Salmonella Effectors: Important players modulating host cell function during infection

doi:10.1111/j.1462-5822.2011.01701.x

Cell Microbiol. Author manuscript; available in PMC 2012 December 1.

Published in final edited form as:

Cell Microbiol. 2011 December; 13(12): 1858–1869.

Published online 2011 October 10. doi: 10.1111/j.1462-5822.2011.01701.x

PMCID: PMC3381885

NIHMSID: NIHMS323366

Salmonella Effectors: Important players modulating host cell function during infection

Terence A. Agbor and Beth A. McCormick^*

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, USA

^*Corresponding Author: Beth A. McCormick, Ph.D. University of Massachusetts Medical School Department of Microbiology and Physiological systems 55 Lake Avenue North Worcester, MA 01655 Tel: 508-856-6048 Fax: 508-856-3355 ; Email: beth.mccormick/at/umassmed.edu

The publisher's final edited version of this article is available at Cell Microbiol

See other articles in PMC that cite the published article.

Summary

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative foodborne pathogen that causes gastroenteritis in humans. This bacterium has evolved a sophisticated machinery to alter host cell function critical to its virulence capabilities. Central to S. Typhimurium pathogenesis are two Type three secretion systems (T3SS) encoded within pathogenicity islands SPI-1 and SPI-2 that are responsible for the secretion and translocation of a set of bacterial proteins termed effectors into host cells with the intention of altering host cell physiology for bacterial entry and survival. Thus, once delivered by the T3SS, the secreted effectors play critical roles in manipulating the host cell to allow for bacteria invasion, induction of inflammatory responses, and the assembly of an intracellular protective niche created for bacterial survival and replication. Emerging evidence indicates that these effectors are modular proteins consisting of distinct functional domains/motifs that are utilized by the bacteria to activate intracellular signaling pathways modifying host cell function. Also, recently reported are the dual functionality of secreted effectors and the concept of “terminal reassortment”. Herein, we highlight some of the nascent concepts regarding Salmonella effectors in the context infection.

Introduction

Salmonella enterica serovar Typhimurium (S. Typhimurium), the causative agent of salmonellosis, is a Gram-negative facultative intracellular bacterial pathogen capable of infecting a number of hosts and causing significant morbidity and mortality globally. Salmonellosis is among the most common food-borne diseases in humans (representing 20% of all food-borne infections), and is a major public health and economical burden worldwide (Kubori et a., 1998; Galan, 1999; Coburn et al., 2007). In the USA alone, there is an estimated 1.4 million cases of non-typhoidal Salmonella annually, resulting in over 1,000 deaths (Scallan et al., 2011). S. Typhimurium infection in humans is typically acquired by ingestion of contaminated food or water leading to acute gastroenteritis with clinical manifestations of diarrhea, abdominal pain, nausea and vomiting (Coburn et al., 2007; McGovern and Slavutin, 1979). However, in persons at risk such as infants/small children, the elderly, and those with suppressed immunity, Salmonella infections can become very serious, leading to severe complications and sometimes death.

Fundamental to Salmonella virulence is its ability to invade and trespass the intestinal mucosal barrier. Key to this success, Salmonella species have evolved sophisticated virulence mechanisms to manipulate host cell functions to their own benefit. Two type III secretion systems (T3SS) encoded within pathogenicity islands SPI-1 and SPI-2 are responsible for the delivery of a series of bacterial effectors into host cells with the intention to reprogram eukaryotic cell functions (Galán and Wolf-Watz, 2006; Coburn et al., 2007b; Schraidt et al., 2010). While the T3SS apparatus is highly conserved, the translocated effectors are unique proteins with very specialized functions critical to virulence. Moreover, considerable evidence indicates that individual effectors secreted by the T3SS are modular proteins composed of functionally distinct domains that may act in different stages of the infection process. This review highlights this important emerging concept.

