scholarly journals Exploiting Prophage-Mediated Lysis for Biotherapeutic Release byLactobacillus reuteri

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Laura M. Alexander ◽  
Jee-Hwan Oh ◽  
Donald S. Stapleton ◽  
Kathryn L. Schueler ◽  
Mark P. Keller ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Secretory Pathway ◽  
Lactobacillus Reuteri ◽  
Gastrointestinal Transit ◽  
Biologically Active ◽  
Content Type ◽  
Extracellular Milieu ◽  
Therapeutic Molecules

ABSTRACTLactobacillus reuterihas the potential to be developed as a microbial therapeutic delivery platform because of an established safety profile, health-promoting properties, and available genome editing tools. Here, we show thatL. reuteriVPL1014 exhibits a low mutation rate compared to other Gram-positive bacteria, which we expect will contribute to the stability of genetically modified strains. VPL1014 encodes two biologically active prophages, which are induced during gastrointestinal transit. We hypothesized that intracellularly accumulated recombinant protein can be released following bacteriophage-mediated lysis. To test this, we engineered VPL1014 to accumulate leptin, our model protein, inside the cell.In vitroprophage induction of recombinant VPL1014 released leptin into the extracellular milieu, which corresponded to bacteriophage production. We also employed a plasmid system that does not require antibiotic in the growth medium for plasmid maintenance. Collectively, these data provide new avenues to exploit native prophages to deliver therapeutic molecules.IMPORTANCELactic acid bacteria (LAB) have been explored as potential biotherapeutic vehicles for the past 20 years. To secrete a therapeutic in the extracellular milieu, one typically relies on the bacterial secretion pathway, i.e., the Sec pathway. Overexpression of a secreted protein can overload the secretory pathway and impact the organism’s fitness, and optimization of the signal peptide is also required to maximize the efficiency of the release of mature protein. Here, we describe a previously unexplored approach to release therapeutics from the probioticLactobacillus reuteri. We demonstrate that an intracellularly accumulated recombinant protein is released following prophage activation. Since we recently demonstrated that prophages are activated during gastrointestinal transit, we propose that this method will provide a straightforward and efficient approach to deliver therapeuticsin vivo.

scholarly journals Prophages in Lactobacillus reuteri Are Associated with Fitness Trade-Offs but Can Increase Competitiveness in the Gut Ecosystem

2019 ◽  
Vol 86 (1) ◽  
Author(s):  
Jee-Hwan Oh ◽  
Xiaoxi B. Lin ◽  
Shenwei Zhang ◽  
Stephanie L. Tollenaar ◽  
Mustafa Özçam ◽  
...  
Keyword(s):  
Competitive Advantage ◽  
Intestinal Tract ◽  
Lactobacillus Reuteri ◽  
Distal Colon ◽  
Biologically Active ◽  
Trade Off ◽  
Content Type ◽  
Trade Offs ◽  
Phage Production

ABSTRACT The gut microbiota harbors a diverse phage population that is largely derived from lysogens, which are bacteria that contain dormant phages in their genome. While the diversity of phages in gut ecosystems is getting increasingly well characterized, knowledge is limited on how phages contribute to the evolution and ecology of their host bacteria. Here, we show that biologically active prophages are widely distributed in phylogenetically diverse strains of the gut symbiont Lactobacillus reuteri. Nearly all human- and rodent-derived strains, but less than half of the tested strains of porcine origin, contain active prophages, suggesting different roles of phages in the evolution of host-specific lineages. To gain insight into the ecological role of L. reuteri phages, we developed L. reuteri strain 6475 as a model to study its phages. After administration to mice, L. reuteri 6475 produces active phages throughout the intestinal tract, with the highest number detected in the distal colon. Inactivation of recA abolished in vivo phage production, which suggests that activation of the SOS response drives phage production in the gut. In conventional mice, phage production reduces bacterial fitness as fewer wild-type bacteria survive gut transit compared to the mutant lacking prophages. However, in gnotobiotic mice, phage production provides L. reuteri with a competitive advantage over a sensitive host. Collectively, we uncovered that the presence of prophages, although associated with a fitness trade-off, can be advantageous for a gut symbiont by killing a competitor strain in its intestinal niche. IMPORTANCE Bacteriophages derived from lysogens are abundant in gut microbiomes. Currently, mechanistic knowledge is lacking on the ecological ramifications of prophage carriage yet is essential to explain the abundance of lysogens in the gut. An extensive screen of the bacterial gut symbiont Lactobacillus reuteri revealed that biologically active prophages are widely distributed in this species. L. reuteri 6475 produces phages throughout the mouse intestinal tract, but phage production is associated with reduced fitness of the lysogen. However, phage production provides a competitive advantage in direct competition with a nonlysogenic strain of L. reuteri that is sensitive to these phages. This combination of increased competition with a fitness trade-off provides a potential explanation for the domination of lysogens in gut ecosystem and how lysogens can coexist with sensitive hosts.

