Published On: Thu, Apr 18th, 2019

Transcriptomic analysis of Spodoptera frugiperda Sf9 cells resistant to Bacillus thuringiensis Cry1Ca toxin reveals that extracellular Ca2+, Mg2+ and production of cAMP are involved in toxicity [RESEARCH ARTICLE]


In this report, we describe Sf9 cells resistant to the Cry1Ca toxin obtained after a selection program. We tested Sf9wt cells for their sensitivity to another Cry toxin (Cry1Ab) at toxin concentrations up to 100 mg/l in the same time conditions as with Cry1Ca and confirmed that they were almost ineffective on these cells, suggesting a peculiar mode of action for Cry1Ca. The LD50 measured with these cells that are specifically sensitive to Cry1Ca were in the same concentration range to what was observed in previous studies (Kwa et al., 1998). Therefore, we started our selection program using LD50 or LD80 Cry1Ca doses and we obtained cell lines with an increasing resistance factor, named Sf9-LD50 and Sf9-LD80.

While the so-called ‘mode 1 resistance’, the most frequently mechanism of Cry toxins resistance, results in toxin receptor binding defects (Gahan et al., 2001; Jurat-Fuentes et al., 2011; Morin et al., 2003) we observed no detectable difference in Cry1Ca binding to the plasma membrane of Sf9 sensitive or resistant cells (Fig. 2). It is important to note that this approach using immuno-fluorescence microscopy could not rule out an undetectable small diminution of receptor affinity or loss of a secondary receptor, which could be sufficient to perturb toxin toxicity onto Sf9-LD80 cells. However, our binding results are strongly correlated with our transcriptomic analysis. Indeed, we did not detect any significant variation of expression of previously described and widely accepted plasma membrane toxin receptor as CADR or APN. All these results strongly suggest that Cry1Ca resistance observed in the Sf9-LD50 or Sf9-LD80 cells is probably not caused by loss or alteration of plasma membrane binding.

Use of differential transcriptomic analysis to understand resistance mechanisms developed by insects to counteract toxicity is a challenging task. It generates a large amount of data and shows that cellular responses are likely diverse and affect many cellular processes as observed in Fig. 3A and in other studies (Nanoth Vellichirammal et al., 2015; Canton et al., 2015). Therefore, we decided to focus on genes differentially expressed in the same way in both selected resistant cells, as they may be key players in resistance processes (Table 1). Hence, we highlighted genes involved in cation-dependent signalling pathways or cation homeostasis. For example, calmodulin-binding transcription activator (CAMTA) is activated after a rise in intracellular calcium concentration ([Ca2+]i). Both Ca2+ and calmodulin (Cam) bind to CAMTA, which translocates into the nucleus and activates its target genes (Finkler et al., 2007). Like CAMTA, the predicted Calcineurin B homologue was differentially expressed in both selected resistant cell lines. Calcineurin is a calcium-activated protein phosphatase that participates in diverse biological functions through interaction with various partners (Li et al., 2011). It is composed of two subunits, a catalytic subunit (CnA) and a regulatory subunit (CnB) containing Ca2+ binding sites. Increased intracellular Ca2+ allows binding of the cations to the regulatory sites, which initiates a series of conformational changes and the activation of calcineurin (Yang and Klee, 2000). Thus, in both examples a rise in [Ca2+]i appears as a crucial event in the activation of our potential key players in Cry1Ca resistance.

Earlier studies showed the involvement of divalent cations in Cry activity. Cry1Ca toxicity increases with the presence of calcium in a dose-dependent manner and the toxin induces a rapid elevation of intracellular calcium in treated cells (Monette et al., 1997). We followed intoxication of Sf9 sensitive cells in the presence of EDTA or EGTA and observed a protective effect for both ion chelators (Fig. 4). Similar to the observations for Cry1Ab binding onto S5 cells (Zhang et al., 2005), neither EDTA nor EGTA affected the interaction of Cry1Ca with the plasma membrane of Sf9wt or Sf9-LD80 cell lines (Fig. 5). The protection obtained with the Ca2+ chelator EGTA was not the result of a loss of toxin binding capacity to Sf9wt cells and could be the result of the failure of the cells to increase their [Ca2+]i in response to the toxin. If pore formation or the signal transduction pathway are the two main models of toxin action leading to cell death, a third model was proposed in which formation of toxin oligomers followed by pore formation due to oligomer insertion in cell membrane could participate in establishment of a signalling pathway (Jurat-Fuentes and Adang, 2006). Pores composed of Cry1Ca toxin do not induce only potassium-selective channels but increase the permeability of the plasma membrane of Sf9 cells to numerous solutes (Guihard et al., 2000; Villalon et al., 1998). The toxin has been shown to trigger an intracellular calcium surge in Sf9 cells and is still toxic when Sf9 cells are incubated in media containing barium, a divalent cation used as potassium channel blocker (Monette et al., 1997; Schwartz et al., 1991). So, the protective effect of calcium chelators could result from blocking Ca2+ entry in the cell through poorly selective channels obtained by toxin membrane insertion and the incapacity of this calcium surge to contribute to a Ca2+-dependent signal pathway, leading to cell death. When we used the Mg2+ chelator EDTA, we observed a stronger decrease of Cry1Ca toxicity compared to protection obtained with EGTA (Fig. 4). Like Zhang and colleagues, who observed that Cry1Ab still binds to the plasma membrane of High five cells in the presence of EDTA (Zhang et al., 2005), we saw no binding loss of Cry1Ca to Sf9wt sensitive or Sf9-LD80 resistant cells when incubated with 5 mM EDTA (Fig. 5). So, as described for Cry1Ab, depletion of Mg2+ using EDTA could prevent Cry1Ca-induced cellular response and stop the establishment of a Mg2+-dependent intracellular pathway critical to promote cell death (Zhang et al., 2006). Since Mg2+ is essential for adenylyl cyclase (AC) to catalyse the formation of cAMP (Tesmer et al., 1999; Pieroni et al., 1995) and since AC activity has been shown by Zhang et al. (2005) to participate in Cry1Ab toxicity, we monitored cAMP production after Sf9 cell intoxication with Cry1Ca (Fig. 6). Treatment of Sf9 sensitive cells with forskolin, used as positive control, produced a 38-fold increase of intracellular [cAMP] while Cry1Ca presented an even stronger stimulation of cAMP production. [cAMP]i increase after toxin treatment was not affected by the presence of EDTA, whereas the cAMP increase was reduced two fold in Sf9wt cells stimulated with forskolin plus EDTA as compared to forskolin alone. In contrast, EGTA affected only Cry1Ca-induced cAMP production. These results suggest the importance of Ca2+ ions in toxin action via formation of cAMP. Since numerous AC activities have be shown to be regulated by [Ca2+]i (Willoughby and Cooper, 2007), Cry1Ca, after formation of pores in the Sf9 membrane, could activate AC through entry in cells of extracellular Ca2+. Näsman’s work showed that applying 1 mM EGTA to Sf9 cell media blocked the raise in [Ca2+]i and drastically reduced the increase of cAMP levels after stimulation with octopamine (Näsman et al., 2002). Thus, the increase in cAMP levels observed in our cells treated with Cry1Ca could be due to the activation of calcium-dependent AC, with EGTA protecting cells by preventing the increase in calcium and AC activation.

If EGTA was the most efficient to reduce cAMP elevation in response to Cry1Ca stimulation, EDTA which preferentially binds Mg2+ appeared the most protective against Cry1Ca toxicity. This effect of EDTA could be explained, on one hand, by the necessity of Mg2+ for catalytic activity of ACs. Indeed, Mg2+ which binds to the active domain of ACs is essential for their activity and therefore for the synthesis of cAMP (Pieroni et al., 1995; Tesmer et al., 1999). On the other hand, Mg2+ could have a more central role than Ca2+ in participating in the activation of numerous proposed other partner proteins involved in toxin-induced signalling pathway, beginning with heterotrimeric G protein activation (Sprang, 1997). However, while in the absence of Mg2+ we observed an effect on cAMP production by forskolin stimulation, EDTA did not seem to prevent the increase of cAMP after the action of the toxin. Cry1Ca might be triggering the production of cAMP by activation of an Mg2+-independent AC whose regulation requires Ca2+ (Braun et al., 1977; Litvin et al., 2003).

It has been proposed that the mechanism of action of the toxin can be independent of cAMP production (Knowles and Farndale, 1988; Portugal et al., 2017). Portugal and colleagues clearly demonstrated that both Cry1Ab and Cry1Ac toxins triggered cell death in CF1 cells via pore formation activity. While they saw neither the activation of PKA nor an increase in intracellular cAMP concentration during Cry1Ab or Cry1Ac intoxication, in our hands Cry1Ca triggered an increase of the [cAMP]i in favour of a peculiar mode of action of this toxin (Fig. 6). Moreover, we observed (Fig. 7) that the Sf9-LD80 resistant cell line was unable to elevate [cAMP]i in response to Cry1Ca treatment. Thus, the [cAMP]i increase seems to be an essential element to the toxicity induced by Cry1Ca but appeared insufficient since forskolin, although it may increase cAMP levels, presents no toxicity onto Sf9 cells. While Zhang and colleagues observed a similar lack of forskolin toxicity, Cry1Ab cytotoxic effects were potentialised when sensitive cells were pre-treated with forskolin (Zhang et al., 2006). In light of these observations, they proposed that if cAMP is a key player in toxin effect through activation of PKA, specific PKA-dependent effector(s) mediating downstream cell death activity might be stimulated by Cry1Ab but not by forskolin. All these observations raise the question of how specificity is maintained in this cAMP/PKA system, activated by both forskolin and toxins (Cry1Ab and Cry1Ca) but where only Bt toxins are capable of generating cellular events leading to cell death. Since we identified Ca2+ as another important molecule involved in the Cry1Ca mode of action, we might think that through toxin pore formation, Ca2+ entry in the cell would stimulates Ca2+-dependent events interacting with the PKA pathway. The protein kinase C (PKC) pathway could be such Ca2+-dependent event which, together with the PKA activation, would allow the toxin to perform its cytotoxic effect. PKC is a large superfamily of protein kinases comprising numerous members activated by elevation of intracellular Ca2+ concentration (Webb et al., 2000). Coordinated activity and cross-talk between the PKA and PKC pathways have been shown in multiple cellular processes (Robinson-White and Stratakis, 2002). It might be possible that such a scenario was encountered in our intoxicated cells where forskolin was not sufficient, and Ca2+ was necessary, to promote cell death using Cry1Ca. It is worth noting that since the intracellular responses to Cry1 toxins studied using cell lines in culture remain controversial, this scenario will have to be validated in larvae of target insects. Finally, we observed in both selected resistant cell lines the upregulation of a gene described as the homologous of S. exigua activated C kinase receptor (RACK1). This PKC-activated binding protein (Mochly-Rosen, 1995) has been shown as a specific binding partner of a cAMP-phosphodiesterase (PDE) and was proposed as a potential point of cross-talk between the cAMP and PKC signalling pathways (Bird et al., 2010; Yarwood et al., 1999). RACK1 and its partners could, after intoxication by Cry1Ca, be the node of convergence of the two signalling pathways mentioned above.

In summary, Sf9-LD80 cells, which were the most resistant to Cry1Ca toxicity, were affected in cAMP production. Furthermore, our results have shown the importance of Ca2+ and Mg2+ ions in the toxicity of Cry1Ca. These ions may participate in different ways to the establishment of a cAMP-dependent signalling pathway following the binding of the toxin to the membrane of Sf9 cells. Both the signalling model and the pore forming model may contribute to Cry1Ca toxicity as was observed in the third model of toxin action proposed by Jurat-Fuentes and colleagues for Cry1A toxin, or more recently for the pore-forming α-Toxin from Clostridium septicum (Chakravorty et al., 2015; Jurat-Fuentes and Adang 2006). At low concentrations, Cry1Ca insertion in the plasma membrane could create pores, allowing Ca2+ entry in the cell and resulting in the activation of a cAMP-dependent cell death pathway. Future studies focusing on the identification and characterization of proteins involved in cAMP turnover, such as PDEs and their regulators, may identify new resistance factors, and contribute to a better understanding of the molecular mode of action of Cry1Ca and insect resistance mechanisms.

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