Engineered biological nanofactories trigger quorum sensing response in targeted bacteria

Fernandes, Rohan; Roy, Varnika; Wu, Hsuan‐Chen; Bentley, William E.

doi:10.1038/nnano.2009.457

Cited by 85 publications

(86 citation statements)

References 25 publications

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“…Certainly, among the beneficial phenotypes attributed to QS, which could also be referred to as the 'currency' of the public good, are bioluminescence (Bassler et al, 1994), virulence (Zhu et al, 2002), biofilm formation (Balestrino et al, 2005;Gonzalez Barrios et al, 2006;Li et al, 2007), cell division, motility and cooperativity (Dandekar et al, 2012). We note that intentional rewiring these QS-regulated systems also benefits biotechnological applications, if not the microbes themselves (Fernandes et al, 2010;Tsao et al, 2010;Wu et al, 2013;Swofford et al, 2015;Thompson et al, 2015). It is therefore intriguing to consider what is the particular quorum and why was it reached.…”

Section: Introductionmentioning

confidence: 90%

Directed assembly of a bacterial quorum

et al. 2015

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Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can 'call' groups neighboring cells that 'travel' and establish a QS-mediated phenotype upon reaching the new locale.

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Section: Introductionmentioning

confidence: 90%

Directed assembly of a bacterial quorum

et al. 2015

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“…Clearly, achieving such a goal of building nanofactories or nanomachines depends on our ability to link various functional units on demand and with high precision (1,3). It is surprising that current efforts in this promising field still rely on linking technologies that were invented several decades ago: biotin-streptavidin pairing, antibody-epitope recognition, and chemical linking through amino-or sulfhydryl groups (4). These approaches are often limiting because of the need to chemically modify recombinant proteins or the complexity of antibody-based techniques.…”

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confidence: 99%

SNARE tagging allows stepwise assembly of a multimodular medicinal toxin

Darios

Niranjan

Ferrari

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Generation of supramolecular architectures through controlled linking of suitable building blocks can offer new perspectives to medicine and applied technologies. Current linking strategies often rely on chemical methods that have limitations and cannot take full advantage of the recombinant technologies. Here we used SNARE proteins, namely, syntaxin, SNAP25, and synaptobrevin, which form stable tetrahelical complexes that drive fusion of intracellular membranes, as versatile tags for irreversible linking of recombinant and synthetic functional units. We show that SNARE tagging allows stepwise production of a functional modular medicinal toxin, namely, botulinum neurotoxin type A, commonly known as BOTOX. This toxin consists of three structurally independent units: Receptor-binding domain (Rbd), Translocation domain (Td), and the Light chain (Lc), the last being a proteolytic enzyme. Fusing the receptor-binding domain with synaptobrevin SNARE motif allowed delivery of the active part of botulinum neurotoxin (Lc-Td), tagged with SNAP25, into neurons. Our data show that SNARE-tagged toxin was able to cleave its intraneuronal molecular target and to inhibit release of neurotransmitters. The reassembled toxin provides a safer alternative to existing botulinum neurotoxin and may offer wider use of this popular research and medical tool. Finally, SNARE tagging allowed the Rbd portion of the toxin to be used to deliver quantum dots and other fluorescent markers into neurons, showing versatility of this unique tagging and self-assembly technique. Together, these results demonstrate that the SNARE tetrahelical coiled-coil allows controlled linking of various building blocks into multifunctional assemblies.botulinum neurotoxin | protein linking | recombinant | self-assembly | coiled coil

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“…With properly designed scaffolded multienzyme systems, one may accelerate conversion by taking advantage of the enhanced local intermediate concentrations resulting from the spatial control over enzyme organization (28). Finally, while this study demonstrated a new scaffoldin concept in yeast, such a technique should be equally applicable for improving protein display on other hosts as novel whole-cell biocatalysts, or even on nonbiological nanoparticles, to assemble nanofactories that comprise multiple functional modules (7).…”

Section: Discussionmentioning

confidence: 87%

Self-Assembled Amyloid-Like Oligomeric-Cohesin Scaffoldin for Augmented Protein Display on the Saccharomyces cerevisiae Cell Surface

Han

Zhang

Wang

et al. 2012

Appl Environ Microbiol

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In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of Saccharomyces cerevisiae is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p 1-80 ). The resulting Ure2p 1-80 -HD-cohesin fusion protein was successfully expressed in Escherichia coli to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of S. cerevisiae engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the Clostridium cellulolyticum CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFPcohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display. Yeast surface display provides a promising platform for protein engineering (9). The ability to anchor and display functional proteins on the yeast cell surface is also being exploited for developing novel whole-cell biocatalysts (11). Although whole-cell biocatalysis using yeast surface display is generally considered an established technology, challenges remain regarding the efficient display of protein complexes and the further increase in the amount of protein that can be displayed on the yeast surface without destabilizing the cell. To this end, unique properties of protein building blocks found in natural higher-order protein complexes may be exploited to create novel synthetic protein chimeras that self-assemble into nano-scaffolds on the yeast surface to display increased levels and varieties of proteins. One such protein building block is the cohesin (Coh) domain, which is found with multiple copies in cellulosomes.The cellulosome is an extracellular multienzyme complex found on the cell surface of anaerobic bacteria such as Clostridium cellulolyticum, facilitating highly efficient hydrolysis of amorphous and crystalline cellulose. It is composed of numerous cellulolytic enzymes and other functional units assembled, via highaffinity interactions between the cohesin and dockerin domains (22), on a protein scaffold (scaffoldin) that contains multiple copies of cohesin. Cellulases and r...

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Engineered biological nanofactories trigger quorum sensing response in targeted bacteria

Cited by 85 publications

References 25 publications

Directed assembly of a bacterial quorum

Directed assembly of a bacterial quorum

SNARE tagging allows stepwise assembly of a multimodular medicinal toxin

Self-Assembled Amyloid-Like Oligomeric-Cohesin Scaffoldin for Augmented Protein Display on the Saccharomyces cerevisiae Cell Surface

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