Circumvention of Learning Increases Intoxication Efficacy of

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Circumvention of increases intoxication efficacy of nematicidal engineered bacteria. Olena R Bracho, Cyril Manchery, Evan C Haskell, Christopher A Blanar, and Robert P Smith ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00192 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 29, 2015

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ACS Synthetic Biology

Circumvention of increases intoxication efficacy of nematicidal engineered bacteria. Olena R Bracho1*, Cyril Manchery1*, Evan C Haskell2, Christopher A Blanar1 and Robert P Smith1

1

Department of Biological Sciences, Halmos College of Natural Sciences and Oceanography,

Nova Southeastern University, Fort Lauderdale FL, 33314 2

Department of Mathematics, Halmos College of Natural Sciences and Oceanography, Nova

Southeastern University, Fort Lauderdale FL, 33314

Correspondence should be addressed to Robert P Smith. E-mail: [email protected] Tel: 954 262 7979

*

denotes co-first authorship

KEYWORDS: synthetic biology, nematodes, quorum sensing, biocontrol, Bt toxins

ACS Paragon Plus Environment

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ABSTRACT Synthetic biology holds promise to engineer systems to treat diseases. One critical, yet underexplored, facet of designing such systems is the interplay between the system and the pathogen. Understanding this interplay may be critical to increasing efficacy and overcoming resistance against the system. Using the principles of synthetic biology, we engineer a strain of Escherichia coli to attract and intoxicate the nematode Caenorhabditis elegans. Our bacteria are engineered with a toxin module, which intoxicates the nematode upon ingestion, and an attraction module, which serves to attract and increase the feeding rate of the nematodes. When independently implemented, these modules successfully intoxicate and attract the worms, respectively. However, in combination, the efficacy of our bacteria is significantly reduced due to aversive associative learning in C. elegans. Guided by mathematical modeling, we dynamically regulate module induction to increase intoxication by circumventing learning. Our results detail the creation of a novel nematicidal bacterium that may have application against nematodes, unravel unique constraints on circuit dynamics that are governed by C. elegans physiology, and add to the growing list of design and implementation considerations associated with synthetic biology.


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INTRODUCTION Synthetic biology holds promise for designing and implementing systems to solve medical problems including infectious diseases, cancer and drug delivery

1, 2

. To date, most

studies have focused primarily on circuit design (e.g., 3), delivery systems (e.g., engineering synthetic individuals to infiltrate a population of like species (e.g.,

5, 6

3, 4

) or

). One critical

challenge when designing systems to treat diseases is the interplay between the engineered system and the pathogen. This interaction is unlikely to be static; in some cases the pathogen will alter its behavior in response to treatment (e.g., 7), which may lower efficacy of treatment or lead to resistance against the engineered system in the long term. An understanding of the dynamic interplay between synthetic systems and their target pathogen is critical to advancing the field of synthetic biology. Parasitic worms are the most common infectious agent in the world

8-10

. It is estimated

that nearly 3.5 billion cases of parasitic worm infection occur annually, 125,000 of which result in death 11. The impact of these infections on the global economy is staggering, given the direct costs to disease sufferers and the indirect costs to society 12, 13. Despite the prevalence of parasitic worms and infections, the methods used to prevent or treat such infections are limited; only four new anthelmintics were developed from 1975 to 2004

8, 9

. Although these drugs have proven to

be effective in the past, there is growing concern that parasitic worms are becoming resistant to anthelmintics

14-16

. Furthermore, pharmaceutical companies have been hesitant to develop

anthelmintics 17 as the cost of such drugs is often too high for families in developing countries 11, 18, 19

. While sanitation programs are becoming increasingly common, they can be difficult to

implement, fund, and maintain

20, 21

. Consequently, the global incidence of infections due to

parasitic worms has remained unchanged over the last 50 years


22

. While systems expressing

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nematicidal proteins in bacteria have been used in vitro

23-25

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and in vivo

26

to kill nematodes,

these systems often lack regulatory ability that may allow interplay between worm behavior and toxin delivery, both of which may be critical in augmenting efficacy and circumventing resistance to the toxin protein 27. There is a dire need to develop novel approaches to preventing infection by parasitic worms. In the following manuscript, we use synthetic biology to engineer a laboratory strain of Escherichia coli to both attract and kill the model nematode, Caenorhabditis elegans. Our engineered bacteria consist of two independently regulated modules. Guided by mathematical modeling, we dynamically regulate the circuit components to increase intoxication efficacy by circumventing learning in C. elegans. Our results highlight a unique design constraint when engineering and implementing synthetic systems that interact with pathogens.

RESULTS AND DISCUSSION Characterization of the toxin and attraction modules We sought to engineer a bacterium that could both attract and intoxicate nematodes using two modules: an attraction module and a toxin module (Figure 1a). As many nematodes are bacteriovorous (Table S1), we implemented our system in E. coli such that our engineered bacteria would deliver the toxin upon ingestion by the nematode. The attraction module consists of an inducible promoter driving the expression of an acylhomoserine lactone (AHL) producing gene, which serves to attract and increase feeding rate of the nematode. The toxin module consists of an inducible promoter driving the expression of a toxin gene, which codes for a toxin protein. We designed both modules of the circuit to respond to different external chemicals, thus allowing independent activation of either AHL or toxin protein expression. We chose to test our


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bacteria using C. elegans as this nematode serves a model system to test novel anthelmintics

28

and as a model of infectious disease 29. To build our toxin module, we engineered E. coli DH5αPRO to express the Cry5B toxin isolated from Bacillus thuringiensis 23. The cry5B gene is under the regulation of a T5 promoter and the lac operator (herein referred to as Plac promoter), which can be induced in a graded manner using IPTG (Figure 1a, Figure S1). We cultured C. elegans N2 (mixed stage culture) on lawns of E. coli that did or did not (empty vector control) contain the cry5B gene. Furthermore, we grew both strains of E. coli in the presence

