Pertussis toxin is a multisubunit AB-toxin exclusively produced by B. pertussis. Through B-subunits, PT binds to any sialic acid-containing glycoprotein at the cellular surface (Witvliet et al., 1989; Saukkonen et al., 1992; Stein et al., 1994), is internalized by endocytosis and follows a retrograde transport pathway through the Golgi complex to the endoplasmic reticulum (Plaut and Carbonetti, 2008; Figure 1), from which the A-subunit translocate to the cytoplasm of host cells (Locht et al., 2011). In the host cell cytosol, the A-subunit exhibits ADP-ribosyltransferase activity, hydrolysing NAD and ADP-ribosylating heteromeric G-proteins of the G_i family (Katada et al., 1983). This modification prevents the interaction of G_i proteins with their cognate G-protein coupled receptors (GPCRs) causing the disruption of downstream cell signaling transduction (Mangmool and Kurose, 2011). Besides the activity of the A domain, B-subunits also display signaling function by activating intracellular signaling cascades in a G_i-protein independent manner (Mangmool and Kurose, 2011).

Pertussis toxin has long been considered as a major B. pertussis virulence factor. Indeed, PT was reported to be the cause of systemic symptoms of pertussis disease such as lymphocytosis and leukocytosis (Morse and Morse, 1976) and was associated with lethal infection by B. pertussis in a neonatal mouse model (Goodwin and Weiss, 1990). However, evidences supporting its role in respiratory infection have only emerged in the last decade. Experiments in the mouse model revealed that the lack of PT confers a defect in B. pertussis colonization at the early stages of infection (Carbonetti et al., 2003, 2005). Interestingly, a PT-deficient strain reaches wild type levels of colonization whenever co-infections with both strains are performed or intranasal inoculation of purified PT precedes infection (Carbonetti et al., 2003). Further studies have shown that depletion of resident airway macrophages leads to exacerbated B. pertussis infection in a PT-independent manner (Carbonetti et al., 2007), indicating that PT targets airway macrophages disrupting their protective activity at early steps of infection (Carbonetti et al., 2007). Intranasal administration of PT resulted in ADP-ribosylation of airway macrophages G_i-proteins (Carbonetti et al., 2007), suggesting that its inhibitory function on macrophages in vivo results from the immunosuppressive activities ascribed to PT in vitro. In particular, in vitro PT was shown to inhibit macrophage and neutrophil migration (Meade et al., 1984), phagocytosis (Mork and Hancock, 1993) and cytokine response (He et al., 1988; Hume and Denkins, 1989).

In vivo, PT plays a dual role in the establishment of the disease. Whereas it has immunosuppressive functions at early stages of infection, later it potentiates inflammatory responses, likely prolonging healing and promoting bacterial dissemination (Eby et al., 2015). Early in infection, PT targets airway macrophages and inhibits neutrophil recruitment to the infection site (Carbonetti et al., 2003, 2005; Kirimanjeswara et al., 2005; Andreasen and Carbonetti, 2008). Neutrophils were reported to play a protective role against B. pertussis only in previously infected mice and in the presence of anti-B. pertussis antibodies (Kirimanjeswara et al., 2005; Andreasen and Carbonetti, 2009), which may suggest that PT delays neutrophil recruitment to the airways avoiding rapid antibody-mediated clearance of the pathogen. Later, at the peak of infection, high numbers of neutrophils are recruited to the lungs of mice infected with wild type strain, but not to those infected with PT-deficient strain (Carbonetti et al., 2005; Andreasen et al., 2009). This recruitment of neutrophils to the lungs correlates with an increase of proinflammatory cytokines such as IL-17, TNFα and IFNγ that appears to be dependent on PT activity (Andreasen et al., 2009). Recently, global transcriptional profiles of mice lungs infected with wild type or PT-deficient B. pertussis revealed that, at the peak of infection, the ADP-ribosylation activity of PT correlates with upregulation of immune and inflammatory response genes (Connelly et al., 2012). In vitro studies suggested that PT directly impairs neutrophil migration through ADP-ribosylation the G_i-proteins associated with surface chemokine receptors (Spangrude et al., 1985; Scott et al., 1988; Figure 1). However, in vivo data showed that besides acting directly on chemokine receptors signaling (Kirimanjeswara et al., 2005), PT also suppresses early neutrophil recruitment by inhibiting the production of neutrophil-attracting chemokines by airway macrophages and lung epithelial cells (Andreasen and Carbonetti, 2008). Transcription of genes expressing CXCL1, CXCL2, and CXCL5 is inhibited in the lungs of mice intranasally infected with wild type B. pertussis, as compared to animals infected with strains deficient for PT production or producing a PT variant devoid of ADP-ribosylation activity (Andreasen and Carbonetti, 2008). Interestingly, PT also blocks chemokine gene expression and early neutrophil recruitment to the airways following intranasal administration of LPS (Andreasen and Carbonetti, 2008), which occurs presumably through modulation of TLR-4 signaling, a G_i-protein independent pathway.