Type III Secretion System (T3SS)

The T3SS is a specialized organelle the principal function of which is to direct the delivery of bacterial proteins into eukaryotic cells. While this secretion system is present in several bacterial species, including animals and plant pathogens, it is exclusive to Gram-negative bacteria (Galan, 2001; Galan and Colmer, 1999). Located on the chromosome of Salmonellae are distinct genetic loci (with base composition distinct from rest of chromosome; low G+C ratio) that encode for a number of virulence determinants. These regions termed Salmonella pathogenicity islands (SPI) have been acquired by horizontal gene transfer. In S. Typhimurium, there are two T3SS systems encoded for by SPI-1 and SPI-2, and these secretion systems are thought to play distinct yet overlapping functions. For example, whereas the T3SS encoded for by SPI-1 plays a key function in bacterial invasion, the T3SS encoded for by SPI-2 is thought to be required for bacterial survival in host cells. However, it has recently been reported that mutations of the S. Typhimurium SPI-2 T3SS induce significant reduction in the expression of several SPI-1 T3SS genes, thereby impairing the ability of the bacteria to invade epithelial cells (Hensel et al., 1998; Deiwick et al., 1998). These observations suggest that SPI-1 and SPI-2 depend on each other for efficient functioning. While the T3SS expressed by S. enterica serovar Typhimurium will be the focus of this review, it should be noted that other pathogenic Salmonella serovars causing distinct diseases express the SPI1 and SPI2-encoded Type three secretion systems, as well.

The main function of the T3SS secretion system is to secret and translocates bacterial effector proteins across host cell membranes. Such secretion systems have the following properties (Galan, 2001; Galan and Collmer, 2006):

i)
the absence of a cleavable signal peptide in the secreted proteins, which is characteristic feature of proteins secreted via the sec-mediated pathway
ii)
the requirement for customized accessory proteins or chaperones for many of the secreted proteins
iii)
the requirement of a host cell contact for full activation of the pathway.

In addition, it has been well documented that the T3SS and the bacterial flagellar export apparatus are related both at the protein sequence level and with regards to higher level-order assemblies, indicating that both systems may have a common origin. Indeed, both systems are known to perform similar functions (that of protein secretion) with the basal body and many of the flagellar proteins secreted in a fashion paralleling the T3SS (Galan, 2001; Hueck, 1998; Van Gijsegem et al., 1993; Galan et al., 1992; Groisman and Ochman, 1993; Ginocchio and Galan, 1995, Michiels et al., 1991; Rosqvist et al., 1995). Likewise, other similarities include, the use of specific chaperones or molecular mechanisms that regulate the length and size of the appendage(s) (e.g., hook in the case of flagella, and needle in case of T3SS).

Components and functions of the T3SS

The Salmonella T3SS (both T3SS-1 and T3SS-2) is composed of approximately 20–30 proteins with a major subset of these proteins performing a structural role – that of forming the supramolecular injection apparatus termed the needle complex (Kubori et al., 2000). Detailed examination of the needle complex by electron microscopy revealed that this structure consists of four (two pairs) membrane-localized rings that span the inner and outer bacterial membranes and are joined by a hollow cylindrical structure that functions as the base for the externally protruding needle-like structure (Kubori et al., 2000; Kimbrough and Miller, 2000). Another set of proteins called the translocons function to form a pore at the host cell membrane called the translocation pore from where bacterial effectors are injected into the host cell cytoplasm. Apart from the structural proteins and the translocon, the remaining proteins secreted by the T3SS are called effectors. Secreted effector proteins translocated into host cells by S. Typhimurium play multiple roles in altering host cell physiology ranging from invasion, induction of inflammation to intracellular survival and replication. A summary of the functions of the secreted/translocated effectors is provided in Table 1.

Table 1

Salmonella secreted effectors and their role in disease

Salmonella effectors mediate bacterial entry into host epithelial cells

Like most Gram-negative bacteria (e.g. Shigella, Yersinia), S. Typhimurium has the discrete capacity to invade non-phagocytic cells, such as the intestinal epithelial cells. This process termed bacterial mediated endocytosis is governed by several of the Salmonella secreted effectors. These effectors include but are not limited to SipA, SipC, SopB, SopE, and SopE2, each of which has distinct as well as redundant functions. As an example, to assist in bacterial entry, both SipA and SipC function not only to bind and bundle actin but also to polymerize actin (Wall et al., 2007; McGhie et al., 2004; Myneni and Zhou, 2010; Hayward and Koronakis, 1999). In general, the principal function of these effectors is to induce the membrane ruffling, which results in the “phagocytosis” of the bacteria.