30

or absence of IPTG. At 24-hour intervals, we

examined the worms for previously established indicators of significant intoxication

23

, which

primarily included arrested movement (Figure 1b and c, Supporting Results). When grown in the presence of both IPTG and cry5B-expressing bacteria, C. elegans showed pronounced signs of intoxication after 24 hours of exposure and time points thereafter. In contrast, worms grown on control E. coli, or in the absence of IPTG, showed no significant intoxication. It appeared that our cry5B-expressing bacteria caused significant intoxication in mixed stage cultures of C. elegans. We used mixed stage nematode cultures, as they are more likely representative of what is observed in the natural setting (as opposed to synchronized or isolated cultures). Note that we could alter the amount of intoxication by changing the concentration of IPTG in the medium (Figure S2a, Supporting Methods and Results). Furthermore, we confirmed that C. elegans was ingesting our bacteria (Figure S2b, Supporting Methods) and that our experimental setup was sufficient to allow for sustained protein expression over the length of our experiments (Figure S2c, Supporting Methods). Previous studies have indicated that C. elegans

31

and other nematodes

move towards chemical signals (chemoattractants) produced by bacteria


32

32

can sense and

. AHLs represent a

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diverse class of chemicals that are secreted by numerous bacterial species

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33

. These chemicals

can readily pass through the cell membrane and wall and can often be implemented in a bacterial strain with the expression of a single gene. We tested the ability of C. elegans to be attracted to various AHLs

31

. We used a choice assay

34

(Figure 2a, Eq. 1) to quantify the attraction of C.

elegans to 3-oxohexanoyl homoserine lactone (3OC6HSL) and N-3-oxododecanoyl homoserine lactone (3OC12HSL), which can be readily produced and secreted by E. coli. We observed that the choice index in response to 3OC6HSL was higher and less variable (Figure 2b) than the choice index in response to 3OC12HSL (Figure 2c, Supporting Results). Next, we engineered E. coli to produce and secrete 3OC6HSL. We placed the anhydrotetracycline (atc) inducible promoter, Ptet 30, upstream of the luxI gene, which catalyzes the formation of 3OC6HSL (Figure S1)

35

. We confirmed that this strain was secreting 3OC6HSL using a detector strain, which

expresses a green fluorescent protein (gfp) in response to 3OC6SHL (Figure S4a, Supporting Methods). We then determined choice index of C. elegans as described above, where E. coli containing an empty vector was the control (Figure 2d). We measured choice index at 1-hour intervals for 4 hours followed by measurements every 24 hours (Figure 2e). We observed that the choice index increased steadily for the first four hours and reached an averaged maximum of 0.60 (±0.14) at 48 hours. Choice index was consistently above 0.5 from 3 hours to 48 hours, demonstrating that luxI expression led to sustained attraction over an extended period of time. Without atc in the medium, C. elegans did not show a preference to either the luxI-expressing strain or the control strain. Furthermore, we observed that C. elegans increases its feeding rate when exposed to 3OC6HSL (Figure 2f, Supporting Methods and Results). Overall, these results indicated that expression of luxI caused sustained attraction, and that the presence of 3OC6HSL increased feeding rate, in C. elegans. Note that we could manipulate choice index by introducing


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various amounts of atc into the medium (Figure S3a, Supporting Methods). Here, increasing the amount of atc produced an increase in the choice index. Furthermore, as a control, we verified that light-induced degradation of atc (due to microscopy) did not significantly impact our choice assays when using atc induced luxI-expressing bacteria (Figure S3b, Supporting Methods).

Combining the toxin and attraction module results in an unexpected decrease in intoxication efficiency To determine if the inclusion of the attraction module increased the intoxication efficacy of the cry5B-expressing bacteria, we combined both modules in E. coli. We exposed a mixed stage culture of C. elegans to this cry5B-/luxI-expressing strain for 72 hours and determined intoxication at 24-hour intervals. With IPTG in the medium, there was no significant difference in intoxication between the cry5B-/luxI-expressing strain and a control strain that contained cry5B and an empty vector (Figure 3a). When both IPTG and atc were included in the medium, there was a significant reduction in the intoxication efficacy. Specifically, intoxication was reduced to nearly half relative to the control after 72 hours. This reduction in intoxication efficacy was not primarily due to a reduction in AHL production (Figure S4a) or a large reduction in growth rate, which may be indicative of a metabolic burden

(Figure S4b,

Supporting Methods and Results). We note, however, that our growth rate analysis cannot completely rule out that co-expression of both cry5B and luxI produces a metabolic burden in the bacteria. Interestingly, C. elegans 36-38, like other nematodes (e.g., 39), evaluate the palatability and potential pathogenicity of their food sources

34, 40, 41

. Some pathogenic bacteria secrete small

molecules such as AHL 31. After feeding on these bacteria, C. elegans uses aversive associative


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learning to link AHL with harmful bacteria, and subsequently avoids them

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31, 34, 41

. Furthermore,

C. elegans will reduce its feeding rate when presented with toxin producing bacteria

42, 43

. It is

possible that C. elegans used aversive associative learning to learn that our cry5B-/luxIexpressing bacteria were toxic and modified its feeding behavior. To explore this hypothesis, we exposed mod-1 and lrn-1 knockout strains to our engineered bacteria. mod-1 and lrn-1 deficient strains of C. elegans are incapable of learning

31, 44

. Both knockout strains were significantly

intoxicated by the cry5B-/luxI-expressing bacteria when atc and IPTG were present in the medium (Figure 3b). The loss of learning increased intoxication indicating that wild type C. elegans was likely learning that our bacteria were toxic and altering its behavior, including decreasing its feeding rate (Figure S4c, Supporting Results).

