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PI3P is present in the SCV. A–D, HeLa cells were transiently transfected with the 2-FYVE-GFP construct 18 h before infection with S. typhimurium SL1344. Cells were fixed at the indicated times by incubation for 1 h with 4% paraformaldehyde, permeabilized, and immunostained with antibodies to S. typhimurium LPS. A and B, cells were incubated for 10 min with Salmonella. C and D, cells were treated with 100 nM wortmannin for 30 min prior to infection. E and F, cells were transiently transfected with the p40PX-GFP construct 18 h before infection with Salmonella and fixed after 10 min. A, C, and E, green (GFP) fluorescence. B, D, and F, red (Salmonella ) fluorescence. Arrows indicate the location of intracellular bacteria. G, time course of the association of PI3P with the SCV, quantified from experiments using 2-FYVE-GFP at the times after invasion indicated on the abscissa. Ordinate, percentage of SCV that contained clearly detectable PI3P. Results shown are the means S.E. of three separate experiments each with at least 50 individual vacuoles counted.
Source publication
Salmonella typhimuriuminvades mammalian cells and replicates within a vacuole that protects it from the host's microbicidal weapons. TheSalmonella-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 phosphatidylinos...
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Citations
... Endosomal membranes were labeled with enhanced green fluorescent protein (EGFP) attached to two tandem FYVE domains of the early endosome autoantigen 1 (EEA1) protein (2FYVE-EGFP). FYVE binds to phosphatidylinositol 3phosphate (PI3P), a phospholipid enriched in early endosome membranes (39). To label endosomal luminal contents, cells were pulsed for 1 h with Alexa Fluor-594 conjugated dextran (10-kDa molecular weight) (40). ...
Significance
Membrane phase behavior in cells permits transient concentration of specific proteins and lipids into dynamic nanoscopic domains. Here, we tested the existence and role of such phase behavior in endoplasmic reticulum (ER) membranes. Employing hypotonic cell swelling, we created large intracellular vesicles (LICVs) from internal organelles. ER LICVs maintained stable interorganelle contacts, with known protein tethers concentrated at the contact sites. Cooled ER LICVs underwent reversible phase separation into microscopically visible domains with different lipid order and membrane fluidity. The phase-separated domains specified sites of contact between the ER and different organelles.
... 77 Phosphatidylinositol-3-phosphate develops an interaction between EEA1 and Rab5 on the SCV. 78 Salmonellainduced filaments (SIF) are membrane tubules in the eukaryotic cell that can provide Salmonella with access to nutrition. 79 Rab5 by means of phosphatidylinositol-3-phosphate facilitates the attachment of EEA1 to SCV, which, in turn, produces fusion with endosome. ...
Salmonella can be categorized into many serotypes, which are specific to known hosts or broadhosts. It makes no difference which one of the serotypes would penetrate the gastrointestinal tract because they all face similar obstacles such as mucus and microbiome. However, following their penetration, some species remain in the gastrointestinal tract; yet, others spread to another organ like gallbladder. Salmonella is required to alter the immune response to sustain its intracellular life. Changing the host response requires particular effector proteins and vehicles to translocate them. To this end, a categorized gene called Salmonella pathogenicity island (SPI) was developed; genes like Salmonella pathogenicity island encode aggressive or modulating proteins. Initially, Salmonella needs to be attached and stabilized via adhesin factor, without which no further steps can be taken. In this review, an attempt has been made to elaborate on each factor attached to the host cell or to modulating and aggressive proteins that evade immune systems. This review includes four sections: (A) attachment factors or T3SS- independent entrance, (B) effector proteins or T3SS-dependent entrance, (c) regulation of invasive genes, and (D) regulation of immune responses.
... We next examined the effects of hypotonic swelling on the shapes of endosomes, lysosomes, mitochondria, peroxisomes and LDs ( Figure 3). Endosomal membranes were labeled with GFP attached to two tandem FYVE (2FYVE-GFP) domains of the early endosome autoantigen 1 (EEA1) protein, which binds strongly to phosphatidylinositol 3-phosphate (PI3P), a phospholipid enriched in early endosomes 35 . To label endosomal lumenal contents, cells were pulsed for 1-hour with AlexaFluor-594 labeled Dextran (10 kDa molecular weight) 36 . ...
