Many bacterial pathogens have developed an array of effector proteins to rewire host signaling networks and down-regulate the immune response 2
). Some effectors mimic host activities, such as the Yersinia pestis
effector YopH, which is a highly active phosphotyrosine phosphatase3
. Other effectors utilize unusual mechanisms, such as the Shigella flexneri
OspF protein, which irreversibly inactivates MAP kinases by catalyzing a β-elimination reaction that removes the hydroxyl group of the key phospho-threonine side chain4
Bacterial effector OspF can block selective MAP kinase pathways in yeast
MAPK pathways play a central role in diverse eukaryotic responses, ranging from immune response to cell fate decisions5,6
. Thus, the ability to tune MAPK response would facilitate engineering cells for diverse therapeutic and biotechnological applications7,8
. Recent work has shown that MAPK signaling dynamics in yeast can be reshaped with synthetic feedback loops that involve controlled expression and targeting of pathway modulators to appropriate signaling complexes9
. Identifying effective pathway modulators is challenging, and thus we hypothesized that pathogen effector proteins may have untapped utility as components for predictably and systematically engineering signaling pathways. Here, we use the effector proteins OspF and YopH to modulate kinase signaling pathways in yeast and in human primary T cells.
We first introduced OspF into yeast. As reported10
, overexpression of OspF led to growth inhibition under standard conditions, hyperosmotic stress conditions (
), and cell wall damaging conditions (Supplementary Fig. 1a
). OspF contains a canonical docking peptide at its N-terminus that allows it to bind multiple MAPK's in yeast11
. We found that expression of an OspF mutant lacking its native docking peptide (ΔN-OspF) yielded normal growth behavior under all conditions (, Supplementary Fig. 1a
). Next we tested whether ΔN-OspF could be redirected to a specific pathway by tagging the protein with a leucine zipper heterodimerization motif, and fusing the complementary interacting motif to Pbs2, the scaffold protein that organizes the osmolarity MAPK pathway. This targeted version of ΔN-OspF only displayed a growth defect under high salt conditions, showing that OspF activity could be engineered to inhibit a specific MAPK (
To further explore re-targeting OspF to specific pathways, we engineered yeast strains in which ΔN-OspF was selectively targeted to either the osmolarity MAPK complex or the mating MAPK complex (by targeting it to the mating pathway scaffold protein, Ste5) (). Targeting of ΔN-OspF to the Pbs2 inhibited the osmolarity response but not the mating response. Conversely, when ΔN-OspF was targeted to Ste5, only the mating response was inhibited. Thus, the inhibitory activity of this effector could be selectively aimed at one of several MAPK pathways in the same cell.
One of the unique aspects of OspF is that it catalyzes an irreversible inactivation of MAPKs (unlike reversible dephosphorylation by a phosphatase). Thus MAPK activity can only be restored through new protein synthesis, which has a much slower timescale than re-phosphorylation (). This longer timescale would be expected to lead to an extended refractory period after OspF action, during which the targeted MAPK pathway could no longer respond to subsequent stimuli.
Tuning frequency dependent response of yeast osmolarity pathway using synthetic OspF feedback loop
Computational simulations indicated that a long refractory period could result in significant changes to the frequency-dependent behavior of pathway response (Supplementary Fig. 2
). There is growing evidence that cells use frequency modulation of diverse molecular events to encode and transmit information12,13
. Our models indicated that with a negative feedback loop (i.e. MAPK activity induced expression of OspF) to “filter out” pathway activation, pathway output would be dampened when input periods are long enough to accumulate significant amounts of the negative effector but shorter than the refractory period (Supplementary Fig. 2
To test if OspF could be used to filter frequency dependent inputs, we constructed a synthetic negative feedback loop in the yeast osmo-response pathway by expressing OspF targeted to the osmo-response signaling complex (ΔN-OspF-zipper) from the Hog1 responsive promoter, pSTL1 ()
. As a comparison, we also engineered an analogous synthetic feedback loop using a reversible Hog1 MAPK inhibitory protein -- the yeast MAPK phosphatase, PTP2. Phospho-Hog1 translocation to the nucleus was used as a fast-timescale output reporter (Supplementary Fig. 3
. To measure integrated output over a longer timescale, we also measured a slower timescale transcriptional reporter -- expression of mCherry from the pSTL1
We found that the OspF-mediated negative feedback circuit altered the osmostress pathway response to intermediate frequency stimulation, but not to continuous stress or to high frequency stimulation (, Supplementary Fig. 4a
). Furthermore, in the course of stimulating cells with pulses of KCl of varying length, we discovered that an input period of ~16 minutes (intermediate frequency) leads to highly divergent transcriptional responses (
). Examination of Hog1 nuclear import in the OspF feedback strain shows that after 3 pulses, the amount of Hog1 competent for nuclear localization has decreased to near zero, consistent with a model in which - in this timeframe - there is sufficient OspF to inactivate the bulk of the Hog1 population and to now render the cells refractory to further pulses of stimulation. The cells containing a PTP2 negative feedback circuit do not show this dramatic filtering at this frequency. With higher frequency stimulation (2 min period), the three strains also do not significantly differ from each other in response, presumably because there is inadequate activation time with each pulse to build up a sufficient concentration of effector17
A broad frequency dependence analysis shows that the wild-type osmo-response pathway functions as a band pass filter, with maximal response at intermediate frequencies, while the engineered pathway more closely resembles a low pass filter, with maximal responses at low frequency (
). These distinct frequency filtering behaviors fit those predicted by computational simulations (Supplementary Fig. 2d
We then sought to test whether these bacterial effectors could be used to rewire signaling in immune cells. Human T cells are an attractive synthetic biology platform because they can be isolated from patients, genetically engineered ex vivo
, and then transferred back into patients to treat cancer and chronic infection18,19
. While promising, the therapeutic application of engineered T cells carry risks of adverse side effects including inadvertent autoimmune-like attack of off-target host tissues20,21
. Thus mechanisms to control the specificity, amplitude and timing of T cell function are critical to balance therapeutic action against off-target toxicity.
Both OspF and YopH can modify the T cell receptor (TCR) pathway (
). OspF inactivates the MAPK ERK, which is a central component of TCR signaling4,22
, while YopH dephosphorylates phospho-tyrosine, including the T cell scaffold proteins LAT and SLP-7623
. Constitutive expression of YopH and OspF in Jurkat
T cells leads to severe inhibition of TCR activation, as measured by an NFAT transcriptional reporter24
) (as well as other reporters of T cell activation –Supplementary Fig. 5a)
. Expression of the catalytic dead versions of YopH and OspF had no effect on the TCR activation (Supplementary Fig. 5b
). In addition, we showed that these two effectors clearly target distinct steps of the T cell activation pathway, since induction of the T cells with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin, which activates the T cell downstream of the PLCγ1-LAT/SLP76 dependent response, bypassed YopH inhibition25
, but was sensitive to OspF inhibition (, Supplementary Fig. 5a
). Thus, distinct pathogen effector proteins can be used to block this pathway at particular steps, much like a specific small molecule inhibitor.
OspF can be used to precisely control T cell activation amplitude and duration in Jurkat T cells
Given the ability of OspF and YopH to modulate T cell responses, we sought to use these proteins to build circuits that could, in principle, improve the safety of therapeutic T cells. In adoptive T cell therapy, a challenge is to limit over-activation or off-target activation of T cells that could lead to killing of host cells or to cytokine storm – a life-threatening immune response. One approach is to incorporate a safety “kill switch”26-28
into the T cells, such as the herpes simplex virus thymidine kinase (HSV TK) gene. This protein converts the pro-drug ganciclovir into an inhibitor of replication, thus killing cells expressing the gene. While HSV TK is currently being tested in a phase III clinical trial for the treatment of graft vs. host disease in bone marrow transplants, this strategy irreversibly destroys the engineered, adoptively transferred cells29
. Thus instead of killing the engineered cells, we sought to design circuits that would limit the amplitude of the T cell response or to temporarily pause T cell activity.
We first tested whether bacterial effectors could be used to limit the response amplitude of Jurkat
T cells. Negative feedback loops can act to limit the maximal amplitude of a response30
, so we engineered a library of negative feedback loops in which the OspF and YopH were expressed from a series of TCR responsive promoters of varying strength (AP1 and NFAT) (, Supplementary Fig. 7
). For further tuning of feedback parameters, we also tagged effectors with degradation sequences (PEST motif) that reduce half-life of the effectors. This series of negative feedback loops led to controlled reduction of the maximal response amplitude of T cell activation ()
. Moreover, the amplitude could be tuned systematically by varying feedback promoter strength and effector stability (Supplementary Fig. 7b
). For example, expression of OspF from the strong feedback (pNFAT
) promoter leads to a very low maximal response amplitude, but this effect could be systematically tuned by destabilizing the OspF effector with a PEST tag.
We also tested whether the bacterial effectors could be used to construct pause switches, which could transiently and reversibly, disable T cells. We placed the effectors under the control of a tetracycline inducible promoter (pTRE
), which allowed external control of the timing of effector expression with the addition of doxycycline. Effectors were fused to a destabilization domain so that they would be rapidly degraded once doxycycline is removed. Using this system, we first showed that transient expression of bacterial effectors can inhibit TCR signaling after the pathway is activated in Jurkat
T cells (
). TCR signaling can be inhibited up to 6 hours after activation using this approach (Supplementary Fig. 8b
). Finally, we showed that engineered T cells can be subjected to cycles of TCR activation, pausing with a short period of induced expression of the bacterial effector, and then reactivated after this pause (Supplementary Fig. 8c
We then tested the pause switch in a clinically important cell type for adoptive immunotherapy - primary human CD4+ T cells (in contrast to the Jurkat
T cell line, which does not require cytokine or TCR activation to stimulate proliferation). We showed that when OspF is induced by the addition of doxycycline, both IL-2 release and cell proliferation were inhibited in a dose-dependent manner (
). Activation of the TCR by anti-CD3/CD28 and antigen presenting cells can also be inhibited by expression of OspF (Supplementary Fig. 9a
). Moreover, after dox is removed, IL-2 release and cell division recovers within 6-18 hours (
). Sustained dox exposure can inhibit T cell activity over the course of several days (
) without having any significant effect on cell viability (Supplementary Fig. 9c
). Thus this work provides a proof of principle for the design of a simple “pause” switch that could allow external control over the timing and level of T cell activation and cytokine release, in order to minimize adverse events associated with adoptive immunotherapy such as cytokine storm.
OspF can be used as a synthetic pause switch to control human primary CD4+ T cell activation
Most work on bacterial pathogen effector proteins has the long-term aim of neutralizing the pathogens’ infectious capabilities. We have shown, however, that bacterial effectors can also be valuable synthetic biology tools, because of their unique biochemical properties. We have employed bacterial effectors to modulate MAPK signaling in yeast to generate novel time-dependent dynamics. We also showed that bacterial effectors can be used to flexibly tune human T cell receptor signaling dynamics, with potential application as safety switches for adoptive immunotherapy. The vast array of bacterial pathogen effector proteins, beyond those studied here, holds promise as a rich and important source of parts for the cellular engineering toolkit.