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MEDFORD, MA, United States

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.

Kim J.S.,University of Otago | Brownjohn P.W.,University of Otago | Dyer B.S.,Glycosyn, Inc. | Dyer B.S.,Callaghan Innovation | And 6 more authors.
Endocrinology | Year: 2015

RFamide-related peptide-3 (RFRP-3) is a recently discovered neuropeptide that has been proposed to play a role in the stress response. We aimed to elucidate the role of RFRP-3 and its receptor, neuropeptide FF (NPFF1R), in modulation of stress and anxiety responses. To achieve this, we characterized a new NPFF1R antagonist because our results showed that the only commercially available putative antagonist, RF9, is in fact an agonist at both NPFF1R and the kisspeptin receptor (KISS1R). We report here the identification and pharmacological characterization of GJ14, a true NPFFR antagonist. In in vivo tests of hypothalamic-pituitary-Adrenal (HPA) axis function, GJ14 completely blocked RFRP-3-induced corticosterone release and neuronal activation in CRH neurons. Furthermore, chronic infusion of GJ14 led to anxiolytic-like behavior, whereas RFRP-3 infusion had anxiogenic effects. Mice receiving chronic RFRP-3 infusion also had higher basal circulating corticosterone levels. These results indicate a stimulatory action of RFRP-3 on the HPA axis, consistent with the dense expression of NPFF1R in the vicinity of CRH neurons. Importantly, coinfusion of RFRP-3 and GJ14 completely reversed the anxiogenic andHPAaxis-stimulatory effects of RFRP-3. Here we have established the role of RFRP-3 as a regulator of stress and anxiety.Wealso show that GJ14 can reverse the effects of RFRP-3 both in vitro and in vivo. Infusion of GJ14 causes anxiolysis, revealing a novel potential target for treating anxiety disorders. © 2015 by the Endocrine Society.

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

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 158.87K | Year: 2008

DESCRIPTION (provided by applicant): Adherence of pathogens to their host cells is the obligatory first step in infection and is frequently mediated by specific molecular interactions [1, 2]. Virulent Campylobacter species 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 which is the cause annual ly of over 2 million deaths worldwide [8]. However the production of a(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 [9-11]. In vitro enzymatic syntheses are also possible but are limited by a requirement for expensive nucleotide-sugar precursors. Glycosyn Inc.'s broad goal is to develop way s to manufacture a(1,2) fucosylated glycans cheaply and in bulk through microbial fermentation, and three classes of potential anti-infective products are envisaged: 1) purified a(1,2) fucosylated oligosaccharides, 2) yeast strains expressing a(1,2) fucosy lated glycans on their cell surface, and 3) purified a(1,2) fucosylated glycoproteins. The goal of the studies outlined in this application are to synthesize, purify, and test as a nutritional supplement, an example of the first of these product classes, n amely a purified a(1,2) fucosylated oligosaccharide, 2-fucosyllactose (2FL). Glycan synthetic pathways in the common dairy yeast Kluyveromyces lactis will be engineered through a combination of endogenous gene manipulation and the introduction of heterolog ous genes encoding desired activities. Specifically, K.lactis will be engineered to synthesize the key precursor sugar, GDP-fucose, and subsequently to make 2-fucosyllactose in the cell cytoplasm. Yields of product will be optimized, and extraction and pur ification schemes will be developed. Purified 2-fucosyllactose produced in K.lactis will be tested for efficacy both in vitro and in vivo in models of infection. PUBLIC HEALTH RELEVANCE: Worldwide, infectious diarrhea [12] is responsible for approxi mately 20% of all mortality in children under the age of 5, and for an estimated 2.5 millions deaths annually [8]. In the United States infectious diarrhea has an annual incidence of over 200 million cases and is responsible for approximately 900,000 hospi talizations and 5,000 deaths per year [3, 13]. Infection by Norwalk-like viruses is by far the single largest cause of infectious diarrhea in the US, with the single largest bacterial cause being infection by Campylobacter species. The surface a(1,2) fucos ylated probiotic K.lactis that is the subject of this application will target both Norwalk-like viruses and C.jejuni, as well as other fucose-binding enteropathogens.

The invention provides compositions and methods for engineering

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