Altogether, these studies show that PT suppresses early inflammation in the respiratory tract and inhibit microbicidal function of inflammatory cells, potentially providing advantage to the pathogen by allowing its rapid growth and establishment within the host at early phases of infection.

Adenylate cyclase toxin is a bi-functional toxin produced by all species of Bordetella that infect mammals. While its N-terminal domain contains an adenylate cyclase activity that converts ATP in cAMP, the C-terminus possesses RTX motifs that bind mammalian cells and form cation-selective pores in the host plasma membrane. Through its combined adenylate cyclase and pore-forming activities, ACT manipulates host cell physiology in two different ways: it interferes with intracellular signaling by increasing the levels of cAMP, and disturbs ion homeostasis by disrupting the permeability barrier of the plasma membrane (Figure 1). ACT specifically binds with high affinity CD11b/CD18 (also known as CR3 or Mac-1) present at the surface of macrophages, neutrophils and dendritic cells (DCs; Guermonprez et al., 2001). Upon binding, ACT integrates the membrane of target cells in two different conformations: a translocation precursor that re-localizes at lipid raft domains from where the adenylate cyclase activity translocates directly to the cell cytoplasm; and a pore precursor that oligomerises and permeabilises the cells causing ion concentration imbalance (Fiser et al., 2007, 2012; Bumba et al., 2010; Figure 1). While the rapid translocation of adenylate cyclase activity across the host plasma membrane does not dependent on endocytosis (Gordon et al., 1988), the clathrin-dependent endocytosis of ACT together with its receptor CD11b/CD18 was reported (Khelef et al., 2001; Martin et al., 2011). Classical mechanisms of membrane repair upon toxin-induced pore formation include the removal of the pores through endocytosis. Interestingly, ACT-translocated molecules control the rate of ACT-pore removal delaying their endocytic uptake, thus exacerbating the permeabilisation of phagocytes and maximizing the cytotoxic action (Fiser et al., 2012). Despite its interaction with a specific receptor, ACT was reported to promiscuously bind and intoxicate many cell types, including CD11b/CD18-negative cells (Ladant and Ullmann, 1999; Paccani et al., 2008; Eby et al., 2012), although the biological relevance of these findings remains unclear. Indeed, while ACT triggers macrophage apoptosis in vitro (Khelef et al., 1993; Khelef and Guiso, 1995) and in vivo (Gueirard et al., 1998), the viability of cells from non-haematopietic origin remains unaffected (Gueirard et al., 1998; Bassinet et al., 2000). In vivo studies support that ACT primarily targets phagocytes such as alveolar macrophages and neutrophils, disrupting the early innate antibacterial host immune response (Harvill et al., 1999; Figure 1).

cAMP is a key second messenger with pleiotropic effects. Increased levels of cAMP severely compromise cellular functions such as the migration of neutrophils, the plasticity of DC responses, the release of cytokines by macrophages and the homeostasis of actin cytoskeleton. Thus, through the uncontrolled production of intracellular cAMP, ACT subverts phagocytic, and bactericidal function of macrophages and neutrophils in a variety of ways (Figure 1). In human alveolar macrophages and neutrophils, as in mouse macrophages, the ACT-mediated cAMP production blocks phagocytosis, chemotaxis, and oxidative burst (Confer and Eaton, 1982; Friedman et al., 1987; Weingart and Weiss, 2000; Kamanova et al., 2008; Eby et al., 2014). High levels of cAMP cause a transient inactivation of RhoA inducing massive actin rearrangements that dramatically decrease macropinocytosis, block complement-mediated phagocytosis (Kamanova et al., 2008) and possibly impair the chemotactic properties of primary monocytes. Recently, ACT-induced cAMP synthesis was shown to trigger pro-apoptotic signaling in phagocytes through the activity of tyrosine phosphatase SHP-1, the accumulation of cytosolic BimEL and the consequent activation of Bax, permeabilisation of the outer mitochondrial membrane and activation of programmed cell death (Ahmad et al., 2015; Cerny et al., 2015). Besides the immediate action of ACT on the ablation of bactericidal functions of phagocytes, ACT activity was also reported to block the release of TNFα and the production of ROS in human monocytes (Njamkepo et al., 2000), to promote incomplete or aberrant maturation of DCs (Skinner et al., 2004; Boyd et al., 2005) and to impair T-cell activation by interfering with immunological synapse signaling (Paccani et al., 2011). ACT was described to suppress the secretion of pro-inflammatory cytokines such as IL-12 and TNFα and favor the production of anti-inflammatory IL-10 molecules (Spensieri et al., 2006; Hickey et al., 2008).

