Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors

Summary

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called “pathogen-associated molecular patterns” (PAMPs). Pathogens use virulence factors to counteract PAMP-directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram-negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP-directed responses and are critical for infection. A plasmid-encoded T3SS in the human-pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innate immune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defenses.

Introduction

Pattern recognition receptors (PRRs) in host cells recognize conserved structural determinants of microbes, termed pathogen associated molecular patters (PAMPs), to activate innate immune responses (Medzhitov, 2007). Pathogens encode virulence factors that function to inhibit PAMP-dependent innate immune responses in order to establish and maintain infection (Finlay et al., 2006). Many Gram-negative bacterial pathogens of animals and plants encode type III secretion systems (T3SSs) that translocate effectors into host cells. The effectors inhibit or subvert PAMP-directed innate immune responses to promote infection. In order to counteract infection by pathogens, host cells can sense perturbations caused by T3SSs and other virulence factors, and adjust innate immune responses accordingly.

In plant cells, recognition of virulence factor activities, including those of translocated effectors, can occur by a process called the “guard hypothesis” (Mackey et al., 2006, Espinosa et al., 2004, Chisholm et al., 2006). In the guard model, a resistance (R) protein (the guard) directly or indirectly recognizes the modification of a key host protein (the guardee) by an effector protein. Immunity in plants triggered by effectors is termed effector triggered immunity (Chisholm et al., 2006). The phrase “microbe-induced molecular pattern” has been introduced to describe the intrinsic activity of an effector that is perceived by an R protein (Mackey et al., 2006). These concepts of innate immune recognition in plants have been extended and broadened to include PAMPs produced selectively by viable bacteria and other types of pathogen-associated activities that can be sensed in animal hosts (Vance et al., 2009, Blander et al., 2012). PAMPs produced uniquely by viable bacteria have been termed “PAMPs-per vita” (PAMPs-PV) (Vance et al., 2009) or “vita-PAMPs” (Blander et al., 2012). Virulence factor activities that are sensed by the innate immune system are called “patterns of pathogenesis” (Vance et al., 2009). Disruption of the plasma membrane when a virulence factor gains access to the cytosol is an example of a perturbation that is sensed as a pattern of pathogenesis, while flagellin protein that is translocated into host cytosol via a T3SS is an example of a PAMP-PV (Vance et al., 2009).

The human-pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica) have been extensively studied with the aim of identifying and characterizing bacterial virulence factors (Viboud et al., 2005). Y. pestis, the agent of plague, has unique factors required for fleaborne transmission and high virulence in mammalian hosts. For example, Y. pestis has LPS that is weakly recognized by the PRR Toll-like receptor 4 (TLR4), while the LPS in enteropathogenic Y. pseudotuberculosis and Y. enterocolitica activates TLR4 signaling (Montminy et al., 2006). Y. pseudotuberculosis and Y. enterocolitica have virulence determinants that are important for their intestinal route of infection (i.e. the adhesin YadA and Invasin), but are absent in Y. pestis. In addition to these species specific determinants, a number of common Yersinia virulence factors have been identified including iron acquisition pathways, mechanisms of resistance to antimicrobial peptides and complement, and a plasmid-encoded T3SS. These common virulence factors likely explain the characteristic tropism of pathogenic Yersinia species to infect and replicate in lymphoid tissues.

