Team:Wageningen UR/Results/Quorum Sensing
Quorum Sensing
Introduction
Mantis is a modular bacterial diagnostic device that will generate a visible fluorescent signal upon detection of antigens of infectious diseases in blood samples of infected patients. When quantifying fluorescence, a low signal-to-noise ratio hinders a proper understanding of the imaging system. Therefore, it is necessary to both increase the sensitivity of the detection and decrease the background signal to allow for a higher accuracy and precision of the quantitative fluorescence measurements [1].
While the fast Mantis reporter module (Signal Transduction and Specific Visualization) focuses on generating a signal in the shortest possible time, this project will focus on getting the most robust detection system. Due to the modular nature of Mantis, it is easy to swap out one system for the other, based on the requirements of the antigen we want to detect. For example, when highly contagious diseases are to be detected, a fast signal would be preferred. However, when the aim is not to detect diseases that are not very contagious, but pose a large health risk if untreated, the least amount of false negatives is desired. In order to achieve a robust signal generation system, a high signal-to-noise ratio is essential. Thus, a higher sensitivity of the fluorescent Green Fluorescent Protein (GFP) detection system and a lower background signal are needed.
The robust Mantis reporter module is based on a spatially separated two-component system. For that, two bacterial cell populations (BCP) will be used, each expressing only one of the two components needed to generate the output signal. Both components will be isolated inside their own BCP so the fluorescent signal is not generated. The two components will only be able to interact with each other after both BCPs lyse, which will be biologically induced after sensing the antigen (Figure 1).
Together with a reduction of the background signal, a higher sensitivity of the system is desired. For that, a quorum sensing mechanism will be implemented in both BCPs so the signal output produced after the detection of the antigen is propagated to all the bacteria present, no matter if they are directly detecting the antigen or not (Figure 2).
In conclusion, when the antigen is detected, a lytic mechanism triggers the lysis of both BCPs. This will allow the interaction of both interdependent components leading to the generation of a fluorescent signal. Provided that both components are separated by both bacterial cell membranes, the background signal is hypothesized to be reduced as the random interaction of those two components is expected to be lower than when present within the same BCP. Therefore, by reducing the background signal which could be generated by the random interaction of both components, the signal-to-noise ratio will be increased (Figure 3).
Quorum sensing mechanism
Quorum sensing (QS) is a cell-to-cell communication system based on chemical molecules which allow bacteria to detect the presence of other bacteria. This mechanism allows bacteria to control gene expression in response to changes in bacteria concentration in the medium [1,4]. In many Gram-negative bacteria quorum sensing is mainly controlled by production and diffusion of Acylated Homoserine Lactone (AHL). AHL is able to freely diffuse from cell to cell. Once AHL is detected, it is able to trigger other bacteria to generate more AHL molecules, creating a positive feedback loop. Our quorum sensing mechanism (originating from V. fischeri) is controlled by two proteins: LuxI (BBa_C0061) and LuxR (BBa_C0062). LuxR is a transcription factor that becomes active when complexed with AHL, leading to the transcription of genes placed under the pLux promoter (BBa_R0062) (Figure 4) [2].
In nature, the LuxR-AHL complex controls transcription of LuxI via a positive feedback loop which increases the AHL production. When the AHL concentration is high in the cell, dimerization with LuxR protein takes place in higher amounts [3]. Both LuxI and LuxR are expressed under the pLux promoter. However, LuxI is under the Lux PR promoter (BBa_R0062) whose expression is upregulated by the LuxR complex and the LuxR gene is under the reverse Lux PL promoter (BBa_R0063) whose expression is downregulated by LuxR complex [4]. Both promoters are the same sequence but inverted, with one of them being upregulated by the LuxR complex while the other one is downregulated.
Quorum sensing takes place in nature under high bacterial concentrations. However, in our case, the quorum sensing mechanism is desired to only become active after the detection of the antigen. For that, the quorum sensing system needs to be repressed in order to prevent quorum sensing from happening unless the antigen is detected.
