Deciphering interplay between Salmonella invasion effectors

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a 'signaling hub' during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cisbinary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen-host interaction.     

Salmonella entry into host cells: the work in concert of type III secreted effector proteins.

Upon contact with intestinal epithelial cells, Salmonella enterica serovar spp. inject a set of bacterial proteins into host cells via the bacterial SPI-1 type III secretion system. SopE, SopE2 and SopB, activate CDC42 and Rac to initiate actin cytoskeleton rearrangements. SipA and SipC, two Salmonella actin-binding proteins, directly modulate host actin dynamics to facilitate bacterial uptake. SptP promotes the recovery of the actin cytoskeleton rearrangements by antagonizing CDC42 and Rac. Therefore, Salmonella-induced reversible actin cytoskeleton rearrangements are the result of two coordinated steps: (i) stimulation of host signal transduction to indirectly promote actin rearrangements and (ii) direct modulation of actin dynamics.     

Salmonella type III secretion-associated chaperones confer secretion-pathway specificity

Type III protein secretion systems (TTSSs) are ancestrally related to the flagellar export system and are essential for the virulence of many bacteria pathogenic for humans, animals and plants. Most proteins destined to travel the TTSS pathway possess at least two domains that specifically target them to the secretion apparatus. One of the domains is located within the amino terminal first approximately 20 amino acids and the second domain, located within the first approximately 140 amino acids, serves as a binding site for specific chaperones. It has been previously proposed that these two secretion signals are capable of operating independently of one another to facilitate secretion into the extracellular environment. We have found that in the absence of their chaperone-binding domains, the Salmonella typhimurium TTSS-secreted proteins SptP and SopE are no longer targeted for secretion through their cognate TTSS and, instead, are secreted through the flagellar export pathway. These results indicate the existence of an 'ancestral' flagellar secretion signal within TTSS-exported proteins that is revealed in the absence of the chaperone-binding domain. Furthermore, we found that secretion into culture supernatants as well as translocation into host cells by the cognate TTSS require both, the amino terminal and chaperone-binding domains. We conclude from these studies that a critical function for the TTSS-associated chaperones is to confer secretion-pathway specificity to their cognate secreted proteins.     

沙门菌侵袭研究进展

鼠伤寒沙门菌表达两个不同的Ⅲ型分泌系统(typeⅢsecretion/translocation systems, TTSS),分别由致病岛1和2(pathogenicityi slands 1 and 2, SPI-1 and SPI-2)编码。细菌依赖TTSS将效应蛋白转运至宿主细胞,通过“触发”机制诱导细菌进入宿主细胞。这些效应蛋白可诱导细胞骨架重排,导致“巨吞饮”,促使细菌入侵。本综述依据多种沙门菌效应蛋白的功能,建立沙门菌侵袭模型。TTSS活化并转运效应蛋白进入宿主细胞发挥功能(Ⅰ)。小G蛋白交换因子SopE和肌醇磷酸酯酶SopB通过激活CDC42和Rac1,诱导内陷相关的蛋白聚集(Ⅱ)。SipA和SipC通过降低肌动蛋白临界浓度、刺激网素成束、稳定纤维状肌动蛋白(fibrousactin, F-actin)以及使肌动蛋白核化等功能,促使细菌入侵(Ⅲ)。SopB可使膜内陷区PIP2的浓度降低以及VAMP8聚集,促使细胞膜分裂(Ⅳ)。这些效应蛋白的联合作用,使膜皱褶在局部向外显著延伸,使沙门菌被细胞内形成的特殊膜结构包裹。沙门菌的另一种效应蛋白SptP,通过刺激小G蛋白内源性GTPase的活性,抑制小G蛋白的活化,使细胞膜恢复至原有状态(Ⅴ)。     

The first 45 amino acids of SopA are necessary for InvB binding and SPI-1 secretion

Salmonella enterica serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE78-240, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of Salmonella SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA1-45, but not SopA1-44, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.     