The T3SS in Y. pestis, Y. pseudotuberculosis and Y. enterocolitica (hereafter collectively called Yersinia) plays a key role in counteracting innate immune responses (Cornelis, 2002). Shifting the growth temperature of Yersinia from environmental temperature to 37°C results in increased expression of the proteins that comprise the three main components of the T3SS (injectisome, translocators and effectors). Injectisomes are assembled in the bacterial envelope and tipped with multimers of the protein LcrV (Cornelis, 2010). Translocators and effectors are synthesized, but generally not secreted, prior to host contact. Upon host cell contact injectisomes are activated and the translocators are secreted, followed by the effectors (Dewoody et al., 2013b). The two hydrophobic translocators, YopB and YopD, insert into plasma membrane, and are believed to form a channel called a translocon (Mueller et al., 2008, Galan et al., 2006). A current model of effector translocation suggests that the LcrV tip complex maintains direct contact with the translocon, providing a sealed conduit for effectors to move through the injectisome and plasma membrane (Mueller et al., 2008). This model explains why probes for membrane integrity do not reveal the presence of open channels or pores in Yersinia-infected cells during the process of effector translocation. Translocons appear to become “unsealed” from injectisomes, resulting in formation of open channels or pores when erythrocytes are infected with Yersinia (Mueller et al., 2008), or when epithelial cells are infected with Yersinia strains lacking effectors (“effectorless mutants”) (Mueller et al., 2008, Viboud et al., 2005). In the latter case, an active host response to translocon insertion is required for pore formation (see below). An alternative two-step mechanism of translocation that utilizes an intermediate stage of surface-localized effectors has recently been proposed (Akopyan et al., 2011). Further work on this two-step model is needed to understand its implications for translocon function.

Six effectors of the T3SS, YopH, YopE, YopT, YpkA (YopO), YopJ (YopP), and YopM directly interact with host proteins, inhibit signaling pathways, and counteract innate immunity (Viboud et al., 2005). For example, four effectors, YopH, YopE, YopT, and YpkA, counteract phagocytosis. YopH is a protein tyrosine phosphatase and YopE and YopT inactivate Rho GTPases. YopJ inhibits nuclear factor kappa B (NF-κB) and mitogen-activated protein (MAP) kinase signaling pathways. A seventh effector, YopK, interacts with translocators to regulate effector delivery (Dewoody et al., 2013b) as well as host responses (see below).

Studies of the Yersinia T3SS have provided key advances in our understanding of how host cells can recognize components of specialized bacterial secretion systems during infection. For example, the initial concept that host cells can sense insertion of translocons into plasma membrane was obtained in this system (Viboud et al., 2005). This review will discuss these and additional advances, including data showing that effectors function to inhibit signals resulting from insertion of translocons into host plasma membrane. Studies showing that YopJ can induce a pro-inflammatory mode of cell death associated with caspase-1 activation during Yersinia infection (for a recent review see (Philip et al., 2012)) will not be covered due to space limitations.

Defining the translocon from the perspective of the host response

When discussing how translocon insertion can activate host responses, it is important to define which features of this process are likely sensed by the host cell. The pores detected in epithelial cells infected with effectorless Yersinia may represent unsealed translocons, like those found in membranes of erythrocytes (Costa et al., 2013, Montagner et al., 2011). However, in the former case, pore formation specifically requires an active host response triggered by translocon insertion (Viboud et al., 2005). Therefore, translocon insertion must generate a primary signal sensed by the host cell. If pores are formed, by unsealing of translocons or opening of host-derived channels, they are a downstream consequence of the host response, but not the source of the primary signal. Translocon insertion may induce primary signals by one of three mechanisms: 1) subtle perturbations of the plasma membrane (e.g. transient ion fluxes) caused by insertion of YopB and YopD into plasma membrane; 2) direct engagement of host signaling proteins by domains of translocators exposed to host cytosol; and 3) translocation of PAMPs. The first two mechanisms could be classified as patterns of pathogenesis, while the third would be an example of a PAMP-PV. These three mechanisms are not mutually exclusive and different combinations could be operating simultaneously depending on the nature of the infected host cell.

