Showing papers by "Todd R. Klaenhammer published in 2011"

Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid

Abstract: Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile-induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4+ T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4+CD45RBhighT cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4+FoxP3+ T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.

Transcriptional and functional analysis of galactooligosaccharide uptake by lacS in Lactobacillus acidophilus

Abstract: Probiotic microbes rely on their ability to survive in the gastrointestinal tract, adhere to mucosal surfaces, and metabolize available energy sources from dietary compounds, including prebiotics. Genome sequencing projects have proposed models for understanding prebiotic catabolism, but mechanisms remain to be elucidated for many prebiotic substrates. Although β-galactooligosaccharides (GOS) are documented prebiotic compounds, little is known about their utilization by lactobacilli. This study aimed to identify genetic loci in Lactobacillus acidophilus NCFM responsible for the transport and catabolism of GOS. Whole-genome oligonucleotide microarrays were used to survey the differential global transcriptome during logarithmic growth of L. acidophilus NCFM using GOS or glucose as a sole source of carbohydrate. Within the 16.6-kbp gal-lac gene cluster, lacS, a galactoside-pentose-hexuronide permease-encoding gene, was up-regulated 5.1-fold in the presence of GOS. In addition, two β-galactosidases, LacA and LacLM, and enzymes in the Leloir pathway were also encoded by genes within this locus and up-regulated by GOS stimulation. Generation of a lacS-deficient mutant enabled phenotypic confirmation of the functional LacS permease not only for the utilization of lactose and GOS but also lactitol, suggesting a prominent role of LacS in the metabolism of a broad range of prebiotic β-galactosides, known to selectively modulate the beneficial gut microbiota.

Construction of vectors for inducible and constitutive gene expression in Lactobacillus

Abstract: Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.

Assessment of Lactobacillus gasseri as a Candidate Oral Vaccine Vector

Abstract: Lactobacillus species are commensal bacteria that have long been recognized as probiotic microbes and are generally regarded as safe (GRAS) for human consumption. We have investigated the use of L. gasseri as a vaccine vector for oral immunization against mucosal pathogens. Recent research has shown that the immune response to different lactobacilli can vary widely depending on the species or subspecies of Lactobacillus being studied. While some lactobacilli seem to induce oral tolerance, others induce an adaptive immune response. This study characterized the systemic and mucosal immune response to wild-type and genetically modified L. gasseri. L. gasseri primarily activates TLR2/6, with additional activation through the TLR2 homodimer. To expand the Toll-like receptor (TLR) activation profile of L. gasseri and the immunogenicity of the vector, a plasmid containing fliC, the gene encoding bacterial flagellin, was introduced which resulted in the strong activation of TLR5. The treatment of human myeloid dendritic cells with recombinant lactobacilli expressing flagellin triggered phenotypic maturation and the release of proinflammatory cytokines. In contrast, bacterial treatment also resulted in a statistically significant increase in IL-10 production. In vivo studies established that treatment with L. gasseri led to a diversification of B-cell populations in the lamina propria of the murine colon. Furthermore, treatment with genetically modified L. gasseri led to a significant decrease in the percentage of FoxP3+ colonic lymphocytes. Taken together, these data clarify the interaction of L. gasseri with the host immune system and support further investigation of the in vivo immunogenicity of L. gasseri expressing both flagellin and candidate vaccine antigens.

Prebiotic formulations and methods of use

Abstract: The invention provides methods and compositions for treating symptoms associated with lactose intolerance and for overall improvement in gastrointestinal health. Described herein are methods and compositions for improving overall gastrointestinal health or for decreasing symptoms of lactose intolerance by administering to subject in need thereof a prebiotic composition, optionally in combination with effective amount of a probiotic microbe or microbes.

Dissimilar Properties of Two Recombinant Lactobacillus acidophilus Strains Displaying Salmonella FliC with Different Anchoring Motifs

Abstract: Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro. The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.

A rendezvous with our microbes.

