Publications by authors named "Fanglei Zuo"

22 Publications

  • Page 1 of 1

Lactate-induced dispersal of microcolonies is mediated by changes in cell density and pilus retraction and is influenced by temperature change.

Infect Immun 2021 Jun 14:IAI0029621. Epub 2021 Jun 14.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.

is the etiologic agent of meningococcal meningitis and sepsis. Initial colonization of meningococci to the upper respiratory tract epithelium is crucial for disease development. The colonization occurs in several steps and expression of type IV pili (Tfp) is essential for both attachment and microcolony formation of encapsulated bacteria. Previously, we have shown that host-derived lactate induces synchronized dispersal of meningococcal microcolonies. In this study, we demonstrated that lactate-induced dispersal is dependent on bacterial concentration but not on the quorum sensing system autoinducer-2 or the two-component systems NarP/NarQ, PilR/PilS, NtrY/NtrX, and MisR/MisS. Further, there were no changes in expression of genes related to assembly, elongation, retraction, and modification of Tfp throughout the time course of lactate induction. By using and mutants, however, we found that lactate-induced dispersal was dependent on PilT-retraction but not on phosphoglycerol-modification of Tfp even though the PptB activity was important for preventing re-aggregation post-dispersal. Furthermore, protein synthesis was required for lactate-induced dispersal. Finally, we found that at a lower temperature, lactate-induced dispersal was delayed and unsynchronized, and bacteria reformed microcolonies. We conclude that lactate-induced microcolony dispersal is dependent on bacterial concentration, PilT-dependent Tfp retraction, and protein synthesis and influenced by environmental temperature.
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http://dx.doi.org/10.1128/IAI.00296-21DOI Listing
June 2021

Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing.

Curr Opin Biotechnol 2021 Aug 11;70:75-82. Epub 2021 Jan 11.

Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden.

Typical traditional probiotics lactobacilli and bifidobacteria are gaining great interest to be developed as living diagnostics and therapeutics for improving human health. However, the mechanistic basis underlying their inherent health beneficial property remain incompletely understood which can slow down the translational pipeline in the functional food and pharmaceutical field. Efficient genome editing will advance the understanding of the molecular mechanism of the probiotics' physiological properties and their interaction with the host and the host microbiota, thereby further promote the development of next-generation designer probiotics with improved robustness and tailored functionalities. With the expansion of genome editing strategies such as CRISPR-Cas-based tools and IPSD assisted genome engineering as well as other synthetic biology technologies, the research and application of these health-promoting bacteria for the food and pharmaceutical industry will be further enhanced.
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http://dx.doi.org/10.1016/j.copbio.2020.12.015DOI Listing
August 2021

Engineer probiotic bifidobacteria for food and biomedical applications - Current status and future prospective.

Biotechnol Adv 2020 12 5;45:107654. Epub 2020 Nov 5.

Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden.

Bifidobacteria are members of the human gut microbiota and have shown to exert beneficial effects on their host. Certain strains have a long history of safe and effective use as probiotics. Due to the lack of efficient genetic tools, however, little is known about the molecular mechanisms on which these health-promoting properties are based, thus limiting the synthetic biology applications in bifidobacteria. Here, we discuss the recent development of genetic tools and their engagement in engineering bifidobacteria for food and biomedical applications, from eliminating antibiotic resistance mobile elements and improving robustness to preventing pathogen infections and delivering therapeutics for cancer treatment. In addition, we highlight the application of emerging genome engineering techniques for manipulating the bifidobacterial genome. Finally, we provide our perspective on the future development of synthetic biology techniques and programmed probiotic bifidobacteria with enhanced robustness and designer functionalities.
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http://dx.doi.org/10.1016/j.biotechadv.2020.107654DOI Listing
December 2020

Role of Sortase A in Kx110A1 Adhesion to Gastric Epithelial Cells and Competitive Exclusion of .