Mathematical modeling reveals that subsequent activation of our circuit modules can increase intoxication efficacy. To explore scenarios that may circumvent learning in C. elegans, and thus increase the intoxication efficacy of our cry5B-/luxI-expressing bacteria, we constructed a mathematical model consisting of five ordinary differential equations (Eq. 2-6, Methods, Supporting Results, Table S2). The model describes the growth of the engineered bacteria (Eq. 2), the production of AHL (Eq. 3) and toxin (Eq. 4), learning in C. elegans (Eq. 5) and the net growth of the C. elegans population including dynamics for population growth and intoxication (Eq. 6). The description of dynamics for C. elegans toxin-induced death incorporates enhancement by the attraction module, which may be realized biologically through increased feeding rate, and evasion of toxin bacteria via aversive associative learning. Our model predicts the trends observed in our experimental analysis including the intoxication efficacy of our cry5B-


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expressing bacteria (Figure 3c) and the reduction in intoxication efficacy via learning when both modules were activated simultaneously (Figure 3d). While our parameters were identified from a range of biologically reasonable values (Table S2), in Figure S5 we show how varying selected parameters affects the central predictions of our mathematical model. We hypothesized that the ability to dynamically regulate the expression of our modules might offer a unique approach to decoupling aversive learning and intoxication. Specifically, we could independently control luxI (attraction) and cry5B (toxin) to increase intoxication efficacy. Our model predicts that activation of the attraction module first, followed by activation of the toxin module, results in a nearly three-fold increase in intoxication efficacy (Figure 4a). On the other hand, activation of the toxin module first, followed by the attraction module, serves to reduce intoxication efficacy. Experimentally, we could achieve dynamic regulation of the modules by sequentially applying inducers (atc and IPTG) to medium containing bacteria. Specifically, by adding either atc or IPTG to solid medium where bacteria were growing for 24 hours, we could activate expression of elements downstream of the Plac or Ptet promoter. To test this, we cultured C. elegans on cry5B-/luxI-expressing bacteria on medium that contained atc. After 24 hours, we added IPTG. We observed negligible intoxication when atc alone was present on the plate (Figure 4b). However, the addition of IPTG served to increase the fraction of intoxicated worms to 0.52 (+/-0.06) after 48 hours, as predicted by our model. This represented a nearly three-fold increase in the percentage of intoxicated worms when C. elegans was exposed to cry5Bexpressing bacteria for 24 hours (Figure 4c). Intoxication was sustained for at least 5 days under this condition (Figure 4d). Sequential activation using IPTG followed by atc resulted in only a moderate increase in intoxicated worms as predicted by our model (Figure 4b and c).


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Furthermore, after 5 days, this condition resulted in large, active worms that appeared unharmed (Figure 4e). Note that we verified that adding either atc or IPTG to M9 agar plates induced expression of the Ptet and Plac promoters, respectively (Figure S6a, Supporting Methods). A learning assay was used to demonstrate that sequential activation circumvented aversive learning (Figure 4f). When IPTG was applied first, followed by atc, C. elegans showed no preference towards 3OC6HSL at 32 hours. Moreover, at 48 hours, C. elegans was repulsed by 3OC6HSL. Conversely, when atc was applied first, followed by IPTG, C. elegans remained attracted to 3OC6HSL at 32 hours, but showed no preference towards it at 40 and 48 hours. Importantly, C. elegans was not repulsed by 3OC6HSL. Note that when the modules were induced in this manner (atc first, IPTG second), significantly fewer worms dispersed from their initial point of inoculation during our learning assays, thus providing support to the strong intoxication ability of our cry5B-/luxI-expressing bacteria (Figure S6b). Note that we verified that the presence of atc or IPTG in the medium did not affect choice in C. elegans, and is thus unlikely to influence learning (Figure S6c, Supporting Methods). Overall, sequential activation of the attraction and toxin modules served to circumvent aversive learning and thus increased the intoxication efficacy of our engineered bacteria. Interestingly, a previous study has also found that E. coli signaling molecules can alter the behavior a second, co-cultured species 45. Herein, we have described a strain of engineered bacteria that can be dynamically regulated to effectively intoxicate the model nematode C. elegans. Such dynamic regulation served to circumvent aversive learning in C. elegans, which, under non-dynamic conditions, significantly reduced intoxication efficacy. While our study served as a starting point to explore dynamic regulation of our circuit, optimization of intoxication will likely require the measurement of additional parameters such as the mean time for learning, the time required for


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C. elegans, the time required to ‘forget’ the AHL signal and Cry5B toxicity

31, 36, 46, 47

, and

recovery time from intoxication by Cry5B 48. Our system has several advantages over currently existing systems that target nematodes. For example, as we can independently control attractant and toxin, as well as circumvent learning, we may be able to minimize resistance to toxin proteins as sustained challenges of biocides

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, including Bt toxin proteins

50

, have previously shown to increase the rate of

resistance. We hypothesize that intoxication efficacy may be increased by expressing our system in a strain of Bacillus that possesses the ability to colonize the intestinal tract of nematodes increasing intoxication efficacy

26

and can be used in mammals to treat infection. It remains

unclear whether cry5B is the best toxic protein for our application as several other toxic proteins have been described to date including those with synergistic ability 23, 28, 51-53. Nevertheless, the framework presented herein establishes proof-of-concept engineered bacteria that could be modified and optimized for various applications and parasitic nematodes that are bacteriovorous. While previous synthetic systems have been designed using multiple ‘species’ of engineered bacteria 54-56 or inter-kingdom engineered species 57, 58, our synthetic system required tuning the implementation of our circuit components to adapt to a non engineered species, which, to our knowledge, has received little attention thus far and may be critical in designing systems to treat pathogenic agents that dynamically respond to treatment. The study of ‘failure modes’ of gene circuits represents a relatively new area in synthetic biology. Previous studies have identified several reasons that can lead to the failure of gene circuit function 59 including the use of interfering genetic components 60, non-optimal growth conditions 61, and interactions between the host and the gene circuit 62. Recent studies have highlighted the importance of developing a mechanistic understanding as to why synthetic systems fail. For example, Gonzalez et al (2015)


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demonstrated that interactions between a stress response gene circuit, the host cell and the environment drive evolution, and thus circuit functionality, in Saccharomyces cerevisiae 63. The research presented herein adds to the growing list (e.g.,62, 64) of non-intuitive engineering and optimization principles that must be taken into account when engineering synthetic gene circuits, including those designed to treat diseases.

METHODS Strains and growth conditions We used E. coli strain DH5αPRO (Clontech, Mountain View, CA) in this study (unless otherwise indicated). All experiments were performed in modified M9 medium [1X M9 salts (48 mM Na2HPO4, 22 mM KH2PO4, 862 mM NaCl, 19 mM NH4Cl) 0.4% glucose, 0.1% casamino acids (Teknova, Hollister, CA), 0.5% thiamine (Calbiochem, San Diego, CA), 2 mM MgSO4, 0.1 mM CaCl2] buffered to pH 7.0 with 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS, Amresco, Solon, OH) with or without 1% agar (Alfa Aesar, Ward Hill, MA). To create overnight cultures, we inoculated single colonies from an agar plate into 3mL of Luria-Bertani (LB) medium (MP Biomedicals, Solon OH). Unless otherwise indicated, all culture medium contained 25 µg/mL chloramphenicol, 50 µg/mL kanamycin or 100 µg/mL ampicillin. The toxin module was activated using isopropyl β-D-1-thiogalactopyranoside (IPTG, Promega, Madison, WI) using 1 mM IPTG (unless otherwise indicated). The attraction module was activated using anhydrotetracycline (atc, Acros Organics, Geel, Belgium) using 100 ng/mL (unless otherwise indicated). C. elegans (N2, Carolina Biological, Burlington, NC) was used throughout this study unless otherwise indicated. C. elegans was grown on Nematode Growth agar (Carolina


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Biological) containing a lawn of E. coli strain MG1655 (F- λ- ilvG- rfb-50 rph-1). Cultures were grown at room temperature and sub-cultured by cutting out small chunks of agar and transferring the worms to a new plate 65. In all cases, we used mixed stage worms from plates that were 7-12 days old. C. elegans knockout strains were received from Caenorhabditis Genetics Center (CGC, University of Minnesota). All cultures used were free of fungal contaminants. In all assays, worms that crawled off the plate were not counted in our analysis.

Intoxication assays To quantify the intoxication rate of our toxin module, we grew cry5B-expressing bacteria overnight and inoculated them on to M9 medium (with 1% agar, with or without 0.5 mM IPTG, and with or without ampicillin) in the wells of a six well plate. As a control, we also plated DH5αPRO containing the pUC19 plasmid (which confers ampicillin resistance). We counted the total number of worms, as well as the total number of worms showing signs of intoxication 66 at 24-hour intervals for 72 hours. We then plotted the total number of intoxicated worms as a function of total worms. Worms that crawled off the plate were not counted in our analysis. We note that the use of carbenicillin as our selection marker did not affect our results (not shown). To quantify intoxication using E. coli that contained both the toxin and attraction module, we grew E. coli expressing cry5B and luxI overnight. Similarly, we grew a control strain (containing the toxin module and plasmid pPROLar (Clontech), which confers kanamycin resistance and is the backbone plasmid for the luxI containing plasmid) overnight at 37oC. The following day, we washed the cells in M9 medium and plated them on plates containing kanamycin and ampicillin. These plates also contained atc, atc and IPTG or were without inducers. Washed cells were plated on these plates and incubated overnight. The following day,


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we added C. elegans to the center of each plate and quantified intoxication at 24-hour intervals for 72 hours. For intoxication assays using the learning deficient mod-1 and lrn-1 knockout strains, we reared all strains (including N2) as the same time and performed the experiments simultaneously as described above. This served to limit the effect of differences in the distribution of worm stages form the C. elegans culture plates. Sequential activation of the toxin and attraction module was achieved by growing bacteria and C. elegans as described above but on media that initially only contained either 100 ng/mL atc or 1 mM IPTG. After 24 hours, the additional inducer was added directly to the agar by pipetting it on top of the bacterial lawn. Plates were immediately dried in a hood to limit ethanol contact (via the addition of atc) with the worms. Intoxication was examined as described above. For long-term studies, cultures were kept for 5 days, whereupon intoxication was examined as described above.

Microscopy C. elegans was viewed using a Leica M80 with a V-lux 1000 light source (Leica Microsystem, Buffalo Grove, IL) at 60X magnification. Intoxicated worms (see Intoxication assay) were determined by examining each worm for ~10 seconds. If the worm did not move during that time frame, the worm was counted as intoxicated 23, 66. Images and videos were taken with an Olympus IX73P2F fluorescent microscope at 25X magnification using a DP-80 camera (Olympus Microscopes, Center Valley, PA).