Translocon insertion induces host responses leading to pore formation and gene expression in Yersinia-infected cells

The first evidence that translocon insertion induces a host response was the finding that YopB-dependent pore formation in epithelial cells infected with Y. pseudotuberculosis required actin polymerization and was inhibited by catalytically active YopE or YopT (Viboud et al., 2005, Viboud et al., 2006). In addition, YopB was required for proinflammatory signaling in epithelial cells infected with Y. pseudotuberculosis (Viboud et al., 2005). This proinflammatory signaling response was linked to activation of the small GTPase Ras, the MAP kinases ERK and JNK, NF-κB and production of the chemokine IL-8. Signaling and IL-8 production was inhibited when epithelial cells were infected with Yersinia expressing catalytically active YopE, YopH, YopT or YopJ (Viboud et al., 2005, Viboud et al., 2006, Bose et al., 2011). Inhibition of actin polymerization prevented pore formation but not IL-8 production (Viboud et al., 2005). These data indicate that translocon insertion in epithelial cells induces a pore formation pathway and a gene expression pathway, and after they are delivered, several effectors function to counteract both responses (Fig.1A). Interestingly, in epithelial cells infected with Y. enterocolitica, YopT activity has been linked to increased expression of transcription factors (Klf2 and Gilz) that are regulators of anti-inflammatory responses (Dach et al., 2009, Koberle et al., 2012). Thus, YopT appears to function in two ways to inhibit pro-inflammatory innate immune responses in epithelial cells.

Fig. 1. Model for gene expression and pore formation responses induced by translocon insertion in epithelial cells or macrophages infected with Yersinia.

(A) In epithelial cells, the major pathway that stimulates host gene expression following translocon insertion results from activation of Rho GTPases. Invasin-β1 integrin signaling cooperates with translocon insertion to activate Rho. Activated Rho stimulates actin polymerization, leading to pore formation. Two possible pathways of pore formation are shown, one leading to opening of translocons and the other leading to opening of host-derived pores. YopE and YopT directly inhibit Rho. YopH and YopJ inhibit steps in the gene expression response pathway downstream of Rho. (B) In macrophages, insertion of translocons leading to perturbation of the plasma membrane or translocation of a PAMP are alternative but not mutually exclusive pathways leading to host gene expression and production of TNFα. YopJ dampens TNFα expression, suggesting that direct YopJ targets such as MAP kinase kinases (MAPKK) or IκB kinases (IKK) transmit signals in the gene expression response pathway.

Effector translocation is reduced by treatment of Yersinia-infected epithelial cells with inhibitors of actin polymerization or toxins that specifically inhibit Rho (Mejia et al., 2008). These data suggest that translocon insertion in plasma membrane induces Rho-mediated changes in the host cell that are required for an efficient translocation process. The subsequent delivery of effectors inhibits Rho activation to not only prevent pore formation and nuclear responses but, importantly, to downregulate the translocation process. These findings indicate that in epithelial cells, stimulation of host gene expression following translocon insertion is a result of a process wherein Yersinia intentionally activates of Rho GTPases to enhance effector translocation (Fig. 1A).

In contrast to epithelial cells, TLR4 plays a major role in activating signaling responses in macrophages infected with Y. pseudotuberculosis (Zhang et al., 2003). Zhang et al. observed that although signaling responses are reduced in TLR4^−/− macrophages, they are not eliminated, suggesting that additional PAMP-sensing mechanisms are operating during Y. pseudotuberculosis infection (Zhang et al., 2003). Auerbuch et al. identified a TLR-independent pathway for activation of NF-κB- and type I interferon (IFN)-regulated genes in macrophages infected with effectorless Y. pseudotuberculosis (Auerbuch et al., 2009). This response, detected in macrophages lacking the TLR adaptors MyD88 and Trif (MyD88^−/−/Trif^−/− macrophages), required translocon insertion (Auerbuch et al., 2009). The pro-inflammatory response, which culminated in the production of cytokines such as TNF-α (Fig. 1B), did not require the cytosolic peptidoglycan sensors Nod1 or Nod2. The authors proposed that Yersinia-infected macrophages sensed translocons by their insertion of pores in plasma membrane and or delivery of an unknown PAMP (Auerbuch et al., 2009). Several effectors modulated the macrophage response: YopJ partially suppressed TNF-α message production (Fig. 1B), and surprisingly, YopT and YopE caused elevated TNF-α message and protein production. Although the mechanism of such signaling enhancement by YopT and YopE remains unknown, it appeared to involve inactivation of Rho GTPases, as the catalytic activity of both effectors was required to increase TNF-α levels.

