Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes

The intracellular pathogen, Salmonella enterica, translocates type III effectors across its vacuolar membrane into host cells. Herein we describe a new Salmonella effector, PipB2, which has sequence similarity to another type III effector, PipB. In phagocytic cells, PipB2 localizes to the Salmonella-containing vacuole (SCV) and tubular extensions from the SCV, Salmonella-induced filaments (Sifs). We used the specific targeting of PipB2 in macrophages to characterize Sifs in phagocytic cells for the first time. In epithelial cells, PipB2 has a unique localization pattern, localizing to SCVs and Sifs and additionally to vesicles at the periphery of infected cells. We further show that the N-terminal 225-amino-acid residues of PipB2 are sufficient for type III translocation and association with SCVs and Sifs, but not peripheral vesicles. Subcellular fractionation demonstrated that both PipB and PipB2 associate with host cell membranes and resist extraction by high salt, high pH and to a significant extent, non-ionic detergent. Furthermore, PipB and PipB2 are enriched in detergent-resistant microdomains (DRMs), also known as lipid rafts, present on membranes of SCVs and Sifs. The enrichment of Salmonella effectors in DRMs on these intracellular membranes probably permits specific interactions with host cell molecules that are concentrated in these signalling platforms.     

Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems

Pathogenicity islands (PAIs) are large DNA segments in the genomes of bacterial pathogens that encode virulence factors. Five PAIs have been identified in the Gram-negative bacterium Salmonella enterica. Two of these PAIs, Salmonella pathogenicity island (SPI)-1 and SPI-2, encode type III secretion systems (TTSS), which are essential virulence determinants. These 'molecular syringes' inject effectors directly into the host cell, whereupon they manipulate host cell functions. These effectors are either encoded with their respective TTSS or scattered elsewhere on the Salmonella chromosome. Importantly, SPI-1 and SPI-2 are expressed under distinct environmental conditions: SPI-1 is induced upon initial contact with the host cell, whereas SPI-2 is induced intracellularly. Here, we demonstrate that a single PAI, in this case SPI-5, can encode effectors that are induced by distinct regulatory cues and targeted to different TTSS. SPI-5 encodes the SPI-1 TTSS translocated effector, SigD/SopB. In contrast, we report that the adjacently encoded effector PipB is part of the SPI-2 regulon. PipB is translocated by the SPI-2 TTSS to the Salmonella-containing vacuole and Salmonella-induced filaments. We also show that regions of SPI-5 are not conserved in all Salmonella spp. Although sigD/sopB is present in all Salmonella spp., pipB is not found in Salmonella bongori, which also lacks a functional SPI-2 TTSS. Thus, we demonstrate a functional and regulatory cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, which has significant implications for the evolution and role of PAIs in bacterial pathogenesis.     

Coiled-coil domains enhance the membrane association of Salmonella type III effectors

Coiled-coil domains in eukaryotic and prokaryotic proteins contribute to diverse structural and regulatory functions. Here we have used in silico analysis to predict which proteins in the proteome of the enteric pathogen, Salmonella enterica serovar Typhimurium, harbour coiled-coil domains. We found that coiled-coil domains are especially prevalent in virulence-associated proteins, including type III effectors. Using SopB as a model coiled-coil domain type III effector, we have investigated the role of this motif in various aspects of effector function including chaperone binding, secretion and translocation, protein stability, localization and biological activity. Compared with wild-type SopB, SopB coiled-coil mutants were unstable, both inside bacteria and after translocation into host cells. In addition, the putative coiled-coil domain was required for the efficient membrane association of SopB in host cells. Since many other Salmonella effectors were predicted to contain coiled-coil domains, we also investigated the role of this motif in their intracellular targeting in mammalian cells. Mutation of the predicted coiled-coil domains in PipB2, SseJ and SopD2 also eliminated their membrane localization in mammalian cells. These findings suggest that coiled-coil domains represent a common membrane-targeting determinant for Salmonella type III effectors.     

Role of 3-phosphoinositides in the maturation of Salmonella-containing vacuoles within host cells

Salmonella typhimurium invades mammalian cells and replicates within a vacuole that protects it from the host's microbicidal weapons. The Salmonella-containing vacuole (SCV) undergoes a remodelling akin to that of the host cell's endocytic pathway, but SCV progression is arrested prior to fusion with lysosomes. We studied the role of phosphatidylinositol 3-kinase (PI3-K) in SCV maturation within HeLa cells. Phosphatidylinositol 3-phosphate (PI3P), monitored in situ using fluorescent conjugates of FYVE or PX domains, was found to accumulate transiently on the SCV. Wortmannin prevented PI3P accumulation and the recruitment of EEA1 but did not affect the association of Rab5 with the SCV. Importantly, inhibition of PI3-K also impaired fusion of the SCV with vesicles containing LAMP-1. Rab7, which is thought to be required for association of LAMP-1 with the SCV, still associated with SCV in wortmannin-treated cells. We have therefore concluded that a 3-phosphoinositide-dependent step exists following recruitment of Rab7 to the SCV. The data also imply that 3-phosphoinositide-dependent effectors of Rab5 are not an absolute requirement for recruitment of Rab7. Despite failure to acquire LAMP-1, the SCV persists and allows effective replication of Salmonella within wortmannin-treated host cells. These findings imply that PI3-K is involved in the development of the SCV but is not essential for intracellular survival and proliferation of Salmonella.     