Attraction assays


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We quantified the ability of 3-oxohexanoyl-homoserine lactone (3OC6HSL) and 3oxododecanoyl-homoserine lactone (3OC12HSL, both from Sigma-Aldrich, Saint Louis, MO) to attract C. elegans by using a modified chemotaxis (choice) assay 31. We placed as single 10 µL aliquot of C. elegans resuspended in M9 medium in the center of an M9 agar plate. After allowing the M9 medium containing the worms to dry, we placed 10 µL of varying concentrations of either acylhomoserine lactone (AHL) on one side of an M9 agar plate, 2 cm away from the C. elegans culture. On the opposite side of the agar plate, and 2 cm away from the C. elegans cultures, we placed 10 µL of ethanol (the carrier solvent for both AHLs and that is not an attractant for C. elegans

31

). The plate was then incubated at room temperature for one hour

whereupon the choice index was calculated. We counted the number of worms on the side of the plate with the AHL and the side of the plate containing ethanol alone (control). We then calculated the choice index as below. Note we counted only worms that had moved away from their spot of inoculation (i.e., the center of the plate). ℎ =

( )

(Eq. 1)

To determine the choice index for E. coli containing the attraction module, we grew E. coli that did, or did not, express luxI. The following day, we diluted each strain 10 fold into fresh M9 medium containing various amounts of atc and shook the cultures at 37oC for 2 hours to induce the circuit. Next, we washed the cultures in fresh M9 medium, concentrated the cells 10 fold and placed 10 µL of both cultures 2 cm apart on an M9 agar plate (in a 6-well plate) containing the atc (i.e., the same concentration of atc that was used to induce the circuit). In between the bacterial cultures, we placed a single 20 µL aliquot of C. elegans resuspended in M9 medium. We allowed the bacterial cultures and C. elegans to dry. We then assessed the choice index at various time intervals. When the assay plates were not being measured, the plates were


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kept in a darkened drawer to minimize atc degradation due to light exposure. Worms that did not move from their initial point of inoculation were not counted in our analysis.

Learning assay We conducted modified learning assays as previously described

34

. We grew C. elegans

on plates containing cry5B/luxI-expressing bacteria and sequentially activated gene expression as described in ‘Intoxication assay.’ At various time points, we removed C. elegans from the plate by washing the surface gently with M9 medium. Next, we centrifuged the worms at 400 RPM for 2 minutes and washed the worms three times with fresh M9 medium to remove residual bacteria. We then placed the worms on M9 medium agar plates equidistant from either 10 µL of ethanol (carrier solvent) or 10 µL of 20 µM 3OC6HSL. After 1 hour, we determined choice index as described in Attraction assays.

Feeding Rate Analysis To examine if the presence of 3OC6HSL increased feeding rate, we grew E. coli strain MG1655 overnight on 0.5 mL of M9 agar in wells of a 24 well plate. The follow day, we added, or did not add, 1 µL of 20 µM 3OC6HSL directly to the plate and allowed it to dry for one hour. C. elegans was isolated from a culture plate and washed twice in liquid M9 medium to remove residual bacteria. ~ 20 C. elegans were then added to each of the wells that contained or did not contain 3OC6HSL. After allowing the worm to disentangle for ~ 10 minutes, we took videos using an Olympus IX73P2F fluorescent microscope. We counted the number of pharyngeal pumps in each worm over multiple 10-second periods as a measure of feeding rate. Videos were observed at 40% normal speed and pharyngeal pumps were counted as one full bob of the


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pharynx. Worms that did not bob at all in the video, or were clearly quiescent, were not scored. We used a minimum of fifteen worms for our analysis. To determine if expression of luxI and cry5B simultaneously decreased feeding rate, we cultured C. elegans for 24 hours on cry5B-/luxI-expressing bacteria without inducers, with IPTG only or with both IPTG and atc. We then removed the worms, washed them twice in M9 medium and placed the worms on M9 medium. We counted pharyngeal pumps as described above. We used a minimum of fifteen worms for our analysis.

Mathematical Modeling We developed a mathematical model to aid in the guidance of our experimental design. The model provides kinetic descriptions for the evolution of the densities of bacterium, AHL, Cry5B, learning and C. elegans incorporating explicit dynamics for toxin induced death with enhancement by the attraction module. The model system is:

'

3

= (1 − )

(Eq.2)

#

= $ − % &

(Eq. 3)

= $( − %( )

(Eq. 4)

*

= + (

-./

, '#

,0#

1, '# 3

− 2 )

(Eq. 5)

= 45 61 − 3 7 − (1 − 2)81 + :;(&; = , )?% ;(); =' , ' )5 (Eq. 6) #

where C represents the density of the engineered bacteria, µ represents the intrinsic growth rate of bacteria; Cm represents the bacterial carrying capacity (normalized to 1); A represents the concentration of AHL; Ka represents the synthesis rate of AHL; Da represents the degradation rate of AHL; B represents the toxin Cry5B; Kb represents the synthesis rate of Cry5B; Db @A

represents the degradation rate of Cry5B; ;(, =, ) = A 1@ A and represents the modulation of a


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maximum rate of x; Hx represents the half maximal level of the modulation of x; n, m and p represents the sharpness of the transition from low to high rate; W represents the density of C. elegans; Wm represents the C. elegans carrying capacity (normalized to 1); γ represents the intrinsic growth rate of C. elegans; Dw represents the intoxication rate of Cry5B; δ represents maximal attraction strength of AHL; L is a lumped term representing learning and avoidance in C. elegans; τ represents the time constant for learning; and t represents time. The right hand term of Eq. 6 represents an approximation of AHL attraction, learning and intoxication of worms. Additional modeling description is described in Supporting Results. Parameter estimation, values and units are described in Supporting Results and Table S2.

Statistical Analysis Unless otherwise indicated, we used a two-tailed t-test for all statistical analyses. We assumed statistical significance with a P value ≤ 0.05.

ACKNOWLEDGEMENTS This research is supported by a Presidents Faculty Research and Development Grant #335318 through Nova Southeastern University and an HBCU/MI Equipment/ Instrumentation from the Department of Defense/Army Research Office (W911NF-14-1-0070). We would like to thank Raffi Aorian for providing the cry5B gene, and Cheemeng Tan, Jaydeep Srimani and Allison Lopatkin for their critical evaluation and comments.