SipC has also been observed to be a dual functioning protein with roles involved in actin bundling as well as in actin nucleation (Hayward and Koronakis, 1999; McGhie et al., 2009). The C-terminus of SipC binds to and bundles F-actin, an interaction that prompts Salmonella invasion (Myeni and Zhou, 2010). The C-terminus of SipA has also been shown to bind to and polymerize actin by decreasing the critical concentration for actin assembly which stabliizes the actin filaments (Wall et al., 2007; McGhie et al., 2009, 2004). SipA also functions to potentiate the actin nucleation and bundling function of SipC, in addition to enhancing T-plastin activity (Hayward and Koronakis, 2002).

Another secreted effector, SopB, is an inositol phosphatase, an enzyme that hydrolyzes a variety of phosphoinositides and inositol phosphates at the host cell membrane during

Salmonella invasion (Norris et al., 1998; Patel and Galan, 2005). In a separate function, SopB also contributes to the remodeling of actin by indirectly activating the exchange factor RhoG through its activation of the SH3-containing guanine nucleotide exchange factor (SGEF), an upstream activator of RhoG (Patel and Galan, 2006). This contributes to the extensive actin remodeling that occurs during Salmonella invasion. SopB dephosphorylates phosphatidyl inositol (PtdIns)(3,4,5) triphosphate to (PtdIns)(3,5)-bisphosphate and hydrolyzes the cellular Inositol (1,3,4,5,6) pentakisphosphate (IP5) to generate Ins(1,4,5,6)P4 (Norris et al., 1998). Mutations of this phosphatase activity abolish SopB-induced cytoskeletal reorganization (Zhou et al., 2001). Moreover, ectopic expression of SopB in COS cells induce localized actin-rich ruffles, similar to those induced upon Salmonella entry (Zhou et al., 2001).

SopE and SopE2, on the other hand, modulate the host actin cytoskeleton by activating the Rho GTPases, which through the actin-related protein 2/3 (Arp2/3) drives actin cytoskeletal assembly (Patel and Galan, 2006). SopE and SopE2 activate distinct substrates, Rac-1 and Cdc42, respectively, both of which function as small Rho GTPases (Patel and Galan, 2006). Cumulatively, these events (e.g. actin bundling, actin polymerization and cytoskeletal rearrangements) instructed by specific Salmonella effectors result in the cellular process of epithelial cell membrane ruffling and the formation of membrane invaginations that foster bacterial uptake and internalization.

Once S. Typhimurium is internalized, the effector protein SptP mediates the recovery of the normal organization of the host cell membrane by reversing the cytoskeletal changes induced by the other effectors (Galan and Zhou, 2000). Thus, SptP functions as a GTPase activating protein (GAP) (Fu and Galan, 1999; Humphreys et al. 2009; Patel and Galan, 2005) whose GAP activity down-regulates Cdc42 and Rac-1, which is required for termination or reversal of membrane ruffling.

Salmonella effectors induce intestinal inflammatory responses

Following Salmonella internalization, the host cell responds by activating pro-inflammatory responses. This response is largely influenced by T3SS-1 secreted effectors, which have the capacity to manipulate host cell physiology and induce intestinal inflammation. For example, the secreted effectors SopE, SopE2, and SopB activate the small GTPases Cdc42 and Rac-1, triggering the activation of different downstream effectors of the Mitogen-activated kinase (MAPK) pathway such as Erk, Jnk and p38 MAPK that in turn leads to the subsequent activation of the transcription factors AP-1 and NF-κB (Haraga et al., 2008; Hobbie et al., 1997, Patel and Galan, 2008). AP-1 and NF-κB play a critical role in the transcription of proinflammatory chemokines such IL-8. IL-8 is a potent neutrophil chemoattractant that promotes the movement of neutrophils across the endothelial cell barrier to the subepithelium of the intestinal mucosa.

SipA, on the other hand, has been shown to be both necessary and sufficient to initiate signal transduction cascades that lead to the directed migration of neutrophils (PMN) across the intestinal epithelial barrier into the lumen during acute states of active intestinal inflammation (Srikanth et al., 2010; Lee et al., 2000; Silva et al., 2004, Criss et al., 2001; Wall et al., 2007). The underlying molecular mechanisms include SipA activating a novel ADP-ribosylation factor (ARF)-6 and phospholipase D lipid-signaling cascade (Criss et al., 2001), which culminates in the activation of protein kinase C (PKC)-α (Silva et al., 2004). Through a mechanism that is incompletely understood, PKC-α activation governs the control of the 12-lipoxygenase pathway, which upon activation leads to the synthesis of another potent neutrophil chemoattract, hepoxilin A3 (HXA₃) (Pazos et al., 2008; Mrsny et al., 2004). Working in concert with IL-8, HXA₃ drives the transepithelial migration of neutrophil across the intestinal epithelial barrier into the lumen, the final step in formation of crypt abscesses.