Kwuan et al. investigated how insertion of the translocon triggers innate immune signaling in MyD88^−/−/Trif^−/− macrophages (Kwuan et al., 2013). An effectorless Y. pseudotuberculosis strain encoding a YopD protein that lacks its predicted transmembrane domain (YopDΔTM) was used. The YopDΔTM mutant, defective for translocation function but retaining normal lytic activity on erythrocytes, did not trigger TNF-α expression in MyD88^−/−/Trif^−/− macrophages (Kwuan et al., 2013). These data are consistent with a model in which delivery of a PAMP into macrophages via the translocon is responsible for the transcriptional response in macrophages (Fig.1B). However, the pores made by the YopDΔTM mutant in macrophages were smaller and appeared with slower kinetics than those made by the wild type, suggesting that translocon insertion was impaired, which could contribute to a dampening of the host transcriptional response.

Translocon insertion impacts Yersinia survival in macrophages

Yersinia grows primarily in an extracellular form in vivo, however these bacteria can survive and grow inside phagocytic cells, which may be important at the early stages of infection (Pujol et al., 2005). The ability of Yersinia to survive in murine macrophages in vitro is influenced by the state of host cell activation and T3SS expression in the bacteria. Interestingly, unlike other bacterial pathogens where T3SS function is necessary for intracellular growth, the Yersinia T3SS can inhibit survival of Y. pseudotuberculosis in macrophages (Roy et al., 2004, Zhang et al., 2008). Roy et al. obtained evidence that during internalization of Salmonella enterica serovar Typhimurium or Y. pseudotuberculosis, the T3SSs of these pathogens stimulated phagolysosome fusion in macrophages (Roy et al., 2004). Phagosome-lysosome fusion required SytVII, a protein that localizes to lysosomes, and regulates vesicle fusion in a Ca²⁺-dependent manner. A Y. pseudotuberculosis YopE⁻ YopH⁻ YopT⁻ mutant (yopEHT) survived better in SytVII^−/− macrophages than in wild-type macrophages, while a T3SS null mutant (T3SS⁻) survived equally in both types of cells (Roy et al., 2004). Additionally, the yopEHT mutant but not the T3SS⁻ mutant caused lysosomal exocytosis in fibroblasts and pore formation in macrophages. From these data it was suggested that after phagocytosis, translocon insertion by the yopEHT mutant resulted in pore formation and Ca²⁺ influx into cytosol, stimulating SytVII-mediated phagosome-lysosome fusion and subsequent bacterial killing. Interestingly, recent data show that a T3SS in Salmonella (T3SS-1) and the Y. pseudotuberculosis T3SS stimulate Ca²⁺-dependent lysosome exocytosis in infected macrophages via a process that requires caspase-1 (Bergsbaken et al., 2011). One can envision that Ca²⁺- and caspase-1-dependent lysosome exocytosis is a general and conserved response to membrane perturbation by translocon insertion, leading to increased killing of intracellular bacteria and release of antimicrobial host factors.

In another study, T3SS-dependent killing of wild-type Y. pseudotuberculosis in macrophages was observed (Zhang et al., 2008). This intracellular growth restriction phenotype was YopB-dependent, indicating a requirement for translocon insertion, and was observed in a wild-type strain and a mutant expressing catalytically inactive YopH, YopE, and YopT. Additional studies are needed to determine if translocon insertion activates the Ca²⁺-SytVII pathway of intracellular killing in macrophages infected with wild-type Y. pseudotuberculosis, and how effectors modulate this response.