Despite the fact that studies in mouse models established ACT as an important virulence factor for B. pertussis infection (Khelef et al., 1992), the effects described above were obtained from in vitro studies, and their significance during in vivo infection requires further investigation. Importantly, recent studies using baboon infection model and clinical samples from humans showed that the concentration of ACT in tissues is much lower than the amount used for in vitro experiments (Eby et al., 2013), which may compromise the relevance of some effects reported in vitro.

Pertussis toxin and ACT have undeniable roles during B. pertussis infection and certainly play key functions in the pathophysiology of pertussis disease. Numerous and wide-ranging effects of the purified toxins on cultured cell lines have been reported, however, establishing the correlation of such effects with the human pathology appeared as an incredible difficult task. In addition, the mouse model only provides limited possibilities to address this issue. Together with analysis of clinical samples from humans, the use of baboons as non-human primate model is expected to shed new light on the mechanisms of action of PT and ACT and on pertussis disease.

Anthrax has for long been described as a toxin-mediated disease, mainly because LT and ET can be lethal for experimental animals and anti-toxin immunization is effective in protecting against infection (Kaur et al., 2013). However, it has recently been proposed that extreme bacteraemia and severe sepsis rather than the anthrax toxins per se are the cause of anthrax-induced lethality (Coggeshall et al., 2013). Indeed, the fact that anthrax toxins act on multiple tissues simultaneously, due to the ubiquitous expression of the anthrax toxin receptors, complicate untangling their effects on the host and delayed the identification of the key tissue targets responsible for its lethal effects. Recently, using cell-type specific CMG2-null mice and the correspondent cell-type specific CMG2-expressing mice, Liu and colleagues have shown that LT-induced mortality requires targeting of cardiomyocytes and vascular smooth muscle cells, whereas ET-induced lethality relies mainly on targeting hepatocytes (Liu et al., 2013). Using myeloid-specific CMG2-null mice, in which both macrophages and neutrophils are insensitive to LT and ET due to their inability to bind and internalize the toxins, the same authors have also clarified the role of macrophages and other myeloid cells in anthrax toxins induced lethality and in B. anthracis infection (Liu et al., 2010). Myeloid-specific CMG2-null mice are fully sensitive to both LT and ET, indicating that lethality does not depend on the targeting of macrophages, neutrophils, and other myeloid cells (Liu et al., 2010). However, they are completely resistant to infection by B. anthracis, indicating that the targeting of myeloid cells by anthrax toxins is required for the establishment of a successful infection (Liu et al., 2010). In what concerns the relative contributions of ET and LT to the establishment of B. anthracis infection, trials with Sterne strains deleted of PA, LF, or EF suggest that LT plays a more prominent role (Liu et al., 2010).