The plasma membrane of cells exhibits phase behavior that allows transient concentration of specific proteins and lipids, giving rise to functionally dynamic and diverse nanoscopic domains. This phase behavior is observable in giant plasma membrane-derived vesicles, in which microscopically visible, liquid-ordered (L o ) and liquid-disordered (L d ) lipid domains form upon a shift to low temperatures. The extent such phase behavior exists in the membrane of the endoplasmic reticulum (ER) of cells remains unclear. To explore the phase behavior of the ER membrane in cells, we used hypotonic cell swelling to generate Large Intra-Cellular Vesicles (LICVs) from the ER in cells. ER LICVs retained their lumenal protein content, could be retubulated into an ER network, and maintained stable inter-organelle contacts, where protein tethers are concentrated at these contacts. Notably, upon temperature reduction, ER LICVs underwent reversible phase separation into microscopically-visible L o and L d lipid domains. The L o lipid domains marked ER contact sites with other organelles. These findings demonstrate that LICVs provide an important model system for studying the biophysical properties of intracellular organelles in cells.
Significance Statement
Prior work has demonstrated that the plasma membrane can phase separate into microscopically visible L o and L d domains with distinct lipid and protein content. However, such behavior on the ER membrane has not been experimentally observed, even though the ER contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner at these contacts. We find here that hypotonic treatment generates Large Intra-Cellular Vesicles from the endoplasmic reticulum and other membrane-bound organelles in cells, enabling the study of phase behavior on the ER membrane. We show that ER membranes can be reversibly phase separated into microscopically-observable, L o and L d domains. ER LICVs also maintained stable inter-organelle contact sites in cells, with organelle tethers concentrated at these contacts.
... The SPI-1 effector SopB (also known as SigD) is a phosphatidylinositol phosphatase that hydrolyzes a wide range of phosphoinositide substrates in vitro (19) with specific phosphatidylinositol 4,5-biphosphate phosphatase [PI(4,5)P 2 ] activity in vivo (20). Although not essential for cell invasion, SopB plays an important role in nascent SCV formation (21)(22)(23). Depletion of PI(4,5)P 2 by SopB promotes fusion between the SCV and vesicles containing Rab5 (23). One of the Rab5 effectors that is associated with the SCV is Vps34, a phosphatidylinositol-3 (PI-3) kinase (21,23). ...
... Depletion of PI(4,5)P 2 by SopB promotes fusion between the SCV and vesicles containing Rab5 (23). One of the Rab5 effectors that is associated with the SCV is Vps34, a phosphatidylinositol-3 (PI-3) kinase (21,23). Vps34 phosphorylation of local pools of phosphatidylinositol (PI) forms phosphatidylinositol 3-phosphate [PI(3)P] that recruits the early endosomal component early-endosome antigen 1 (EEA1) to the SCV membrane (21,23). ...
... One of the Rab5 effectors that is associated with the SCV is Vps34, a phosphatidylinositol-3 (PI-3) kinase (21,23). Vps34 phosphorylation of local pools of phosphatidylinositol (PI) forms phosphatidylinositol 3-phosphate [PI(3)P] that recruits the early endosomal component early-endosome antigen 1 (EEA1) to the SCV membrane (21,23). Therefore, the early biogenesis of the SCV is dependent on the activity of SopB, which promotes the acquisition of Rab5, PI(3)P, and EEA1. ...
The Role of the Type III Secretion System in the Intracellular Lifestyle of Enteric Pathogens, Page 1 of 2
Abstract
Many bacterial pathogens have evolved to infect host cells from the inside. In fact, some bacteria, such as Rickettsia spp. and Coxiella spp., are entirely reliant on host intracellular resources to propagate ( 1 ). The adaptation of bacteria to an intracellular lifecycle is thought to confer a means to avoid the harsh extracellular milieu (low pH, physical stress, host defenses), to gain access to a nutrient-rich environment, and to facilitate the spread of the pathogen to neighboring host tissues ( 2 , 3 ).
... Furthermore, expression of SopB alone in cells can induce formation of macropinosomes from the PM that migrate to the perinuclear region (Hernandez et al. 2004). SopB expression is required for multiple stages of SCV maturation, including initial PtdIns3P enrichment and subsequent recruitment of endosomal markers such as EEA1 and Rab7 (Scott et al. 2002;Mallo et al. 2008) as well as inhibition of fusion between SCVs and lysosomes (Dukes et al. 2006;Bakowski et al. 2010). Most intriguing is its ability to directly metabolize PIs through its phosphatidylinositol-5-phosphatase activity. ...
Signalling through phosphoinositide lipids is essential for regulating many cellular processes, including endosomal trafficking. A number of intracellular pathogens have found ways to subvert host trafficking pathways via exploitation of endosomal phosphoinositides. This review will discuss how pathogens such as bacteria, viruses, and eukaryotic parasites depend on endosomal phosphoinositides for infection, as well as the mechanisms through which some are able to actively manipulate these signalling lipids to facilitate invasion, survival, replication, and immune evasion.
... As also seen in Fig. 3, the chloroquine-mediated inhibition of intracellular growth remained the same at both 4 and 24 h post-infection, indicating no "hyperreplication" of intracellular Salmonella which has been reported for cytosolic growth (37). These results are consistent with other studies showing recruitment of other host proteins to the SCV membrane, despite the absence of LAMP-1, the latter of which is an indicator of SCV membrane integrity (39,40). More recent studies have also shown that intracellular Salmonellainduced filaments can be associated with membrane which does not contain LAMP-1 (41,42). ...
Salmonella enterica are invasive, intracellular pathogens which replicate within a membrane-bound compartment inside infected host cells known as the Salmonella-containing vacuole (SCV). How Salmonella obtains nutrients for growth within this intracellular niche despite the apparent isolation is currently not known. Recent studies have indicated the importance of glucose and related carbon sources for tissue colonisation and intracellular proliferation within host cells during Salmonella infections, although none have been found to be essential. We found that wild-type Salmonella are capable of replicating within infected host cells in the absence of both exogenous sugars and/or amino acids. Furthermore, mutants defective in glucose uptake or dependent upon peptides for growth also showed no significant loss in intracellular replication, suggesting host-derived peptides can supply both carbon units and amino acids. Here, we show that intracellular Salmonella recruit the host proteins LAMP-2A and Hsc73, key components of the host protein turnover pathway known as chaperone-mediated autophagy (CMA) involved in transport of cytosolic proteins to the lysosome for degradation. Host-derived peptides are shown to provide a significant contribution towards the intracellular growth of Salmonella. The results reveal a means whereby intracellular Salmonella gain access to the host cell cytosol from within its membrane-bound compartment to acquire nutrients. Furthermore, this study provides an explanation as to how Salmonella evades activation of autophagy mechanisms as part of the innate immune response.
... Early in development, the SCV associates with organelles of the endosomal system, acquiring markers such as EEA1, SNX1 (Bujny et al., 2008), PI(3)P, and Rab5 (Dai et al., 2007;Bakowski et al., 2010). PI(3)P in particular is crucial to the stability and integrity of the SCV as intracellular Salmonella treated with PI(3)-kinase inhibitor, wortmannin, escape from the SCV and replicate within the cytoplasm unchallenged (Brumell et al., 2002;Scott et al., 2002). In the later stages of infection, SCV maturation is characterized by the formation of tubular protrusions called Salmonella Induced Filaments (SIFs), as well as the loss of PI(3)P and the acquisition of late endosomal markers such as LAMP1 and Rab7 (Knodler and Steele-Mortimer, 2005). ...
... During Salmonella infection, PI(3)P has been observed to first accumulate on membrane ruffles and subsequently remains associated with the SCV throughout its early biogenesis (Scott et al., 2002). To determine if MTMR4 is associated with the nascent SCV, A431 cells were transfected with GFP-MTMR4 or GFP-MTMR3 for 18 h before being infected with a wildtype S. typhimurium strain (SL1344) expressing RFP (RFP-SL1344) at a MOI of 1. Time-lapse video microscopy demonstrated that GFP-MTMR4 but not GFP-MTMR3 associated with the nascent SCV as early as 15 min p.i. (Figure 3A). ...
The intracellular pathogen Salmonella enterica servovar Typhimurium (S.typhimurium) modulates the host cell's phosphoinositide (PI) metabolism to establish its intracellular replicative niche, the Salmonella-containing vacuole (SCV). Upon invasion, phosphoinositide 3-phosphate (PI(3)P) and other early endosomal markers are rapidly recruited to and remain associated with the SCV throughout its early maturation. While the phosphoinositide 3-phosphatase myotubularin 4 (MTMR4) has an established role in regulating autophagy and cellular PI(3)P-content, two processes associated with the intracellular survival of S. typhimurium, a direct role for MTMR4 in Salmonella biology has not been examined. Here we demonstrate that GFP-tagged MTMR4 is recruited to the SCV and infection of cells depleted of endogenous MTMR4 results in a decrease in viable intracellular Salmonella. This reflects a significant increase in the proportion of SCVs with compromised integrity, which targets the compartment for autophagy and consequent bacterial cell death. These findings highlight the importance of PI(3)P regulation to the integrity of the SCV and reveal a novel role for the myotubularins in bacterial pathogenesis.