SipA also harbors distinct functional motifs that account for its induction of PMN transcellular signals and the binding to actin (as described above). Prior truncation analysis of SipA revealed that the actin binding function of SipA is localized to a C-terminal fragment (amino acids 426–684), termed SipAb, that consists of two actin binding arms, extending from a central globular domain (Wall et al., 2007, Mitra et al., 2000). Additionally, our laboratory discovered that an N-terminal fragment of the SipA effector protein (amino acids 2–425), termed SipAa, harbors the functional domain that accounts for the activation of the epithelial signaling pathway, which in turn, promotes PMN transepithelial migration (Wall et al., 2007).

SopB through its inositol phosphatase activity generates inositol 1,4,5,6 tetrakisphosphate by hydrolyzing inositol 1,3,4,5,6 pentakisphosphate (Steele-Mortimer et al., 2000; Norris et al., 1998), which promotes cellular chloride ion secretion, and ion flux, thereby inducing diarrhea (Bertelsen et al., 2004; Layton and Glyov, 2007). Furthermore, SipA, SopB, SopE and SopE2 through their activation of the small Rho GTPases were recently reported to be involved in the disruption of the structure and function of the epithelial cell tight junction, a characteristic feature of Salmonella induced enteritis (Boyle et al., 2006). Transient disruptions in epithelial barrier function also facilitate the transepithelial migration of PMNs across the epithelial barrier into the lumen. Additionally, SipB modulates the intestinal inflammatory response by increasing the production of IL-1β and IL-18 through its ability to activate caspase-1; SipB binds and activates the caspase-1 inflammasome (Hersh et al., 1999). SopA and SopD are also involved in Salmonella induced enteritis in calves (Haraga et al., 2008; Wood et al., 2000).

Given that S. Typhimurium is a self-limited illness, a few secreted effectors have been recognized to antagonize host cellular immune responses. For instance, SptP, through both its GTP activating protein (GAP) and tyrosine phosphatase activities inhibits ERK/MAP kinase activation (Murli et al., 2001; Lin et al., 2003). Additionally, the phosphothreonine lyase activity of SpvC also inhibits host Erk, Jnk and p38 MAP kinases (Mazurkiewicz et al., 2008; Li et al., 2007). In addition, another effector, AvrA, possesses acetyltransferase activity toward specific mitogen-activated protein kinase kinases (MAPKKs) and potently inhibits c-jun N-terminal kinase (JNK) and nuclear factor kappa-B (NF-κB) signaling pathways in both transgenic Drosophila and mouse models of S. Typhimurium infection (Jones et al., 2008). Lastly, the SPI-2 T3SS effectors, SseL and SspH1, have recently been reported to inhibit the NF-κB pathway through different mechanisms (Le Negrate et al., 2008; Rohde et al., 2007), thereby antagonizing host cellular immune responses and prolonging intracellular bacterial survival.

Salmonella effectors mediate the intracellular survival of bacteria in host cells (formation and biogenesis of the Salmonella containing vacuole (SCV))

Immediately following the internalization of the Salmonellae into the host cells, the bacteria resides in a membrane bound compartment called the Salmonella containing vacuole (SCV). The SCV is a vacuolar microenvironment formed and adapted for intracellular survival and replication of the bacteria through the direct activities of both SPI-1 and SPI-2 T3SS effector proteins.