Front Microbiol 2019 3;10:2770. Epub 2019 Dec 3.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

We have previously shown that Kx110A1, a human stomach isolate, can colonize mouse stomach and reduce the initial colonization of . Here, we investigated the role of sortase-dependent proteins (SDPs) involved in these functions by the construction of a mutant for , the gene encoding the housekeeping sortase that covalently anchors SDPs to the cell surface. The mutant showed a decrease in hydrophobicity and autoaggregation under acidic conditions, indicating the effect of SDPs on cell surface properties. Correspondingly, the mutant lost the capacity to adhere to gastric epithelial cells, thus resulting in an inability to provide a physical barrier to prevent adherence. These results indicate that sortase A is a key determinant of the cell surface properties of Kx110A1 and contributes to -mediated exclusion of . Understanding the molecular mechanisms by which lactobacilli antagonize might contribute to the development of novel therapeutic strategies that take advantage of health-promoting bacteria and reduce the burden of antibiotic resistance.
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http://dx.doi.org/10.3389/fmicb.2019.02770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902081PMC
December 2019

An exploratory pilot study evaluating the supplementation of standard antibiotic therapy with probiotic lactobacilli in south African women with bacterial vaginosis.

BMC Infect Dis 2019 Sep 18;19(1):824. Epub 2019 Sep 18.

Perinatal HIV Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, 1862, South Africa.

Background: To reduce acquisition and relapse of bacterial vaginosis (BV), lactobacilli must be maintained in the vaginal microbiome. Probiotic lactobacilli may aid this purpose. We investigated whether vaginal probiotics (containing Lactobacillus rhamnosus DSM 14870 and Lactobacillus gasseri DSM 14869) would result in vaginal colonisation with lactobacilli in women with and without BV.

Methods: This prospective, partially randomised, exploratory pilot study was conducted in Soweto, South Africa. Thirty-nine sexually-active, HIV negative women were enrolled from October 2014 to May 2016 into three arms. Women who did not have BV (Group 1, n = 13) self-administered probiotic capsules vaginally once daily for 30 days, then once a week until Day 190. Women diagnosed with BV were randomized into Group 2 (n = 12) or Group 3 (n = 14) and treated with the triple oral antibiotic combination for vaginal discharge syndrome per South African guidelines (cefixime 400 mg stat, doxycycline 100 mg BD for 7 days and metronidazole 2 g stat). Immediately after antibiotic treatment, women in Group 2 self-administered probiotic capsules vaginally once daily for 30 days then vaginally once a week until Day 190. Women in Group 3 were not given lactobacilli.

Results: During the study, L. rhamnosus DSM 14870 or L. gasseri DSM 14869, were isolated in 5/13 (38.5%) women in Group 1 compared to 10/12 (83.3%) women in Group 2 (p = 0.041). The 1-month and 6-month BV cure rates were similar (P >  0.05) between Group 2 (42 and 25%) compared to Group 3 (36 and 25%). In Group 2, no correlation was observed between the frequency of isolation of the two Lactobacillus strains and the 1-month or 6-month cure rate.

Conclusions: Supplementation with vaginal probiotic capsules resulted in colonisation of the vagina by the Lactobacillus strains (L. rhamnosus DSM 14870 and L. gasseri DSM 14869) contained in the capsules. We observed low initial cure rates of BV after a stat dose of metronidazole and that the probiotic did not improve BV cure rates or alleviate recurrence which could be due to treatment failure or very limited power of the study.

Trial Registration: Registered at the Pan African Clinical Trial Registry ( www.pactr.org ) on April 13, 2018 (retrospectively registered). Trial identification number: PACTR201804003327269.
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http://dx.doi.org/10.1186/s12879-019-4425-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751625PMC
September 2019

Putative Adhesion Factors in Vaginal Lactobacillus gasseri DSM 14869: Functional Characterization.

Appl Environ Microbiol 2019 10 17;85(19). Epub 2019 Sep 17.

Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital, Huddinge, Stockholm, Sweden

Lactobacilli play an important role in the maintenance of a healthy vaginal microbiota, and some select species are widely used as probiotics. Vaginal isolates of DSM 14869 and DSM 14870 were previously selected to develop the probiotic EcoVag capsules and showed therapeutic effects in women with bacterial vaginosis (BV). However, the molecular mechanisms involved in their probiotic activity are largely unknown. In this study, we identified three cell surface molecules in DSM 14869 that promote adhesion to vaginal epithelial cells (VEC) by constructing dedicated knockout mutants, including exopolysaccharides (EPSs), a protein containing MucBP-like domains (N506_1778), and a putative novel adhesin (N506_1709) with rib/alpha-like domain repeats. EPS knockout mutants revealed 20-fold and 14-fold increases in adhesion to Caco-2 and HeLa cells, respectively, compared with wild type, while the adhesion to VEC was reduced 30% by the mutation, suggesting that EPSs might mediate tissue tropism for vaginal cells. A significant decrease in adhesion to Caco-2 cells, HeLa cells, and VEC was observed in the N506_1778 knockout mutant. The N506_1709 mutant showed no significant difference for the adhesion to Caco-2 and HeLa cells compared with wild type (WT); in contrast, the adhesion to VEC revealed a significant decrease (42%), suggesting that N506_1709 might mediate specific binding to stratified squamous epithelial cells, and this putative novel adhesin was annotated as vaginal epithelium adhesin (LVEA). Thus, we have discovered an important role for EPSs and a novel adhesin, LVEA, in the adhesive capacity of a vaginal probiotic strain. Lactobacilli are known to contribute to the maintenance of a healthy vaginal microbiota and some are selected as probiotics for the prevention or treatment of urogenital diseases, such as bacterial vaginosis. However, the molecular mechanisms for these health-promoting effects are not fully understood. Here, we functionally identified three cell surface factors of a strain potentially involved in its adhesion to vaginal epithelial cells, including exopolysaccharides (EPSs) and two sortase-dependent proteins (N506_1778 and N506_1709). We could demonstrate the tissue-specific adhesion of EPSs to vaginal cells and that N506_1709 might be a novel adhesin specifically mediating bacterial binding to stratified squamous epithelial cells. The results provide important new information on the molecular mechanisms of vaginal spp. adhesion.
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http://dx.doi.org/10.1128/AEM.00800-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752014PMC
October 2019

Inducible Plasmid Self-Destruction (IPSD) Assisted Genome Engineering in Lactobacilli and Bifidobacteria.

ACS Synth Biol 2019 08 12;8(8):1723-1729. Epub 2019 Jul 12.

Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine , Karolinska Institutet at Karolinska University Hospital Huddinge , Stockholm SE-141 86 , Sweden.

Genome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria, but still have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called inducible plasmid self-destruction (IPSD) was successfully used to perform gene knockout and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.
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http://dx.doi.org/10.1021/acssynbio.9b00114DOI Listing
August 2019

Exopolysaccharide-Producing Strains with Similar Adhesion Property Induce Differential Regulation of Inflammatory Immune Response in Treg/Th17 Axis of DSS-Colitis Mice.

Nutrients 2019 Apr 4;11(4). Epub 2019 Apr 4.

Key Laboratory of Functional Dairy Science, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.