On the other hand, the T3SS in Y. enterocolitica has been shown to inhibit β1-integrin mediated bacterial internalization, protecting the bacteria from an autophagy-dependent pathway of killing inside macrophages (Deuretzbacher et al., 2009). Autophagy may play a species specific role in survival of Yersinia in macrophages, because Moreau et al. demonstrated that autophagosomes supported Y. pseudotuberculosis replication in macrophages, while inhibition of autophagy resulted in bacterial killing (Moreau et al., 2010). It is important to note that in the experiments of Moreau et al. the expression of T3SS was not induced before infection. Therefore, the effect of the T3SS on bacterial survival in macrophages or modulation of autophagy was not explored. A better understanding of how the T3SS modulates autophagy and survival of the different Yersinia species in macrophages will require more investigation.

Translocon insertion activates inflammasomes

In addition to triggering the host responses described above, insertion of the Yersinia T3SS translocon induces inflammasome activation (Bergsbaken et al., 2007, Brodsky et al., 2010, Schotte et al., 2004, Shin et al., 2007). Inflammasomes are multiprotein complexes that are assembled in response to numerous infectious and stress-associated stimuli (Martinon et al., 2007, Brodsky et al., 2009, Vladimer et al., 2013). Inflammasome assembly results in processing of pro-caspase-1 to its cleaved, active form. Active caspase-1 in turn mediates cleavage and secretion of the cytokines, IL-1β and IL-18, and a pro-inflammatory form of cell death termed pyroptosis associated with pore formation (Bergsbaken et al., 2009). Both pyroptosis and secretion of caspase-1-dependent cytokines play an important role in immunity to infection. Nod-like receptors (NLRs) are essential for inflammasome activation, and distinct NLRs are required for inflammasome activation in response to particular stimuli (Mariathasan et al., 2007). Nod-like Receptor Card domain 4 (NLRC4) is required for inflammasome activation in response to cytosolic delivery of bacterial flagellin, which occurs through the activity of T3SS or type IV secretion systems of various pathogens (Martinon et al., 2007, Brodsky et al., 2009, Vladimer et al., 2013). In addition to bacterial flagellin, NLRC4 detects the inner rod protein subunit of injectisomes, thereby enabling detection of bacteria that downregulate or do not express flagellin, but still possess T3SSs (Miao et al., 2010, Vladimer et al., 2013). Nod-like Receptor Pyrin domain 3 (NLRP3) induces inflammasome activation in response to a variety of stimuli including bacterial toxins (Martinon et al., 2007, Brodsky et al., 2009, Vladimer et al., 2013).

Effectorless Yersinia strains have facilitated the dissection of translocon-induced innate immune responses leading to inflammasome activation in macrophages. Effectorless Y. pseudotuberculosis induces rapid activation of caspase-1 in both naïve and LPS-primed macrophages through the NLRP3 inflammasome (Brodsky et al., 2010). This activation requires translocon insertion, as YopB and YopD are essential (Brodsky et al., 2010, Kwuan et al., 2013). Interestingly, in the presence of NLRP3, NLRC4 is not required for inflammasome activation in response to translocon insertion. However, in cells lacking NLRP3, a reduced but still significant level of IL-1β release and caspase-1 activation is mediated by NLRC4 (Brodsky et al., 2010), indicating that NLRC4 also contributes to inflammasome activation in response to translocon insertion, perhaps by detecting YscI, the inner rod protein of the T3SS. These data suggest that either sensing of the inserted translocon, or the translocation of unknown substrate(s) induces NLRP3 inflammasome activation in Yersinia-infected macrophages (Brodsky et al., 2010) (Fig. 2). Indeed, recent studies that utilized the translocation defective Y. pseudotuberculosis YopDΔTM mutant are consistent with the possibility that translocation of an as-yet undefined substrate is responsible for inflammasome activation in macrophages (Kwuan et al., 2013). However, the precise mechanism of inflammasome activation in response to translocon insertion remains to be determined.

Fig. 2. Model for inflammasome activation induced by translocon insertion in macrophages infected with Yersinia.