SopB mediates the initial formation of the SCV at the cell membrane during Salmonella internalization into host cells through its phosphoinositol (PI) phosphatase activity. Through its phosphatase activity, SopB hydrolyzes PtdIns(3,5)P2 and PI(3,4,5)P3 to PI(3)P which activates protein kinase B (or Akt). It also promotes macropinosome and phagosome formation by depleting the cellular levels of PI(4,5)P2 (Steele-Mortimer et al., 2000; Terebiznik et al., 2002; Malik-Kale et al., 2011). In this role, SopB also contributes to SCV maturation, and thus plays an important role in the establishment of the Salmonella intracellular niche (Terebiznik et al., 2002, Schlmberger et al., 2006). Furthermore, SopB recruits the endosomal/lysosomal protein, GTPase Rab5 (a marker of early endosome and lysosome), to the SCV membrane and via its interacting partner, Vps34, generates PtdIns(3)P at the SCV membrane (Mallo et al., 2008). PtdIns(3)P is necessary for the recruitment of the early endosome associated protein 1 (EEA1) to the SCV (McGhie et al., 2009). This effector also inhibits the degradation of the epidermal growth factor receptor (EGFR) by lysosomes, and recently it was reported that SopB contributes in the disappearance of the late endosomal/lysosomal markers from the maturing SCV through their recruitment of sorting nexin-1 (SNX1) and SNX3 (Dukes et al., 2006; Bujny et al., 2008; Braun et al., 2010). SNX 3 depletion results in impairment in the delivery of the endosomal proteins Rab7 and Lamp1, implying that SNX(s) are an important requirement for SCV maturation (Braun et al., 2010). Together, these observations demonstrate that phosphoinositol metabolism catalyzed by SopB plays a crucial role in formation and early maturation of the SCV. Another effector protein important in SCV biogenesis is SptP. SptP is a bifunctional effector working both as a tyrosine phosphatase and a GTPase activating protein (GAP) (Fu and Galan, 1999; Humphreys et al., 2009; Malik-Kale et al., 2011). SptP dephosphorylates valosin-containing protein (VCP), a member of the AAA protein family required for efficient SCV maturation (Humphreys et al., 2009; Kubori and Galan, 2003; Malik-Kale et al., 2011).

Once formed, the SCV localizes to the perinuclear region at the microtubule-organizing center (MTOC), a location crucial for its accessibility to cellular and/or bacterial components that travel along microtubules using the dynein-mediated transport system (Wileman, 2007; Malik-Kale et al., 2011). During migration to the perinuclear region, the SCV transiently recruits the Rab7-interacting lysosomal protein (RILP) which interacts with the minus end-directed microtubule motor, dynein. Subsequently, SifA, a SPI-2 secreted effector induces the formation of tubular structures called Sifs (Salmonella-induced filaments). SifA also regulates the location and intracellular replication of the bacteria within the SCVs (Stein et al., 1996; Malik-Kale et al., 2011). The absence of SifA results in a compromised SCV membrane accompanied by the release of Salmonella into the cytosol (Dumont et al., 2010). SifA directly recruits the host protein SKIP (SifA and kinesin interacting protein), which in turns binds to and recruits kinesin-1 (Dumont et al., 2010; Boucrot et al., 2005), an interaction that is required for SCV maturation (Leone and Meresse, 2011). The interplay between SifA and Rab7 promotes Sif extensions by uncoupling Rab7 from RILP and prevents dynein recruitment to Sifs (Ramsden et al., 2007, McGhie et al., 2009)

SipA also cooperates with SifA in SCV biogenesis and maturation as cells infected with Salmonella deficient in SipA results in mis-localization of SifA and a peripheral distribution of the SCV (Brawn et al., 2007). The SCV positioning function of SipA is located on its N-terminal domain while the C-terminal actin-binding domain of SipA is also required for F-actin accumulation around the SCV, and thus SCV maturation. The actin polymerizing function of SipA also plays a critical role in the localization of SCV as depolymerization of the actin cytoskeleton in the host cells results in the redistribution of the SCV away from the perinuclear region to the periphery (Wasylnka et al., 2008; Malik-Kale et al., 2011).

The SPI-2 effectors, SseG and SseF, have been reported to maintain the SCV near the perinuclear region, forming a complex that tethers the SCV to the Golgi apparatus and manipulates dynein activity. Golgi localization of the SCV is necessary for efficient replication of bacteria as disruption of the Golgi network and loss of SseG results in diminished bacteria growth (Salcedo et al., 2003, Hallstrom and McCormick, 2011). These effectors also augment Sif formation by modulating the aggregation of the endosomal components since Salmonella lacking the sseF, sseG and/or sopD2 genes induce fewer Sifs compared to wild-type bacteria (McGhie et al., 2009; Ramsden et al., 2008).