Intestinal bifidobacteria benefit human health by promoting and modulating the gut flora, and boosting therapeutic efficiency for chronic metabolic diseases and cancer. Recently, strains with high adhesion to intestinal epithelial cells were associated with induction of T-helper 17 (Th17) cells in humans and rodents. Here, two strains with similar adhesive ability but different aggregation properties were investigated for specific immunoregulatory effects, including the underlying cellular pathway, on macrophage and T-regulatory (Treg)/Th17 axis activation in vitro and in the colon of dextran sodium sulfate (DSS)-colitis mice in vivo. In-vitro, the auto-aggregative . strain IF1-11 induced significantly higher IL-6 and lower IL-10 secretion from immune cells, and it induced abundant Th17 cells. The non-aggregating strain IF1-03 induced significantly higher IL-10, less IL-6 and a high proportion of Treg/Th17 cells compared to total T cells. In vivo, orally administered IF1-03 protected DSS-colitis mice via activation of dendritic cells or macrophages and skewing of Treg/Th17 cells, consistent with Treg cell induction in vitro. IF1-03 exopolysaccharides showed a functional recognition pattern similar to IF1-03 for IL-10 cytokine secretion and Treg cell-differentiation induction, both dependent on the toll-like receptor 2-ERK/p38 MAPK-signaling cascade for macrophage activation. We suggest that . exopolysaccharide-associated enterocyte adhesion/aggregation phenotypes determine strain-specific adaptive immune responses in the gut via the macrophage-regulated Treg/Th17 axis.
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http://dx.doi.org/10.3390/nu11040782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520857PMC
April 2019

Deletion of D-Lactate Dehydrogenase A in Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release.

Front Microbiol 2019 5;10:422. Epub 2019 Mar 5.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences microcolony dispersal. Here, we examined the ability of mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A () mutant had an increased level of biofilm formation. Deletion of increased the cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the -deficient mutant, suggesting a critical role for eDNA. Compared to wild-type, the -deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while , which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of in promotes biofilm formation and aggregation through autolysis-mediated DNA release.
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http://dx.doi.org/10.3389/fmicb.2019.00422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411758PMC
March 2019

Transcriptomic analysis of Bifidobacterium longum subsp. longum BBMN68 in response to oxidative shock.

Sci Rep 2018 11 20;8(1):17085. Epub 2018 Nov 20.

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, P. R. China.

Bifidobacterium longum strain BBMN68 is sensitive to low concentrations of oxygen. A transcriptomic study was performed to identify candidate genes for B. longum BBMN68's response to oxygen treatment (3%, v/v). Expression of genes and pathways of B. longum BBMN68 involved in nucleotide metabolism, amino acid transport, protein turnover and chaperones increased, and that of carbohydrate metabolism, translation and biogenesis decreased to adapt to the oxidative stress. Notably, expression of two classes of ribonucleotide reductase (RNR), which are important for deoxyribonucleotide biosynthesis, was rapidly and persistently induced. First, the class Ib RNR NrdHIEF was immediately upregulated after 5 min oxygen exposure, followed by the class III RNR NrdDG, which was upregulated after 20 min of exposure. The upregulated expression of branched-chain amino acids and tetrahydrofolate biosynthesis-related genes occurred in bifidobacteria in response to oxidative stress. These change toward to compensate for DNA and protein damaged by reactive oxygen species (ROS). In addition, oxidative stress resulted in improved B. longum BBMN68 cell hydrophobicity and autoaggregation. These results provide a rich resource for our understanding of the response mechanisms to oxidative stress in bifidobacteria.
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http://dx.doi.org/10.1038/s41598-018-35286-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244367PMC
November 2018

Characterization and complete genome sequences of L. rhamnosus DSM 14870 and L. gasseri DSM 14869 contained in the EcoVag probiotic vaginal capsules.

Microbiol Res 2017 Dec 14;205:88-98. Epub 2017 Aug 14.

Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, 14186, Stockholm, Sweden.