LPS-TLR4 signaling results in expression of pro-IL-1β and inflammasome components (not shown). Perturbation of the plasma membrane by translocon insertion generates a signal or the translocon delivers a PAMP. One or both events lead to activation of NLRP3 or NLRC4 inflammasomes, activation of caspase-1, maturation of IL-1β, pore formation and pyroptosis. YopK inhibits translocon-proximal signals in naïve or LPS-primed macrophages. YopM directly inhibits caspase-1 in LPS-activated macrophages.

Recently a non-canonical inflammasome that involves caspase-11 was found to induce macrophage cell death and release of IL-1α independently of caspase-1 and NLRP3 in response to Gram-negative bacteria and certain pore-forming toxins (Kayagaki et al., 2011, Broz et al., 2013). This non-canonical inflammasome requires macrophage priming through the TLR4-Trif and type I IFN signaling axis, and occurs with a kinetic delay relative to rapid canonical NLRP3 inflammasome activation (Rathinam et al., 2012, Gurung et al., 2012, Broz et al., 2013). Interestingly, the macrophage response to the Yersinia translocon involves both canonical inflammasomes and the non-canonical caspase-11 inflammasome, but does not require priming by a TLR4-Trif pathway, indicating that the presence of specialized secretion systems can induce rapid activation of the non-canonical inflammasome (Casson et al., 2013). The bacterial and host signals involved in both canonical and non-canonical NLRP3 inflammasome activation in response to specialized virulence-associated secretion systems of bacterial pathogens remain enigmatic.

YopK associates with the translocon and inhibits inflammasome activation

During infection of macrophages by wild-type Yersinia, inflammasome activation in response to translocon insertion is inhibited by two mechanisms – one mediated by the effector YopK, which limits translocation of other effectors and prevents recognition of the translocon by the host inflammasome machinery (Brodsky et al., 2010), and a second by the effector YopM (see below). YopK is required for Yersinia virulence in animal models of infection (Holmstrom et al., 1995, Thorslund et al., 2012). YopK has no primary sequence homology with other proteins in public databases, and the precise role of YopK in mediating Yersinia virulence remains to be determined. Several recent studies have investigated the molecular interactions between YopK and both bacterial and host proteins, providing new insight into its function. Initial studies found that yopK mutant strains exhibited a hypertranslocation phenotype, indicating that YopK regulated the translocation of other Yops (Holmstrom et al., 1997). These data imply that appropriately regulating translocation is critical for Yersinia to avoid triggering host inflammatory responses. The mechanism by which YopK regulates translocation is not entirely clear. YopK itself is translocated into host cells (Brodsky et al., 2010, Garcia et al., 2006), and this translocation is required for YopK to exert its regulatory function (Dewoody et al., 2013a, Dewoody et al., 2013b). YopK deficiency is also associated with increased lysis of erythrocytes, which typically correlates with pore size, suggesting that YopK somehow modulates the size or structural properties of the translocon (Holmstrom et al., 1997). Consistent with this model, YopK associates with YopB and YopD in Yersinia-infected cells, suggesting that YopK physically interacts with the translocon (Dewoody et al., 2013a, Brodsky et al., 2010). Furthermore, the absence of YopK results in increased levels of YopB and YopD insertion into erythrocyte membranes, and alters the molar ratio of YopB and YopD present in membranes (Thorslund et al., 2011). Notably, YopK prevents inflammasome activation in response to translocon insertion by Yersinia both in vitro and in vivo (Brodsky et al., 2010), but whether this is due to the effect of YopK on limiting translocation of a bacterial molecule that triggers inflammasome activation, or limiting translocon-mediated perturbations in the plasma membranes of infected cells is currently unknown (Fig. 2). The host kinase RACK1 has been shown to bind to YopK (Thorslund et al., 2011). The importance of RACK1 for regulation of translocation and inflammasome activation by YopK remains to be determined (Dewoody et al., 2013a, Thorslund et al., 2011)

