INTRODUCTION
Bacterial infections are a leading cause of morbidity and mortality worldwide, and bacteria can cause infections in nearly all host tissues. Furthermore, health care-associated urinary tract infections, pneumonia, skin and soft tissue infections, invasive bloodstream infections, and surgical-wound infections are increasingly common (1, 2). The usual method of treating bacterial infections is by local or systemic administration of broad-spectrum antibiotics. Excessive use of antibiotics is, however, common practice in many countries and is a leading cause of the rise of multidrug-resistant pathogenic bacterial strains (3).
Several well-known pathogenic bacteria have developed into highly antibiotic-resistant strains. Examples are Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Mycobacterium tuberculosis (1, 2). A common denominator of these drugresistant strains, as well as of many other major pathogenic bacteria, is that they employ pore-forming toxins (PFTs) as virulence factors.
PFTs are common among bacteria, and about 25 to 30% of cytotoxic bacterial proteins are PFTs, making them the single largest category of virulence factors (4, 5).
Because of their nearly universal presence in bacterial pathogens, PFTs are a unique and important target for research into novel, broadly applicable antimicrobial prophylactics and therapeutics. PFTs function to perforate membranes of host cells, predominantly the plasma membrane but also intracellular organelle membranes (6).
They are classically hypothesized to do so in order to directly kill target cells, for intracellular delivery of other bacterial or external factors (7, 8), to release nutrients (9), or for phagosomal escape in the case of intracellularly acting PFTs (10). Loss of their PFTs generally causes pathogenic bacteria to be less virulent or completely avirulent (see below and Table 1). Conversely, transgenic expression of a PFT can turn an otherwise harmless bacterium into a parasite or a pathogen (224, 225).
PFT Mechanism of Action
PFTs are generally secreted as water-soluble molecules. Recognition and binding to a specific receptor cause them to associate with the target membrane, form multimers, and undergo a conformational change, leading to the formation of an aqueous pore in the membrane (16, 226) (Fig. 1).
FIG 1 Generalized mechanism of pore formation by PFTs. Soluble PFTs bind membrane receptors, which leads to oligomerization and insertion of an aqueous pore into the plasma membrane (5). Note that during the oligomerization step, some PFTs remain associated with their receptor, whereas others have already disassociated at this point.
PFTs can be classified based on the secondary structure of the regions that penetrate the host cell plasma membrane, which generally consist of either α-helices or -barrels, and the specific toxins may be referred to as α -PFTs or β -PFTs. The majority of bacterial PFTs are β -PFTs, which also form the most-studied group (227).
The large-pore-forming cholesterol-dependent cytolysins (CDCs), produced by Gram-positive and some Gram-negative bacteria, are a β -PFT subclass (4, 227, 228). The small-pore-forming repeat in toxin (RTX) toxins, produced by Gram-negative bacteria, form a large group of PFTs, but their classification and mechanism of pore formation remain unclear (4). Another useful form of classification, especially with regard to host defenses, is by the size of the pore that is formed (227). Bacterial PFTs generally form either small (0.5 to 5 nm) or large (20 to 100 nm) pores, and host cellular defenses against the different classes only partially overlap (229–232). Examples of the different classes of PFTs are shown in Fig. 2. Various host cell receptors for PFTs have been identified, including glycosylphosphatidylinositol (GPI)-anchored proteins, other membrane proteins (e.g., ADAM10 and CCR5), lipids, and cholesterol (5, 233, 234).
The target cell tropism of PFTs varies widely, one important cause of which is their various receptor specificities, and host or cell type specificity can be altered in vitro by genetic modification of PFTs (235–237). Many bacteria produce toxins that are presumed to be PFTs based on their sequence or properties. A number of reviews have been written that extensively cover the biochemical properties and in vitro effects of bacterial and eukaryotic PFTs (4–6, 9, 16, 227, 229, 238, 239). For numerous human-pathogenic bacteria, as well as several economically important bacteria that are pathogenic to animals, there is direct proof or considerable circumstantial evidence that a PFT is expressed during infection and contributes significantly to virulence in vivo. We have listed such bacteria in Table 1. For a smaller group of bacteria, there are also in vivo data on the mechanisms through which their PFTs contribute to infection. After a summary of known PFT defense mechanisms, this review focuses on the contributions of PFTs to infections by 10 of the most-studied PFT-wielding bacterial pathogens, as determined by the study of in vivo and ex vivo infection models and clinical and epidemiological data. Although effects of PFTs are highly diverse, a number of common themes could be identified and provide the structure for this review. Relevant background information on the 10 bacteria is provided in Table 2.