Role of the ESCRT‐III complex in controlling integrity of the Salmonella‐containing vacuole

Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2‐encoded type III secretion system (SPI2‐T3SS). Fusion events of endosomal compartments with the Salmonella‐containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella‐modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT‐III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT‐III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2‐T3SS‐deficient mutant strain was partially abrogated. ESCRT‐dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT‐III recruitment in Salmonella‐infected host cells.     

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella‐containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane‐bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP‐bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63‐containing late endosomes. Nischarin is recruited to the SCV in a Rab14‐dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners—Rac1, Rab14 and Rab9 GTPases—reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.     

Salmonella-containing vacuoles: directing traffic and nesting to grow

Salmonella enterica serovar Typhimurium (S. typhimurium) is a gram-negative facultative intracellular pathogen that can infect a broad range of mammalian hosts. Following invasion of host cells, the majority of S. typhimurium are known to reside in a membrane-bound compartment known as the Salmonella-containing vacuole (SCV). S. typhimurium actively remodels this compartment using bacterial virulence proteins, called effectors, to establish a protected niche where it can replicate. S. typhimurium delivers more than 30 effectors into the host cell cytosol by bacterial type three secretion systems, encoded by Salmonella pathogenicity island 1 or 2 (SPI-1 or SPI-2). Recent studies have revealed a critical role for the SPI-1 effector SopB in 'directing traffic' at early stages of infection, allowing the bacteria to control SCV maturation by modulating its interaction with the endocytic system. At later stages of infection, the SCV establishes a 'nest' near the Golgi where optimal bacterial growth takes place. In this study, we highlight these recent developments in our understanding of SCV trafficking.     

Dynein-mediated vesicle transport controls intracellular Salmonella replication

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.     

Anchoring of bacterial effectors to host membranes through host-mediated lipidation by prenylation: a common paradigm

Post-translational lipidation by prenylation of the CaaX-box C-terminal motif in eukaryotic proteins facilitates anchoring of hydrophilic proteins, such as Ras and Rab, to membranes. A large cadre of bacterial effectors injected into host cells is anchored to host membranes by unknown mechanisms. As already documented for Legionella and Salmonella, we propose a common paradigm of microbial exploitation of the host prenylation machinery for anchoring of injected effectors to host membranes. This is supported by numerous potential microbial CaaX-box-containing proteins identified using refined bioinformatic tools. We also propose utilization of the CaaX motif as a membrane-targeting tag for proteins expressed in eukaryotic cells to facilitate deciphering of biological function.     

Growth control in the Salmonella-containing vacuole

Salmonella enterica is an intracellular bacterial pathogen that inhabits membrane-bound vacuoles of eukaryotic cells. Coined as the 'Salmonella-containing vacuole' (SCV), this compartment has been studied for two decades as a replicative niche. Recent findings reveal, however, marked differences in the lifestyle of bacteria enclosed in the SCV of varied host cell types. In fibroblasts, the emerging view supports a model of bacteria facing in the SCV a 'to grow' or 'not to grow' dilemma, which is solved by entering in a dormancy-like state. Fine-tuning of host cell defense/survival routes, drastic metabolic shift down, adaptation to hypoxia conditions, and attenuation of own virulence systems emerge as strategies used by Salmonella to intentionally reduce the growth rate inside the SCV.     

SseG, a virulence protein that targets Salmonella to the Golgi network

Intracellular replication of the bacterial pathogen Salmonella enterica occurs in membrane-bound compartments called Salmonella-containing vacuoles (SCVs). Maturation of the SCV has been shown to occur by selective interactions with the endocytic pathway. We show here that after invasion of epithelial cells and migration to a perinuclear location, the majority of SCVs become surrounded by membranes of the Golgi network. This process is dependent on the Salmonella pathogenicity island 2 type III secretion system effector SseG. In infected cells, SseG was associated with the SCV and peripheral punctate structures. Only bacterial cells closely associated with the Golgi network were able to multiply; furthermore, mutation of sseG or disruption of the Golgi network inhibited intracellular bacterial growth. When expressed in epithelial cells, SseG co-localized extensively with markers of the trans-Golgi network. We identify a Golgi-targeting domain within SseG, and other regions of the protein that are required for localization of bacteria to the Golgi network. Therefore, replication of Salmonella in epithelial cells is dependent on simultaneous and selective interactions with both endocytic and secretory pathways.     