YopM inhibits inflammasome assembly and caspase-1 in activated macrophages

The effector YopM has been functionally linked to suppression of innate immune responses in mice infected with Y. pestis (Straley, 2012, Viboud et al., 2005). In mice infected with Y. pseudotuberculosis, YopM function was associated with systemic induction of the immunosuppressive cytokine IL-10 (McPhee et al., 2010, McPhee et al., 2012). YopM consists of an N-terminal secretion signal, followed by two α-helices that serve to initiate the folding of the leucine-rich repeat (LRR) region that makes up the majority of the protein (Evdokimov et al., 2001). The LRR region, which contains between 13 and 21 repeats in different Yersinia strains, is followed by an unstructured tail that is conserved in different YopM isoforms. The LRR region of YopM adopts an overall horseshoe-like structure and this fold is predicted to serve as a binding platform for host cell proteins (Evdokimov et al., 2001). In the host cytosol YopM binds to and activates two protein kinases, RSK1 and PRK2 (McDonald et al., 2003, Hentschke et al., 2010). The C-terminal tail of YopM is required for RSK1 binding in macrophages and virulence of Yersinia in mice (McCoy et al., 2010, McPhee et al., 2010). It remains unknown how complexes of YopM and RSK1 or PRK2 contribute to Yersinia virulence.

LaRock and Cookson recently reported that a specific isoform of YopM found in Y. pestis and some Y. pseudotuberculosis strains binds to and inhibits caspase-1 in macrophages fully activated by exposure to LPS for 18 hr before infection (LaRock et al., 2012). YopM inhibited recruitment of caspase-1 to inflammasomes, caspase-1 cleavage, secretion of IL-1β, pyroptosis and lysosome exocytosis in LPS-activated macrophages infected with Y. pseudotuberculosis. It is important to point out that translocon insertion by wild-type Y. pseudotuberculosis activates caspase-1 in LPS-activated macrophages (Bergsbaken et al., 2007), but with delayed kinetics compared to a yopM mutant (LaRock et al., 2012). Purified YopM bound to cleaved caspase-1 and inhibited caspase-1 activity in vitro. Binding to and inhibition of caspase-1 required Asp residue 271 located in the 10^th LRR of YopM. This Asp residue is located in a consensus caspase-1 cleavage motif (YLTD), however caspase-1 did not cleave YopM. The data suggest that the YLTD motif in YopM acts as a pseudosubstrate inhibitor of caspase-1, similar to endogenous inhibitors such as Flightless-1 (LaRock et al., 2012, Li et al., 2008). Importantly, evidence that inhibition of caspase-1 is of major importance for YopM virulence function was obtained by infection of caspase-1 deficient mice: the virulence defect of a Y. pseudotuberculosis yopM mutant was fully rescued in these mice (LaRock et al., 2012). The above data provide the first molecular understanding of how YopM inhibits pro-inflammatory responses, and also raise a number of questions that will need to be addressed in future studies. For example, do YopM isoforms that lack the YLTD motif also inhibit caspase-1? Is YopM a bifunctional effector, with the LRR region acting as a caspase-1 inhibitor and the C-terminal tail working in conjunction with RSK1? It will also be interesting to determine if inhibition of caspase-1 is linked to the remarkable demonstration that purified Y. enterocolitica YopM can penetrate into cultured cells and inhibit transcription of pro-inflammatory cytokine genes (Ruter et al., 2010).

Conclusions and future perspectives

Several features of the Yersinia T3SS have made it an ideal system to study how translocators activate and effectors inhibit innate immune responses. These features include the genetic tractability of Yersinia, the availability of natural rodent hosts as infection models, and the small number of effectors (seven). From the analysis of effectorless Yersinia mutants, evidence has been obtained that insertion of YopB and YopD into the plasma membrane to form a translocon is sensed by the host cell, resulting in innate immune responses that differ according to the host cell type.