AUTHOR CONTRIBUTIONS


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All authors conceived and designed research. ORB, CM, CAB, and RPS performed experimental research. ECH and RPS performed modeling. All authors contributed to the writing of the manuscript. All authors approve of the manuscript.

COMPETING INTEREST STATEMENT The authors declare that they have no competing financial interests.

SUPPORTING INFORMATION The Supporting Information contains Supporting Methods, Supporting Results, Figures S1-S6 and Tables S1-S2.


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FIGURE LEGENDS

Figure 1: A synthetic circuit that leads to intoxication of C. elegans. a) Our circuit consists of a toxin module (red shaded) and an attraction module (green shaded). The toxin module consists of the T5 promoter with tandem copies of lacO driving expression of a toxin gene, which can intoxicate C. elegans upon ingestion. The attraction module consists of the Ptet promoter driving the expression of an acylhomoserine lactone (AHL) producing gene (attract). The AHL, which can readily pass through the bacterial membrane and cell wall, is secreted by E. coli. Red diamonds = toxin protein. Green circles = AHL. b) We cultured C. elegans in the presence of cry5B-expressing bacteria (blue and red circles) or a control strain of bacteria (empty vector control, orange and grey circles), which lacked cry5B. Furthermore, we grew the bacteria in medium that did or did not contain IPTG. We observed that when cry5b was expressed there was significant mortality of worms at 24 hours and time points thereafter (P < 0.003, two-tailed t-test). There were very few worms that exhibited signs of intoxication when fed the control strain or without the toxin module activated (red circles). Standard deviation from three replicates (within radius of data points). c) During our assay to determine intoxication, we took images using an X73P2F fluorescent microscope at 25X magnification. These panels illustrate canonical worms that we observed in our experiments.

Figure 2: Using AHL to attract and increase the feeding rate of C. elegans.


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a) Using a choice assay to assess the ability of AHL of attract C. elegans. In our choice assay, we placed C. elegans in the middle of an M9 agar plate. 2cm on either side of center (dotted line) we placed either increasing concentration of AHL or the neutral carrier solvent, ethanol. After 1 hour, we counted the number worms on either side of the plate and calculated choice index (defined as the total percentage of worms on the side of the plate containing AHL). b) We observed a bisphasic relationship between choice index (Figure S3a) and the concentration of the AHL 3OC6HSL. Specifically, C. elegans was attracted to 3OC6HSL at concentrations between 1 µM and 20 µM (P < 0.011, two-tailed t-test). In panels b and c, standard deviation from three replicates. c) C. elegans was not reliably attracted to the AHL 3OC12HSL within an hour as noted by the large standard deviation associated with the data points in this panel (P > 0.4, twotailed t-test). d) For choice assays involving bacteria, we used a 6-well plate where C. elegans was separated from either luxI-expressing bacteria or control bacteria by ~1cm. Choice index was calculated as the total percentage of worms on the side of the plate containing luxIexpressing bacteria (Eq. 1). e) We engineered E. coli to express luxI, which creates 3OC6HSL, under the regulation of an anhydrotetracycline (atc) inducible promoter. We used this luxI-expressing strain in a choice assay. We observed that, with atc in the medium, C. elegans preferentially migrated towards the luxI-expressing bacteria in as little as an hour (blue circles). This preference remained over the course of 48 hours, whereas at 72 hours the worms had no preference for either bacteria. Without atc, C. elegans showed no preference at all time


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points measured (red circles). P < 0.05 for all data points expect 0 and 72 hour measurements (where P > 0.5, two tailed t-test). Standard deviation from minimum of 3 replicates. f) The presence of 3OC6HSL increases feeding rate in C. elegans. We counted pharyngeal pumps of worms feeding on E. coli with and without 3OC6HSL. We observed that, when presented with E. coli and 3OC6HSL, the pumping rate of the worms was significantly higher as compared to when the worms were only presented with E. coli (P = 0.0009, two-tailed t-test). Standard deviation plotted from a minimum for fifteen worms.

Figure 3:

Concurrent activation of toxin and attraction modules does not increase

intoxication efficacy of our engineered bacteria. a) We combined the attraction and the toxin modules (cry5B-/luxI-expressing strain) and assessed the ability of this bacterial strain to intoxicate C. elegans. When grown with IPTG (orange circles), the fraction of intoxicated worms was similar to when worms were fed cry5B-expressing bacteria (red circles). However, when grown with both IPTG and atc (blue circles), the fraction of intoxicated worms was significantly decreased at 48 and 72 hours (P < 0.003, two tailed t-test) indicating that expression of luxI served to decrease intoxication. Without inducers, there was no increase in intoxication (grey circles). Standard deviation from three replicates. b) Learning deficient strains of C. elegans do not demonstrate a reduction in intoxication when exposed to bacteria simultaneously expressing cry5B and luxI. We grew mod-1 (green shapes) and lrn-1 (red shapes) knockout strains in the presence of cry5B-/luxIexpressing bacteria with IPTG and atc (circles) or without inducers (triangles). We


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observed that the knockout strains were significantly intoxicated when both atc and IPTG were contained in the medium. P < 0.001 for all data points at 24 hours and thereafter compared between induced and non-induced bacteria (two tailed t-test). P ≤ 0.005 when comparing mod-1 and lrn-1 to the wildtype C. elegans strain (blue shapes) at 24 hours and thereafter (except P = 0.15 between mod-1 and wildtype C. elegans at 72 hours). Standard deviation from a minimum of three replicates. c) Our model (Eq. 2-6) predicts that ~60% of C. elegans worms should be intoxicated after 72 hours of exposure to cry5B-expressing bacteria (circuit ON, blue line, circuit OFF, red line). For both panels, initial value of C = 0.9, initial value of W = 0.1. d) The incorporation of a learning parameter in our model reduces intoxication efficacy. Without the learning term (green line), intoxication is very rapid (near ~100% after 24 hours). However, with the learning term (blue line) our mathematical prediction matches our experimental data; only 25% of the worms are intoxicated when both modules are activated simultaneously.

