[Show abstract][Hide abstract] ABSTRACT: 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. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.
PLoS ONE 10/2015; 10(10):e0141713. DOI:10.1371/journal.pone.0141713 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Lactobacillus acidophilus homology group are Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they vary in ecological lifestyles ranging from dairy and food fermentations, to allochthonous probiotics, and autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex are distinguished in two clades by the presence or absence of S-layers, which are semi-porous, crystalline arrays of self-assembling, proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer associated proteins (SLAPs) in the exoproteomes of various S-layer forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions, encoding six putative SLAPs, were conserved in the S-layer forming Lactobacillus species, but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences of the cell surface properties between S-layer and non-S-layer forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa.
Applied and Environmental Microbiology 10/2015; DOI:10.1128/AEM.01968-15 · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.
[Show abstract][Hide abstract] ABSTRACT: Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes, which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbor 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. In this study, 17 L. gasseri strains were investigated and 6 harbored a Type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the Protospacer Adjacent Motif (PAM) sequence, determined to be 5'-NTAA-3' flanking the 3'end of protospacer. The system in L. gasseri JV-V03 and NCK1342, interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native Type II-A CRISPR-Cas system in the commensal species, L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.
[Show abstract][Hide abstract] ABSTRACT: 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. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac(-) survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.
Proceedings of the National Academy of Sciences 06/2015; 112(26). DOI:10.1073/pnas.1508525112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent insights into the relationship between the human gut and its resident microbiota have revolutionized our appreciation of this symbiosis and its impact on health and disease development. Accumulating evidence on probiotic and prebiotic interventions has demonstrated promising effects on promoting gastrointestinal health by modulating the microbiota toward the enrichment of beneficial microorganisms. However, the precise mechanisms of how prebiotic nondigestible oligosaccharides are metabolized by these beneficial microbes in vivo remain largely unknown. Genome sequencing of probiotic lactobacilli and bifidobacteria has revealed versatile carbohydrate metabolic gene repertoires dedicated to the catabolism of various oligosaccharides. In this review, we highlight recent findings on the genetic mechanisms involved in the utilization of prebiotic fructooligosaccharides, β-galactooligosaccharides, human milk oligosaccharides, and other prebiotic candidates by these probiotic microbes. Expected final online publication date for the Annual Review of Food Science and Technology Volume 6 is February 28, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Review of Food Science and Technology 12/2014; 6(1). DOI:10.1146/annurev-food-022814-015706 · 6.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Surface proteins of probiotic microbes, including Lactobacillus acidophilus and Lactobacillus gasseri, are believed to promote retention in the gut and mediate host-bacterial communications. Sortase, an enzyme that covalently couples a subset of extracellular proteins containing an LPXTG motif to the cell surface, is of particular interest in characterizing bacterial adherence and communication with the mucosal immune system. A sortase gene, srtA, was identified in L. acidophilus NCFM (LBA1244) and L. gasseri ATCC 33323 (LGAS_0825). Additionally, eight and six intact sortase-dependant proteins were predicted in L. acidophilus and L. gasseri, respectively. Due to the role of sortase in coupling these proteins to the cell wall, DsrtA deletion mutants of L. acidophilus and L. gasseri were created using the upp-based counterselective gene replacement system. Inactivation of sortase did not cause significant alteration in growth or survival in simulated gastrointestinal juices. Meanwhile, both ΔsrtA mutants showed decreased adhesion to porcine mucin in vitro. Murine dendritic cells exposed to the ΔsrtA mutant of L. acidophilus or L. gasseri induced lower levels of pro-inflammatory cytokines TNF-α and IL-12, respectively, compared to the parent strains. In vivo co-colonization of the L. acidophilus DsrtA mutant and its parent strain in germ-free 129S6/SvEv mice resulted in a significant one log reduction of the DsrtA mutant population. Additionally, a similar reduction of the DsrtA mutant was observed in the cecum. This study shows for the first time that sortase-dependant proteins contribute to gut retention of probiotic microbes in the gastrointestinal tract.