In epithelial cells, translocon insertion somehow activates Rho and actin polymerization to promote efficient translocation. This pathway has a built-in negative feedback mechanism: YopE and YopT inhibit Rho after they are translocated. In the absence of YopE and YopT, Rho activates a transcriptional response, and, via actin polymerization, stimulates pore formation (Fig. 1A). Even in the absence of YopE and YopT, the transcriptional response is dampened by the activities of YopH and YopJ, presumably acting at steps in signaling pathways that are downstream of Rho (Fig. 1A). Activation of Rho by translocon insertion can be considered a pattern of pathogenesis as the resulting transcriptional response produces IL-8 and the cytosolic response produces pores, both of which are highly proinflammatory (Fig. 1A). It is not known how translocon insertion activates Rho, although it may involve direct interaction of host proteins with domains of YopB or YopD exposed to cytosol. It has been shown that β1 integrin signaling triggered by Yersinia Invasin cooperates with translocon insertion to activate Rho (Mejia et al., 2008) (Fig. 1A). Additional studies are needed to understand how actin polymerization causes pore formation, and the nature of the pores formed under these conditions.

In macrophages, translocon insertion presumably activates Rho, but this pathway may be difficult to detect due to other more prominent innate sensing mechanisms that exist in these cells. Translocon insertion into macrophage plasma membrane triggers a transcriptional response (Fig. 1B), activation of inflammasomes (Fig. 2), and increased killing of Yersinia in phagosomes. In all three cases the nature of the primary signal generated by translocon insertion in macrophages is unknown, and is an important and active area of investigation. Translocon insertion may cause a perturbation of the membrane that can be sensed as a pattern of pathogenesis, or inserted translocons may also allow a PAMP-PV, such as the inner rod of the injectisome, to gain access to the cytosol of the macrophage. It is also possible that both processes occur simultaneously. The use of strains encoding YopD variants that are selectively defective for producing one signal, e.g. translocation of a PAMP-PV, is a promising approach to address the question of which process is important (Kwuan et al., 2013). The YopDΔTM mutant is defective for translocation, but also has a partial defect in pore formation, suggesting that finer scale changes in the protein sequence (e.g. single amino acid substitutions) may be needed to separate distinct YopD functions. Along these lines, Costa et al. recently reported that introduction of single amino acid changes into a predicted alpha-helix in the C-terminus of YopD caused a defect in pore formation in infected erythrocytes but no impairment of translocation into epithelial cells (Costa et al., 2013).

YopK and YopM represent interesting examples of effectors that function to inhibit inflammasome activation in macrophages. Both effectors counteract inflammasome activation that occurs downstream of translocon insertion (Fig. 2), but their underlying mechanisms are distinct. YopK inhibits inflammasome activation in macrophages that are naïve, LPS-primed or LPS-activated. In contrast, YopM seems to inhibit caspase-1 activation only in LPS-activated macrophages. YopK is positioned in contact with the translocon and may inhibit proximal events, e.g. dampening membrane perturbations or delivery of a PAMP-PV. YopM inhibits terminal steps in inflammasome activation, including recruitment of caspase-1 to these structures and directly inhibiting its enzymatic activity (Fig. 2). It is conceivable that YopK and YopM act simultaneously in LPS-activated macrophages to inhibit distinct inflammasome pathways. Thus, caspase-1 activation processes in host cells infected with pathogens appear to be highly complex, as even within one cell type such as macrophages, several distinct mechanisms can be used, and the outcome will depend on which pathways are activated during infection in response to the specific repertoire of PAMPs and virulence factors expressed by the microbe. Future studies that reveal how YopK and YopM function are likely to lead to new insights into inflammasome activation mechanisms.

The T3SS endows Yersinia with a high degree of virulence because it allows the pathogen to bypass PRR-directed host responses and host responses to translocon insertion. Future research will need to address the possibility that there are additional innate immune responses to the T3SS. Y. pseudotuberculosis or Y. pestis strains that express hyperactive forms of YopJ are attenuated in vivo (Zauberman et al., 2009, Philip et al., 2012), suggesting that a form of effector-triggered immunity can result from excessive YopJ activity. The activities of effectors are likely fine-tuned to avoid activating immune responses, and therefore it may be necessary to overexpress hyperactive alleles of effectors in Yersinia to reveal and study novel types of effector-triggered immunity.