Available data indicate that LT and ET act in concert to inhibit macrophage activation as well as the activation and recruitment of other immune cells, such as neutrophils, early in infection (Baldari et al., 2006; Tournier et al., 2009; Liu et al., 2014). This favors bacterial escape and multiplication, and contributes to the severe bacteraemia observed in terminal disease. Macrophage activation requires signaling through MAPK cascades, including JNK and p38 pathways, which are central for induction of inflammatory molecules, including cytokines and chemoattractants, as well as Cox-2 and iNOS. LT interrupts MAPK cascades by directly cleaving MAPKK, whereas ET inhibits MAPK-dependent gene expression by interfering with multiple PKA-related pathways (Baldari et al., 2006). Several reports show that LT inhibits the secretion of pro-inflammatory cytokines by macrophages as well as by DCs in vitro and in vivo (Pellizzari et al., 1999; Erwin et al., 2001; Agrawal et al., 2003; Alileche et al., 2005; Bergman et al., 2005; Brittingham et al., 2005; Tournier et al., 2005; Ribot et al., 2006). ET has been shown to suppress secretion of inflammatory mediators by DCs (Tournier et al., 2005). Whereas LT inhibits IL-10 secretion by these cells, ET inhibits IL12p70 production (Tournier et al., 2005). Interestingly, LT and ET have a cumulative suppressive effect upon TNFα secretion (Tournier et al., 2005). In addition to suppress pro-inflammatory cytokine secretion by macrophages, LT is also able to trigger programmed cell death in these cells in vitro. Indeed, it has been reported that LT induced apoptosis of RAW264.7 cells (Popov et al., 2002). Furthermore, although treatment of three different human monocytic cell lines (HL-60, THP-1, and U937) did not result in cell death, upon differentiation into macrophage-like phenotypes, the cells become susceptible to a cell death program that has features of apoptosis but apparently does not requires the activity of effector caspases (Kassam et al., 2005). The apoptogenic activity of LT toward macrophages likely relates to LT-dependent disruption of survival signals triggered by TLR4 and mediated by p38 MAPK (Park et al., 2002, 2005; Hsu et al., 2004) that activates the NF-kB-dependent expression of pro-survival genes. By cleaving the upstream MAPKK MKK3, LT blocks p38 MAPK and NF-kB activation, leading to macrophage apoptosis (Huang et al., 2004; Park et al., 2005).

Lethal toxin has also been shown to induce a rapid and lytic form of caspase-1-dependent cell death, called pyroptosis, in macrophages from specific rat and mice strains. The susceptibility of macrophages to pyroptosis has been linked to polymorphisms of the Nlrp1b gene in mice (Boyden and Dietrich, 2006) and of the orthologous Nlrp1 gene in rat (Newman et al., 2010). The ability of LT to induce macrophage pyroptosis was initially interpreted as a virulence mechanism of B. anthracis (Muehlbauer et al., 2007). It was speculated that the destruction of macrophages by LT compromised their role in restricting B. anthracis infection and that the cytokine burst associated to LT-induced macrophage lysis contributed to LT-dependent pathological effects by aggravating the vascular damage occurring in anthrax (Muehlbauer et al., 2007). More recently, it has been shown that LT is able to directly cleave mouse Nlrp1b and rat Nlrp1 close to their N-terminus (Hellmich et al., 2012; Levinsohn et al., 2012; Chavarria-Smith and Vance, 2013) resulting in the activation of Nlrp1 inflammasomes in rat and in mice with LT-sensitive macrophages. Ultimately, this leads to caspase-1 activation and pyroptosis accompanied by the release of the inflammatory cytokines IL-1β and IL-18, which induces a strong innate immune response that is protective against B. anthracis infection (Moayeri et al., 2010; Terra et al., 2010). Therefore, the current view is that LT-mediated activation of Nlrp1 that leads to inflammasome activation and macrophage pyroptosis is not a virulence mechanism used by B. anthracis to promote infection, but rather a protective host-response against anthrax (Chavarria-Smith and Vance, 2015).

Neutrophils play a major role in controlling B. anthracis infection and anthrax-toxin mediated neutralization of neutrophil functions is essential for successful infection (Liu et al., 2010). In vitro studies suggest that by reducing F-actin formation, LT and ET cooperate to inhibit neutrophil chemotaxis, chemokinesis, and ability to polarize (During et al., 2005, 2007; Szarowicz et al., 2009). It was also reported that neutrophils intoxicated with ET have reduced phagocytic activity (O’Brien et al., 1985). Additionally, LT has been shown to suppress cytokine production by neutrophils in vitro (Barson et al., 2008) and LT and ET were found to block neutrophil priming by LPS or muramyl dipeptide, thereby dampening the oxidative burst normally elicited by bacterial products and required for full antimicrobial activity (Wright and Mandell, 1986). In the case of ET, it has been shown that the inhibition of superoxide production results from an impairment of the activation of the neutrophil NADPH oxidase, an effect that likely results from the activity of ET as an adenylate cyclase (Crawford et al., 2006). Although it has been proposed that these effects are due to the ET-induced rise in cAMP leading to phosphorylation (activation) of PKA (Szarowicz et al., 2009), the downstream targets remain to be identified.