SseJ, a SPI-2 secreted effector has homology with the glycophospholipid-cholesterol acyltransferases, which are enzymes involved in cholesterol esterification and lipid body formation (Lossi et al., 2008). SseJ localizes to the SCV and has been shown to esterify cholesterol in vitro and during infection in the SCV, a process required for full virulence of S. Typhimurium during systemic infection in mice (Ohlson et al., 2005; Lossi et al., 2008). In addition, SopD2, PipB, PipB2, SseG, SifA, SseJ all localize to the SCV and have been documented to modulate SCV tubulation (Ohlson et al., 2008; Malik-Kale et al., 2011). Taken together, both SPI-1 and SPI-2 T3SS effectors play an essential role in the formation of the SCV, an important intracellular niche for bacterial replication.

Observation of Effectors with dual function

Over the past decade, progress has been made in understanding the molecular mechanisms by which effector proteins mediate their function. Amongst the findings, several lines of evidence suggest that certain effectors have evolved in such a way that they harbor distinct functional domains on either termini of the protein. As an example, the SPI-1 effector protein SipA has been reported to contain two functional domains: the C-terminal domain has been reported to be involved in actin binding (Mitra et al., 2000), whereas the N-terminal domain has been demonstrated to be essential for inducing transcellular signals that guide PMN transepithelial migration (figure 1) (Wall et al., 2007).

Figure 1

Salmonella effectors with dual functions

SifA is another effector protein that has been reported to have a dual function. This effector plays a critical role in SCV formation and maintenance, especially regarding Sif formation. The C-termini domain of SifA contains a WxxxE motif, a protein motif present in bacterial effectors that directly mimic activated GTPases (Alto et al., 2006; Ohlson et al., 2008) while the N-termini binds to SKIP, a cellular protein that binds to the motor protein kinesin and is required for SCV tubulation (figure 1) (Ohlson et al., 2008). Therefore, SifA has both a GTPase activity located at its C-termini, as well as a motor function (by binding to SKIP (kinesin)) located at its N-termini. Furthermore, SptP, also functions both as a tyrosine phosphatase and a GTPase activating protein (GAP) (Fu and Galan, 1999; Humphreys et al. 2009; Malik-Kale et al., 2010).

Two interesting hypotheses have recently been postulated regarding the evolution of the dual functionality exhibited by effector proteins: i) “terminal reaasortment” hypothesis postulated by the Guttman lab, and ii) the “Caspase-3 processing” hypothesis postulated and discovered in our laboratory (Stavrinides et al., 2006; Srikanth et al., 2010).

Terminal Reassortment in the evolution of effector proteins

One of the most prominent features amongst T3SS effectors is their modular nature wherein these proteins consist of well-defined regions (called domains) that confer a specific function. These domains (within the same effector) usually mediate different and unrelated functions suggesting that these domains evolved independent of each other and combined to form a chimeric effector (Stavrinides et al., 2006). This process, termed “terminal reassortment”, is a common theme not only among several Salmonella effectors but also other bacteria effectors as well.

A conventional example is the case of the Salmonella effector, SptP whose N-terminal domain has sequence homology with ExoS and YopE proteins of Pseudomonas and Yersinia spp., respectively, while its C-terminal is highly homologous to another Yersina protein, YopH (Kaniga et al., 1996; Dean, 2011). Described as design-by-recombination, “terminal reassortment” explains the high diversity amongst bacterial effectors and together with horizontal gene transfer and genomic shuffling, it results in the fusion of these functional effector modules with the C-termini of the different effectors (Dean, 2011). This establishes the newly formed chimeric protein with a new C-termini and the original N-terminal domain, giving rise to a single step evolution mechanism that yields new effector proteins (Dean, 2011; Stavrinides et al., 2011). Four Salmonella effectors SifA, SspH1, SseI and SseJ all share a homologous N-terminal domain but different C-terminal regions, further substantiating the evidence and significance of the terminal reassortement hypothesis (Hansen-Wester et al., 2002, Dean et al., 2011). Strengthening this hypothesis is the fact that ~32% of T3SS effector families in bacterial species are chimeric proteins, which is much greater than in any other protein family analyzed to date, and highlights the importance of this process in the evolution of bacteria virulence factors (Stavrinides et al., 2006; Dean, 2011). Thus, it is likely that terminal reassortment plays a role in effector diversity by the addition of a new function to an already existing effector protein.