The robust Mantis reporter module implements a quorum sensing mechanism in such a way that after the binding of the antigen to one cell, AHL is produced and the detection signal is spread to other cells. In order to assess the expression of the quorum sensing mechanism, the quorum sensing plasmid (BBa_K1913005) which encodes for GFP under the pLux promoter, was used (Figure 5). Therefore, in our system, when both AHL and LuxR are present in the cell, the dimer is formed and GFP is expressed as a reporter. Two negative controls were constructed to perform this experiment. Negative control 1 encodes only GFP under the pLux promoter and no LuxR nor LuxI (Figure 5). Provided that no AHL nor LuxR will be generated, the formation of the [AHL-LuxR] dimer will not take place and no fluorescence will be generated. Negative control 2 encodes for GFP and LuxR under the pLux promoter but no LuxI (Figure 5). In this case, as no AHL can be formed, the dimer wont be formed and therefore GFP can’t be generated.
LuxI expression leads to the formation of an AHL synthase, producing AHL which can diffuse through the cell membrane from cell to cell. LuxR encodes for a protein which dimerizes with AHL. Once the dimer is formed, the genes under the pLux promoter will be expressed, hence fluorescence is generated (Figure 6). It can be seen in the graph that negative control 1 is slightly more active, while the LuxR protein is not even in the cell. A possible explanation for this could be that the uncomplexed luxR has a certain repressing effect to some extent (Figure 6).
The robust Mantis reporter module aims to change the spontaneously activating quorum sensing system (Figure 6) into a system with proper antigen-dependent signaling behavior by keeping under control the quorum sensing mechanism. To achieve this, the aiiA enzyme (BBa_K2387070) which catalyzes the degradation of N-acyl-homoserine lactones (AHLs), will be constitutively expressed.
In order to test the mechanism, a strain containing a construct expressing GFP, LuxI and LuxR under the pLux promoter and the aiiA molecule under the pTet promoter, whose inhibitor TetR was produced constitutively under the pLac promoter, was constructed (Figure 7).
In this mechanism, the quorum sensing mechanism was hypothesized to be active under no induction. When induced by anhydrotetracycline (analogue of tetracycline) (aTc), the aiiA should be expressed as the inhibitor is captured by aTc leading to the expression of the gene downstream. Therefore, a decrease in fluorescence should be observed when aTc is added to the medium (Figure 8).
The anhydrotetracycline-inducible mechanism was tested together with the Reporter quorum sensing, Negative control 1 and Negative control 2 constructs. After triggering the system by adding aTc, a decrease in fluorescence was observed while comparing both induced and non-induced aiiA-expressing constructs (Figure 9). No difference was observed while comparing both induced and non-induced non-expressing aiiA (Reporter quorum sensing strain) (Figure 10).
Fluorescence is modestly decreased when inducing the Inducible QS with high aTc concentrations in comparison to its non-induced state, suggesting that the aiiA mechanism is working. This can indicate that aiiA works as expected and that AHL degradation might have been reduced as well leading to a recovery of fluorescence. However, the results seen do not show a fluorescence recovery comparable of the fluorescence level achieved with the Reporter QS which might imply that leaky expression of the pTet could be causing expression of aiiA also in the induced state (Figure 8).
Furthermore, it seems that overall fluorescence is still very high compared to the negative controls (Figure 10). Moreover, the Inducible QS strain showed a modest increase in fluorescence which is not in agreement of the expected decrease in fluorescence under induction. The reason for this is the low induction of the system, under higher aTc inductions, the decrease in fluorescence can be seen (Figure 9). However, higher aTc inductions are deadly for the cells and therefore the assessments cannot be performed. This suggested that it was needed to increase the repressing effect of aiiA gene in order to avoid the generation of a signal in the absence of the triggering signal. By doing so, the sensitivity of the system could be increased. by allowing a base no signal level.