It is now unquestionable that anthrax toxins are crucial in anthrax pathogenesis, but their precise roles during human anthrax infections remain to be further clarified. Following the discovery of the anthrax toxins more than half a century ago, a myriad of studies were developed aiming at defining their role in anthrax disease and lethality. However, most of the infection studies were performed in animal models that may not completely reflect the events occurring during human infections. Concerning the data documenting the immunomodulatory effects of the anthrax toxins, most were obtained in vitro, often in experimental set-ups that involve the use of purified toxins and do not allow examining intoxication in the context of infection. Therefore, the available data need to be validated in the context of relevant animal models of infections before being extrapolated to the human disease scenario.

AIP56 is the founding and the only characterized member of a continuously growing family of bacterial proteins identified in different organisms, mainly marine Vibrio species and Arsenophonus nasoniae. It is an AB-type toxin, possessing a catalytic A domain at its N-terminal region and a B domain involved in binding/internalization into target cells at its C-terminal region (Silva et al., 2013; Figure 3). The catalytic domain of AIP56 is a zinc-dependent metalloprotease that cleaves the p65 subunit of NF-κB (Silva et al., 2013), an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes, playing a key role in host responses to microbial pathogen invasion. AIP56 has likely originated from a fusion of two components: its A domain is related to NleC, a type III secreted effector present in several enteric pathogenic bacteria (Yen et al., 2010; Baruch et al., 2011; Pearson et al., 2011; Sham et al., 2011; Hodgson et al., 2015) that are associated with severe human illness and death worldwide, whereas its B domain is related to a protein of unknown function from the lambda-like bacteriophage APSE2, a phage that infects Hamiltonella defensa (Degnan et al., 2009).

Although mammals are not susceptible to Phdp infection, likely due to temperature and osmolality restrictions, AIP56 is able to intoxicate mouse bone marrow derived macrophages (mBMDM; Pereira et al., 2014), through a mechanism similar to the one operating during intoxication of fish cells. Upon encountering susceptible cells, AIP56 binds to a still unidentified cell-surface receptor and is internalized through clathrin-mediated endocytosis (Pereira et al., 2014; Figure 3). Once in early endosomes, the toxin either follows the recycling pathway back to the extracellular medium or undergoes low pH-induced translocation across the endosomal membrane into the cytosol to display its toxic activity (Pereira et al., 2014). AIP56 cleaves an evolutionarily conserved peptide bond of the Rel homology domain of NF-κB p65, removing residues crucial for p65-DNA interaction and compromising NF-κB activity (Silva et al., 2013; Figure 3). During intoxication, the proteolytic activity of AIP56 results in a complete depletion of p65 and leads to the apoptotic death of cells (Silva et al., 2013; Pereira et al., 2014) through a process involving quick activation of caspases-8, -9 and -3, loss of mitochondrial membrane potential, translocation of cytochrome c to the cytosol and overproduction of ROS (do Vale et al., 2007; Reis et al., 2007a,b, 2010; Costa-Ramos et al., 2011).

AIP56 plays a pivotal role in the establishment of Phdp infection and in the development of the infection-associated pathology. In the initial phase of Phdp infection, when local multiplication of Phdp becomes detectable in infected tissues, extensive infiltration of macrophages and neutrophils occurs (do Vale et al., 2007). As the infection progresses, the pathogen extensively multiplies and disseminates systemically, which leads to a septicemic situation paralleled by the occurrence of AIP56 in the systemic circulation (do Vale et al., 2007). The presence of circulating toxin correlates with the appearance of high numbers of apoptotic macrophages and neutrophils in the peripheral blood, in the spleen, liver, and head–kidney vasculature, as well as in the splenic and head-kidney parenchyma and gut lamina propria (do Vale et al., 2007). This systemic apoptotic destruction of macrophages and neutrophils triggered by AIP56 explains the extensive phagocyte depletion observed in advanced Phdp infections (do Vale et al., 2007). The ability of the toxin to neutralize the main players responsible for the phagocytic defense of the host is a very effective pathogenicity strategy that contributes to the severity of Phdp infections by promoting survival of the pathogen and its unrestricted extracellular multiplication. Concomitantly, the AIP56-induced apoptosis of both professional phagocytes leads to tissue damage with deleterious consequences for the host. In fact, the destruction of macrophages, the cells with the crucial role of eliminating apoptotic cells (Parnaik et al., 2000; Wood et al., 2000), compromises the efficient clearance of apoptotic phagocytes and leads to their lysis by secondary necrosis (do Vale et al., 2003) with release of their cytotoxic intracellular contents (do Vale et al., 2007). This has particularly serious consequences in the case of neutrophils, due to their richness in highly cytotoxic molecules, which damage many cell types and produce tissue injury, thus contributing to the genesis of the Phdp-associated cytopathology.