scholarly journals Lactobacillus reuteri 100-23 Modulates Urea Hydrolysis in the Murine Stomach

2014 ◽  
Vol 80 (19) ◽  
pp. 6104-6113 ◽  
Author(s):  
Charlotte M. Wilson ◽  
Diane Loach ◽  
Blair Lawley ◽  
Tracey Bell ◽  
Ian M. Sims ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Lactobacillus Reuteri ◽  
Urine Analysis ◽  
Acid Tolerance ◽  
Laboratory Culture ◽  
Urea Hydrolysis ◽  
Content Type ◽  
Urease Production

ABSTRACTComparisons ofin vivo(mouse stomach) andin vitro(laboratory culture) transcriptomes ofLactobacillus reuteristrain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of theureCgene reduced the acid tolerance of strain 100-23in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent inLactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta ofLactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of bothLactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact ofLactobacilluscolonization on urea hydrolysis in the murine gut.

scholarly journals Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Monchaya Rattanaprasert ◽  
Jan-Peter van Pijkeren ◽  
Amanda E. Ramer-Tait ◽  
Maria Quintero ◽  
Car Reen Kok ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Lactobacillus Reuteri ◽  
Selective Advantage ◽  
Wild Type ◽  
Carbohydrate Source ◽  
Content Type ◽  
Gnotobiotic Mice

ABSTRACT Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo. In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two β-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway. IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

scholarly journals Persistence, Immune Response, and Antigenic Variation of Mycoplasma genitalium in an Experimentally Infected Pig-Tailed Macaque (Macaca nemestrina)

2013 ◽  
Vol 81 (8) ◽  
pp. 2938-2951 ◽  
Author(s):  
Gwendolyn E. Wood ◽  
Stefanie L. Iverson-Cabral ◽  
Dorothy L. Patton ◽  
Peter K. Cummings ◽  
Yvonne T. Cosgrove Sweeney ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Antigenic Variation ◽  
Immune Escape ◽  
Variable Region ◽  
Mycoplasma Genitalium ◽  
Macaca Nemestrina ◽  
Suitable Model ◽  
Content Type

ABSTRACTMycoplasma genitaliumis a sexually transmitted pathogen associated with several acute and chronic reproductive tract disease syndromes in men and women. To evaluate the suitability of a pig-tailed macaque model ofM. genitaliuminfection, we inoculated a pilot animal withM. genitaliumstrain G37 in the uterine cervix and in salpingeal pockets generated by transplanting autologous Fallopian tube tissue subcutaneously. Viable organisms were recovered throughout the 8-week experiment in cervicovaginal specimens and up to 2 weeks postinfection in salpingeal pockets. Humoral and cervicovaginal antibodies reacting to MgpB were induced postinoculation and persisted throughout the infection. The immunodominance of the MgpB adhesin and the accumulation ofmgpBsequence diversity previously observed in persistent human infections prompted us to evaluate sequence variation in this animal model. We found that after 8 weeks of infection, sequences withinmgpBvariable region B were replaced by novel sequences generated by reciprocal recombination with an archived variant sequence located elsewhere on the chromosome. In contrast,mgpBregion B of the same inoculum propagated for 8 weeksin vitroremained unchanged. Notably, serum IgG reacted strongly with a recombinant protein spanning MgpB region B of the inoculum, while reactivity to a recombinant protein representing the week 8 variant was reduced, suggesting that antibodies were involved in the clearance of bacteria expressing the original infecting sequence. Together these results suggest that the pig-tailed macaque is a suitable model to studyM. genitaliumpathogenesis, antibody-mediated selection of antigenic variantsin vivo, and immune escape.