[Show abstract][Hide abstract] ABSTRACT: In prokaryotic species equipped with glycogen metabolism machinery, the co-regulation of glycogen biosynthesis and degradation has been associated with the synthesis of energy storage compounds and various crucial physiological functions, including global cellular processes such as carbon and nitrogen metabolism, energy sensing and production, stress response and cell-cell communication. In addition, the glycogen metabolic pathway was proposed to serve as a carbon capacitor that regulates downstream carbon fluxes, and in some microorganisms the ability to synthesize intracellular glycogen has been implicated in host persistence. Among lactobacilli, complete glycogen metabolic pathway genes are present only in select species predominantly associated with mammalian hosts or natural environments. This observation highlights the potential involvement of glycogen biosynthesis in probiotic activities and persistence of intestinal lactobacilli in the human gastrointestinal tract. In this review, we summarize recent findings on (i) the presence and potential ecological distribution of glycogen metabolic pathways among lactobacilli, (ii) influence of carbon substrates and growth phases on glycogen metabolic gene expression and glycogen accumulation in L. acidophilus, and (iii) the involvement of glycogen metabolism on growth, sugar utilization and bile tolerance. Our present in vivo studies established the significance of glycogen biosynthesis on the competitive retention of L. acidophilus in the mouse intestinal tract, demonstrating for the first time that the ability to synthesize intracellular glycogen contributes to gut fitness and retention among probiotic microorganisms.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0094-3) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: α-Glucans from bacterial exo-polysaccharides or diet, e.g., resistant starch, legumes and honey are abundant in the human gut and fermentation of resistant fractions of these α-glucans by probiotic lactobacilli and bifidobacteria impacts human health positively. The ability to degrade polymeric α-glucans is confined to few strains encoding extracellular amylolytic activities of glycoside hydrolase (GH) family 13. Debranching pullulanases of the subfamily GH13_14 are the most common extracellular GH13 enzymes in lactobacilli, whereas corresponding enzymes are mainly α-amylases and amylopullulanases in bifidobacteria. Extracellular GH13 enzymes from both genera are frequently modular and possess starch binding domains, which are important for efficient catalysis and possibly to mediate attachment of cells to starch granules. α-1,6-Linked glucans, e.g., isomalto-oligosaccharides are potential prebiotics. The enzymes targeting these glucans are the most abundant intracellular GHs in bifidobacteria and lactobacilli. A phosphoenolpyruvate-dependent phosphotransferase system and a GH4 phospho-α-glucosidase are likely involved in metabolism of isomaltose and isomaltulose in probiotic lactobacilli based on transcriptional analysis. This specificity within GH4 is unique for lactobacilli, whereas canonical GH13 31 α-1,6-glucosidases active on longer α-1,6-gluco-oligosaccharides are ubiquitous in bifidobacteria and lactobacilli. Malto-oligosaccharide utilization operons encode more complex, diverse, and less biochemically understood activities in bifidobacteria compared to lactobacilli, where important members have been recently described at the molecular level. This review presents some aspects of α-glucan metabolism in probiotic bacteria and highlights vague issues that merit experimental effort, especially oligosaccharide uptake and the functionally unassigned enzymes, featuring in this important facet of glycan turnover by members of the gut microbiota.
[Show abstract][Hide abstract] ABSTRACT: Lactobacillus gasseri ATCC 33323 is a member of the acidophilus-complex group, microbes of human origin with significant potential for impacting human health based on niche-specific traits. In order to facilitate functional analysis of this important species, a upp-based counterselective chromosomal integration system was established and employed for knockout mutagenesis of the lipoteichoic acid synthase gene in L. gasseri ATCC 33323. No isogenic mutants baring the deletion genotype were recovered, but an integration knockout mutant was generated in the phosphoglycerol transferase gene (ltaS) responsible for building the glycerol chain of lipoteichoic acid (LTA). The ltaS deficient derivative exhibited an altered cellular morphology and significantly reduced ability to adhere to Caco-2 intestinal cell monolayers, relative to the wild-type parent strain.
Gut Microbes 05/2014; 5(3). DOI:10.4161/gmic.29101
[Show abstract][Hide abstract] ABSTRACT: For thousands of years, humans have safely consumed microorganisms through fermented foods. Many of these bacteria are considered probiotics, which act through diverse mechanisms to confer a health benefit to the host. However, it was not until the availability of whole-genome sequencing and the era of genomics that mechanisms of probiotic efficacy could be discovered. In this review, we explore the history of the probiotic concept and the current standard of integrated genomic techniques to discern the complex, beneficial relationships between probiotic microbes and their hosts.
Antonie van Leeuwenhoek 04/2014; 106(1). DOI:10.1007/s10482-014-0171-y · 1.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lactobacillus gasseri is an endogenous species of the human gastrointestinal tract and vagina. With recent advances in microbial taxonomy, phylogenetics, and genomics, L. gasseri is recognized as an important commensal, and is increasingly being used in probiotic formulations. L. gasseri ADH is lysogenic, and harbors two inducible prophages. In this study, prophage adh was found to spontaneously induce in broth cultures to populations of ∼10(7) PFU/ml by stationary phase. The adh prophage-cured ADH derivative, NCK102, was found to harbor a new, second inducible phage, vB_Lga_jlb1 (jlb1). Phage jlb1 was sequenced and found highly similar to the closely-related phage LgaI, which resides as two tandem prophages in the neotype strain, L. gasseri ATCC 33323. The common occurrence of multiple prophages in L. gasseri genomes, their propensity for spontaneous induction, and the high degree of homology amongst phages within multiple species of Lactobacillus suggest that temperate bacteriophages likely contribute to horizontal gene transfer (HGT) in commensal lactobacilli. In this study, the host ranges of phages adh and jlb1 were determined against 16 L. gasseri strains. The transduction range and the rate of spontaneous transduction were investigated in co-culture experiments to ascertain the degree to which prophages can promote HGT among a variety of commensal and probiotic lactobacilli. Both adh and jlb1 particles were confirmed to mediate plasmid transfer. As many as ∼10(3) spontaneous transductants/ml were obtained. HGT by transducing phages of commensal lactobacilli may have a significant impact on the evolution of bacteria within the human microbiota.