The chaperone binding domain of SopE inhibits transport via flagellar and SPI-1 TTSS in the absence of InvB

Type III secretion systems (TTSS) are used by many Gram-negative pathogens for transporting effector proteins into eukaryotic host cells. Two modes of type III effector protein transport can be distinguished: transport into the surrounding medium (secretion) and cell-contact induced injection of effector proteins directly into the host cell cytosol (translocation). Two domains within the N-terminal regions of effector proteins determine the mode of transport. The amino terminal approximately 20 amino acids (N-terminal secretion signal, NSS) mediate secretion. In contrast, translocation generally requires the NSS, the adjacent approximately 100 amino acids (chaperone binding domain, CBD) and binding of the cognate chaperone to this CBD. TTSS are phylogenetically related to flagellar systems. Because both systems are expressed in Salmonella Typhimurium, correct effector protein transport involves at least two decisions: transport via the Salmonella pathogenicity island 1 (SPI-1) but not the flagellar TTSS (= specificity) and translocation into the host cell instead of secretion into the surrounding media (= transport mode). The mechanisms guiding these decisions are poorly understood. We have studied the S. Typhimurium effector protein SopE, which is specifically transported via the SPI-1 TTSS. Secretion and translocation strictly require the cognate chaperone InvB. Alanine replacement of amino acids 30-42 (and to some extent 44-54) abolished tight InvB binding, abolished translocation into the host cell and led to secretion of SopE via both, the flagellar and the SPI-1 TTSS. In clear contrast to wild-type SopE, secretion of SopE(Ala30-42) and SopE(Ala44-54) via the SPI-1 and the flagellar export system did not require InvB. These data reveal a novel function of the CBD: the CBD inhibits secretion of wild-type SopE via the flagellar and the SPI-1 TTSS in the absence of the chaperone InvB. Our data provide new insights into mechanisms ensuring specific effector protein transport by TTSS.     

The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton

The Salmonella typhimurium protein tyrosine phosphatase SptP is a target of the centisome 63 type III protein secretion system. This system is essential for the interaction of these bacteria with host cells. We have shown here by a combination of biochemical and microscopy techniques that S . typhimurium directs the translocation of SptP into cultured epithelial cells. Translocation requires the function of the secreted proteins, SipB, SipC and SipD, as strains carrying mutations in any of the genes encoding these proteins fail to translocate SptP. Microinjection of purified GST–SptP into cultured cells results in the disruption of the actin cytoskeleton and the disappearance of stress fibres. These changes are reversible, as microinjected cells regain the normal appearance of their actin cytoskeleton upon prolonged incubation. Microinjection of the catalytically inactive GST–SptP(C481S) protein results in changes similar to those induced by the wild-type toxin. Furthermore, microinjection of a fusion protein between GST and the first 285 amino acids of SptP also leads to identical disruption of the host cell actin cytoskeleton, indicating that the amino-terminal half of SptP is sufficient to mediate this effect. However, microinjection of a fusion protein between GST and the last 259 amino acids of SptP also disrupted the normal appearance of the cytoskeleton. These results support the hypothesis that SptP is an effector protein arranged in modular domains that may co-operate with each other to exert related functions.     

The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay

Salmonella species trigger host membrane ruffling to force their internalization into non-phagocytic intestinal epithelial cells. This requires bacterial effector protein delivery into the target cell via a type III secretion system. Six translocated effectors manipulate cellular actin dynamics, but how their direct and indirect activities are spatially and temporally co-ordinated to promote productive cytoskeletal rearrangements remains essentially unexplored. To gain further insight into this process, we applied mechanical cell fractionation and immunofluorescence microscopy to systematically investigate the subcellular localization of epitope-tagged effectors in transiently transfected and Salmonella-infected cultured cells. Although five effectors contain no apparent membrane-targeting domains, all six localized exclusively in the target cell plasma membrane fraction and correspondingly were visualized at the cell periphery, from where they induced distinct effects on the actin cytoskeleton. Unexpectedly, no translocated effector pool was detectable in the cell cytosol. Using parallel in vitro assays, we demonstrate that the prenylated cellular GTPase Cdc42 is necessary and sufficient for membrane association of the Salmonella GTP exchange factor and GTPase-activating protein mimics SopE and SptP, which have no intrinsic lipid affinity. The data show that the host plasma membrane is a critical interface for effector-target interaction, and establish versatile systems to further dissect effector interplay.     

Role of the Salmonella pathogenicity island 1 (SPI-1) protein InvB in type III secretion of SopE and SopE2, two Salmonella effector proteins encoded outside of SPI-1

Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.     