Abstract: On November 2–3, 2009 an international group of scientists representing multiple disciplines gathered to consider the current state of our understanding of the symbiotic and beneficial relationships between microbes and humans and to define the challenges, gaps in knowledge, and opportunities that this exciting field of study now offers. A number of adjectives come to mind when describing the subject of microbes and health, ranging from ancient and historic , to integrative and interdisciplinary , to timely and pressing . Coexistence and coevolution with microbes has been a theme of life on Earth for all metazoans past and present. Historically, the discovery that microbes are an integral part of us was made as soon as Antonie van Leeuwenhoek peered through his microscope and examined dental plaque sampled from himself and others (without institutional review board approval!). His sense of awe and his early appreciation of the diversity our microbial partners were evident in the words he chose for a letter written to the Royal Society of London in September 1683: > “Though my teeth are kept usually very clean, nevertheless, when I view them in a magnifying glass, I find growing between them a little white matter as thick as wetted flower: in this substance though I could not perceive any motion, I judged there might probably be living creatures. I therefore took some of this flower and mixed it either with pure rain water wherein were no animals, or else with some of my spittle (having no air bubbles to cause a motion in it) and then to my great surprise perceived that the aforesaid matter maintained very many small living animals, which moved themselves very extravagantly. ….The spittle of an old man that had lived soberly, had no animals in it; but the substance upon and between his teeth … [↵][1]1To whom correspondence should be addressed. E-mail: jgordon{at}wustl.edu. [1]: #xref-corresp-1-1

The impact of omic technologies on the study of food microbes.

Abstract: The advent of the molecular biology era in the 1950s and the subsequent emergence of new technologies positively impacted on all areas of biology. New discoveries in molecular biology and experimental tools were developed over the next 60 years that have revolutionized the study of food microbiology. Previously, food microbiology relied on classic microbiology techniques, which had remained relatively unchanged since the discoveries of Louis Pasteur in the 1800s. More recently, new advances resulting in "omic" technologies have exploded the areas of genomics, transcriptomics, and proteomics and revealed many fundamental processes driven by both pathogens and commensals. This review outlines advances in omic technologies and how these have impacted food microbiology through providing examples of recently published landmark work.

Lipoteichoic acid-deficient Lactobacillus acidophilus regulates downstream signals.

Abstract: The trillions of microbes residing within the intestine induce critical signals that either regulate or stimulate host immunity via their bacterial products. To better understand the immune regulation elicited by lipoteichoic acid (LTA)-deficient Lactobacillus acidophilus NCFM in steady state and induced inflammation, we deleted phosphoglycerol transferase gene, which synthesizes LTA in L. acidophilus NCFM. In vitro and in vivo experiments were conducted in order to compare the immune regulatory properties of the L. acidophilus strain deficient in LTA (NCK2025) with its wild-type parent (NCK56) in C57BL/6, C57BL/6 recombination-activation gene 1-deficient (Rag1 -/-) and C57BL/6 Rag1-/-IL-10-/- mice. We demonstrate that NCK2025 significantly activates the phosphorylation of Erk1/2 but downregulates the phosphorylation of Akt1, cytosolic group IV PLA2 and p38 in mouse dendritic cells. Similarly, mice treated orally with NCK2025 exhibit decreased phosphorylation of inflammatory signals (Akt1, cytosolic group...

Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM.

Abstract: Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β-d-glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3-negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression.

Probiotic and prebiotic claims in Europe : seeking a clear roadmap.

Abstract: In 2008, the European Food Safety Authority (EFSA) began reviewing the proposed health benefit claims on all foods. To date, none of the 164 claims of the benefits of probiotic or prebiotic products submitted to EFSA and reviewed by the Panel on Dietetic Products, Nutrition, and Allergies (NDA) have been accepted (see Table 1). Those who are not aware of either the research supporting specific probiotics and prebiotics or the NDA review process may come to the fallacious conclusion that probiotics or prebiotics have not been shown to have health benefits. Without doubt, fraudulent or exaggerated claims are being made for some products. However, the scientists and clinical investigators belonging to the Board of Directors of the International Scientific Association for Probiotics and Prebiotics (ISAPP) are concerned that claims supported by solid scientific evidence are also being rejected. They are further concerned that there is a lack of clarity regarding the criteria – from study design through wording of the claim – for a dossier suitable for a positive regulatory opinion. One unintended consequence of the current review process may well be that the responsible companies studying the physiological effects of their probiotic or prebiotic products will decide that continued investment into this line of research is not cost-effective if, in the end, evidence supporting product benefits deemed valid by the scientific community cannot be communicated to the consumer. Certainly, evaluation of evidence to support claims is not a simple process. The NDA scientists must implement challenging legislation and assess a flood of dossiers providing evidence, which in the nature of all research could always be improved. But the process is difficult for industry scientists, too, who must prepare a dossier in support of a claim with only general guidance from the NDA. A successful dossier requires not only compelling studies on efficacy, but also specification of a physiological effect that will be considered by the NDA as beneficial and a claim that is worded to accurately reflect the science but also be in compliance with regulations. Some recent documents have been drafted by the NDA to provide guidance on their interpretation of what constitutes beneficial effects and acceptable outcome measures (http:// www.efsa.europa.eu/en/consultationsclosed/call/nda100928. pdf), but many questions remain unanswered. This opinion is reflected in a letter (http://www.gut-health.eu) by the European scientists expressing dissatisfaction with the process. As of 23 February 2011, 148 scientists have signed this letter. One overriding concern with the review process is the standard of evidence required by the NDA. The legislation states that ‘Health claims should only be authorised for use in the Community after a scientific assessment of the highest possible standard’. However, this seems to be interpreted by the NDA to mean that the evidence (as opposed to the assessment) must meet the highest possible standard. A more realistic standard is expressed in article 6 of the EC Regulation 1924/2006, which states that health claims shall be based on and substantiated by ‘generally accepted scientific evidence’. Thus, regulators have indicated a definite roadmap: generally accepted scientific evidence is not the same as the notion that evidence must be based on a restrictive number of criteria established by a closed group of individuals. Generally accepted scientific evidence is a well-established concept, and is the basis for the peer review process of scientific journals, evaluation of grant applications or scientific productivity of researchers, and grading recommendations in evidencebased medicine. In the latter case, this means that findings of a single randomised control study with narrow CI can constitute level 1b of evidence and invoke a recommendation of top, Grade A, intervention. In practice, this means that the recommendation should be applied unless there is a specific reason for not doing it. An example of implementing the ‘highest possible standard’ is apparent when the NDA rejected the validity of an independently conducted study published in the British Medical Journal (2) to support a claim that a probiotic food could reduce Clostridium difficile toxin in the gut and reduce the risk of acute diarrhoea in patients receiving antibiotics. One concern expressed by the NDA panel judgement of the trial was with study blinding. Although the products were not identified to the patients, the bottle shapes were different for the placebo and the test product, but only for product sent home with a subset of discharged patients. It is unclear how the NDA expected this small imperfection to influence the level of C. difficile toxin in faeces. Importantly, the staff who conducted the toxic analysis on the stool samples from patients who had diarrhoea remained fully blinded to the test group assignment. An additional criticism of the study was that C. difficile toxin was measured only in patients with diarrhoea and not in all study participants. However, it is common practice in a hospital environment to assay toxin only when diarrhoea occurs. The authors of this study concluded ‘Consumption of a probiotic drink. . . can reduce the incidence of antibiotic-associated diarrhoea and C. difficile-associated diarrhoea. This has the potential to decrease morbidity, healthcare costs and mortality if used routinely in patients British Journal of Nutrition (2011), 106, 1765–1767 q The Authors 2011

Compositions and methods for the delivery of therapeutic peptides

Abstract: Methods and compositions for targeted delivery of biotherapeutics are provided. The compositions comprise bile-sensitive St. thermophilus bacteria modified to release a biotherapeutic agent following bile exposure. Biotherapeutic agents released by the St. thermophilus bacteria disclosed herein include AQ and AQR rich peptides. Methods of the invention comprise administering to a subject a St. thermophilus bacterium modified to release a biotherapeutic agent following bile exposure. Administration of the St. thermophilus bacterium promotes a desired therapeutic response. The bacterium may be modified to express and release AQ or AQR rich peptides which subsequently inhibit cellular apoptosis or reduce mucosal damage. Thus, methods of the invention find use in treating or preventing a variety of gastrointestinal disorders including C. difficile infection and antibiotic-associated diarrhea.

Integrative Food Grade Expression System for Lactic Acid Bacteria

Abstract: Lactobacillus acidophilus NCFM is a probiotic microbe with the ability to survive passage to the -gastrointestinal tract, interact intimately with the host and induce immune responses. The genome of NCFM has been determined and the bacterium is genetically accessible. Therefore, L. acidophilus has excellent potential for use as a vaccine delivery vehicle to express antigens at mucosal surfaces. Plasmids, commonly used to carry antigen encoding genes, are inherently unstable and require constant selection by antibiotics, which can be problematic for in vivo studies and clinical trials. Chromosomal expression of gene cassettes encoding antigens offers enhanced genetic stability by eliminating requirements for marker selection. This work illustrates the integration and inducible expression of the reporter gene gusA3, -encoding a β-glucuronidase (GusA3), in the L. acidophilus chromosome. A previously described upp-counterselectable gene replacement system was used to direct insertion of the gusA3 gene into an intergenic chromosomal location downstream of lacZ (LBA1462), encoding a β-galactosidase. The transcriptional activity of integrated gusA3 was evaluated by GUS activity assays using 4-methyl-umbelliferyl-β-D: -glucuronide (MUG) and was determined to be one to two orders of magnitude higher than the GusA3-negative parent, NCK1909. The successful chromosomal integration and expression of GusA3 demonstrate the potential of this method for higher levels of inducible gene expression in L. acidophilus.

Group-specific comparison of four lactobacilli isolated from human sources using differential blast analysis

Abstract: Lactic acid bacteria (LAB) have been used in fermentation processes for centuries. More recent applications including the use of LAB as probiotics have significantly increased industrial interest. Here we present a comparative genomic analysis of four completely sequenced Lactobacillus strains, isolated from the human gastrointestinal tract, versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Lactobacillus acidophilus NCFM, Lactobacillus johnsonii NCC533, Lactobacillus gasseri ATCC33323, and Lactobacillus plantarum WCFS1are all considered probiotic and widely used in industrial applications. Using Differential Blast Analysis (DBA), each genome was compared to the respective remaining three other Lactobacillus and 25 other LAB genomes. DBA highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Initial comparative analyses highlighted a significant number of genes involved in cell adhesion, stress responses, DNA repair and modification, and metabolic capabilities. Furthermore, the range of the recently identified potential autonomous units (PAUs) was broadened significantly, indicating the possibility of distinct families within this genetic element. Based on in silico results obtained for the model organism L. acidophilus NCFM, DBA proved to be a valuable tool to identify new key genetic regions for functional genomics and also suggested re-classification of previously annotated genes.

Recombinant lactobacillus with decreased lipoteichoic acid to reduce inflammatory responses

Abstract: Methods and compositions for treating or preventing inflammatory disorders are provided. The compositions of the invention comprise a recombinant bacterium genetically modified to decrease the display of lipoteichoic acid on the cell surface. Methods of the invention comprise administering to a subject a recombinant bacterium modified to decrease the display of lipoteichoic acid on the cell surface. Administration of the recombinant bacterium promotes a desired therapeutic response. The recombinant bacterium may be administered in a single dose or series of doses. Methods of the invention find use in treating or preventing a variety of inflammatory disorders including, for example, treating or preventing inflammatory bowel disease, colitis, or Crohn's disease.

Compositions et procédés pour l'administration de peptides thérapeutiques

Abstract: L'invention porte sur des procedes et sur des compositions pour l'administration ciblee d'agents biotherapeutiques. Les compositions comprennent des bacteries St. thermophilus sensibles a la bile, modifiees pour liberer un agent biotherapeutique a la suite d'une exposition a la bile. Les agents biotherapeutiques liberes par les bacteries St. thermophilus decrits dans la presente invention comprennent des peptides riches en AQ et AQR. Les procedes de l'invention comprennent l'administration a un sujet d'une bacterie St. thermophilus modifiee pour secreter un agent biotherapeutique a la suite d'une exposition a la bile. L'administration de la bacterie St. thermophilus favorise une reponse therapeutique souhaitee. La bacterie peut etre modifiee pour exprimer et liberer des peptides riches en AQ ou AQR qui, ulterieurement, inhibent l'apoptose cellulaire ou reduisent les dommages des muqueuses. Ainsi, les procedes de l'invention trouvent une utilisation dans le traitement ou la prevention de divers troubles gastro-intestinaux comprenant l'infection par C. difficile et les diarrhees associees aux antibiotiques.

Formulations prébiotiques et méthodes d'utilisation

Abstract: La presente invention a pour objet des methodes et des compositions pharmaceutiques destinees au traitement des symptomes associes a l'intolerance au lactose et a une amelioration globale de la sante gastro-intestinale. L'invention concerne des methodes et des compositions pharmaceutiques destinees a une amelioration globale de la sante gastro-intestinale ou a la diminution des symptomes de l'intolerance au lactose par l'administration au sujet en ayant besoin d'une composition pharmaceutique contenant un prebiotique, eventuellement en association avec une quantite efficace d'un microbe ou de microbes prebiotique(s).

Genetically modified Streptococcus thermophilus bacterium

Abstract: Methods and compositions for targeted delivery of biotherapeutics are provided. The compositions comprise bile-sensitive St. thermophilus bacteria modified to release a biotherapeutic agent following bile exposure. Biotherapeutic agents released by the St. thermophilus bacteria disclosed herein include AQ and AQR rich peptides. Methods of the invention comprise administering to a subject a St. thermophilus bacterium modified to release a biotherapeutic agent following bile exposure. Administration of the St. thermophilus bacterium promotes a desired therapeutic response. The bacterium may be modified to express and release AQ or AQR rich peptides which subsequently inhibit cellular apoptosis or reduce mucosal damage. Thus, methods of the invention find use in treating or preventing a variety of gastrointestinal disorders including C. difficile infection and antibiotic-associated diarrhea.