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Damron F.H.,Marshall University | Damron F.H.,PROGENESIS TECHNOLOGIES | Damron F.H.,University of Virginia | Owings J.P.,University of Virginia | And 7 more authors.
Journal of Bacteriology | Year: 2012

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ 22; AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ 54). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy. © 2012, American Society for Microbiology.


Damron F.H.,Marshall University | Damron F.H.,PROGENESIS TECHNOLOGIES | Damron F.H.,University of Virginia | Davis Jr. M.R.,University of Virginia | And 6 more authors.
Molecular Microbiology | Year: 2011

Alginate overproduction by P. aeruginosa strains, also known as mucoidy, is associated with chronic lung infections in cystic fibrosis (CF). It is not clear how alginate induction occurs in the wild-type (wt) mucA strains. When grown on Pseudomonas isolation agar (PIA), P. aeruginosa strains PAO1 and PA14 are non-mucoid, producing minimal amounts of alginate. Here we report the addition of ammonium metavanadate (AMV), a phosphatase inhibitor, to PIA (PIA-AMV) induced mucoidy in both these laboratory strains and early lung colonizing non-mucoid isolates with a wt mucA. This phenotypic switch was reversible depending on the availability of vanadate salts and triclosan, a component of PIA. Alginate induction in PAO1 on PIA-AMV was correlated with increased proteolytic degradation of MucA, and required envelope proteases AlgW or MucP, and a two-component phosphate regulator, PhoP. Other changes included the addition of palmitate to lipid A, a phenotype also observed in chronic CF isolates. Proteomic analysis revealed the upregulation of stress chaperones, which was confirmed by increased expression of the chaperone/protease MucD. Altogether, these findings suggest a model of alginate induction and the PIA-AMV medium may be suitable for examining early lung colonization phenotypes in CF before the selection of the mucA mutants. © 2011 Blackwell Publishing Ltd.


de Regt A.K.,Massachusetts Institute of Technology | Yin Y.,Marshall University | Yin Y.,Zhejiang Academy of Agricultural Sciences | Withers T.R.,Marshall University | And 5 more authors.
Molecular Microbiology | Year: 2014

In Pseudomonas aeruginosa, alginate overproduction, also known as mucoidy, is negatively regulated by the transmembrane protein MucA, which sequesters the alternative sigma factor AlgU. MucA is degraded via a proteolysis pathway that frees AlgU from sequestration, activating alginate biosynthesis. Initiation of this pathway normally requires two signals: peptide sequences in unassembled outer-membrane proteins (OMPs) activate the AlgW protease, and unassembled lipopolysaccharides bind periplasmic MucB, releasing MucA and facilitating its proteolysis by activated AlgW. To search for novel alginate regulators, we screened a transposon library in the non-mucoid reference strain PAO1, and identified a mutant that confers mucoidy through overexpression of a protein encoded by the chaperone-usher pathway gene cupB5. CupB5-dependent mucoidy occurs through the AlgU pathway and can be reversed by overexpression of MucA or MucB. In the presence of activating OMP peptides, peptides corresponding to a region of CupB5 needed for mucoidy further stimulated AlgW cleavage of MucAin vitro. Moreover, the CupB5 peptide allowed OMP-activated AlgW cleavage of MucA in the presence of the MucB inhibitor. These results support a novel mechanism for conversion to mucoidy in which the proteolytic activity of AlgW and its ability to compete with MucB for MucA is mediated by independent peptide signals. © 2014 John Wiley & Sons Ltd.


Withers T.R.,Marshall University | Yin Y.,Marshall University | Yu H.D.,Marshall University | Yu H.D.,PROGENESIS TECHNOLOGIES
Pathogens and Disease | Year: 2014

In this study, we performed whole-genome complementation using a PAO1-derived cosmid library, coupled with in vitro transposon mutagenesis, to identify gene locus PA1494 as a novel inhibitor of alginate overproduction in P. aeruginosa strains possessing a wild-type mucA. We report identification and characterization of a novel inhibitor of a mucoid biofilm produced by laboratory and clinical strains of Pseudomonas aeruginosa with a wild type MucA. © 2013 Federation of European Microbiological Societies.