In general, upon pathogen recognition, pro-inflammatory signals released by resident macrophages promote the adhesion of circulating neutrophils and further extravasation across capillary endothelium to the site of infection. This process relies on interactions between the endothelial surface receptors (e.g., selectins and ICAM1) and their respective ligands on the surface of neutrophils (e.g., PSGL1 and β2-integrin) (Spaan et al., 2013b). To inhibit neutrophil recruitment to the infected tissues, S. aureus secretes two anti-inflammatory factors that prevent neutrophil adhesion to the blood vessels and further transmigration (Figure 4A). The staphylococcal SSL5 binds PSGL1 in a glycan dependent manner at the surface of neutrophils, blocking its interaction with P-selectin expressed by endothelial cells and abrogating the early steps of neutrophil attachment (Bestebroer et al., 2007). In addition, SSL5 was shown to inactivate matrix metalloproteinase from human neutrophils, accounting for the limited capacity of neutrophils to transmigrate into infected tissues (Itoh et al., 2010). The extracellular adherence protein (Eap) recognizes endothelial ICAM1, preventing its interaction with β2-integrins at the surface of neutrophils and further inhibiting extravasation (Chavakis et al., 2002).

Staphylococcus aureus also secretes a number of antagonists of neutrophil receptors interfering with chemokine signaling and limiting neutrophil recruitment (Figure 4A). In particular, SSL5 directly binds the N-terminus of G-protein coupled chemokine receptors (GPCRs) inhibiting calcium mobilization and actin polymerization, thus impairing neutrophil responses to a huge diversity of chemokines (e.g., CXCL8, CXCL1, CCL2, and CCL5) and to complement fragments C3a and C5a (Bestebroer et al., 2009). Similarly to the recognition of PSGL1 by SSL5, binding to GPCRs relies on the presence of sialic acid residues (Baker et al., 2007; Bestebroer et al., 2007, 2009). In addition, SSL5 was shown to bind to platelet glycoproteins, inducing platelet activation and aggregation, which could be important for colonization and immune evasion by S. aureus (de Haas et al., 2009). SSL10 inhibits CXCL12-mediated responses by targeting CXCR4 (Walenkamp et al., 2009) and SSL3 binds to TLR2, hindering immune recognition of staphylococcal lipoproteins and peptidoglycan (Yokoyama et al., 2012).

Phenol-soluble modulins are produced by all S. aureus strains and have multiple roles in staphylococcal pathogenesis (Cheung et al., 2014a). In particular, PSMs are potent pro-inflammatory molecules that interact with human formyl peptide receptor 2 (FPR2) (Kretschmer et al., 2010), a GPCR involved in the recognition of pathogens. At nanomolar concentrations, PSMs bind to and activate FPRs, with the strongest activation occurring through FPR2, stimulating several FPRs effector functions such as chemotaxis and pro-inflammatory cytokine production (e.g., CXCL8; Fu et al., 2006). However, human isolates of S. aureus evolved other strategies to counter neutrophil chemotaxis by directly interfering with FPRs signaling and limiting cytotoxicity while promoting bacterial replication (McCarthy and Lindsay, 2013; Cheung et al., 2014b). Inhibition of FPRs-mediated pro-inflammatory signaling occurs via the secretion of the chemotaxis-inhibitor protein of S. aureus (CHIPS) (de Haas et al., 2004) and the FPR2/ALS-inhibitory protein (FLIPr) and its homologue FLIPrL (Prat et al., 2006, 2009; Figure 4A). While CHIPS binds and inhibits FPR1 and C5aR, FLIPr and FLIPrL block FRP2-mediated signaling, thus avoiding recognition of PSMs secreted by S. aureus, impairing pro-inflammatory response and reducing neutrophil recruitment (Haas et al., 2004, 2005; Prat et al., 2006, 2009). Clearly, CHIPS, FLIPr and FLIPrL, and PSMs have opposite effects on FPRs activation. Thus the production/secretion of these bacterial molecules is likely to be under strict control to allow the establishment of the infection and to evade immune recognition.