Recognition of bacteria in the cytosol of Mammalian cells by the ubiquitin system

Recent studies have suggested the existence of innate host surveillance systems for the detection of bacteria in the cytosol of mammalian cells. The molecular details of how bacteria are recognized in the cytosol, however, remain unclear. Here we examined the fate of Salmonella typhimurium, a gram-negative bacterial pathogen that can infect a variety of hosts, in the cytosol of mammalian cells. These bacteria typically occupy a membrane bound compartment, the Salmonella-containing vacuole (SCV), in host cells. We show that some wild-type bacteria escape invasion vacuoles and are released into the cytosol. Subsequently, polyubiquitinated proteins accumulate on the bacterial surface, a response that was witnessed in several cell types. In macrophages but not epithelial cells, the proteasome was observed to undergo a dramatic subcellular relocalization and become associated with the surface of bacteria in the cytosol. Proteasome inhibition promoted replication of S. typhimurium in the cytosol of both cell types, in part through destabilization of the SCV. Surprisingly, the cytosol-adapted pathogen Listeria monocytogenes avoided recognition by the ubiquitin system by using actin-based motility. Our findings indicate that the ubiquitin system plays a major role in the recognition of bacterial pathogens in the cytosol of mammalian cells.     

Esterification of cholesterol by a type III secretion effector during intracellular Salmonella infection

Survival of Salmonella typhimurium within a vacuole in host cells depends on secreted virulence factors encoded by the Salmonella pathogenicity island 2 (SPI-2). High levels of cholesterol are detected at the Salmonella -containing vacuole (SCV). Here we show that the SPI-2 effector SseJ esterifies cholesterol in vitro , in cells and during infection. Intracellular infections with wild-type as compared with Δ sseJ bacteria led to higher levels of cholesterol ester production in HeLa cells and RAW macrophages and were shown to increase levels of lipid droplets (structures enriched in cholesterol esters). Ectopic expression of SseJ reduced cholesterol levels in cellular membranes and antagonized a major membrane activity of a second bacterial effector known to be important to the stability of the SCV. Previous studies in mouse models of infection have established a virulence defect in Δ sseJ bacteria and have suggested a role for SseJ in regulating SCV dynamics. Our data indicating the molecular activity of SseJ suggest that cholesterol and its esterification at the SCV are functionally important for intracellular bacterial survival.     

Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system

Salmonella enterica uses two functionally distinct type III secretion systems encoded on the pathogenicity islands SPI-1 and SPI-2 to transfer effector proteins into host cells. A major function of the SPI-1 secretion system is to enable bacterial invasion of epithelial cells and the principal role of SPI-2 is to facilitate the replication of intracellular bacteria within membrane-bound Salmonella-containing vacuoles (SCVs). Studies of mutant bacteria defective for SPI-2-dependent secretion have revealed a variety of functions that can be attributed to this secretion system. These include an inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidase-dependent killing, the induction of a delayed apoptosis-like host cell death, the control of SCV membrane dynamics, the assembly of a meshwork of F-actin around the SCV, an accumulation of cholesterol around the SCV and interference with the localization of inducible nitric oxide synthase to the SCV. Several effector proteins that are translocated across the vacuolar membrane in a SPI-2-dependent manner have now been identified. These are encoded both within and outside SPI-2. The characteristics of these effectors, and their relationship to the physiological functions listed above, are the subject of this review. The emerging picture is of a multifunctional system, whose activities are explained in part by effectors that control interactions between the SCV and intracellular membrane compartments.     

Salmonella SPI1 effector SipA persists after entry and cooperates with a SPI2 effector to regulate phagosome maturation and intracellular replication

Salmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.     

Membrane dynamics and spatial distribution of Salmonella-containing vacuoles

Salmonella enterica are facultative intracellular bacteria that cause intestinal and systemic diseases, and replicate within host cells in a membrane-bound compartment, the Salmonella-containing vacuole. Intravacuolar bacterial replication depends on spatiotemporal regulated interactions with host cell vesicular compartments. Recent studies have shown that type III secretion effector proteins control both the vacuolar membrane dynamics and intracellular positioning of bacterial vacuoles. The functions of these effectors, which are beginning to be understood, disclose a complex hijacking of host cell microtubule motors--kinesins and dynein--and regulators of their function, and suggest interactions with the Golgi complex. Here, we discuss current models describing the mode of action of Salmonella type III secretion effector proteins involved in these processes.     

SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34

Salmonella colonizes a vacuolar niche in host cells during infection. Maturation of the Salmonella-containing vacuole (SCV) involves the formation of phosphatidylinositol 3-phosphate (PI(3)P) on its outer leaflet. SopB, a bacterial virulence factor with phosphoinositide phosphatase activity, was proposed to generate PI(3)P by dephosphorylating PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3. Here, we examine the mechanism of PI(3)P formation during Salmonella infection. SopB is required to form PI(3,4)P2/PI(3,4,5)P3 at invasion ruffles and PI(3)P on nascent SCVs. However, we uncouple these events experimentally and reveal that SopB does not dephosphorylate PI(3,4)P2/PI(3,4,5)P3 to produce PI(3)P. Instead, the phosphatase activity of SopB is required for Rab5 recruitment to the SCV. Vps34, a PI3-kinase that associates with active Rab5, is responsible for PI(3)P formation on SCVs. Therefore, SopB mediates PI(3)P production on the SCV indirectly through recruitment of Rab5 and its effector Vps34. These findings reveal a link between phosphoinositide phosphatase activity and the recruitment of Rab5 to phagosomes.     

Functional dissection of SseF, a type III effector protein involved in positioning the salmonella-containing vacuole

Intracellular replication of Salmonella enterica requires the formation of a unique organelle termed Salmonella-containing vacuole (SCV). The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2-T3SS) has a crucial role in the formation and maintenance of the SCV. The SPI2-T3SS translocates a large number of effector proteins that interfere with host cell functions such as microtubule-dependent transport. We investigated the function of the effector SseF and observed that this protein is required to maintain the SCV in a juxtanuclear position in infected epithelial cells. The formation of juxtanuclear clusters of replicating Salmonella required the recruitment of dynein to the SCV but SseF-deficient strains were highly reduced in dynein recruitment to the SCV. We performed a functional dissection of SseF and defined domains that were important for translocation and the specific effector functions of this protein. Of particular importance was a hydrophobic domain in the C-terminal half that contains three putative transmembrane (TM) helices. Deletion of one of these TM helices ablated the effector functions of SseF. We observed that this domain was essential for the proper intracellular positioning of the SCV to a juxtanuclear, Golgi-associated localization. These data show that SseF, in concert with the effector proteins SifA and SseG mediate the precise positioning of the SCV by differentially modulating the recruitment of microtubule motor proteins to the SCV.     

Salmonella trafficking is defined by continuous dynamic interactions with the endolysosomal system

Following invasion of non-phagocytic host cells, Salmonella enterica survives and replicates within a phagosome-like compartment known as the Salmonella-containing vacuole (SCV). It is now well established that SCV biogenesis, like phagosome biogenesis, involves sequential interactions with the endocytic pathway. However, Salmonella is believed to limit these interactions and, in particular, to avoid fusion of terminal lysosomes with the SCV. In this study, we reassessed this process using a high-resolution live-cell imaging approach and found an unanticipated level of interaction between the SCV and the endocytic pathway. Direct interactions, in which late endosomal/lysosomal content was transferred to SCVs, were detected within 30 min of invasion and continued for several hours. Mechanistically, these interactions were very similar to phagosome-lysosome fusion because they were accompanied by rapid acidification of the SCV, could be blocked by chemical perturbation of microtubules or vacuolar acidification and involved the smallGTPase Rab7. In comparison with vacuoles containing internalized Escherichia coli or heat-killed Salmonella, SCVs did show some delay of fusion and acidification, although, this appeared to be independent of either type III secretion system. These results provide compelling evidence that inhibition of SCV-lysosome fusion is not the major determinant in establishment of the Salmonella replicative niche in epithelial cells.     

PipB2 is a substrate of the Salmonella pathogenicity island 1-encoded type III secretion system

Salmonella harbors two type III secretion systems, T3SS1 and T3SS2, encoded on the pathogenicity islands SPI1 and SPI2, respectively. Several effector proteins are secreted through these systems into the eukaryotic host cells. PipB2 is a T3SS2 effector that contributes to the modulation of kinesin-1 motor complex activity. Here, we show that PipB2 is also a substrate of T3SS1. This result was obtained infecting human epithelial HeLa cells for 2 h and was confirmed in murine RAW264.7 macrophages, and rat NRK fibroblasts. Analysis at different time points after infection revealed that translocation of PipB2 is T3SS1-dependent in epithelial cells throughout the infection. In contrast, translocation into macrophages is T3SS1-dependent during invasion but T3SS2-dependent at later time points. The N-terminal 10 amino acid residues contain the signal necessary for translocation through both systems. These results confirm the functional overlap between these virulence-related secretion systems and suggest a new role for the effector PipB2.