PFT EFFECTS AND CELLULAR DEFENSE MECHANISMS
The search for mechanisms of action shared by PFTs has involved predominantly in vitro studies on simplified target systems (lipid bilayers, cultured cells, and primary cells) and studies on the in vivo model involving the nematode Caenorhabditis elegans and the Bacillus thuringiensis crystal toxin PFT Cry5B. These studies have led to an understanding of the molecular requirements for attack, oligomerization, and pore formation. In addition, a number of important host defense and cell death pathways and membrane repair mechanisms have been identified. Nonetheless, several caveats deserve consideration in interpreting these studies, some of which extend to studies discussed in the section on in vivo PFT effects. First, because these studies use purified toxin, cells may be exposed to artificially high doses of toxin. Such doses may not be physiologically relevant and hence could result in responses that are not reflective of those seen during an infection. Second, purified PFTs may behave differently in isolation compared to their behavior in the presence of their pathogen or additional virulence factors. Third, PFTs can affect expression of other genes in their source bacteria (143) and hence may play roles that extend beyond their cytotoxic properties. Lastly, it can be unclear whether an observed response benefits the host, the pathogen, or both.
MAPK Pathways
Using C. elegans and Cry5B, the first functional molecular PFT defense pathways were identified, involving p38 mitogen-activated protein kinase (MAPK) and the c-Jun N-terminal (JNK)- like MAPK KGB-1 (309). p38 MAPK was shown to be important in mammalian cells in defense against the Aeromonas hydrophila PFT aerolysin and S. aureus alpha-toxin (230, 309). p38 activation is seen in vivo with B. thuringiensis Cry toxins and in vitro with numerous PFTs, including S. pneumoniae pneumolysin (PLY), S. aureus alpha-toxin, group A streptococcus (GAS) streptolysin O (SLO), Bacillus anthracis anthrolysin O (ALO) (230, 310), Gardnerella vaginalis vaginolysin (VLY) (104), A. hydrophila aerolysin (309), Listeria monocytogeneslisteriolysin O (LLO) (311), and Lactobacillus iners inerolysin (ILY) (312). p38 was further shown to be activated by and required for defense against Cry toxin in vivo in the lepidopteran Manduca sexta and the dipteran Aedes aegypti (313). The activation of p38 in response to PFT is thus evolutionarily strongly conserved. Activity of JNK and extracellular signalregulated kinase (ERK) MAPKs was also identified on several occasions in vitro (M. tuberculosis 6-kDa early secretory antigenic target [ESAT-6] [314], S. pneumoniae PLY [315], GAS SLO [316], A. hydrophila aerolysin, and L. monocytogenes LLO [311]) and in vivo, in C. elegans (with B. thuringiensis Cry5B) (231). Activator protein 1 (AP-1; Fos/Jun), functioning downstream of JNK, is involved in C. elegans in defenses against PFTs that form small pores (Cry5B) as well as against PFTs that form large pores (SLO). A role for AP-1 in defense against SLO was confirmed in vitro in mammalian cells (231). LLO and B. anthracis protective antigen (PA; a component of anthrax toxin and a PFT [Table 2]) can activate ERK, and both p38 and ERK can function to restore potassium homeostasis in cells damaged by PFTs (311). Thus, the p38, JNK, and perhaps ERK MAPK pathways are arguably the main mediators of physiological PFT defense pathways.
Potassium Efflux-Dependent Defenses, Including Inflammasome Activation
An important consequence of pore formation by PFTs is the efflux of cellular potassium. Aerolysin-induced potassium efflux was found to induce the activation of the Nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome and cysteine-aspartic protease 1 (caspase-1). Caspase-1 activates sterol regulatory element-binding proteins (SREBPs), which are central regulators of membrane lipid biogenesis, contributing to cellular survival (317). PFTs can also trigger apoptosis, which in the case of S. aureus alpha-toxin and aerolysin is dependent upon caspase-2. Preventing the PFT-associated efflux of potassium inactivated caspase-2, and inhibition of caspase-2 inhibited PFT-induced apoptosis (318). Potassium efflux was also found to be required for PFT-induced autophagy (311, 319) and to mediate p38 MAPK activation by S. aureus alpha-toxin, Vibrio cholerae cytolysin (VCC), SLO, and E. coli hemolysin A (HlyA) (320). As mentioned above, p38 and ERK activation promotes the recovery of disturbed potassium levels (311).
