modulation of the colonic bacteria to metabolize lactose effectively is a potentially useful approach to improve lactose digestion and tolerance. A randomized, double-blind, multisite placebo-controlled trial conducted in human subjects demonstrated that administration of a highly purified (>95%) short-chain galactooligosaccharide (GOS), designated "RP-G28," significantly improved clinical outcomes for lactose digestion and tolerance. In these individuals, stool samples were collected pretreatment (day 0), after GOS treatment (day 36), and 30 d after GOS feeding stopped and consumption of dairy products was encouraged (day 66).

Stuck in the Middle: Fibronectin-Binding Proteins in Gram-Positive Bacteria.

Fibronectin is a multidomain glycoprotein found ubiquitously in human body fluids and extracellular matrices of a variety of cell types from all human tissues and organs, including intestinal epithelial cells. Fibronectin plays a major role in the regulation of cell migration, tissue repair, and cell adhesion. Importantly, fibronectin also serves as a common target for bacterial adhesins in the gastrointestinal tract.

Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus.

Unlabelled: Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure.

AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM.

Unlabelled: Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L.

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.

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain.

Characterization of bacterial isolates from the microbiota of mothers' breast milk and their infants.

This investigation assessed the potential of isolating novel probiotics from mothers and their infants. A subset of 21 isolates among 126 unique bacteria from breast milk and infant stools from 15 mother-infant pairs were examined for simulated GI transit survival, adherence to Caco-2 cells, bacteriocin production, and lack of antibiotic resistance. Of the 21 selected isolates a Lactobacillus crispatus isolate and 3 Lactobacillus gasseri isolates demonstrated good profiles of in vitro GI transit tolerance and Caco-2 cell adherence.

Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic.

The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants.

Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein.

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions.

Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli.

The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L.

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera.

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria.

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes.

CRISPR-based screening of genomic island excision events in bacteria.

Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus.