Proteomic analysis of the Simkania‐containing vacuole: the central role of retrograde transport

Simkania negevensis is an obligate intracellular bacterial pathogen that grows in amoeba or human cells within a membrane‐bound vacuole forming endoplasmic reticulum (ER) contact sites. The membrane of this Simkania‐containing vacuole (SnCV) is a critical host–pathogen interface whose origin and molecular interactions with cellular organelles remain poorly defined. We performed proteomic analysis of purified ER‐SnCV‐membranes using label free LC‐MS² to define the pathogen‐containing organelle composition. Of the 1,178 proteins of human and 302 proteins of Simkania origin identified by this strategy, 51 host cell proteins were enriched or depleted by infection and 57 proteins were associated with host endosomal transport pathways. Chemical inhibitors that selectively interfere with trafficking at the early endosome‐to‐trans‐Golgi network (TGN) interface (retrograde transport) affected SnCV formation, morphology and lipid transport. Our data demonstrate that Simkania exploits early endosome‐to‐TGN transport for nutrient acquisition and growth.     

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.     

The COPII complex and lysosomal VAMP7 determine intracellular Salmonella localization and growth

Salmonella invades epithelial cells and survives within a membrane‐bound compartment, the Salmonella‐containing vacuole (SCV). We isolated and determined the host protein composition of the SCV at 30 min and 3 h of infection to identify and characterize novel regulators of intracellular bacterial localization and growth. Quantitation of the SCV protein content revealed 392 host proteins specifically enriched at SCVs, out of which 173 associated exclusively with early SCVs, 124 with maturing SCV and 95 proteins during both time‐points. Vacuole interactions with endoplasmic reticulum‐derived coat protein complex II vesicles modulate early steps of SCV maturation, promoting SCV rupture and bacterial hyper‐replication within the host cytosol. On the other hand, SCV interactions with VAMP7‐positive lysosome‐like vesicles promote Salmonella‐induced filament formation and bacterial growth within the late SCV. Our results reveal that the dynamic communication between the SCV and distinct host organelles affects both intracellular Salmonella localization and growth at successive steps of host cell invasion.     

SopB‐ and SifA‐dependent shaping of the Salmonella‐containing vacuole proteome in the social amoeba Dictyostelium discoideum

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella‐containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type‐3 secretion systems encoded in SPI‐1 and SPI‐2 (T3SS‐1 and T3SS‐2, respectively). Recently, we reported that S. Typhimurium requires T3SS‐1 and T3SS‐2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.     

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.     

Unusual intracellular trafficking of Salmonella typhimurium in human melanoma cells

Salmonella spp. are enterobacteria capable of invading and replicating in both professional and non‐professional phagocytes. Here, we investigate the fate of S. typhimurium in human melanoma MelJuSo cells. The bacterium entered MelJuSo cells by a trigger mechanism and resided within a unique organelle, the Salmonella‐containing vacuole (SCV). The SCV acquired early endosomal markers transiently and then underwent a series of membrane modifications. In HeLa cells, vacuole maturation is characterized by the simultaneous acquisition of the lysosomal membrane glycoproteins (Lgps) Lamp1, CD63 and vacuolar (v)‐ATPase; in MelJuSo cells, however, acquisition of CD63 and v‐ATPase preceded that of Lamp1. A very striking event in MelJuSo cells was the arrest of bacterial septation starting from 8 h after infection. Bacteria nevertheless continued to elongate, remained morphologically intact and viable and were eventually exocytosed. This original feature was observed in several skin‐related cells including melanocytes, suggesting that it may provide the basis for an efficient host defence mechanism against Salmonella infection.     

Chlamydiae interaction with the endoplasmic reticulum: contact,function and consequences

Chlamydiae and chlamydiae‐related organisms are obligate intracellular bacterial pathogens. They reside in a membrane‐bound compartment termed the inclusion and have evolved sophisticated mechanisms to interact with cellular organelles. This review focuses on the nature, the function(s) and the consequences of chlamydiae–inclusion interaction with the endoplasmic reticulum (ER). The inclusion membrane establishes very close contact with the ER at specific sites termed ER–inclusion membrane contact sites (MCSs). These MCSs are constituted of a specific set of factors, including the C. trachomatis effector protein IncD and the host cell proteins CERT and VAPA/B. Because CERT and VAPA/B have a demonstrated role in the non‐vesicular trafficking of lipids between the ER and the Golgi, it was proposed that Chlamydia establish MCSs with the ER to acquire host lipids. However, the recruitment of additional factors to ER–inclusion MCSs, such as the ER calcium sensor STIM1, may suggest additional functions unrelated to lipid acquisition. Finally, chlamydiae interaction with the ER appears to induce the ER stress response, but this response is quickly dampened by chlamydiae to promote host cell survival.     

Take the tube: remodelling of the endosomal system by intracellular Salmonella enterica

Salmonella enterica is a facultative intracellular pathogen residing in a unique host cell‐derived membrane compartment, termed Salmonella‐containing vacuole or SCV. By the activity of effector proteins translocated by the SPI2‐endoced type III secretion system (T3SS), the biogenesis of the SCV is manipulated to generate a habitat permissive for intracellular proliferation. By taking control of the host cell vesicle fusion machinery, intracellular Salmonella creates an extensive interconnected system of tubular membranes arising from vesicles of various origins, collectively termed Salmonella‐induced tubules (SIT). Recent work investigated the dynamic properties of these manipulations. New host cell targets of SPI2‐T3SS effector proteins were identified. By applying combinations of live cell imaging and ultrastructural analyses, the detailed organization of membrane compartments inhabited and modified by intracellular Salmonella is now available. These studies provided unexpected new details on the intracellular environments of Salmonella. For example, one kind of SIT, the LAMP1‐positive Salmonella‐induced filaments (SIF), are composed of double‐membrane tubules, with an inner lumen containing host cell cytosol and cytoskeletal filaments, and an outer lumen containing endocytosed cargo. The novel findings call for new models for the biogenesis of SCV and SIT and give raise to many open questions we discuss in this review.     

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii is a gram‐negative intracellular bacterium that forms a large, lysosome‐like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol‐binding protein‐related protein 1 long (ORP1L) is a mammalian lipid‐binding protein that plays a dual role in cholesterol‐dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N‐terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co‐localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L‐depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.     

Chlamydiae Assemble a Pathogen Synapse to Hijack the Host Endoplasmic Reticulum

Chlamydiae are obligate intracellular bacterial pathogens that replicate within a specialized membrane‐bound compartment, termed an ‘inclusion’. The inclusion membrane is a critical host–pathogen interface, yet the extent of its interaction with cellular organelles and the origin of this membrane remain poorly defined. Here we show that the host endoplasmic reticulum (ER) is specifically recruited to the inclusion, and that key rough ER (rER) proteins are enriched on and translocated into the inclusion. rER recruitment is a Chlamydia‐orchestrated process that occurs independently of host trafficking. Generation of infectious progeny requires an intact ER, since ER vacuolation early during infection stalls inclusion development, whereas disruption post ER recruitment bursts the inclusion. Electron tomography and immunolabelling of Chlamydia‐infected cells reveal ‘pathogen synapses’ at which ordered arrays of chlamydial type III secretion complexes connect to the inclusion membrane only at rER contact sites. Our data show a supramolecular assembly involved in pathogen hijack of a key host organelle.     

A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella‐containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi‐function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion‐associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra‐cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.     

Anaplasma phagocytophilum Rab10‐dependent parasitism of the trans‐Golgi network is critical for completion of the infection cycle

Anaplasma phagocytophilum is an emerging human pathogen and obligate intracellular bacterium. It inhabits a host cell‐derived vacuole and cycles between replicative reticulate cell (RC) and infectious dense‐cored (DC) morphotypes. Host–pathogen interactions that are critical for RC‐to‐DC conversion are undefined. We previously reported that A. phagocytophilum recruits green fluorescent protein (GFP)‐tagged Rab10, a GTPase that directs exocytic traffic from the sphingolipid‐rich trans‐Golgi network (TGN) to its vacuole in a guanine nucleotide‐independent manner. Here, we demonstrate that endogenous Rab10‐positive TGN vesicles are not only routed to but also delivered into the A. phagocytophilum‐occupied vacuole (ApV). Consistent with this finding, A. phagocytophilum incorporates sphingolipids while intracellular and retains them when naturally released from host cells. TGN vesicle delivery into the ApV is Rab10 dependent, up‐regulates expression of the DC‐specific marker, APH1235, and is critical for the production of infectious progeny. The A. phagocytophilum surface protein, uridine monophosphate kinase, was identified as a guanine nucleotide‐independent, Rab10‐specific ligand. These data delineate why Rab10 is important for the A. phagocytophilum infection cycle and expand the understanding of the benefits that exploiting host cell membrane traffic affords intracellular bacterial pathogens.     

Tracking the dynamic interplay between bacterial and host factors during pathogen‐induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen‐induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram‐negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin‐3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high‐content and high‐throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.     

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.     

Salmonella modulation of the phagosome membrane,role of SseJ

Salmonellae have the ability to invade, persist and replicate within an intracellular phagosome termed the Salmonella‐containing vacuole (SCV). Salmonellae alter lipid and protein content of the SCV membrane and manipulate cytoskeletal elements in contact with the SCV using the Salmonella pathogenicity island 1 (SPI‐2) type III secretion system effectors. These modifications result in microtubular‐based movement and morphological changes, which include endosomal tubulation of the SCV membrane. SseJ is a SPI‐2 effector that localizes to the cytoplasmic face of the SCV and esterifies cholesterol through its glycerophospholipid : cholesterol acyltransferase activity. SseJ enzymatic activity as well as localization to the SCV are determined by binding to the small mammalian GTPase, RhoA. This review will focus on current knowledge about the role of SseJ in SCV membrane modification and will discuss how the hypothesis that a major role of SPI‐2 effectors is to modify SCV protein and lipid content to promote bacterial intracellular survival.     

The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55

Intracellular, pathogenic Salmonella typhimurium avoids phago-lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella-containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the construction of a three-dimensional space-filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol-rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)-anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol-rich early endocytic pathways during intracellular bacterial replication.     

Lipid droplet biogenesis is spatially coordinated at ER–vacuole contacts under nutritional stress

Eukaryotic cells store lipids in cytosolic organelles known as lipid droplets (LDs). Lipid droplet bud from the endoplasmic reticulum (ER), and may be harvested by the vacuole for energy during prolonged periods of starvation. How cells spatially coordinate LD production is poorly understood. Here, we demonstrate that yeast ER–vacuole contact sites (NVJs) physically expand in response to metabolic stress, and serve as sites for LD production. NVJ tether Mdm1 demarcates sites of LD budding, and interacts with fatty acyl‐CoA synthases at the NVJ periphery. Artificially expanding the NVJ through over‐expressing Mdm1 is sufficient to drive NVJ‐associated LD production, whereas ablating the NVJ induces defects in fatty acid‐to‐triglyceride production. Collectively, our data suggest a tight metabolic link between nutritional stress and LD biogenesis that is spatially coordinated at ER–vacuole contact sites.     

Presence of SopE and mode of infection result in increased Salmonella‐containing vacuole damage and cytosolic release during host cell infection by Salmonella enterica

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that has evolved sophisticated molecular mechanisms to establish an intracellular niche within a specialised vesicular compartment, the Salmonella‐containing vacuole (SCV). The loss of the SCV and release of STM into the cytosol of infected host cells was observed, and a bimodal intracellular lifestyle of STM in the SCV versus life in the cytosol is currently discussed. We set out to investigate the parameters affecting SCV integrity and cytosolic release. A fluorescent protein‐based cytosolic reporter approach was established to quantify, time‐resolved, and on a single cell level, the release of STM into the cytosol of host cells. We observed that the extent of SCV damage and cytosolic release is highly dependent on experimental conditions such as multiplicity of infection, type of host cell line, and STM strain background. Trigger invasion mediated by the Salmonella Pathogenicity Island 1‐encoded type III secretion system (SPI1‐T3SS) and its effector proteins promoted cytosolic release, whereas cytosolic bacteria were rarely observed if entry was mediated by zipper invasion. Presence of SPI1‐T3SS effector SopE was identified as major factor for damage of the SCV in the early phase after STM invasion and sopE‐expressing strains showed higher levels of cytosolic release.