Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching

This work sets out to provide a self-assembled biopolymer capsule activated with a multi-functional enzyme for localized delivery. This enzyme, SsoPox, which is a lactonase and phosphotriesterase, provides a means of interrupting bacterial communication pathways that have been shown to mediate pathogenicity. Here we demonstrate the capability to express, purify and attach SsoPox to the natural biopolymer chitosan, preserving its activity to “neutralize” long-chain autoinducer-1 (AI-1) communication molecules. Attachment is shown via non-specific binding and by engineering tyrosine and glutamine affinity ‘tags’ at the C-terminus for covalent linkage. Subsequent degradation of AI-1, in this case N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), serves to “quench” bacterial quorum sensing (QS), silencing intraspecies communication. By attaching enzymes to pH-responsive chitosan that, in turn, can be assembled into various forms, we demonstrate device-based flexibility for enzyme delivery. Specifically, we have assembled quorum-quenching capsules consisting of an alginate inner core and an enzyme “decorated” chitosan shell that are shown to preclude bacterial QS crosstalk, minimizing QS mediated behaviors.

[1]  Jessica L. Terrell,et al.  Incorporating LsrK AI‐2 quorum quenching capability in a functionalized biopolymer capsule , 2018, Biotechnology and bioengineering.

[2]  M. Elias,et al.  Biotechnological applications of quorum quenching enzymes. , 2017, Chemico-biological interactions.

[3]  Jessica L. Terrell,et al.  Constructing “quantized quorums” to guide emergent phenotypes through quorum quenching capsules , 2017, Biotechnology and bioengineering.

[4]  W. Bentley,et al.  A Facile Two-Step Enzymatic Approach for Conjugating Proteins to Polysaccharide Chitosan at an Electrode Interface , 2016, Cellular and Molecular Bioengineering.

[5]  W. Bentley,et al.  Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase. , 2016, Metabolic engineering.

[6]  W. Bentley,et al.  Autoinducer-2 analogs and electric fields - an antibiotic-free bacterial biofilm combination treatment , 2016, Biomedical microdevices.

[7]  W. Bentley,et al.  Data on biochemical fluxes generated from biofabricated enzyme complexes assembled through engineered tags and microbial transglutaminase , 2016, Data in brief.

[8]  Giuseppe Manco,et al.  An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination , 2016, Biotechnology and bioengineering.

[9]  Jessica L. Terrell,et al.  Directed assembly of a bacterial quorum , 2015, The ISME Journal.

[10]  Tom Coenye,et al.  Quorum sensing inhibitors as anti-biofilm agents. , 2014, Current pharmaceutical design.

[11]  M. Elias,et al.  Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox , 2013, PloS one.

[12]  Jun Huang,et al.  Activated carbons and amine-modified materials for carbon dioxide capture — a review , 2013, Frontiers of Environmental Science & Engineering.

[13]  Jessica L. Terrell,et al.  Encapsulated fusion protein confers “sense and respond” activity to chitosan–alginate capsules to manipulate bacterial quorum sensing , 2013, Biotechnology and bioengineering.

[14]  M. Elias,et al.  Characterisation of the organophosphate hydrolase catalytic activity of SsoPox , 2012, Scientific Reports.

[15]  Reza Ghodssi,et al.  AI-2 analogs and antibiotics: a synergistic approach to reduce bacterial biofilms , 2012, Applied Microbiology and Biotechnology.

[16]  F. Raushel,et al.  Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents. , 2012, Biochemistry.

[17]  Colin J Jackson,et al.  Reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active-site loop remodeling. , 2012, Biochemistry.

[18]  B. Sarmento,et al.  Chitosan-based systems for biopharmaceuticals : delivery, targeting, and polymer therapeutics , 2012 .

[19]  Dan S. Tawfik,et al.  Divergence and Convergence in Enzyme Evolution: Parallel Evolution of Paraoxonases from Quorum-quenching Lactonases* , 2011, The Journal of Biological Chemistry.

[20]  Filomena Ng,et al.  Characterization of a Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus and Its Immobilization for Disruption of Quorum Sensing , 2010, Applied and Environmental Microbiology.