scholarly journals Draft Genome Sequence of Lactobacillus reuteri CECT8605

2017 ◽  
Vol 5 (18) ◽  
Author(s):  
Ana I. Sañudo ◽  
Mónica M. Olivares ◽  
Oscar Bañuelos
Keyword(s):  
Genome Sequence ◽  
Genetic Stability ◽  
Lactobacillus Reuteri ◽  
Draft Genome ◽  
Human Consumption ◽  
Probiotic Properties ◽  
Content Type ◽  
General Aspects

ABSTRACT Lactobacillus reuteri CECT8605 has shown potential probiotic properties in both in vitro and in vivo assays. Besides its beneficial characteristics, general aspects concerning genetic stability and safety for human consumption have been studied. Its genome sequence has been a useful tool to support preliminary conclusions based on empirical observations.

scholarly journals Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

2011 ◽  
Vol 193 (22) ◽  
pp. 6315-6322 ◽  
Author(s):  
Daniel J. Schu ◽  
Revathy Ramachandran ◽  
Jared S. Geissinger ◽  
Ann M. Stevens
Keyword(s):  
Conformational Changes ◽  
Homoserine Lactone ◽  
Biologically Active ◽  
Acyl Homoserine Lactone ◽  
Content Type ◽  
Pantoea Stewartii ◽  
Cell Densities ◽  
The Impact

The quorum-sensing regulator EsaR fromPantoea stewartiisubsp.stewartiiis a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogenP. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to studyin vitrobecause AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling anin vitroanalysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stablein vivoin the absence or presence of AHL. Limitedin vitroproteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.

Bioactive peptides from egg: a review

2015 ◽  
Vol 45 (2) ◽  
pp. 190-212 ◽  
Author(s):  
Z. F. Bhat ◽  
Sunil Kumar ◽  
Hina Fayaz Bhat
Keyword(s):  
Bioactive Peptides ◽  
Antioxidant Activities ◽  
Positive Impact ◽  
Antimicrobial Properties ◽  
Specific Protein ◽  
Biologically Active ◽  
Content Type ◽  
Treatment And Prevention

Purpose – The aim of the article was to focus on various peptides identified in the egg and their probable application as novel ingredients in the development of functional food products. Bioactive peptides of egg origin have attracted increasing interest as one of the prominent candidates for development of various health-promoting functional and designer foods. Design/methodology/approach – Traditionally known as a source of highly valuable proteins in human nutrition, eggs are nowadays also considered as an important source of many bioactive peptides which may find wide application in medicine and food production. These specific protein fragments from egg proteins which, above and beyond their nutritional capabilities, have a positive impact on the body’s function or condition by affecting the digestive, endocrine, cardiovascular, immune and nervous systems, and may ultimately influence health. Findings – Several peptides that are released in vitro or in vivo from egg proteins have been attributed to different health effects, including antihypertensive effects, antimicrobial properties, antioxidant activities, anticancer activity, immunomodulating activity, antiadhesive properties and enhancement of nutrient absorption and/or bioavailability. Extensive research has been undertaken to identify and characterize these biologically active peptides of egg origin which has changed the image of egg as a new source of biologically active ingredients for the development of functional foods with specific benefits for human health and treatment and prevention of diseases. Originality/value – The paper mainly describes the above-stated properties of bioactive peptides derived from egg proteins.