From the parallel modelling project (Quorum sensing and cell lysis), it was suggested that either increasing LuxR degradation or increasing the aiiA concentration, which degrades AHL, will allow for a stronger control of the quorum sensing mechanism. Therefore, the aiiA gene was expressed in the pSB1C3 high copy number plasmid backbone and the quorum sensing mechanism was expressed in the pSB4K5 low copy number backbone. By doing so, the ratio of aiiA to LuxI and LuxR was aimed to be increased, hypothetically leading to a decreased auto-induction (Figure 13).
The aiiA gene was then expressed under the pTet promoter (BBa_R0040) whose inhibition takes place when TetR (BBa_C0040) is expressed. TetR expression is in turn controlled by the pBAD promoter and therefore aiiA expression gets inhibited under the induction of L-Arabinose (Figure 12).
When L-Arabinose is added to the medium, pBAD triggers the expression of tetR which leads to the inhibition of pTet and hence represses the expression of aiiA which is under the control of pTet. Therefore, if unactivated, aiiA is constitutively expressed and a non-fluorescent state is expected.
Fluorescence is decreased in comparison to both negative controls, suggesting that the QS system is repressed. The results seem to imply that by inducing the system with L-Arabinose, AHL is able to complex again LuxR triggering both the further expression of quorum sensing as well as the expression of GFP (Figure 13). The expression of aiiA gene in a higher copy number backbone (pSB1C3) resulted in a clearer change in fluorescence after inducing the system (Figure 13) when compared with the change caused when expressed in a low copy number backbone (pSB4K5) (Figure 9).
In the complete version of the robust Mantis reporter module, it will express a lytic mechanism under the pLux promoter which becomes activated by the LuxR-AHL dimer. After the detection of the antigen, the quorum sensing will be triggered, allowing both interdependent components to interact so the signal output gets generated. Moreover, AHL is able to diffuse from cell to cell, as soon as the QS gets activated in some of the bacteria, the lysis will be triggered in the rest of the cells via the diffusion of AHL from the directly QS activated to the non-directly activated ones [4].
Therefore, this will allow the system to move from a state in which no lysis is generated to another one in which all the bacteria present in the medium will trigger lysis, no matter if they are directly detecting the antigen or not. This means that the sensitivity of the signal will be increased as a result of the implementation of QS which allows the generation of an output signal by all the cells in the medium. In the future, LuxR degradation could be increased in order to achieve a better control of the system. As predicted by the parallel modelling part (https://2017.igem.org/Team:Wageningen_UR/Model/QS), either increasing LuxR degradation or increasing aiiA concentrations will keep quorum sensing under control in a non-induced state. It has been already proven (Figure 13) that an increase in aiiA expression leads to a steeper change in fluorescence emission while comparing the non-induced versus the induced state of the Inducible quorum system (when compared with the previous system (Figure 10) where no change can be observed).
Two interdependent components system
In order to generate a fluorescent output signal, three different systems were constructed and tested. The three proposed systems are composed by two components which do not generate the output signal independently unless they interact between each other.
Bimolecular Fluorescence Complementation System
This system is based on the generation of two BCPs, each encoding for a complementary part of a fluorescent molecule. In this system, both BCPs will be combined in equal concentrations and a lytic process will be induced. This should lead to the fusion of both halves and hence generation of fluorescence. In order to confirm that both complementary fluorescent halves are able to fuse and lead to fluorescence, a third strain used as a positive control which was expressing both complementary halves in the same cell was also generated.
The three BCPs were generated and the GFP fluorescence generated by combining BCP1 and BCP2 and inducing a lytic process was measured. This was compared with the fluorescence generated by the positive control consisting of both halves expressed under the same constitutive promoter. In parallel, a second positive control was created where an entire non-split fluorescent protein was expressed. By comparing the fluorescence generated to the two positive controls. Both the difference in fluorescence efficiency because of the in vitro reassembly and the decrease in efficiency caused by the split of the fluorescent protein can be evaluated.