The repertoire of secreted molecules by S. aureus to evade the early steps of immune response also include Stathopain A (ScpA), a cysteine protease that specifically cleaves the N-terminus of human CXCR2 (Laarman et al., 2012), a GPCR responding to several chemokines (e.g., CXCL1-3 and CXCL5-8). Stathopain A inhibits CXCR2-mediated calcium mobilization, migration, intracellular signaling and activation of neutrophils (Laarman et al., 2012), appearing as an important immunomodulatory molecule causing neutrophil unresponsiveness to several chemokines and blocking their recruitment to the site of infection.

Staphylococcus aureus display intrinsic physical features (e.g. thick peptidoglycan layer and capsule) that confer resistance to complement-mediated killing and neutrophil phagocytosis. In addition, S. aureus secretes a variety of highly specific proteins with complement-modulating functions (Figure 4B), thereby delaying the innate immune attack and generating a window of opportunity to replicate and establish within the host (Spaan et al., 2013b; Thammavongsa et al., 2015). Several of these secreted molecules target C3 or C3 convertases, both central molecules in the complement activation cascade. Aureolysin is a metalloproteinase that cleaves C3, generating a modified C3b fragment that is further degraded instead of being covalently linked to the bacterial surface where it would promote the generation of the chemoattractant molecule C5a (Laarman et al., 2011). In addition, this proteinase degrades human antimicrobial peptides highly potent against S. aureus (Sieprawska-Lupa et al., 2004). Thus, aureolysin activity promotes infection by blocking the complement cascade impairing bacterial C3b opsonization, phagocytosis, and neutrophil-mediated killing (Sieprawska-Lupa et al., 2004; Laarman et al., 2011). Staphylococcal component inhibitor (SCIN) specifically binds to human C3 convertase and blocks its activity thereby preventing the production of C3a, C3b, and C5a, thus interfering with complement activation and with neutrophil-mediated killing of S. aureus (Rooijakkers et al., 2005a, 2009; Figure 4B). In addition, S. aureus secreted extracellular fibrinogen-binding protein (Efb) together with its homologue extracellular complement-binding protein (Ecb), were shown to inhibit both C3 and C5 convertases (Jongerius et al., 2010a,b, 2007). Other S. aureus secreted proteins target both C3 and immunoglobulin binding, thus impairing complement activation and opsonization, this is the case of staphylococcal binder of immunoglobulin (Sbi; Haupt et al., 2008) and staphylokinase (Rooijakkers et al., 2005b).

Besides their role in blocking neutrophil extravasation and chemotaxis (described above), some SSLs also hinder complement activation and phagocytosis. SSL7 binds IgA and complement C5, inhibiting the production of C5a and further phagocytosis and bacterial clearance during in vivo infection (Bestebroer et al., 2010). SSL10 binds to IgG1 preventing recognition by Fc Receptors (FcR), thus impairing IgG1 opsonization and phagocytosis (Patel et al., 2010; Figure 4B).

In addition to phagocytosis and intracellular killing, neutrophils evolved an alternative defense mechanism to trap extracellular pathogens and prevent their dissemination. This strategy relies on the release of nuclear content together with antimicrobial cytosolic and granular proteins to form neutrophil extracellular traps (NETs), which are scaffolds that act as physical barriers to pathogen progression protecting host tissues from damage (Papayannopoulos and Zychlinsky, 2009). A secreted staphylococcal nuclease (Nuc) has the capacity to degrade NETs thereby allowing S. aureus to resist their bactericidal activities in vitro as well as in a respiratory tract infection model (Berends et al., 2010). NET degradation by Nuc leads to the production of monophosphate nucleotides that are further converted into deoxyadenosine through the activity of adenosine synthase (AdsA), another S. aureus secreted protein. Interestingly, the accumulation of deoxyadenosine generated by AdsA activity promotes the autocleavage of pro-caspase-3, triggering caspase-3-induced apoptosis of infiltrating macrophages (Thammavongsa et al., 2013). Together, staphylococcal Nuc and AdsA act in a concerted mode to promote bacterial survival in S. aureus abscesses, by excluding macrophages from the infection foci.