Lactobacillus rhamnosus DSM 14870 and Lactobacillus gasseri DSM 14869 were previously isolated from the vaginal epithelial cells (VEC) of healthy women and selected for the development of the vaginal EcoVag probiotic capsules. EcoVag was subsequently shown to provide long-term cure and reduce relapse of bacterial vaginosis (BV) as an adjunct to antibiotic therapy. To identify genes potentially involved in probiotic activity, we performed genome sequencing and characterization of the two strains. The complete genome analysis of both strains revealed the presence of genes encoding functions related to adhesion, exopolysaccharide (EPS) biosynthesis, antimicrobial activity, and CRISPR adaptive immunity but absence of antibiotic resistance genes. Interesting features of L. rhamnosus DSM 14870 genome include the presence of the spaCBA-srtC gene encoding spaCBA pili and interruption of the gene cluster encoding long galactose-rich EPS by integrases. Unique to L. gasseri DSM 14869 genome was the presence of a gene encoding a putative (1456 amino acid) new adhesin containing two rib/alpha-like repeats. L. rhamnosus DSM 14870 and L. gasseri DSM 14869 showed acidification of the culture medium (to pH 3.8) and a strong adhesion capability to the Caco-2 cell line and VEC. L. gasseri DSM 14869 could produce a thick (40nm) EPS layer and hydrogen peroxide. L. rhamnosus DSM 14870 was shown to produce SpaCBA pili and a 20nm EPS layer, and could inhibit the growth of Gardnerella vaginalis, a bacterium commonly associated with BV. The genome sequences provide a basis for further elucidation of the molecular basis for their probiotic functions.
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http://dx.doi.org/10.1016/j.micres.2017.08.003DOI Listing
December 2017

Characterization of a lactose-responsive promoter of ATP-binding cassette (ABC) transporter gene from Lactobacillus acidophilus 05-172.

FEMS Microbiol Lett 2017 Sep;364(16)

Key Laboratory of Functional Dairy Science of the Chinese Ministry of Education and Municipal Government of Beijing, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China.

A novel lactose-responsive promoter of the ATP-binding cassette (ABC) transporter gene Lba1680 of Lactobacillus acidophilus strain 05-172 isolated from a traditionally fermented dairy product koumiss was characterized. In L. acidophilus 05-172, expression of Lba1680 was induced by lactose, with lactose-induced transcription of Lba1680 being 6.1-fold higher than that induced by glucose. This is in contrast to L. acidophilus NCFM, a strain isolated from human feces, in which expression of Lba1680 and Lba1679 is induced by glucose. Both gene expression and enzyme activity assays in L. paracasei transformed with a vector containing the inducible Lba1680 promoter (PLba1680) of strain 05-172 and a heme-dependent catalase gene as reporter confirmed that PLba1680 is specifically induced by lactose. Its regulatory expression could not be repressed by glucose, and was independent of cAMP receptor protein. This lactose-responsive promoter might be used in the expression of functional genes in L. paracasei incorporated into a lactose-rich environment, such as dairy products.
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http://dx.doi.org/10.1093/femsle/fnx167DOI Listing
September 2017

Recombinant Lactococcus lactis expressing bioactive exendin-4 to promote insulin secretion and beta-cell proliferation in vitro.

Appl Microbiol Biotechnol 2017 Oct 21;101(19):7177-7186. Epub 2017 Aug 21.

Key Laboratory of Functional Dairy, Department of Food Science and Engineering, College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghua East Road, Haidian District, Beijing, 100083, People's Republic of China.

In recent years, therapeutic peptides have garnered great interest in the pharmaceutical industry for the treatment of diabetes. Lactic acid bacteria (LAB) are an appealing vehicle for safe and convenient oral delivery of bioactive peptide and protein drugs. Exendin-4 (Exd4) is a glucagon-like protein-1 (GLP-1) receptor agonist that is considered an excellent therapeutic peptide drug for type 2 diabetes due to its longer-lasting bioactivity, resulting from resistance to dipeptidyl peptidase 4. We explored Lactococcus lactis with the nisin-controlled gene expression (NICE) system as an oral delivery system for recombinant (r) Exd4 peptide in situ. Heterologous expression and secretion of rExd4 by L. lactis NZ9000/pNZ8048-rExd4 were successful and efficient under the NICE system. In vitro treatment with rExd4 significantly enhanced insulin secretion of INS-1 cells and activated the PI3-K/AKT signal pathway with protein levels of AKT and p-AKT increasing 1.6- to 1.8-fold compared to negative controls, similar to the positive GLP-1 controls. INS-1 cells treated with rExd4 also showed enhanced proliferation and inhibited apoptosis, corresponding with the effects of the standard Exd4 and GLP-1 treatments. Our data suggest that the rExd4 secreted by L. lactis is a bioactive insulinotropic peptide and functional GLP-1 receptor agonist that enhances glucose-dependent insulin secretion and activates the PI3-K/AKT signal pathway; furthermore, it may be involved in improving proliferation and inhibiting apoptosis of INS-1 cells in in vitro treatments. Therefore, L. lactis producing rExd4 may potentially serve as a novel strategy for oral treatment of diabetes.
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http://dx.doi.org/10.1007/s00253-017-8410-6DOI Listing
October 2017