... Mycobacterium tuberculosis (35,36) and Legionella pneumophila (37,38) use effectors to deplete PI(3)P at the surface of their vacuoles to block phagosomal maturation and avoid fusion with degradative compartments. On the contrary, Salmonella enterica serovar Typhimurium uses the SPI-1 substrate SopB to maintain elevated levels of PI(3)P at the surface of Salmonella-containing vacuoles, favoring their biogenesis (39)(40)(41). Based on previous reports, the main source of PI(3)P-positive membranes for CCVs are early endosomes and autophagosomes (4). ...
Significance
The biogenesis of a replicative vacuole is an essential step of Coxiella burnetii infections and involves the hijack of several host membrane trafficking pathways. Here we describe Coxiella vacuolar protein B (CvpB) as a Coxiella effector that interacts with phosphoinositides on host cell membranes and manipulates phosphatidylinositol 3-phosphate [PI(3)P] metabolism for optimal Coxiella -containing vacuole (CCV) development. This is achieved by perturbing the activity of the phosphatidylinositol 5-kinase PIKfyve, leading to an enrichment of PI(3)P on CCV membranes, which is required for the autophagy machinery to mediate CCV homotypic fusion. The importance of this process is highlighted by a homotypic fusion defect between CCVs in cells infected with CvpB Coxiella mutants, which translates into an attenuated virulence in the insect model Galleria mellonella .
... Arl8b was subcloned from Arl8b-GFP (Kaniuk et al., 2011) into pmCherry-N1 (Takara Bio Inc.). The generation of plasmids used for the expression of p50 dynamitin–EGFP (Harrison et al., 2003), FYCO1-mCherry (Pankiv et al., 2010), Rab5-EGFP (Scott et al., 2002), EEA1-EGFP (Gillooly et al., 2000), EGFP-Rab11a (Choudhury et al., 2002), EGFP-Rab7 (Bucci et al., 2000), EGFP-RILP-C33 (Colucci et al., 2005), LAMP1-RFP (Sherer et al., 2003), and P3X-FLAG-EGFP-pericentrin-myc (provided by V. Menella, Hospital for Sick Children, Toronto, Canada; Kim and Rhee, 2014) is described in the references provided. pEGFP-N1 was from Takara Bio Inc. LIMP2-GFP cDNA was a gift from D. Neculai (Hospital for Sick Children, Toronto, Canada). ...
We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H
+
–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.
... As an obligate intracellular pathogen, C. trachomatis passes its entire developmental cycle within an intracellular membrane-bound vacuole called the inclusion. The capacity to create and maintain such a specialised replicative niche is an important virulence property for many intracellular pathogens (Scott et al., 2002;Vergne et al., 2005;Dupont et al., 2009). To facilitate the growth of the inclusion and to ensure delivery of nutrients to the pathogen, Chlamydia manipulates the membrane trafficking pathways of the host cell (Elwell and Engel, 2012). ...
Chlamydia species are obligate intracellular pathogens that have a major impact on human health. The pathogen replicates within an intracellular niche called an inclusion and is thought to rely heavily on host-derived proteins and lipids, including ceramide. Sortilin is a transmembrane receptor implicated in the trafficking of acid sphingomyelinase, which is responsible for catalysing the breakdown of sphingomyelin to ceramide. In this study, we examined the role of sortilin in Chlamydia trachomatis L2 development. Western immunoblotting and immunocytochemistry analysis revealed that endogenous sortilin is not only associated with the inclusion, but that protein levels increase in infected cells. RNAi-mediated depletion of sortilin, however, had no detectable impact on ceramide delivery to the inclusion or the production of infectious progeny. This study demonstrates that whilst Chlamydia redirects sortilin trafficking to the chlamydial inclusion, RNAi knockdown of sortilin expression is insufficient to determine if this pathway is requisite for the development of the pathogen.