The Salmonella Typhimurium effector protein SopE transiently localizes to the early SCV and contributes to intracellular replication

Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non‐phagocytic cells by a Type III secretion system (TTSS) and cognate effector proteins. Upon host cell entry, S. Tm expresses a second TTSS and subverts intracellular trafficking to create a replicative niche – the Salmonella‐containing vacuole (SCV). SopE, a guanidyl exchange factor (GEF) for Rac1 and Cdc42, is translocated by the TTSS‐1 upon host cell contact and promotes entry through triggering of actin‐dependent ruffles. After host cell entry, the bulk of SopE undergoes proteasomal degradation. Here we show that a subfraction is however detectable on the nascent SCV membrane up to ～ 6 h post infection. Membrane localization of SopE and the closely related SopE2 differentially depend on the Rho‐GTPase‐binding GEF domain, and to some extent involves also the unstructured N‐terminus. SopE localizes transiently to the early SCV, dependent on continuous synthesis and secretion by the TTSS‐1 during the intracellular state. Mutant strains lacking SopE or SopE2 are attenuated in early intracellular replication, while complementation restores this defect. Hence, the present study reveals an unanticipated role for SopE and SopE2 in establishing the Salmonella replicative niche, and further emphasizes the importance of entry effectors in later stages of host‐cell manipulation.     

A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization

A central feature of Salmonella pathogenicity is the bacterium's ability to enter into non-phagocytic cells. Bacterial internalization is the consequence of cellular responses characterized by Cdc42- and Rac-dependent actin cytoskeleton rearrangements. These responses are triggered by the co-ordinated function of bacterial proteins delivered into the host cell by a specialized protein secretion system termed type III. We report here that SopB, a Salmonella inositol polyphosphatase delivered to the host cell by this secretion system, mediates actin cytoskeleton rearrangements and bacterial entry in a Cdc42-dependent manner. SopB exhibits overlapping functions with two other effectors of bacterial entry, the Rho family GTPase exchange factors SopE and SopE2. Thus, Salmonella strains deficient in any one of these proteins can enter into cells at high efficiency, whereas a strain lacking all three effectors is completely defective for entry. Consistent with an important role for inositol phosphate metabolism in Salmonella-induced cellular responses, a catalytically defective mutant of SopB failed to stimulate actin cytoskeleton rearrangements and bacterial entry. Furthermore, bacterial infection of intestinal cells resulted in a marked increase in Ins(1,4,5,6)P4, a consumption of InsP5 and the activation of phospholipase C. In agreement with the in vivo findings, purified SopB specifically dephosphorylated InsP5 to Ins(1,4,5,6)P4 in vitro. Surprisingly, the inositol phosphate fluxes induced by Salmonella were not caused exclusively by SopB. We show that the SopB-independent inositol phosphate fluxes are the consequence of the SopE-dependent activation of an endogenous inositol phosphatase. The ability of Salmonella to stimulate Rho GTPases signalling and inositol phosphate metabolism through alternative mechanisms is an example of the remarkable ability of this bacterial pathogen to manipulate host cellular functions.     

Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell

Salmonella typhimurium translocates effector proteins into host cells via the SPI1 type III secretion system to induce responses such as membrane ruffling and internalization by non-phagocytic cells. Activation of the host cellular RhoGTPase Cdc42 is thought to be a key event during internalization. The translocated Salmonella protein SopE is an activator for Cdc42. Because SopE is absent from most S. typhimurium strains it remains unclear whether all S. typhimurium strains rely on activation of Cdc42 to invade host cells. We have identified SopE2, a translocated effector protein common to all S. typhimurium strains. SopE2 is a guanine nucleotide exchange factor for Cdc42 and shows 69% sequence similarity to SopE. Analysis of S. typhimurium mutants demonstrated that SopE2 plays a role in recruitment of the actin-nucleating Arp2/3 complex to the membrane ruffles and in efficient host cell invasion. Transfection experiments showed that SopE2 is sufficient to activate host cellular Cdc42, to recruit the actin-nucleating Arp2/3 complex and to induce actin cytoskeletal rearrangements and internalization. In conclusion, as a result of SopE2 all S. typhimurium strains tested have the capacity to activate Cdc42 signalling inside host cells which is important to ensure efficient entry.     

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.     

SptP106–136 plays a role in the complex formation with SptP-specific chaperone SicP

SptP is a virulence effector protein of Salmonella that is involved in bacterial invasion into a host cell. For effective secretion, SptP forms a complex with SptP-specific chaperone SicP through its chaperone-binding domain, residues 35–139. Here, we suggest the possibility that residues 106–136 of SptP are important for complex formation with SicP by in vitro reconstitution experiments.     

