MEDFORD, MA, United States
MEDFORD, MA, United States

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The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.


The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.


Yu Z.-T.,Harvard University | Yu Z.-T.,Boston College | Chen C.,Harvard University | Chen C.,Boston College | And 8 more authors.
Glycobiology | Year: 2013

Breast-fed infant microbiota is typically rich in bifidobacteria. Herein, major human milk oligosaccharides (HMOS) are assessed for their ability to promote the growth of bifidobacteria and to acidify their environment, key features of prebiotics. During in vitro anaerobic fermentation of infant microbiota, supplementation by HMOS significantly decreased the pH even greater than supplementation by fructooligosaccharide (FOS), a prebiotic positive control. HMOS elevated lactate concentrations, increased the proportion of Bifidobacterium spp. in culture, and through their fermentation into organic acids, decreased the proportion of Escherichia and Clostridium perfringens. Three principal components of HMOS, 2′-fucosyllactose, lactodifucotetraose and 3-fucosyllactose, were consumed in these cultures. These three principal oligosaccharides of human milk were then individually tested as supplements for in vitro growth of four individual representative strains of infant gut microbes. Bifidobacterium longum JCM7007 and B. longum ATCC15697 efficiently consumed oligosaccharides and produced abundant lactate and short-chain fatty acids, resulting in significant pH reduction. The specificity of fermentation differed by microbe species and strain and by oligosaccharide structure. Escherichia coli K12 and C. perfringens did not utilize appreciable fucosylated oligosaccharides, and a typical mixture of organic acid fermentation products inhibited their growth. In summary, 2′-fucosyllactose, lactodifucotetraose, and 3-fucosyllactose, when cultured with B. longum JCM7007 and B. longum ATCC15697, exhibit key characteristics of a prebiotic in vitro. If these bifidobacteria are representative of pioneering or keystone species for human microbiota, fucosylated HMOS could strongly promote colonization and maintenance of a mutualist symbiotic microbiome. Thus, these simple glycans could mediate beneficial effects of human milk on infant health. © 2012 The Author.


Balasubramanian D.,Florida International University | Schneper L.,Florida International University | Merighi M.,Harvard University | Merighi M.,Glycosyn, Inc. | And 5 more authors.
PLoS ONE | Year: 2012

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. In addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, we compared the transcriptional profile generated using DNA microarrays of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought, with the deletion of ampR influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Other virulence mechanisms including biofilm formation and QS-regulated acute virulence factors are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the microarray data. Further, using a Caenorhabditis elegans model, we demonstrate that a functional AmpR is required for P. aeruginosa pathogenicity. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. Further, we show differential regulation of other transcriptional regulators and sigma factors by AmpR, accounting for the extensive AmpR regulon. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating biofilm formation, a chronic infection phenotype. Unraveling this complex regulatory circuit will provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors in response to antibiotic exposure. © 2012 Balasubramanian et al.


Skurnik D.,Harvard University | Merighi M.,Harvard University | Merighi M.,Glycosyn, Inc. | Grout M.,Harvard University | And 8 more authors.
Journal of Clinical Investigation | Year: 2010

New prophylactic approaches are needed to control infection with the Gram-positive bacterium Staphylococcus aureus, which is a major cause of nosocomial and community-acquired infections. To develop these, greater understanding of protective immunity against S. aureus infection is needed. Human immunity to extracellular Gram-positive bacterial pathogens is primarily mediated by opsonic killing (OPK) via antibodies specific for surface polysaccharides. S. aureus expresses two such antigens, capsular polysaccharide (CP) and poly-N-acetyl glucosamine (PNAG). Here, we have shown that immunization-induced polyclonal animal antisera and monoclonal antibodies specific for either CP or PNAG antigens have excellent in vitro OPK activity in human blood but that when mixed together they show potent interference in OPK activity. In addition, reductions in antibody binding to the bacterial surface, complement deposition, and passive protection were seen in two mouse models of S. aureus infection. Electron microscopy, isothermal calorimetry, and surface plasmon resonance indicated that antibodies to CP and PNAG bound together via an apparent idiotype-anti-idiotype interaction. This interaction was also found in sera from humans with S. aureus bacteremia. These findings suggest that the lack of effective immunity to S. aureus infections in humans could be due, in part, to interference in OPK when antibodies to CP and PNAG antigens are both present. This information could be used to better design S. aureus vaccine components.


The invention provides compositions and methods for engineering bacteria to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.


The invention provides compositions and methods for engineering bacteria to produce sialylated and N-acetylglucosamine-containing oligosaccharides, and the use thereof in the prevention or treatment of infection.


The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.