Heterologous Expression and Delivery of Biologically Active Exendin-4 by Lactobacillus paracasei L14.

PLoS One 2016 20;11(10):e0165130. Epub 2016 Oct 20.

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, P. R. China.

Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, is an excellent therapeutic peptide drug for type 2 diabetes due to longer lasting biological activity compared to GLP-1. This study explored the feasibility of using probiotic Lactobacillus paracasei as an oral vector for recombinant exendin-4 peptide delivery, an alternative to costly chemical synthesis and inconvenient administration by injection. L. paracasei transformed with a plasmid encoding the exendin-4 gene (L. paracasei L14/pMG76e-exendin-4) with a constitutive promotor was successfully constructed and showed efficient secretion of exendin-4. The secreted exendin-4 significantly enhanced insulin secretion of INS-1 β-cells, along with an increment in their proliferation and inhibition of their apoptosis, corresponding to the effect of GLP-1 on these cells. The transcription level of the pancreatic duodenal homeobox-1 gene (PDX-1), a key transcription factor for cellular insulin synthesis and secretion, was upregulated by the treatment with secreted exendin-4, paralleling the upregulation of insulin gene expression. Caco-2 cell monolayer permeability assay showed a 34-fold increase in the transport of exendin-4 delivered by L. paracasei vs. that of free exendin-4 (control), suggesting effective facilitation of exendin-4 transport across the intestinal barrier by this delivery system. This study demonstrates that the probiotic Lactobacillus can be engineered to secrete bioactive exendin-4 and facilitate its transport through the intestinal barrier, providing a novel strategy for oral exendin-4 delivery using this lactic acid bacterium.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165130PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072737PMC
June 2017

Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells.

Microb Cell Fact 2016 Jun 10;15(1):102. Epub 2016 Jun 10.

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghua East Road, Haidian District, Beijing, 100083, People's Republic of China.

Background: Proteinaceous bioactive substances and pharmaceuticals are most conveniently administered orally. However, the facing problems are the side effects of proteolytic degradation and denaturation in the gastrointestinal tract. In recent years, lactic acid bacteria (LAB) have been verified to be a promising delivery vector for susceptible functional proteins and drugs. KiSS-1 peptide, a cancer suppressor, plays a critical role in inhibiting cancer metastasis and its activity has been confirmed by direct administration. However, whether this peptide can be functionally expressed in LAB and exert activity on cancer cells, thus providing a potential alternative administration manner in the future, has not been demonstrated.

Results: A recombinant Lactococcus lactis strain NZ9000-401-kiss1 harboring a plasmid containing the gene of the tumor metastasis-inhibiting peptide KiSS1 was constructed. After optimization of the nisin induction conditions, the recombinant strain efficiently secreted KiSS1 with a maximum detectable amount of 27.9 μg/ml in Dulbecco's Modified Eagle medium. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and would healing assays, respectively, indicated that the secreted KiSS1 peptide remarkably inhibited HT-29 cell proliferation and migration. Furthermore, the expressed KiSS1 was shown to induce HT-29 cell morphological changes, apoptosis and reduce the expression of matrix metalloproteinase 9 (MMP-9) at both mRNA and protein levels.