Other Cellular Defenses
The endoplasmic reticulum (ER) unfolded protein response (UPR) pathway is a key functional downstream factor of p38 MAPK in C. elegans defense against Cry5B, and the ER UPR was also activated in mammalian cells in response to aerolysin (321). The UPR may function to arrest protein synthesis, which has also been observed in vitro in response to L. monocytogenes LLO and B. anthracis PA, although in those cases the inhibition of protein synthesis occured independent of the UPR (311). The hypoxia response pathway is also involved in C. elegans defense against B. thuringiensis Cry toxins (203). This pathway involves downregulation of hypoxia inducible factor 1 (HIF-1 in C. elegans) by prolyl hydroxylase, Von Hippel-Lindau tumor suppressor protein, and regulator of hypoxia-inducible factor (EGL-9, VHL-1, and RHI-1, respectively, in C. elegans). Mutations in egl-9, vhl-1, and rhi-1, which increase the activity of the hypoxia pathway, lead to resistance to Cry toxins, whereas a mutation in hif-1, which decreases pathway activity, leads to Cry toxin hypersensitivity. These results extended to the V. cholerae PFT cytolysin (VCC). Interestingly, however, whereas activity of the hypoxia pathway protected against a V. cholerae strain with VCC, it caused hypersensitivity to V. cholerae lacking VCC. Thus, whereas host factors may protect against one type of virulence factor, they may cause hypersensitivity to others (203), causing the host to face a difficult challenge. VCC was also shown to cause formation of vacuoles in C. elegans intestinal cells, consistent with earlier in vitro observations (204). The role of these vacuoles remains unclear. Another pathway found to play a role in PFT defense is the insulin/insulin-like growth factor 1 (IGF-1) pathway. Loss of the insulin receptor, DAF-2, causes C. elegans to become resistant to Cry5B. This effect was found to depend not only on the canonical downstream forkhead transcription factor DAF-16 but also on a novel pathway arm involving WW domain protein 1 (WWP-1) (322). α -Defensins have been found to function in PFT defense in vitro (323).
Calcium-Dependent Membrane Repair Mechanisms
In addition to an efflux of potassium, PFT membrane pores often result in an influx of calcium. Ca2 influx is a known trigger of apoptosis (324), a PFT response that has been observed in various cell types (238), and it can affect the vesicle trafficking machinery. GAS SLO-induced calcium influx triggers the exocytosis of lysosomes and extracellular release of the lysosomal enzyme acid sphingomyelinase. Acid sphingomyelinase was found to subsequently induce endocytosis, which contributed to membrane repair (325, 326). During endocytosis, PFT pores are taken up into the cells, ubiquitinated, and then, through activity of the ESCRT machinery, targeted to lysosomes for degradation (327). S. aureus alpha-toxin also enters cells via endocytosis, is transported via late endosomes, and then disappears from the cells. Alpha-toxin multimers, however, were not broken down in acidic compartments but were expelled from cells via exosome-like vesicles called toxosomes (328). A recent study showed that vesicle trafficking pathways also protect cells against PFTs in vivo. Intoxication of C. elegans by Cry5B and V. cholerae VCC was found to trigger increased rates of endocytosis in intestinal cells. Loss of either of the two key Rab proteins (RAB-5 and RAB-11), master regulators of early endosome and recycling endosome functions, resulted in significant decreases in Cry5B-induced endocytosis in intestinal cells. Loss of RAB-5 and RAB-11 furthermore resulted in strong hypersensitivity of C. elegans to Cry5B, and both were required to restore the integrity of the plasma membranes of intestinal cells following Cry5B attack. This demonstrates a correlation between RAB-5, RAB-11, PFT-induced endocytosis, restoration of plasma membrane integrity, and survival of the whole organism. RAB-11 was additionally found to be required for PFT-induced expulsion of microvilli from the enterocyte cell surface, which is hypothesized to be part of the membrane repair mechanism and is also observed in vitro with other PFTs, including Vibrio parahaemolyticus thermostable direct hemolysin and GAS SLO (329–331).