In this study, we have unveiled a signaling cascade activated by several bacteria during infection that links K+
efflux to dePH3. We had previously shown that LLO and other CDC toxins induce dePH3 but had not identified the underlying mechanism (11
). Here, we first showed that aerolysin, which is known to induce K+
efflux, also dephosphorylates H3. We then demonstrated that LLO pores are also permeable to K+
ions and that blocking either pore formation or K+
efflux abrogated LLO-dependent dePH3. We have also demonstrated that LLO-induced K+
efflux is a signal activating the inflammasome during L. monocytogenes
infection and that this cascade is unlinked to dePH3. Therefore, K+
efflux is a potent signal, induced by different bacteria during infection, which triggers at least two separate pathways (see model in B).
Our results unexpectedly show that aerolysin and CDCs, which have distinct modes of action and, most importantly, form different-size pores, have the same effect on phosphorylated H3. We had previously reported that cells treated with LLO (6 nM for 20 min) modulate the transcription of approximately 200 genes that correlated with changes in modified histones at their promoter (11
). Thus, only a specific subset of genes shows histone modifications. That aerolysin also induces dePH3 through a K+
efflux-dependent mechanism could suggest that a similar transcriptional response would be imposed. Interestingly, the genes identified as modulated upon aerolysin treatment were not modulated upon LLO treatment (10
), suggesting that a level of specificity exists, although its basis is unknown. Furthermore, although K+
efflux is sufficient to trigger dePH3, the decrease observed upon valinomycin treatment is not as important as that obtained upon LLO treatment, suggesting that LLO may use another factor to trigger a full response. Therefore, although K+
efflux can be generated under different conditions, the effects on histone H3 are not the same for all conditions, and specificity appears to be imposed by unidentified factors.
efflux has not been previously linked to histone modifications. We hypothesize that similarly to Ca2+
signaling, an intracellular K+
sensor/receptor is probably involved in histone modifications (see model in B). Of note, the crystal structures of several histone deacetylases identified a K+
ion in their structure (5
). Furthermore, K+
regulates the activity of histone deacetylase 8, rendering this enzyme's activity sensitive to changes in the cellular K+
). Interestingly, we have shown that LLO-mediated dePH3 correlates with a deacetylation of histone H4, and therefore, the two events could be linked (11
). Therefore, histone deacetylases, which modify many intracellular proteins, might be important K+
Our results show that LLO-induced K+
efflux is a signal that contributes to inflammasome activation during infection with L. monocytogenes
. It has been suggested that LLO activation of caspase-1 is due to bacterial release from the vacuole exposing the bacteria to intracellular sensors of the inflammasome (7
). However, we show here that the inflammasome may be activated prior to listerial invasion of cells, through LLO pore formation at the plasma membrane and K+
efflux. Our findings could explain conflicting published findings on which inflammasome sensor, NLP3 or AIM2, is involved in responding to L. monocytogenes
). Indeed, in light of what we show here, we propose that several waves of inflammasome activation occur, depending on the signal sensed. Extracellular L. monocytogenes
secreting LLO would activate the NLP3 inflammasome (a sensor of membrane damage), and once bacteria have reached the host cytosol, the AIM2 inflammasome would be solicited (a sensor of foreign DNA). The implications of such a hypothesis are 2-fold. First, since LLO is a secreted and diffusible factor, a large number of cells may sense and respond to the “LLO signal,” compared to cells that are infected with L. monocytogenes
. Second, cells that are responding only to the extracellular “LLO signal” might exhibit a different immune response than those infected with intracellular bacteria.
In this study, we have reinforced two important aspects of LLO's mechanism of action which are still poorly documented and understood. First, we demonstrate that inflammasome activation and histone modifications occur through the action of LLO on the plasma membrane. Although studies have shown that LLO's maximal activity occurs at acidic pH, which are the conditions found inside the internalization vacuole (29
), we clearly see pore formation activity during infection prior to bacterial invasion or with purified LLO added to the culture supernatant. In agreement with these observations, many reports show the activity of LLO from the outside the cell (15
), suggesting that LLO acts in a manner similar to that of other CDCs secreted by extracellular bacteria. Second, we show that the commonly used method of preincubating LLO with cholesterol to block pore formation does not prevent the formation of small ion-permeable pores. We do see that large pores, such as those that release LDH, are blocked by this technique; however, we show here that small pores, such as those permeable to ions, are still formed. We observe that this is the case even if we use a saturating amount of cholesterol (15-fold molar excess compared to LLO). In fact, small pores have been observed in the membrane of L. monocytogenes
-containing vacuoles (30
). What cholesterol is doing at the molecular level to limit the action of LLO remains unknown and should be considered as inhibiting only large pores.
In conclusion, our results define K+ efflux as occurring during infection with L. monocytogenes and as a signal leading to caspase-1 activation and dePH3. As schematically represented in B, histone modifications and inflammasome activation, because caspase-1 is not required for dePH3, are two independent pathways. Since none of the components of the inflammasome have been shown to have a link with histone modifications, it is unlikely, although not completely ruled out, that other inflammasome components besides caspase-1 are involved in histone modifications. What arises from our data, as shown in our model, is that a certain degree of specificity must occur in the host cell in response to K+ efflux; this specificity could be conferred by an intracellular K+ sensor. We highlight here a novel signaling cascade triggered by K+ efflux and leading to histone modifications whose cellular components contributing to specificity remain to be uncovered.