[21]  J. Handelsman,et al.  Identification of Synthetic Inducers and Inhibitors of the Quorum-Sensing Regulator LasR in Pseudomonas aeruginosa by High-Throughput Screening , 2010, Applied and Environmental Microbiology.

[22]  W. Bentley,et al.  Synthetic analogs tailor native AI-2 signaling across bacterial species. , 2010, Journal of the American Chemical Society.

[23]  S. Takka,et al.  Evaluation of Chitosan/Alginate Beads Using Experimental Design: Formulation and In Vitro Characterization , 2010, AAPS PharmSciTech.

[24]  William E Bentley,et al.  Cross species quorum quenching using a native AI-2 processing enzyme. , 2010, ACS chemical biology.

[25]  W. Bentley,et al.  Biofabrication of antibodies and antigens via IgG‐binding domain engineered with activatable pentatyrosine pro‐tag , 2009, Biotechnology and bioengineering.

[26]  L. Achenie,et al.  Quorum quenching analysis in Pseudomonas aeruginosa and Escherichia coli: network topology and inhibition mechanism effect on the optimized inhibitor dose , 2009, Bioprocess and biosystems engineering.

[27]  M. Cámara,et al.  Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. , 2009, Current opinion in microbiology.

[28]  G. Manco,et al.  Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus , 2009, Extremophiles.

[29]  P. Oger,et al.  Quorum Sensing and Quorum Quenching: The Yin and Yang of Bacterial Communication , 2009, Chembiochem : a European journal of chemical biology.

[30]  Giuseppe Manco,et al.  Structural basis for natural lactonase and promiscuous phosphotriesterase activities. , 2008, Journal of molecular biology.

[31]  Hyunmin Yi,et al.  Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro‐tag , 2008, Biotechnology and bioengineering.

[32]  L. Costa.  Current issues in organophosphate toxicology. , 2006, Clinica chimica acta; international journal of clinical chemistry.

[33]  B. Ahmer,et al.  Effect of sdiA on Biosensors of N-Acylhomoserine Lactones , 2005, Journal of bacteriology.

[34]  P. Sokol,et al.  Identification of N-acylhomoserine lactones in mucopurulent respiratory secretions from cystic fibrosis patients. , 2005, FEMS microbiology letters.

[35]  Lian-Hui Zhang,et al.  Quorum sensing and quorum-quenching enzymes. , 2005, Journal of microbiology.

[36]  F. Raushel,et al.  Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase. , 2004, Biochemistry.

[37]  M. Hentzer,et al.  Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. , 2003, The Journal of clinical investigation.

[38]  F. Raushel.  Bacterial detoxification of organophosphate nerve agents. , 2002, Current opinion in microbiology.

[39]  Lian-Hui Zhang,et al.  Identification of Quorum-Quenching N-Acyl Homoserine Lactonases from Bacillus Species , 2002, Applied and Environmental Microbiology.

[40]  M. A. Holgado,et al.  Alginate/chitosan particulate systems for sodium diclofenac release. , 2002, International journal of pharmaceutics.

[41]  Lian-Hui Zhang,et al.  Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase , 2001, Nature.

[42]  D. Wheeler,et al.  The Pseudomonas aeruginosaQuorum-Sensing Signal MoleculeN-(3-Oxododecanoyl)-l-Homoserine Lactone Has Immunomodulatory Activity , 1998, Infection and Immunity.

[43]  M. Aschner,et al.  Comparative study on short- and long-term behavioral consequences of organophosphate exposure: relationship to AChE mRNA expression. , 2014, Neurotoxicology.

[44]  S. Rajamani,et al.  Luminescent reporters and their applications for the characterization of signals and signal-mimics that alter LasR-mediated quorum sensing. , 2011, Methods in molecular biology.

[45]  M. Schuster.  Inaugural Article: Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR , 2004 .

[46]  Angela T. Lewandowski,et al.  ABSTRACT Title of thesis: TYROSINE-BASED “ACTIVATABLE PRO-TAG”: ENZYME- CATALYZED PROTEIN CAPTURE AND RELEASE , 2004 .

[47]  Leo Eberl,et al.  Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. , 2002, Microbiology.