Caspase-3 processing of effector proteins

As described above, a critical feature of Salmonella effectors is the presence of functional modules that comprise domains or motifs that confer an array of functions within the eukaryotic cell. These domains/motifs represent a fascinating repertoire of molecular determinants with important roles during infection (Dean, 2011). Here we will discuss some of the recent findings on the understanding of some Salmonella effector motifs and their role in infection, which may be linked to the terminal reassortment hypothesis.

In examining the bi-functional properties of SipA, we discovered that SipA contains a caspase-3 recognition and cleavage site (DEVD) at amino acid position 431, which is precisely located at the junction between the SipAa N-terminal domain and the SipAb C-terminal domain (Srikanth et al., 2010). This motif is physiologically significant, as a single amino acid substitution to a sequence not recognized by caspase-3 profoundly attenuates the virulence of this pathogen in both in vitro and in vivo models of salmonellosis (Srikanth et al., 2010). Conversely, knocking out the caspase-3 gene in mice resulted in a significantly less virulent Salmonella infection, and dramatically reduced the severity of intestinal inflammation (Srikanth et al., 2010). Notably, SipA, itself, was found to be necessary and sufficient for early caspase-3 activation, but in a process independent from the apoptotic cascade. Further analysis of the S. Typhimurium T3SS revealed the presence of caspase-3 cleavage motifs in other secreted effectors with known bi-functional properties (i.e., SopA, SifA) (Srikanth et al., 2010), indicating this phenomenon is not isolated to SipA. Remarkably, no caspase-3 cleavage motifs were identified in the structural proteins, chaperones, or transcriptional regulators that together comprise the T3SS.

The identification of caspase-3 cleavage motifs in secreted effectors introduces a novel concept that certain effector proteins of S. Typhimurium exist in a pro-form that requires processing to become functionally active (or perhaps inactive). Since caspase-3 cleavage motifs detected in SPI-1 and SPI-2 T3SS are restricted to secreted effectors, it is tempting to speculate that caspase-3 cleavage maybe a general subversion strategy employed by Salmonella for processing of its secreted effectors. Furthermore, while bacteria have previously been described to interact with caspase-3, our study is the first to document that a pathogenic organism is able to sabotage a major host death pathway in the absence of apoptosis, thus directly exploiting the host enzyme, caspase-3, to facilitate infection and pathogenesis.

Based on these new concepts, we hypothesize that terminal reassortment precedes caspase-3 processing in the generation of new bi-functional effectors. First, through terminal reassortment, a new functional motif is added to an “existing” effector protein to form a chimeric protein (as may have been the case with SipA domains described above). Second, upon infection, the bacteria utilize the host cell protein, caspase-3 to process the chimeric effector yielding the two functional motifs, which may play an important role in bacterial pathogenesis.

WEK(I/M)xxFF motif

Another important motif identified in Salmonella secreted effectors is the WEK(I/M)xxFF motif. This motif is located on the N-termini of SifA, SopD2, SseJ and SspH2 and is required for Golgi targeting of these effectors (Brown et al., 2006). Other functional motifs have also been identified in other effector proteins and their putative functions have been suggested (Dean, 2011).

Conclusions

To invade and survive in the host cells, S. Typhimurium have developed important mechanisms to manipulate host cell physiology to their advantage. Upon contact with host cells, S. Typhimurium T3SS-1 and T3SS-2 are activated leading to the secretion/tranlocation of effectors. These effectors have been shown to work independently or in concert with each other to facilitate host cell invasion, induction of inflammation as well as intracellular survival and replication within the SCV. The mechanisms by which effector proteins execute their function is only beginning to be completely deciphered. The identification of terminal domains on effectors with independent functions within the same effector (dual functions) has greatly increased our understanding of not only effector structure and function, but also the mechanisms by which secreted effectors alter host cell physiology. Recent observations of the presence of functional motifs on effectors such as the caspase-3 cleavage sites is only beginning to be understood and further studies will be necessary to completely dissect these molecular pathways. Presumably, more examples of effectors with multiple functions and functional motifs will be identified. The identification of such domains/motifs will further enrich our understanding of the interactions between the host and pathogen.

Acknowledgements

The research was supported by grants from the National Institutes of Health (DK56754 and DK084984), and the Crohn's and Colitis Foundation of America to B.A.M.

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