Cells have evolved different ways to cope with bacterial toxins. Attack on cell membranes by PFTs is a conserved mechanism and due to the widespread nature of PFTs, many of the host cellular responses overlap. Yet, some remain cell and toxin specific, additionally dependent on toxin concentration and time of exposure. When toxin concentration is high, cells die quickly as a result of rapid and irreversible membrane damage. However when the dose is sub-lytic, cells often produce signals promoting survival and repair.
Research has shown that one of the conserved responses to various PFTs is the phosphorylation of p38 mitogen-activated protein kinase (MAPK) (Ratner et al., 2006).
The MAPK protein family comprises highly conserved serine/threonine kinases that are activated in response to extracellular signals and stresses, like osmotic shock, UV radiation, heat shock, ischemia, or DNA damage. MAP kinases can be classified into four subgroups: ERK (extracellular signal regulated kinase), JNK/SAPK (c-Jun N-terminal kinases or stress activated protein kinases), BMK1 (ERK/big MAP kinase 1) and p38.
MAP kinases are activated by MAP kinase kinases (MKKs), with MKK3 and MKK6 being known p38 activators (Zarubin and Han, 2005). p38 is present in four isoforms: α, β, ٧, and δ, but only α and β are ubiquitously expressed. Following stress, cytosolic p38 upon activation is translocated into the nucleus to access its nuclear substrates (Gong et al., 2010). When activated, p38 phosphorylates a broad range of proteins. The downstream targets may be as many as 200-300 and include: cytokines (TNF-α, interleukins), nuclear transcription factors and extracellular receptors (Zarubin and Han, 2005).
Phosphorylation of p38 was previously reported for: Streptococcus aureus streptolysin O, Gardnerella vaginalis vanginolysin, B. anthracis anthrolysin O, S.
pneumonia pneumolysin, Staphylococcus aureus α-hemolysin, Aeromonas hydrophyla proaerolysin, Listeriolysin O, Vibrio cholera cytolysin, Escherichia coli hemolysin A (Fickl et al., 2005, Gelber et al., 2008, Huffman et al., 2004, Kloft et al., 2009, Ratner et al., 2006, Gonzalez et al., 2011b). p38 pathway was activated in experiments involving C.
elegans treated with low doses of Cry5B toxin (Huffman et al., 2004). Activation of p38 MAPK was also shown in lepidopteran M. sexta and dipteran A. aegypti exposed to Cry1Ab and Cry11Aa respectively (Cancino-Rodezno et al., 2010).
In the studies with C. elegans, M. sexta and A. aegypti silencing of p38 MAPK pathway resulted in hypersensitivity to Cry toxins (Cancino-Rodezno et al., 2010, Huffman et al., 2004), suggesting a role in cellular defence. However, published data are contrasting. In a study by Husmann et al., it has been shown using the same target cell line that inhibition of p38 MAPK pathway impeded cellular recovery in cells exposed to S. aureus α toxin but not in cells exposed to streptolysin O (Husmann et al., 2006). Also, a specific p38 inhibitor prevented cell death in neural cells exposed to pneumolysin, implicating a role of p38 in facilitating apoptosis (Stringaris et al., 2002).
Therefore, the p38 MAPK pathway - depending on a cell and toxin type - may exert different biological effects. Interestingly, a recent study by Guo et al. showed that MAPK genes can trans-regulate expression levels of ALP and ABCC genes (encoding Cry toxin receptors) in Bt resistant P. xylostella larvae. The authors suggested that MAPK signalling pathway could play an important role in insect resistance by altering expression levels of Cry toxin receptors (Guo et al., 2015).
The unfolded protein response (UPR) of the endoplasmic reticulum (ER) was identified as one of the downstream targets of p38 MAPK pathway in C. elegans (Bischof et al., 2008, Huffman et al., 2004). Under stressful conditions, like nutrient deprivation, disruption of Ca2+ or redox homeostasis, protein folding in the ER may be impaired, leading to accumulation of misfolded or unfolded proteins. Cells have evolved to deal with this threat by activating pathways that on the transcriptional and translational levels lead to a decrease in protein translation, degradation of misfolded proteins, and promotion of correct folding (Zhang and Kaufman, 2004). Loss of this pathway was demonstrated to cause hypersensitivity of C. elegans and mammalian cells to the attack of Cry5B and aerolysin, respectively and was functionally linked to upstream components of p38 pathway (Bischof et al., 2008).
It was shown that in addition to the p38 pathway some PFTs like proaerolysin and listeriolysin O specifically triggered activation of the ERK pathway. It was suggested that these two pathways helped to recover ion homeostasis as inhibition of either p38 or ERK by specific inhibitors significantly diminished the recovery of intracellular K+ (Gonzalez et al., 2011b). Furthermore JNK MAPK was activated in experiments involving C. elegans and Cry5B toxin (Huffman et al., 2004). JNK MAPK was later shown to protect nematode cells from both small pore (Cry5B) and large pore (streptolysin O) inducing PFTs. Based on a genome-wide RNAi screen results, JNK and p38 MAPKs were suggested to play a paramount role in cellular protection of C. elegans against Cry5B (Kao et al., 2011).
Efflux of intracellular potassium, arising as a consequence of transmembrane pores, was shown to mediate various cellular responses. It was demonstrated that
potassium efflux promoted p38 activation after exposure to S. aureus α-toxin, V.
cholerae cytolysin, streptolysin O, and E. coli hemolysin A (Kloft et al., 2009). Potassium efflux induced activation of the inflammasome and caspase-1 in aerolysin treated Chinese hamster ovary cells. Because caspase-1 positively regulates the Sterol Regulatory Element Binding Proteins (SREBPs) involved in membrane biogenesis, it is thought that this pathway promotes cell survival by assisting in membrane repair (Gurcel et al., 2006). Another consequence of potassium efflux is autophagy, which was observed in aerolysin treated HT29 cells. Although it did not restore ion homeostasis or help in membrane repair, its function was predicted to benefit cells in a quiescent state, where recycling cellular molecules and organelles via autophagy would help surviving low energy consumption state following a PFT attack (Gonzalez et al., 2011b). Autophagy was also observed in cells exposed to S. aureus α-toxin, V.
cholerae cytolysin, E. coli haemolysin and streptolysin O. A similar conclusion was drawn, that autophagy promotes energy maintenance during recovery in cells affected by pore formation (Kloft et al., 2010). A significant increase in lipid droplet formation was observed in HeLa cells following cell treatment with: proaerolysin, listeriolysin O, streptolysin O, S. aureus α-haemolysin. Lipid droplets were beneficial to cells and their formation was abolished by ERK inhibitor in aerolysin treated cells. The authors suggested that - along with an arrest in protein synthesis - storing energy in lipid droplets represents a mechanism that puts the cells into a low energy consumption mode (Gonzalez et al., 2011b).
Hypoxia (low oxygen levels) and hypoxia response was identified as another protective mechanism in C. elegans against Cry21A, Cry5B and V. cholerae cytolysin.
Loss of function of the main hypoxia response effector HIF-1 (hypoxia inducible factor
1) led to hypersensitivity of previously resistant nematodes. Moreover, the hypoxia pathway was shown to work upstream of UPR. The role of low amount of oxygen in pathogenicity and the role of UPR being the downstream effector of hypoxia response was suggested (Bellier et al., 2009).
Additionally, activation of the nuclear factor kappa B (NF-κB) pathway and production of inflammatory molecules was observed in immune cells exposed to the following PFTs: S. aureus α-toxin, aerolysin, pneumolysin, listeriolysin O, streptolysin O (Aroian and van der Goot, 2007). Antimicrobial peptides were released in Spodoptera larvae in response to Cry1Ca and Vip3 (Herrero et al., 2016). In other studies extensive cell vacuolization was noted after treatment with aerolysin or S. marcescens hemolysin (Abrami et al., 1998, Hertle et al., 1999). Listeriolysin O, perfringolysin O and pneumolysin caused Ubc9 degradation, decreasing the levels of protein SUMOylation and increasing at the same time infection efficiency. De-SUMOylation was shown to be independent of the calcium influx or MAPKs (Ribet et al., 2010).
Another pathway activated by PFTs is apoptosis, often mediated by uncontrolled influx of Ca2+ ions – a known apoptosis inducer. An apoptotic pathway was triggered by: S. aureus α-toxin in epithelial cells (Imre et al., 2012) and lymphocytes (Jonas et al., 1994), by listeriolysin in dendric cells (Guzmán et al., 1996), aerolysin in lymphocytes (Nelson et al., 1999), PS-2 like protein in HepG2 (Brasseur et al., 2015b), and PS-1 in HeLa cells (Katayama et al., 2007), however the pore forming nature of PS-1 (from strain A1190) has not been demonstrated.
Other mechanisms attributed to PFT-induced Ca2+ influx is a repair process based on exo- and endocytosis. The studies suggest that after plasma membrane injury
and calcium entry, lysosomal enzyme acid sphingomyelinase is released during exocytosis of lysosomes, where it converts sphingomyelin into ceramide. Ceramides form microdomains which in turn facilitate endosome formation. Endosomes internalise transmembrane pores by disruption of actin cytoskeleton and target them for degradation contributing to membrane repair. The model is based on experiments with cells permeabilized with streptolysin O, but similar processes were observed in mechanically injured cells (Idone et al., 2008, Tam et al., 2010). Vesicle trafficking pathways were also noted for S. aureus α-toxin, Cry5B and V. cholerae cytolysin;
reviewed by Los et al. (Los et al., 2013).
Cellular processes triggered by PFTs are graphically presented in Figure 14.
Figure 14 Cellular responses to PFTs.
Schematic diagram represents signalling pathways and cellular mechanisms triggered by pore formation.
In summary, despite the deceptively simple mechanism of action of PFTs, cellular responses upon pore formation remain complex and not fully characterised. Disrupting protective barrier of plasma membrane initiates numerous pathways arising mostly as a result of uncontrolled flow of ions.