[Show abstract][Hide abstract] ABSTRACT: Commercial probiotic strains for food or supplement use can be altered in different ways for a variety of purposes. Production conditions for the strain or final product may be changed to address probiotic yield, functionality, or stability. Final food products may be modified to improve flavor and other sensory properties, provide new product formats, or respond to market opportunities. Such changes can alter the expression of physiological traits owing to the live nature of probiotics. In addition, genetic approaches may be used to improve strain attributes. This review explores whether genetic or phenotypic changes, by accident or design, might affect the efficacy or safety of commercial probiotics. We highlight key issues important to determining the need to re-confirm efficacy or safety after strain improvement, process optimization, or product formulation changes. Research pinpointing the mechanisms of action for probiotic function and the development of assays to measure them are greatly needed to better understand if such changes have a substantive impact on probiotic efficacy.
Annals of the New York Academy of Sciences 02/2014; 1309(1):1-18. DOI:10.1111/nyas.12363 · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lactose intolerance (LI) is a common medical problem with limited treatment options. The primary symptoms are abdominal pain, diarrhea, bloating, flatulence, and cramping. Limiting dairy foods to reduce symptoms contributes to low calcium intake and the risk for chronic disease. Adaptation of the colon bacteria to effectively metabolize lactose is a novel and potentially useful approach to improve lactose digestion and tolerance. RP-G28 is a proprietary GOS mixture being investigated to improve lactose digestion and the symptoms of lactose intolerance in affected patients.
A randomized, double-blind, parallel group, placebo-controlled study was conducted at 2 sites in the United States. RP-G28 or placebo was administered to 85 patients with LI for 35 days. Post-treatment, subjects reintroduced dairy into their daily diets and were followed for 30 additional days to evaluate lactose digestion as measured by hydrogen production and symptom improvements via a patient-reported symptom assessment instrument.
Lactose digestion and symptoms of LI trended toward improvement on RP-G28 at the end of treatment and 30 days post-treatment. A reduction in abdominal pain was also demonstrated in the study results. Fifty percent of RP-G28 subjects with abdominal pain at baseline were free from pain at the end of treatment and 30 days post treatment (p = 0.0190). RP-G28 subjects were also six times more likely to claim lactose tolerance post-treatment once dairy foods had been re-introduced into their diets (p = 0.0389).
Efficacy trends and favorable safety/tolerability findings suggest that RP-G28 appears to be a potentially useful approach for improving lactose digestion and LI symptoms. The concurrent reduction in abdominal pain and improved overall tolerance could be a meaningful benefit to lactose intolerant individuals.Study registration: ClinicalTrials.gov NCT01113619.
[Show abstract][Hide abstract] ABSTRACT: Recent advances in our understanding of the community structure and function of the human microbiome have implications for the potential role of probiotics and prebiotics in promoting human health. A group of experts recently met to review the latest advances in microbiota/microbiome research and discuss the implications for development of probiotics and prebiotics, primarily as they relate to effects mediated via the intestine. The goals of the meeting were to share recent advances in research on the microbiota, microbiome, probiotics, and prebiotics, and to discuss these findings in the contexts of regulatory barriers, evolving healthcare environments, and potential effects on a variety of health topics, including the development of obesity and diabetes; the long-term consequences of exposure to antibiotics early in life to the gastrointestinal (GI) microbiota; lactose intolerance; and the relationship between the GI microbiota and the central nervous system, with implications for depression, cognition, satiety, and mental health for people living in developed and developing countries. This report provides an overview of these discussions.
Annals of the New York Academy of Sciences 11/2013; 1306(1). DOI:10.1111/nyas.12303 · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer forming bacterium, Lactobacillus acidophilus NCFM expresses three major surface layer proteins: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride (LiCl) S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion; 24 are predicted to be extracellular; 6 are lipid-anchored; 3 have N-terminal hydrophobic membrane spanning regions; and 4 are intracellular, potentially moonlighting proteins. Some of these proteins, designated Surface-Layer Associated Proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified novel extracellular proteins and putative SLAPs of L. acidophilus NCFM using liquid chromatography-tandem mass spectrometry (LC-MS/MS). SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers.
[Show abstract][Hide abstract] ABSTRACT: In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota.
In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics, enzymology and protein characterization proved fruitful in elucidating the protein transport and catabolic machinery conferring the utilization of glucosides, galactosides, and xylosides in the two clinically validated probiotic strains Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Importantly, we have been able to identify and in some cases validate the specificity of several transport systems, which are otherwise poorly annotated. Further, we have demonstrated for the first time that non-naturally occurring tri- and tetra-saccharides are internalized and efficiently utilized by probiotic bacteria in some cases better than well-established natural prebiotics.
Selected highlights of these data are presented, emphasising the importance and the diversity of oligosaccharide transport in probiotic bacteria.
Biocatalysis and Biotransformation 08/2013; 31(4). DOI:10.3109/10242422.2013.828048 · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of L. acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early-log phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early-log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared to trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicates that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.