The invention provides compositions and methods utilizing an improved strain of bacteria, a Lac+ Lactobacillus rhamnosus strain of bacteria.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.00M | Year: 2010

DESCRIPTION (provided by applicant): Adherence of pathogens to their host cells is the obligatory first step of infection and is frequently mediated by specific molecular interactions [1][2]. Virulent Campylobacter species, Vibrio cholerae, enteropathogenic E.coli (EPEC), enterohemorrhagic E.coli (EHEC) and pathogenic strains of Norwalk virus, the leading bacterial and viral causes of human infectious diarrhea [3], adhere to gut epithelial surfaces through binding to 1(1,2) fucosylated cellular receptors[4][5]. 1(1,2) fucosylated glycans, which are abundant in human breast milk[6][7], have been shown both in vitro and in vivo effectively to prevent binding and infection by these pathogens[4][5]. These molecules therefore represent a new class of agent with potential to prevent infectious diarrhea, a condition that is the cause annually of over 2 million deaths worldwide [8]. However the production of 1(1,2) fucosylated glycans as anti-infective agents in sufficient quantities to impact global diarrhea incidence remains a significant challenge. Chemical syntheses are possible, but are limited by stereo-specificity issues, product impurities, and high overall cost[9][10][11]. In vitro enzymatic syntheses are also possible but are limited by a requirement for expensive nucleotide-sugar precursors. Glycosyn Inc.lt s broad goal is to develop ways to manufacture 1(1,2) fucosylated glycans cheaply and in bulk through microbial fermentation, and three classes of potential anti-infective products are envisaged: 1) purified 1(1,2) fucosylated oligosaccharides, 2) yeast strains expressing 1(1,2) fucosylated glycans on their cell surface, and 3) purified 1(1,2) fucosylated glycoproteins. [[[The goal of the studies outlined in this application are to produce in the dairy yeast Kluyveromyces lactis an example of the first of these product classes, namely a purified 1(1,2) fucosylated oligosaccharide, 22-fucosyllactose (22-FL), in sufficient amounts to test this moleculelt s efficacy as a single agent in in vitro and in vivo infection models. Glycan synthetic pathways in Kluyveromyces lactis will be engineered through a combination of endogenous gene manipulation and the introduction of heterologous genes encoding desired activities. Specifically, K.lactis will be engineered to synthesize the key precursor sugar, GDP-fucose, and subsequently to make 22-fucosyllactose in the cell cytoplasm. A subsequent goal is to increase the yield of 2lt -fucolsyllactose recovered from K.lactis to approach levels that will be required for commercialization. To achieve this the production of 2lt fucosyllactose will be increased by manipulating cellular levels of synthetic enzymes and precursor pools, balancing 2;-FL production with overall cell viability and growth performance under bioreactor conditions.]]] PUBLIC HEALTH RELEVANCE: Worldwide, infectious diarrhea[12] is responsible for approximately 20% of all mortality in children under the age of 5, and for an estimated 2 million deaths annually[8]. In the developing world bacterial infections cause more than 50% of all cases of diarrhea, and of these, infections by Campylobacter and diarrheagenic E.coli together account for about half. Campylobacter is the most common cause of culture-proven bacterial gastroenteritis in both developed and developing countries, and is responsible for 400 to 500 million cases of diarrhea each year. By far the highest incidence of Campylobacter infections is in children lt 5 yrs of age[13][14][15]. Unfortunately, prevention and treatment options for bacterial diarrhea are limited. Vaccines are currently unavailable, and if developed, would be costly and of limited availability in rural poor populations where unmet need is highest. Moreover vaccines are typically pathogen-specific, but infectious diarrhea can be caused by numerous diverse pathogens. The use of antibiotics for treatment of diarrhea is also becoming increasingly problematic, since such use is driving the emergence of resistant strains. For example, clinical isolates of Campylobacter are now often resistant to quinolones [16] and erythromycin-resistant strains are rapidly emerging [17]. Conventional antimicrobial agents are designed to inhibit a pathogenlt s replication and growth, yet they do nothing to make a pathogenlt s environmental niche unavailable. Thus emerging resistant strains are readily able to proliferate and spread. Research is needed to develop new classes of anti-infective agent that are both broad-acting and that use a different approach to avoid the development of resistance; for example novel anti-adhesion agents such as those described in this application. The anti-adhesive 1(1,2) fucosylated oligosaccharides described here will not drive resistance, since they exert no selective pressure on pathogens by merely depriving them of their environmental niche. Moreover these anti-infectives will simultaneously target multiple enteropathogens, including C.jejuni [4], ETEC E.coli [18], Vibrio cholerae [19][4] and others [5][20].

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