Two BCPs coding for the respective halves of the GFP molecule were generated (Choosing the best reporter). Each BCP expresses half of the GFP protein with a leucine zipper attached to it (Choosing the best reporter) under a constitutive promoter. A negative control encoding for both halves and second negative control encoding for the entire GFP were constructed under control of the same constitutive promoter were used as controls. In order to assess the recombination of both halves, all the BCPs were lysed using sonication (Figure 14).
However, the results showed that Bimolecular Fluorescence Complementation was not able to take place in vitro. Even though the recombination of both GFP halves was taking place in the in vivo positive control, when the BCPs expressing each of the GFP halves were mixed and sonicated no fluorescence could be detected (Choosing the best reporter). The reason underlying the incapability of recombination in vitro is unknown.
As a second attempt and in order to simulate biological conditions as much as possible, all the strains were co-transformed with the LysE7 lytic mechanism (BBa_K2387066). In this experiment, the lysis was induced biologically via the addition of L-Arabinose (Figure 15).
However, it was concluded that Bimolecular Fluorescence Complementation neither took place in vitro when lysis was triggered biologically. The fusion of both halves took place in vivo as it can be seen for the strain expressing both halves of the GFP, suggesting that the Bimolecular Fluorescence Complementation works, but is prevented from happening when both halves are not contained within a cell.
Proteolytic systems
Because of the split GFP system not working in vivo, an alternative system which has been reported to work in vitro was tested as well [5]. Both systems are based on the generation of two BCPs, the first encoding for a dark quenched GFP molecule and the second one for a constitutively expressed TEV protease (BBa_K2387071).
FRET (Förster Resonance Energy Transfer) system
In this approach, GFP was quenched by a REACh2 dark quencher (BBa_K1319004), the GFP quenching process takes place in this system via a FRET system. The REACh2 quenching protein (BBa_K1319002) fused to GFP reduces the observed fluorescence of GFP. This protein is a non-fluorescent but still absorbent version of EYFP [6]. GFP and REACh2 are fused with a linker (BBa_K1319016) which includes a specific TEV protease (BBa_K1319004) cleavage site. The fused protein brings GFP and REACh2 in proximity to each other which allows both GFP and REACh2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is absorbed by REACh2 leading to a reduction in the GFP fluorescence.
Hydrophobic Quenching System
In this second approach, the GFP molecule is quenched by a hydrophobic peptide from a mutated version of the M2 fragment of influenza virus (Figure 17) and a second one able to constitutively express the TEV protease. The GFP is completely quenched by the binding of the hydrophobic peptide which tetramerizes the GFP disabling the maturation of the chromophore [3].
GFP fluorescence can be recovered after the removal of the quenching peptide, leading to the generation of a robust reporter as a result of a proteolytic process. This proteolytic process is performed by the TEV protease which performs the cut of the linker fusing GFP to the hydrophobic peptide. This linker (BBa_K1319016) includes a specific TEV protease (BBa_K1319004) cleavage site.
Assessment of REACh2 and mutated M2 fragment quenching systems
In both system, both BCPs were co-transformed with the LysE7 lytic mechanism to allow cell lysis after the induction of L-Arabinose. Both BCPs were combined in equal concentrations and a lytic process was induced, which should allow the TEV protease to cut the linker fusing both GFP to its quenching molecule (REACh2 or the mutated M2 fragment). Once both proteins are separated, the quenching interaction is prevented resulting in an increase in GFP fluorescence. The fact that also GFP quenched by REACh2 in the absence of the TEV protease producing strains lead to fluorescence could be explained by a breakage of the linker connecting REACh2 to the GFP as a result of the lytic mechanism. (Figure 21)
The induction of lysis via the addition of L-Arabinose lead to the breakage of the bacterial cell membranes (Figure 21). Fluorescence is increased in comparison to the non co-transformed strains which are not able to lyse, suggesting that the the lytic process leads to the recovery of the quenched GFP. This can indicate that both REACh2 and the mutated M2 fragment work as expected and that its quenching effect might be inhibited after the linker binding it to the GFP is cut, allowing GFP to recover its fluorescence (Figure 17).