Figure 4: Sequential activation of circuit modules increases intoxication efficacy by circumventing learning. a) Our mathematical model predicts that sequential activation of the circuit modules will increase intoxication efficacy. Here, activation of the attraction module prior to the toxin module increases intoxication efficacy after 24 hours (red line). Activation of the toxin module prior to the attraction module limits intoxication efficacy (blue line). Initial value of C = 0.9, initial value of W = 0.1.


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b) We grew C. elegans in the presence of cry5B-/luxI-expressing bacteria. We first activated expression of luxI (atc first, red circles) or cry5B (IPTG first, blue circles) for 24 hours, followed by expression of the additional gene, cry5B or luxI, respectively. We observed that activation of luxI followed by cry5B resulted in a significant increase in intoxicated worms at 40 and 48 hours (P ≤ 0.04, two tailed t-test). Activation of cry5B followed by luxI resulted in a moderate increase in the fraction of intoxicated worms once luxI was activated. Standard deviation from three replicates. c) Fraction of intoxicated worms normalized to the number of intoxicated worms observed during concurrent activation of cry5B and luxI. Through subsequent activation of luxI and cry5B (red bar), we achieved a ~ 3 fold increase in intoxicated worms after 24 hours of cry5B activation. d) Long-term consequences of sequential activation of circuit components. After 5 days, we imaged M9 agar plates containing C. elegans and cry5B-/luxI-expressing bacteria. Activation of luxI, followed by cry5B, resulted in populations of worms that were small, slow moving and had few eggs. Representative image shown. e) Sequential activation of cry5B followed by luxI resulted in worms that were large, fast moving, and with plates that had numerous eggs. Representative image shown. f) A learning assay demonstrates that sequential activation of luxI followed by cry5B circumvents learning. We removed C. elegans at 8-hour intervals after activation of the second module in the experiment described in B). We presented these worms with either 3OC6HSL or ethanol (carrier solvent) in a choice assay. At 24 hours, both patterns of sequential activation led to worms that were attracted to 3OC6HSL (P ≤ 0.04, compared to a choice index of 0, P = 0.83 when compared to each other). If cry5B was expressed


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first (blue bars), C. elegans did not show a preference towards 3OC6HSL at 32 hours (P = 0.86) and showed repulsion towards 3OC6HSL at 48 hours (blue bars, P = 0.04, compared to a choice index of 0 in both cases). If luxI was expressed first (red bars), C. elegans remained attracted to 3OC6HSL at 32 hours (P = 0.048) but did not show a preference to either 3OC6HSL or carrier solvent at 40 and 48 hours (P ≥ 0.41, compared to a choice index of 0 at both time points). Standard deviation from three replicates.


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Figure 1: A synthetic circuit that leads to intoxication of C. elegans. a) Our circuit consists of a toxin module (red shaded) and an attraction module (green shaded). The toxin module consists of the T5 promoter with tandem copies of lacO driving expression of a toxin gene, which can intoxicate C. elegans upon ingestion. The attraction module consists of the Ptet promoter driving the expression of an acylhomoserine lactone (AHL) producing gene (attract). The AHL, which can readily pass through the bacterial membrane and cell wall, is secreted by E. coli. Red diamonds = toxin protein. Green circles = AHL. b) We cultured C. elegans in the presence of cry5B-expressing bacteria (blue and red circles) or a control strain of bacteria (empty vector control, orange and grey circles), which lacked cry5B. Furthermore, we grew the bacteria in medium that did or did not contain IPTG. We observed that when cry5b was expressed there was significant mortality of worms at 24 hours and time points thereafter (P < 0.003, two-tailed ttest). There were very few worms that exhibited signs of intoxication when fed the control strain or without the toxin module activated (red circles). Standard deviation from three replicates (within radius of data points). c) During our assay to determine intoxication, we took images using an X73P2F fluorescent microscope at 25X magnification. These panels illustrate canonical worms that we observed in our experiments. 254x190mm (300 x 300 DPI)



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Figure 2: Using AHL to attract and increase the feeding rate of C. elegans. a) Using a choice assay to assess the ability of AHL of attract C. elegans. In our choice assay, we placed C. elegans in the middle of an M9 agar plate. 2cm on either side of center (dotted line) we placed either increasing concentration of AHL or the neutral carrier solvent, ethanol. After 1 hour, we counted the number worms on either side of the plate and calculated choice index (defined as the total percentage of worms on the side of the plate containing AHL). b) We observed a bisphasic relationship between choice index (Figure S3a) and the concentration of the AHL 3OC6HSL. Specifically, C. elegans was attracted to 3OC6HSL at concentrations between 1 µM and 20 µM (P < 0.011, two-tailed t-test). In panels b and c, standard deviation from three replicates. c) C. elegans was not reliably attracted to the AHL 3OC12HSL within an hour as noted by the large standard deviation associated with the data points in this panel (P > 0.4, two-tailed t-test). d) For choice assays involving bacteria, we used a 6-well plate where C. elegans was separated from either luxI-expressing bacteria or control bacteria by ~1cm. Choice index was calculated as the total percentage of worms on the side of the plate containing luxI-expressing bacteria (Eq. 1). e) We engineered E. coli to express luxI, which creates 3OC6HSL, under the regulation of an anhydrotetracycline (atc) inducible promoter. We used this luxI-expressing strain in a choice assay. We observed that, with atc in the medium, C. elegans preferentially migrated towards the luxI-expressing bacteria in as little as an hour (blue circles). This preference remained over the course of 48 hours, whereas at 72 hours the worms had no preference for either bacteria. Without atc, C. elegans showed no preference at all time points measured (red circles). P < 0.05 for all data points expect 0 and 72 hour measurements (where P > 0.5, two tailed t-test). Standard deviation from minimum of 3 replicates. f) The presence of 3OC6HSL increases feeding rate in C. elegans. We counted pharyngeal pumps of worms feeding on E. coli with and without 3OC6HSL. We observed that, when presented with E. coli and 3OC6HSL, the pumping rate of the worms was significantly higher as compared to when the worms were only presented with E. coli (P = 0.0009, two-tailed t-test). Standard deviation plotted from a minimum for fifteen worms. 254x190mm (300 x 300 DPI)