Conclusions: A recombinant L. lactis NZ9000-401-kiss1 successfully expressing the human kiss1 was constructed. The secreted KiSS1 peptide inhibited human HT-29 cells' proliferation and migration probably by invoking, or mediating the cell-apoptosis pathway and by down regulating MMP-9 expression, respectively. Our results suggest that L. lactis is an ideal cell factory for secretory expression of tumor metastasis-inhibiting peptide KiSS1, and the KiSS1-producing L. lactis strain may serve as a new tool for cancer therapy in the future.
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http://dx.doi.org/10.1186/s12934-016-0506-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901401PMC
June 2016

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

Sci Rep 2015 Sep 15;5:14109. Epub 2015 Sep 15.

Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, P. R. China.

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.
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http://dx.doi.org/10.1038/srep14109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572922PMC
September 2015

Overexpression of Small Heat Shock Protein Enhances Heat- and Salt-Stress Tolerance of Bifidobacterium longum NCC2705.

Curr Microbiol 2015 Jul 5;71(1):8-15. Epub 2015 Apr 5.

Key Laboratory of Functional Dairy Science of Chinese Ministry of Education and Municipal Government of Beijing, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, People's Republic of China.

Bifidobacteria are probiotics that are incorporated live into various dairy products. They confer health-promotive effects via gastrointestinal tract colonization. However, to provide their health-beneficial properties, they must battle the various abiotic stresses in that environment, such as bile salts, acids, oxygen, and heat. In this study, Bifidobacterium longum salt- and heat-stress tolerance was enhanced by homologous overexpression of a small heat shock protein (sHsp). A positive contribution of overproduced sHsp to abiotic stress tolerance was observed when the bacterium was exposed to heat and salt stresses. Significantly higher survival of B. l ongum NCC2705 overexpressing sHsp was observed at 30 and 60 min into heat (55 °C) and salt (5 M NaCl) treatment, respectively. Thermotolerance analysis at 47 °C with sampling every 2 h also revealed the great potential tolerance of the engineered strain. Cell density and acid production rate increased for the sHsp-overexpressing strain after 8 and 10 h of both heat and salt stresses. In addition, tolerance to bile salts, low pH (3.5) and low temperature (4 °C) was also increased by homologous overexpression of the sHsp hsp20 in B. l ongum. Results revealed that hsp20 overexpression in B longum NCC2705 plays a positive cross-protective role in upregulating abiotic responses, ensuring the organism's tolerance to various stress conditions; therefore, sHsp-overexpressing B. l ongum is advised for fermented dairy foods and other probiotic product applications.
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http://dx.doi.org/10.1007/s00284-015-0811-0DOI Listing
July 2015

Homologous overexpression of alkyl hydroperoxide reductase subunit C (ahpC) protects Bifidobacterium longum strain NCC2705 from oxidative stress.

Res Microbiol 2014 Sep 19;165(7):581-9. Epub 2014 Jun 19.

Key Laboratory of Functional Dairy Science of Chinese Ministry of Education and Municipal Government of Beijing, Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghua East Road, Haidian District, Beijing 100083, PR China; Beijing Agricultural Function Microbial Engineering and Technology Center, No. 1 Li'ersi Industrial Area, Zhangjiawan Town, Tongzhou District, Beijing 101113, PR China. Electronic address:

The ability to manage reactive oxygen species (ROS) effectively is crucial for the survival of gut bifidobacteria under conditions of oxidative stress. Alkyl hydroperoxide reductase catalytic subunit C (ahpC) of Bifidobacterium longum responds to various oxidative stresses. In this study, an ahpC-overexpressing transformant of B. longum strain NCC2705 was constructed to investigate the role and function of ahpC in oxidative stresses inflicted by treatments with hydrogen peroxide (H2O2), cumene hydroperoxide, and aerobic oxygen. Results indicated that in B. longum, AhpC is the primary scavenger of endogenous H2O2 generated by aerobic metabolism, but it is unable to detoxify high concentrations of exogenous H2O2. The ahpC-overexpressing B. longum strain showed increased resistance to organic hydroperoxide killing, increased viability under aerobic growth, but decreased resistance to exogenous H2O2 in comparison to the control strain. Analysis of genes from the oxidative stress-defense pathway encoding oxygen-independent coproporphyrinogen III oxidase (HemN), NADH oxidase (Nox) and thioredoxin reductase-like protein (TrxB) showed increased transcript levels in the ahpC-overexpressing vs. control strain. These findings suggest that elevated ahpC expression facilitates or activates the different electron donor-dependent ROS-elimination pathways in B. longum's response to oxidative stress.
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http://dx.doi.org/10.1016/j.resmic.2014.05.040DOI Listing
September 2014

Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress.

Appl Microbiol Biotechnol 2014 Sep 6;98(17):7523-34. Epub 2014 Jun 6.

Key Laboratory of Functional Dairy Science of Chinese Ministry of Education and Municipal Government of Beijing, College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghua East Road, Haidian District, Beijing, 100083, China.

Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H2O2 and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn(2+)-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.
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http://dx.doi.org/10.1007/s00253-014-5851-zDOI Listing
September 2014

Oxidative stress-related responses of Bifidobacterium longum subsp. longum BBMN68 at the proteomic level after exposure to oxygen.

Microbiology (Reading) 2011 Jun 24;157(Pt 6):1573-1588. Epub 2011 Feb 24.

College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, PR China.

Bifidobacterium longum subsp. longum BBMN68, an anaerobic probiotic isolated from healthy centenarian faeces, shows low oxygen (3 %, v/v) tolerance. To understand the effects of oxidative stress and the mechanisms protecting against it in this strain, a proteomic approach was taken to analyse changes in the cellular protein profiles of BBMN68 under the following oxygen-stress conditions. Mid-exponential phase BBMN68 cells grown in MRS broth at 37 °C were exposed to 3 % O(2) for 1 h (I) or 9 h (II), and stationary phase cells were subjected to 3 % O(2) for 1 h (III). Respective controls were grown under identical conditions but were not exposed to O(2). A total of 51 spots with significant changes after exposure to oxygen were identified, including the oxidative stress-protective proteins alkyl hydroperoxide reductase C22 (AhpC) and pyridine nucleotide-disulfide reductase (PNDR), and the DNA oxidative damage-protective proteins DNA-binding ferritin-like protein (Dps), ribonucleotide reductase (NrdA) and nucleotide triphosphate (NTP) pyrophosphohydrolases (MutT1). Changes in polynucleotide phosphorylase (PNPase) plus enolase, which may play important roles in scavenging oxidatively damaged RNA, were also found. Following validation at the transcriptional level of differentially expressed proteins, the physiological and biochemical functions of BBMN68 Dps were further proven by in vitro and in vivo tests under oxidative stress. Our results reveal the key oxidative stress-protective proteins and DNA oxidative damage-protective proteins involved in the defence strategy of BBMN68 against oxygen, and provide the first proteomic information toward understanding the responses of Bifidobacterium and other anaerobes to oxygen stress.
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http://dx.doi.org/10.1099/mic.0.044297-0DOI Listing
June 2011

Complete genome sequence of Bifidobacterium longum subsp. longum BBMN68, a new strain from a healthy chinese centenarian.

J Bacteriol 2011 Feb 19;193(3):787-8. Epub 2010 Nov 19.

Key Laboratory of Functional Dairy Science of Beijing and Chinese Ministry of Education, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.

Bifidobacterium longum subsp. longum BBMN68 was isolated from the feces of a healthy centenarian living in an area of BaMa, Guangxi, China, known for longevity. Here we report the main genome features of B. longum strain BBMN68 and the identification of several predicted proteins associated with the ecological niche of longevity.
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http://dx.doi.org/10.1128/JB.01213-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021241PMC
February 2011
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