scholarly journals NovelN-Benzoyl-2-Hydroxybenzamide Disrupts Unique Parasite Secretory Pathway

2012 ◽  
Vol 56 (5) ◽  
pp. 2666-2682 ◽  
Author(s):  
Alina Fomovska ◽  
Qingqing Huang ◽  
Kamal El Bissati ◽  
Ernest J. Mui ◽  
William H. Witola ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protozoan Parasite ◽  
Secretory Protein ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Dense Granules ◽  
Genome Wide ◽  
Nanomolar Range

ABSTRACTToxoplasma gondiiis a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery ofN-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range againstT. gondii in vitroandin vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors ofT. gondii. Our genome-wide investigations reveal a specific mechanism of resistance toN-benzoyl-2-hydroxybenzamides mediated by adaptin-3β, a large protein from the secretory protein complex.N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs).N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistantPlasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway ofT. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.

scholarly journals Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Xuemei Lu ◽  
Xiaobao Jin ◽  
Yanting Huang ◽  
Jie Wang ◽  
Juan Shen ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Current Treatment ◽  
Biologically Active ◽  
Liver Targeting ◽  
E Coli ◽  
Final Yield ◽  
Pcr Method ◽  
Overlapping Extension Pcr

Interferon alpha (IFNα) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFNαis the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFNαmay account for its lowin vivobiological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFNα2b with aPlasmodiumregion I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression inE. coliBL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentialityin vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.

scholarly journals Vaginal Epithelial Cell-Derived S100 Alarmins Induced by Candida albicans via Pattern Recognition Receptor Interactions Are Sufficient but Not Necessary for the Acute Neutrophil Response during Experimental Vaginal Candidiasis

2013 ◽  
Vol 82 (2) ◽  
pp. 783-792 ◽  
Author(s):  
Junko Yano ◽  
Glen E. Palmer ◽  
Karen E. Eberle ◽  
Brian M. Peters ◽  
Thomas Vogl ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Candida Albicans ◽  
Epithelial Cells ◽  
Biologically Active ◽  
Polymorphonuclear Neutrophil ◽  
Vaginal Candidiasis ◽  
Content Type ◽  
Vaginal Epithelial Cells

ABSTRACTVulvovaginal candidiasis (VVC), caused byCandida albicans, affects women worldwide. Animal and clinical studies suggest that the immunopathogenic inflammatory condition of VVC is initiated by S100 alarmins in response toC. albicans, which stimulate polymorphonuclear neutrophil (PMN) migration to the vagina. The purpose of this study was to extend previousin vitrodata and determine the requirement for the alarmin S100A8 in the PMN response and to evaluate pattern recognition receptors (PRRs) that initiate the response. For the former, PMN migration was evaluatedin vitroorin vivoin the presence or absence of S100 alarmins initiated by several approaches. For the latter, vaginal epithelial cells were evaluated for PRR expression andC. albicans-induced S100A8 and S100A9 mRNAs, followed by evaluation of the PMN response in inoculated PRR-deficient mice. Results revealed that, consistent with previously reportedin vitrodata, eukaryote-derived S100A8, but not prokaryote-derived recombinant S100A8, induced significant PMN chemotaxisin vivo. Conversely, a lack of biologically active S100A8 alarmin, achieved by antibody neutralization or by using S100A9−/−mice, had no effect on the PMN responsein vivo. In PRR analyses, whereas Toll-like receptor 4 (TLR4)- and SIGNR1-deficient vaginal epithelial cells showed a dramatic reduction inC. albicans-induced S100A8/S100A9 mRNAsin vitro, inoculated mice deficient in these PRRs showed PMN migration similar to that in wild-type controls. These results suggest that S100A8 alarmin is sufficient, but not necessary, to induce PMN migration during VVC and that the vaginal PMN response toC. albicansinvolves PRRs in addition to SIGNR1 and TLR4, or other induction pathways.

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