Addressing the effects of Salmonella internalization in host cell signaling on a reverse‐phase protein array

Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island‐1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re‐programming host cell pathways, we used ZeptoMARK reverse‐phase protein array technology, which allows printing 32‐sample lysate arrays that can be analyzed with phospho‐specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS‐deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen‐activated protein kinases, mainly dependent on SopE/E2, as well as SigD‐dependent phosphorylation of PKB/Akt and its targets GSK‐3β and FKHR/FoxO. This is the first time that reverse‐phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.     

SptP,a Salmonella typhimurium type III-secreted protein,inhibits the mitogen-activated protein kinase pathway by inhibiting Raf activation

Salmonella has developed ways to modulate host cellular response in order to survive. Although the steps required for such modulation have been incompletely characterized, there is increasing evidence for a role for SptP, a type III secretion protein. In part, the actions of SptP are thought to be mediated through its reported inhibition of the extracellular-regulated kinase (ERK) MAP kinase pathway. In the present studies, a series of transfections were performed in which various constitutively activated components of the MAP kinase pathway were co-transfected with SptP in order to determine the mechanism by which SptP inhibits this MAP kinase activation. SptP was found to inhibit the activation of ERK stimulated by both a constitutively active form of Ras and a partially activated form of Raf-1 containing a phospho-mimetic mutation (Raf Y340D). In contrast, the activation of ERK by constitutively active forms of MAP kinase kinase (MEK) was not inhibited, suggesting that the actions of SptP were mediated by Raf-1. In order to determine how SptP might interfere with activation of Raf, we utilized a membrane-localized form of Raf. Constitutive membrane-localization of Raf (RafCAAX), resulting in partial activation, did not prevent inhibition by SptP. However, introduction of an additional, partially activating (Y340D) phospho-mimetic mutation, to RafCAAX, dramatically reduced the ability of SptP to inhibit Raf action. Comparison of SptP mutants, lacking either GTPase-activating protein (GAP) activity or tyrosine phosphatase activity, further suggested that SptP inhibits both the membrane localization and subsequent phosphorylation required for activation of Raf. Both tyrosine phosphatase activity and GAP activity were responsible for SptP inhibition of Raf(Y340D)-induced ERK activation, but only GAP activity was responsible for inhibition of the membrane localized forms of Raf-1. To assess the biological significance of SptP, we examined tumour necrosis factor (TNF)-alpha induction following Salmonella infection. SptP gene deletion enhanced the capacity of Salmonella to induce TNF-alpha secretion following infection of J774A.1 macrophage cells.     

Type III secretion of the Salmonella effector protein SopE is mediated via an N-terminal amino acid signal and not an mRNA sequence

Type III secretion systems (TTSS) are virulence-associated components of many gram-negative bacteria that translocate bacterial proteins directly from the bacterial cytoplasm into the host cell. The Salmonella translocated effector protein SopE has no consensus cleavable amino-terminal secretion sequence, and the mechanism leading to its secretion through the Salmonella pathogenicity island 1 (SPI-1) TTSS is still not fully understood. There is evidence from other bacteria which suggests that the TTSS signal may reside within the 5' untranslated region (UTR) of the mRNA of secreted effectors. We investigated the role of the 5' UTR in the SPI-1 TTSS-mediated secretion of SopE using promoter fusions and obtained data indicating that the mRNA sequence is not involved in the secretion process. To clarify the proteinaceous versus RNA nature of the signal, we constructed frameshift mutations in the amino-terminal region of SopE of Salmonella enterica serovar Typhimurium SL1344. Only constructs with the native amino acid sequence were secreted, highlighting the importance of the amino acid sequence versus the mRNA sequence for secretion. Additionally, we obtained frameshift mutation data suggesting that the first 15 amino acids are important for secretion of SopE independent of the presence of the chaperone binding site. These data shed light on the nature of the signal for SopE secretion and highlight the importance of the amino-terminal amino acids for correct targeting and secretion of SopE via the SPI-1-encoded TTSS during host cell invasion.     

SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell.

The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but not with Rac1. Affinity precipitation of Cdc42.GTP and Rac1.GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allow S. typhimurium to specifically activate different sets of RhoGTPase signaling cascades.     

A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators

Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes.