they are primarily produced by the spore - forming bacterium clostridium botulinum and , in rare cases , by some strains of clostridium butyricum and clostridium baratii [ 2 , 3 ] . intoxication with one of the seven distinct serotypes of bont ( a  g ) causes botulism . one of 4 serotypes of bont ( a , b , e , and f ) is usually the cause of human botulism . exposure to type a neurotoxin ( bont / a ) causes the majority of food - borne outbreaks and has been observed to cause more severe symptoms with a higher mortality . bonts are zinc proteases that cleave and inactivate cellular proteins essential for the release of the neurotransmitter acetylcholine . bont / a , -c , and -e cleave the peripheral plasma membrane protein soluble n - ethylmaleimide - sensitive factor attachment protein of 25  kda ( snap-25 ) ; bont / b , -d , -f , and -g cleave synaptobrevin 2 , also called as vesicle - associated membrane protein-2 ( vamp-2 ) . in addition to cleaving snap-25 , bont - c also cleaves the integral plasma membrane protein , syntaxin . the soluble n - ethylmaleimide sensitive factor attachment protein receptor ( snare ) superfamily has become , since its discovery , the most intensively studied element of the protein machinery involved in intracellular trafficking . currently , the only accepted assay with which to detect active clostridium botulinum neurotoxin is an in vivo mouse bioassay . however , the mouse bioassay is slow and not practical in many settings , and it results in the death of animals . several in vitro assays have been developed to detect the activities of the different bont serotypes [ 613 ] . this approach has led to forester resonance energy transfer ( fret ) methods that are based on the natural substrates tagged with fluorescence dyes and their cleaved fluorescence products by bonts , which are then used to detect toxin activity [ 1416 ] . dong and coworkers described two recombinant reporters containing residues 141206 and 3394 of neuronal snare proteins snap-25 and synaptobrevin 2 , respectively . the substrates were expressed as fusions of cyan fluorescent protein ( cfp ) and yellow fluorescent protein ( yfp ) , enabling the detection of bont proteolysis activity by fret . advances in the development and optimization of fret - based assays that detect six of seven bont serotypes have also been reported [ 17 , 18 ] . the fret reporters were found to be sensitive to bont serotypes and able to detect picomolar concentrations of the toxins in real - time assay . the main objective of our study was to develop a cell - based assay for the detection of bont / a and bont / e , which could be automated and applied to many laboratory settings . here , we report a cellular sensor utilizing advanced fret - based substrates expressed as fusions of green fluorescence protein ( gfp ) and discosoma sp . the resulting reporters are stably expressed in living cells , and the neurotoxin cleavage activity is detected either by measuring the loss of fret or by destruction of the c - terminal fragment . these fret - based reporters are currently being evaluated for the analysis of different food matrices and also exploring new immuno - magnetic bead separation methods to counter the food matrix interference and to increase the sensitivity of the detection . even though the assay is thousandfold less sensitive than the mouse bioassay further refinement of such assay will present an alternative to the mouse assay , and the ease of use could generate the confidence in field or reference laboratories capable of performing bont detection , leading to reduction in animal use . bont / a ( hall a strain ) and bont / e ( alaska strain ) were obtained from metabiologics ( madison , wi ) . pacgfp1-c1 and pdsred - monomer - c1 were purchased from clontech laboratories ( mountain view , ca ) . full - length snap-25 cdna and pc12 transplantable rat pheochromocytoma cells were purchased from atcc ( manassas , va ) . antibodies targeting snap-25 were purchased from santa cruz biotechnology ( santa cruz , ca ) . the hrp - conjugated secondary antibody was purchased from millipore ( billerica , ma ) . louis , mo ) . to build a neurotoxin sensor that can detect cleavage of snap-25 in living cells and to report toxin activity , we linked acgfpc1 and dsred together by using full - length snap-25 that can be cleaved by the appropriate bonts ( figure 1(d ) ) . the construct was engineered to encode full - length snap-25 sequence as a linker between acgfp1 and dsred - monomer using standard cloning techniques . pacgfp - c1 was inserted in to pdsred - monomer - c1 by using xhoi / ecori restriction enzymes , resulting in acgfp1-dsred - monomer construct . full - length rat snap-25 cdna was inserted in to the acgfp1-dsred - monomer vector resulting in an acgfp - snap-25-dsred construct . pc12 cells were cultured according to standard cell culture methods . a day before transfection , cells ( ~1  10/ml ) were seeded onto collagen - coated tissue culture dish and transfected with fret sensor ( acgfp - snap-25-dsred ) , donor only ( acgfpc1 ) , and acceptor only ( dsred - monomer ) cdna constructs using a lipid - based transfection reagent according to manufacturer 's instructions ( lipofectin / life technologies ) . after incubation for six hours , the transfection medium was exchanged to dulbecco 's modified eagle medium ( dmem ) plus 10% fetal bovine serum ( fbs ) to grow cells . transfected cells were plated ( ~2.0  10 cells per well ) onto 4-well labtek ii chamber slides and maintained at 37c , 5% co2 in a tissue culture incubator , 2448 hours prior to imaging . thirty minutes prior to imaging , the culture medium was replaced with dmem medium without fbs . images were acquired using a nikon eclipse ti - e confocal microscope ( nikon inc . , usa ) with a 1.4 numerical aperture , 100 oil - immersion objective . images ( 12 bit ) of multitrack channels were with the following configuration : an argon/2 laser ( 25  mw , t1 and t3 = 10% of laser exposure ) for the green channel ( donor excitation / donor emission : green ex / em ) , fret channel ( donor excitation / acceptor emission : fret ex / em ) with excitation at 488  nm , and hene 1 laser ( t2 = 100% ) for the red channel ( acceptor excitation / acceptor emission : red ex / em ) with excitation at 543  nm . all images were acquired with exactly the same settings ( 4  4 binning , 200  msec exposure time ) . the background ( from areas that did not contain cells ) was subtracted from each raw image . pc12 cells transfected with gfp or dsred alone were first tested to obtain the cross - talk / bleed - through values for these filter sets . image capture and calculations were performed by using nis - elements software ( microsoft corporation ) . for experiments involving toxin treatment , different concentrations of holotoxin ( bont / a , trypsin - treated bont / e ) toxin complexes were added to the cell culture media , and cells were then analyzed as described earlier . also , 24  h after pc12 cells were transfected with reporter constructs , growth medium was exchanged for 0.05  mg / ml g418 selection , and incubation continued until single clones appeared . cells were transferred progressively from 24 well plates to 6 well plates as they reached confluence ( 1 - 2 weeks ) . when an assay was required , cells were collected and plated onto microtiter plates ( nunc , rochester , ny ) and allowed to expand for 24  h. the cells were then incubated with samples containing bont / a in the assay buffer ( 100  l of 50  nm hepes - naoh ( ph 7.1 ) , 5  mm nacl , 0.1% tween-20 , 5  mm dithiothreitol ( dtt ) , and 10  nm zncl2 ) and diluted and reconstituted with the culture medium for the duration of 2472 hours to reach desired sensitivity . cells were then washed with phosphate - buffered saline ( pbs ) to reduce background before collecting fret emissions . the data was captured by measuring the total dsred emission , converted into a ratio metric value and plotted as a function of bont concentration . alternatively , fret measurements were also taken on a synergy 2 multimode plate reader ( biotek instruments , winooski , vt ) . for all line graphs , data shown are averages from triplicate determinations with bars indicating standard deviations . pc12 cells grown on a culture slide were fixed in 4% paraformaldehyde and permeabilized with 1% triton x-100 . anti - snap-25 antibody diluted in blocking buffer ( 1  :  250 dilution ) was added to the slides and incubated for 1  h at 4c and washed . fitc labeled , goat anti - rabbit igg antibody was used in blocking buffer and added to the plates . after slides were incubated at 37c for 60  min , they were washed three times , and images were acquired using a nikon confocal microscope with fitc filter . to visualize the cleavage products of neurotoxin activity on an immune blot , pc12 cells grown on culture dishes were treated with bont / a ( 10  nm ) at 37c for 48  h. cells were then harvested and lysed with ripa ( radio - immunoprecipitation assay ) buffer . anti - snap-25 antibody ( c-18 , santa cruz biotechnology ) directed against the c - terminus of the protein was used to recognize the botulinum neurotoxins a and e cleavage products . peroxidase - labeled antibodies diluted in blocking buffers were used to detect cleavage products and then visualized using a gel imager ( syngene , usa ) . we identified transplantable rat pheochromocytoma neuronal cell line ( pc12 cells ) ( figure 1(a ) ) derived from rat brain medulla for developing stable cell lines expressing fret sensor . pc12 cells are sensitive to bont / a with sensitivities equivalent to primary cord neurons , as observed by the significant cleavage of snap-25 at bont / a concentration of 1  nm ( figure 1(b ) ) . previous reports have confirmed that the bont / a light chain contains a membrane localization signal and is targeted to the plasma membrane in differentiated pc12 cells [ 19 , 20 ] . the immunofluorescence staining of methanol - fixed pc12 cells shows the localization of membrane - bound snap-25 ( figure 1(c ) ) . if the gfp and dsred are close enough to each other , excitation of the gfp moiety will result in the sensitized emission from the dsred moiety as a consequence of fret . cleavage of the linker sequence between acgfp and dsred separates them and abolishes fret ( figure 1(d ) ) . in fret , the energy is transferred nonradioactively from donor protein to the acceptor protein when they are in very close proximity ( about 50   ) and when the emission spectrum of the donor protein overlaps with the excitation spectrum of the acceptor protein . we generated stable cell