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Figure 3: Concurrent activation of toxin and attraction modules does not increase intoxication efficacy of our engineered bacteria. a) We combined the attraction and the toxin modules (cry5B-/luxI-expressing strain) and assessed the ability of this bacterial strain to intoxicate C. elegans. When grown with IPTG (orange circles), the fraction of intoxicated worms was similar to when worms were fed cry5B-expressing bacteria (red circles). However, when grown with both IPTG and atc (blue circles), the fraction of intoxicated worms was significantly decreased at 48 and 72 hours (P < 0.003, two tailed t-test) indicating that expression of luxI served to decrease intoxication. Without inducers, there was no increase in intoxication (grey circles). Standard deviation from three replicates. b) Learning deficient strains of C. elegans do not demonstrate a reduction in intoxication when exposed to bacteria simultaneously expressing cry5B and luxI. We grew mod-1 (green shapes) and lrn-1 (red shapes) knockout strains in the presence of cry5B-/luxI-expressing bacteria with IPTG and atc (circles) or without inducers (triangles). We observed that the knockout strains were significantly intoxicated when both atc and IPTG were contained in the medium. P < 0.001 for all data points at 24 hours and thereafter compared between induced and non-induced bacteria (two tailed t-test). P ≤ 0.005 when comparing mod-1 and lrn-1 to the wildtype C. elegans strain (blue shapes) at 24 hours and thereafter (except P = 0.15 between mod-1 and wildtype C. elegans at 72 hours). Standard deviation from a minimum of three replicates. c) Our model (Eq. 2-6) predicts that ~60% of C. elegans worms should be intoxicated after 72 hours of exposure to cry5B-expressing bacteria (circuit ON, blue line, circuit OFF, red line). For both panels, initial value of C = 0.9, initial value of W = 0.1. d) The incorporation of a learning parameter in our model reduces intoxication efficacy. Without the learning term (green line), intoxication is very rapid (near ~100% after 24 hours). However, with the learning term (blue line) our mathematical prediction matches our experimental data; only 25% of the worms are intoxicated when both modules are activated simultaneously. 254x190mm (300 x 300 DPI)


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Figure 4: Sequential activation of circuit modules increases intoxication efficacy by circumventing learning. a) Our mathematical model predicts that sequential activation of the circuit modules will increase intoxication efficacy. Here, activation of the attraction module prior to the toxin module increases intoxication efficacy after 24 hours (red line). Activation of the toxin module prior to the attraction module limits intoxication efficacy (blue line). Initial value of C = 0.9, initial value of W = 0.1. b) We grew C. elegans in the presence of cry5B-/luxI-expressing bacteria. We first activated expression of luxI (atc first, red circles) or cry5B (IPTG first, blue circles) for 24 hours, followed by expression of the additional gene, cry5B or luxI, respectively. We observed that activation of luxI followed by cry5B resulted in a significant increase in intoxicated worms at 40 and 48 hours (P ≤ 0.04, two tailed t-test). Activation of cry5B followed by luxI resulted in a moderate increase in the fraction of intoxicated worms once luxI was activated. Standard deviation from three replicates. c) Fraction of intoxicated worms normalized to the number of intoxicated worms observed during concurrent activation of cry5B and luxI. Through subsequent activation of luxI and cry5B (red bar), we achieved a ~ 3 fold increase in intoxicated worms after 24 hours of cry5B activation. d) Long-term consequences of sequential activation of circuit components. After 5 days, we imaged M9 agar plates containing C. elegans and cry5B-/luxI-expressing bacteria. Activation of luxI, followed by cry5B, resulted in populations of worms that were small, slow moving and had few eggs. Representative image shown. e) Sequential activation of cry5B followed by luxI resulted in worms that were large, fast moving, and with plates that had numerous eggs. Representative image shown. f) A learning assay demonstrates that sequential activation of luxI followed by cry5B circumvents learning. We removed C. elegans at 8-hour intervals after activation of the second module in the experiment described in B). We presented these worms with either 3OC6HSL or ethanol (carrier solvent) in a choice assay. At 24 hours, both patterns of sequential activation led to worms that were attracted to 3OC6HSL (P ≤ 0.04, compared to a choice index of 0, P = 0.83 when compared to each other). If cry5B was expressed first (blue bars), C. elegans did not show a preference towards 3OC6HSL at 32 hours (P = 0.86) and showed repulsion towards 3OC6HSL at 48 hours (blue bars, P = 0.04, compared to a choice index of 0 in both cases). If luxI was expressed first (red bars), C. elegans remained attracted to 3OC6HSL at 32 hours (P = 0.048) but did not show a preference to either 3OC6HSL or carrier solvent at 40 and 48 hours (P ≥ 0.41, compared to a choice index of 0 at both time points). Standard deviation from three replicates.



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Circumvention of Learning Increases Intoxication Efficacy of

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