In line with its ability to evade almost every step of the innate immune response, S. aureus induce the death of innate immune cells, trough the secretion of PSMs and several other toxins. As mentioned above, PSMs are S. aureus secreted molecules with multiples roles in infection (Wang et al., 2007; Cheung et al., 2014a). They trigger inflammatory responses by interacting with FPR2 and display, at higher concentrations, FPR2-independent cytolytic activity likely through membrane insertion and pore formation (Kretschmer et al., 2010; Figure 4A). In particular, PSMα peptides are able to trigger the lysis of the phagosome after neutrophil ingestion allowing intracellular bacterial replication, and ultimately are responsible for lysis of neutrophils promoting bacterial survival and escape to the extracellular milieu (Wang et al., 2007; Geiger et al., 2012; Chatterjee et al., 2013; Grosz et al., 2014). In vitro, expression levels of PSMs correlate with levels of cytotoxicity (Rasigade et al., 2013). Moreover, mutants deficient for PSMα production are perturbed in biofilm formation (Peschel and Otto, 2013) and attenuated in the mouse bloodstream infection model (Wang et al., 2007). Altogether, these observations strongly suggest that PSMα-triggered effects may play a key role in in vivo S. aureus infection.

In addition to PSMs whose cytolytic activity is receptor-independent, S. aureus secretes other cytolytic toxins that interact with specific receptors at the surface of eukaryotic cells, oligomerize and form pores inducing cell leakage and ultimately total lysis. S. aureus-produced toxins targeting white blood cells belong to the beta-barrel pore-forming toxins and comprise hemolysin-α (Hla, also called α-toxin) and bicompetent leukocidins (Otto, 2014; Thammavongsa et al., 2015; Figure 4A). In vivo infection studies have shown that Hla is required for several S. aureus-associated pathologies, such as pneumonia and severe skin infections (Bubeck Wardenburg et al., 2007; Bubeck Wardenburg and Schneewind, 2008; Kennedy et al., 2010). Hla interacts with high affinity with ADAM10 at the surface of host cells (Wilke and Bubeck Wardenburg, 2010) to damage epithelial, endothelial and immune cells (Berube and Bubeck Wardenburg, 2013; Figure 4A). Mice lacking ADAM10 expression in the lung epithelium resist to lethal pneumonia (Becker et al., 2014), whereas animals lacking ADAM10 specifically on myeloid lineage develop exacerbated skin infections (Inoshima et al., 2011). Although these results suggest that the outcome of Hla-mediated effects may dependent on the infected tissue, the role of Hla on tissue-specific innate immunity requires further analysis.

Leukocidins are composed by two distinct and independently secreted subunits that form heteromultimeric pores in the membrane of host myeloid cells (Otto, 2014). S. aureus produce different arrays of leukocidins with different species and cell type specificities, which are mainly dictated by their interaction with host GPCRs (Spaan et al., 2013b; Otto, 2014; Thammavongsa et al., 2015; Figure 4A). In vitro assays with purified proteins, as well as ex-vivo infections with S. aureus, have shown that leukocidin AB (LukAB, also called LukGH) kills human, but not mouse, neutrophils upon binding to CD11b (DuMont et al., 2013a). In addition, purified LukAB induce the release of NETs (Malachowa et al., 2013) and also promotes the escape from the phagosome in neutrophils, thus enabling S. aureus replication (DuMont et al., 2013b). Gamma-hemolysin HlgAB and HlgCB bind chemokine receptors (e.g., CXCR1, CXCR2, and CCR2) and complement receptors (e.g., C5aR), respectively (Spaan et al., 2014), and promote the lysis of human neutrophils and macrophages in vitro. LukED, produced by a large majority of clinical isolates of S. aureus, triggers the lysis of neutrophils and macrophages of different vertebrates by binding chemokine receptors such as CCR5, CXCR1 and CXCR2 (Alonzo et al., 2013; Reyes-Robles et al., 2013). Importantly, LukED was shown to play a critical role in S. aureus systemic infections in mice, by promoting bacterial replication in vivo through direct killing of neutrophils (Alonzo et al., 2012). Lastly, Panton-Valentine leukocidin (PVL), which is secreted by a small percentage of S. aureus isolates, binds C5aR on neutrophils and macrophages and has a restricted activity toward human and rabbit cells (Spaan et al., 2013a, 2015). Despite the several attempts to evaluate the exact contribution of each of these toxins to S. aureus-associated pathologies, their vast species and cell type specificities have rendered this analysis highly challenging. In this context, the data obtained from commonly used animal infection models (e.g., mice and rats) should be interpreted with caution.