lines that stably express the reporter protein over many passages , and the data from the p15 for nine clones are shown in figure 2(a ) . out of nine subclones ( 3a1 , 3a4 , 3a9 , 3a14 , 3a17 , 3a21 , 5a3 , 5a5 , and 5a9 ) tested for stability , only two subclones , 3a14 and 5a3 , constitutively expressed acgfpc1-snap-25-dsred reporter and responsive to bont / a at 1 and 10  nm ( figure 2(b ) ) . to carry out cell - based studies , clones 3a14 and 5a3 were treated with 110  nm bont / a for 7296 hours . the fret signals in living cells were acquired by using three - filter set method as shown in figure 3(a ) , and details are provided in the materials and methods . a progressive decrease in the fret ratio was observed over time . this sensor yielded significant fret ( upper  corrected fret  ) , which was abolished after cells were treated with bont / a for 72  h ( lower  corrected fret  ) . we also tested the cleavage activity of bont / e on these clones ( 3a14 and 5a3 ) , which also resulted in the abolishment of fret activity as indicated by the reduction in the red florescence in the cell population as shown in figure 4(a ) . bont / e response of the clone 3a14 is shown in figure 4(a ) , treated at 1 and 10  nm of bont / e concentration for 72  h , and it was captured using fluorescence microscopy . pseudocolored images indicate the distribution of the gfp / dsred fluorescence ratio within the cells . when 5a3 clones at passage 15 were incubated with bont / a , the toxin is internalized resulting in the release of the bont / a light chain into the cytosol . the bont / a light chain then cleaves the reporter resulting in the release of a c - terminal reporter fragment into the cytosol that contained residues 198206 of snap-25 and dsred . the data in these studies were captured by measuring the total dsred emission of the cells using directly excited gfp ( figure 3(b ) ) . this will be an alternative toxin cleavage measurement in addition to fret fluorescence measurement explained earlier in the study . thus , dsred emissions are divided by gfp emissions giving a ratio metric assay readout . emissions were plotted as a function of bont / e concentration as shown in figure 4(b ) . in addition to holotoxin , we also checked the multiprotein complex form of the toxins bont / a and bont / e responses for the sensor assay . pc12 cells were grown in plates treated with 0.0330  nm of bont / a and bont / e complex for different intervals ( up to 72 hours ) to show the toxin activity . there was only a moderate reduction in the fret activity observed with a bont / a complex as compared to holotoxin treatments . at a higher concentration ( 30  nm ) of bont / a treatment , there was a gradual reduction in the dsred fluorescence indicating the action of toxin . this demonstrated that the bont / a and bont / e in their complex form are functionally less active in this detection method compared to holotoxin form of the toxin . the gfp and dsred emissions were collected by fluorescence microplate reader ( biotek instrument , winooski , vt ) . emissions were plotted as a function of bont / a holotoxin and complex as shown in figure 5(a ) and bont / e holotoxin and complex concentration as shown in figure 5(b ) . the cell - based assay developed here has the potential to be a rapid screening method to confirm the presence of functional botulinum toxin types a and e. the fret - based reporter approach could be adapted to screen other bont serotypes with unique cleavage sites by using synthetic substrates labeled with different dyes . cell - based reporters make it possible to gain further insights into toxin substrate recognition and cleavage in cells and to understand the bont cell biology . here , we tested pc12 cell lines , which are large adherent cells , resist early apoptosis , and can be maintained easily with commercially available tissue culture medium and antibiotics . these stable clones could be produced in large quantities and are reproducible . in a single experiment , sufficient stable cells can easily be generated to populate twenty 96-well dishes . alternatively , cells can also be cryopreserved with 50% viability upon thawing to provide lot - to - lot consistency . even though the assay is of low sensitivity we are currently exploring methods to increase the sensitivity of the assay and the available immuno - magnetic separation methods to counter the food matrix interference .
the standard procedure for definitive detection of bont - producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay ( mba ) . 
 the mouse bioassay is highly sensitive and specific , but it is expensive and time - consuming , and there are ethical concerns due to use of laboratory animals . 
 cell - based assays provide an alternative to the mba in screening for bont - producing clostridia . here 
 , we describe a cell - based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ . 
 our data indicates that the assay can detect as little as 100  pm bont / a activity within living cells , and the assay is currently being evaluated for the analysis of bont in food matrices . among available in vitro assays , we believe that cell - based assays are widely applicable in high - throughput screenings and have the potential to at least reduce and refine animal assays if not replace it .