Yin Y.,Marshall University | Yin Y.,Zhejiang Academy of Agricultural Sciences | Withers T.R.,Marshall University | Wang X.,Zhejiang Academy of Agricultural Sciences | And 2 more authors.
PLoS ONE | Year: 2013

Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU (AlgUA61V). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgUA61V, 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ70). Induction of AlgUA61V in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgUA61V is functional in activating alginate production. Furthermore, the level of AlgUA61V was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgUA61V had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ70 orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (PssrA) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ70 factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD. © 2013 Yin et al.


A specialized culture medium for the promotion of alginate production by stable mucoid Pseudomonas aeruginosa bacterial strains and methods for the production and purification of industrial, commercial, and pharmaceutical grade alginate from bacteriological sources are provided herein. Alginate produced using the media and methods disclosed herein is structurally uniform and substantially free of bacterial cell contaminants, including endotoxin.


A specialized culture medium for the promotion of alginate production by stable mucoid Pseudomonas aeruginosa bacterial strains and methods for the production and purification of industrial, commercial, and pharmaceutical grade alginate from bacteriological sources are provided herein. Alginate produced using the media and methods disclosed herein is structurally uniform and substantially free of bacterial cell contaminants, including endotoxin.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 144.00K | Year: 2015

DESCRIPTION provided by applicant Alginate is a commercially viable anionic biopolymer consisting of two monomeric subunits D mannuronate and a L guluronate Alginate is used in a variety of applications including textile manufacturing waste water remediation food production and a variety of medical applications such as wound care and drug delivery Currently commercial alginate is harvested from various genera of brown seaweeds However seaweed derived alginate has several limitations including seasonal growth inconsistencies in yield composition and quality between harvests supply side instability and the use of economically and environmentally costly processing methods These limitations impact seaweed derived alginateandapos s use for biomedical and pharmaceutical application Alginate is also produced by bacteria in the genera Azotobacter and Pseudomonas Moreover bacteria derived alginate has several advantages specifically it is relatively easy inexpensive and environmentally friendly to cultivate and harvest Additionally the bacteria can be easily manipulated to produce large amounts of alginate with specific customized properties for biomedical and pharmaceutical applications However there is little ongoing research to develop bacterial strains for commercial alginate production Progenesis Technologies LLC has patented a stable high alginate producing strain of Pseudomonas aeruginosa for use in recombinant alginate production At a pre petition consultation the United States Federal Drug Administration FDA suggested abrogating the function of the secreted virulence factors exotoxin A and pyocyanin to decrease the relative pathogenicity of VE The FDA also suggested using the currently approved bacterial strains for alginate production To date we have abrogated the function of the virulence factors exotoxin A pyocyanin phospholipase C and endotoxin In this proposal we intended to design and evaluate a non pathogenic bacterial strain for the production of the biopolymer alginate We propose to accomplish this through the completion of two Specific Aims In Aim we will evaluate the pathogenicity of our recently modified P aeruginosa strain PGN using a murine model In Aim we will determine the feasibility of producing alginate using currently approved E coli strains At the conclusion of ths project we will seek the appropriate federal approval to use our newly acquired non pathogenic bacterial strain as a platform for the production of biomedical and pharmaceutical grade alginate for use in various medical applications PUBLIC HEALTH RELEVANCE The primary goal of Progenesis Technologies LLC is to genetically engineer bacteria to produce the biopolymer alginate at a cost that is competitive with seaweed derived alginate Our technology will lead to the development of a product commonly used in a variety of biomedical and pharmaceutical applications which has enhanced performance due to our ability to alter the composition of the biopolymer and is manufactured in an environmentally friendly manner Our genetically engineered alginates can have a positive impact on public health by aiding in the care and recovery of patients that require specialized wound care topical medications and time released drug delivery


PubMed | University of Kentucky, Marshall University and PROGENESIS TECHNOLOGIES
Type: Journal Article | Journal: Bioorganic & medicinal chemistry letters | Year: 2016

Pseudomonas aeruginosa is a common biofilm-forming bacterial pathogen implicated in diseases of the lungs. The extracellular polymeric substances (EPS) of respiratory Pseudomonas biofilms are largely comprised of anionic molecules such as rhamnolipids and alginate that promote a mucoid phenotype. In this Letter, we examine the ability of negatively-charged fluoroquinolones to transverse the EPS and inhibit the growth of mucoid P. aeruginosa. Anionic fluoroquinolones were further compared with standard antibiotics via a novel microdiffusion assay to evaluate drug penetration through pseudomonal alginate and respiratory mucus from a patient with cystic fibrosis.

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