In the case of GFP quenched by REACh2, the results seen do not show an increase in fluorescence when comparing the lysed cells with the non lysed ones as was seen in the mutated M2 approach (Figure 17). The reason for this is the already high fluorescence level of GFP when quenched by REACh2. The explanation for this comes from the fact that REACh2 is not preventing the maturation of the fluorophore as M2 was doing but is absorbing the emitted fluorescence of GFP. Therefore the REACh2 quencher emits fluorescence in a close emission range as GFP as it is a version of YFP. However, if the fluorescence is measured overtime, an increase in GFP fluorescence could be seen (Figure 18).
Lytic mechanisms
Four different lytic mechanisms were designed to be triggered by the antigen, although for the ease of testing in this project L-Arabinose is used to induce the signal in all constructs. In order to assess and evaluate these mechanisms, they were expressed under the inducible pBAD promoter (BBa_I0500) in two different backbones: a high copy number backbone (pSB1C3) and a low copy number backbone (pSB4K5).
Inducible versions of Colicin E7 (BBa_K2387066), T4 Holin (BBa_K2387068), T4 Endolysin (BBa_K2387069) and a double lytic mechanism (T4 Holin + T4 Endolysin) (BBa_K2387079) were tested in E. coli DH5α (Figure 20). Instead of directly quantifying cell lysis, the optical density at 600 nm was used.
Endolysins are phage-encoded peptidoglycan hydrolases (PGHs) which are used by bacteriophages to enzymatically degrade the peptidoglycan (PG) layer of the host bacterium from the inside at the end of their lytic cycle. By breaking the PG layer, an osmotic lysis can be favored leading to cell death. However, host cell lysis is strictly regulated and controlled by the help and action of T4 Holin (BBa_K112000). T4 Holin constitutes a second component of a lysis cassette of phages. Holins oligomerize and create holes in the cytoplasmic membrane allowing the cytoplasm accumulated endolysins to degrade PG substrate [7].
From the assessment it was concluded that T4 Holin and T4 Endolysin + T4 Holin were successfully achieving bacterial cell lysis. T4 Endolysin alone was not able to trigger the lysis of bacterial cells. However, when combined with T4 Holin, the lytic effect achieved was even higher than the one achieved with T4 Holin alone (Figure 21). The observed lytic function of T4 Endolysin when acting together with T4 Holin in the double lysis gene cassette is understandable as it requires the action of T4 Holin to make the initial holes in the membrane [7]. T4 Holin starts the membrane degradation process and after holes are made in the membrane, T4 Endolysin is able to further increase bacterial membrane lysis (Figure 21).
An SOS response operon regulates the expression of LysE7. This SOS response operon is formed by three genes (ceaE7, ceiE7 and celE7) whose products are ColE7, ImE7 and LysE7, respectively [8]. LysE7 can be inhibited by complexing with the immunity protein ImE7. The inducible LysE7 system (BBa_K2387066) codes for a L-Arabinose inducing lysis gene (BBa_K117000) encodes for the lysis protein of 4872 Da in molecular weight in those colicin-producing strains of bacteria. Once this gene becomes activated, two main functions of Colicin E7 system are covered by the lysis protein. The activation of the gene leads to the partial lysis of the host cell membrane. This first function leads then to the lysis of host cell, uncovering the intracellular environment of the cell and thus allowing for LysE7 proteins free diffusion. Moreover, the lysis gene also removes the immunity protein from colicin protein, resulting in the activation of the endonuclease activity of colicin protein. In this case, the detachment of LysE7 from the Immunity protein is triggered, activating this the LysE7 endonuclease activity.
The BBa_K11700 codes for the lysis gene but not for the LysE7 or ImE7 proteins suggesting this that a specific E. coli strain (able to synthesize colicin) might be required. However, it was hypothesized that the only expression of E7 lysis gene (BBa_K117000) by E. coli DH5α , will cause partially lysis. The results indicated that E7 lysis protein might also have function in noncolicin-secreted strain of E. coli as the lysis was fully achieved after inducing the system (Figure 21) with L-Arabinose (BBa_K2387066). Future approaches could study the differences in the lytic effect by comparing the lysis achieved when inserting the pK317 plasmid which contains the entire LysE7 system and the inducible LysE7 system (BBa_K2387066).
Based on the results, it was concluded that already low inductions of L-Arabinose are able to induce a complete lysis of the bacterial cells (Figure 21). Either LysE7 system, T4 Endolysin + T4 Holin system or T4 Holin system alone could be implemented in the robust Mantis module (Figure 3). Based on the simplicity of the Inducible LysE7 system (BBa_K2387066) which only requires one gene to work compared with the double lytic casette (BBa_K2387079), this system (BBa_K2387066) was the chosen one when co-transforming strains in the proteolytic quenching experiments and the Bimolecular Fluorescence recombination.
Robust Mantis module
The final mechanism will be constituted by four modules (Figure 23) expressed by two different BCPs. Both BCPs will express the four modules, being all the modules the same except module 2 which will encode for component 1 in BCP1 and for component 2 in BCP2. In the system, aiiA will be constitutively expressed under no induction preventing quorum sensing to happen. In an induced state (antigen detected or L-Arabinose added to the medium), pBAD is activated leading to the expression of TetR which inhibits the expression of pTet resulting in the downregulation of aiiA. As a result, AHL degradation is decreased and the quorum sensing mechanism becomes active. The activation of the quorum sensing mechanisms leads to both the activation of the lytic mechanism as well as the activation of a positive feedback performed by the diffusion of AHL from cell to cell. In a final state, all the cells in the medium will lysed allowing component A to B to interact and generate a fluorescent signal.
References
- [1] M. Welvaert and Y. Rosseel, “On the definition of signal-to-noise ratio and contrast-to-noise ratio for FMRI data.,” PLoS One, vol. 8, no. 11, p. e77089, 2013.
- [2] W.-L. Ng and B. L. Bassler, “Bacterial quorum-sensing network architectures.,” Annu. Rev. Genet., vol. 43, pp. 197–222, 2009.
- [3] “Characterization of a quorum sensing device for synthetic biology design: Experimental and modeling validation,” Chem. Eng. Sci., vol. 103, pp. 91–99, Nov. 2013.
- [4] B. Pearson, K. H. Lau, W. DeLoache, P. Penumetcha, V. G. Rinker, A. Allen, R. Cool, E. Feeney, J. Igo, T. T. Eckdahl, X. Zhu, M. Parra, K. Muscalino, A. Lewis, A. Gordon, K. Davis, J. Barron, K. Roland, J. L. Poet, L. J. Heyer, A. Malcolm,Campbell, and A. M. Campbell, “Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter,” Interdiscip. Bio Cent., vol. 3, no. 3, pp. 1–8, Jul. 2011.
- [5] S. B. Nicholls, J. Chu, G. Abbruzzese, K. D. Tremblay, and J. A. Hardy, “Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity,” J. Biol. Chem., vol. 286, no. 28, pp. 24977–24986, Jul. 2011.
- [6] S. Ganesan, S. M. Ameer-Beg, T. T. C. Ng, B. Vojnovic, and F. S. Wouters, “A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP.,” Proc. Natl. Acad. Sci. U. S. A., vol. 103, no. 11, pp. 4089–94, Mar. 2006.
- [7] L. H. Burch, L. Zhang, F. G. Chao, H. Xu, and J. W. Drake, “The bacteriophage T4 rapid-lysis genes and their mutational proclivities.,” J. Bacteriol., vol. 193, no. 14, pp. 3537–45, Jul. 2011.
- [8]K.-F. Chak, W.-S. Kuo, f. –. Lu, and R. James, “Cloning and characterization of the LysE7 plasmid,” J. Gen. Microbiol., vol. 137, no. 1, pp. 91–100, Jan. 1991.
