Formin-mediated actin polymerization promotes Salmonella invasion.

Salmonella invade host cells using Type 3 secreted effectors, which modulate host cellular targets to promote actin rearrangements at the cell surface that drive bacterial uptake. The Arp2/3 complex contributes to Salmonella invasion but is not essential, indicating other actin regulatory factors are involved. Here, we show a novel role for FHOD1, a formin family member, in Salmonella invasion. FHOD1 and Arp2/3 occupy distinct microdomains at the invasion site and control distinct aspects of membrane protrusion formation. FHOD1 is phosphorylated during infection and this modification is required for promoting bacterial uptake by host cells. ROCK II, but not ROCK I, is recruited to the invasion site and is required for FHOD1 phosphorylation and for Salmonella invasion. Together, our studies revealan important phospho-dependent FHOD1 actin polymerization pathway in Salmonella invasion.

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium.

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome-lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

Salmonella exploits Arl8B-directed kinesin activity to promote endosome tubulation and cell-to-cell transfer.

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium establishes a replicative niche, the Salmonella-containing vacuole (SCV), in host cells. Here we demonstrate that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm. Arl8B(+) tubules partially colocalized with LAMP1 and SCAMP3. Formation of LAMP1(+) tubules (the Salmonella-induced filaments phenotype; SIFs) required Arl8B expression. SIFs formation is known to require the activity of kinesin-1. Here we find that Arl8B is required for kinesin-1 recruitment to SCVs. We have previously shown that SCVs undergo centrifugal movement to the cell periphery at 24 h post infection and undergo cell-to-cell transfer to infect neighbouring cells, and that both phenotypes require kinesin-1 activity. Here we demonstrate that Arl8B is required for migration of the SCV to the cell periphery 24 h after infection and for cell-to-cell transfer of bacteria to neighbouring cells. These results reveal a novel host factor co-opted by S. Typhimurium to manipulate the host endocytic pathway and to promote the spread of infection within a host.

Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella-containing vacuole.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella-containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3-phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30-60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella-induced filaments (Sifs) was altered by SNX3 knock-down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs.

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.

AP-1 and ARF1 control endosomal dynamics at sites of FcR mediated phagocytosis.

Phagocytosis, the mechanism of ingestion of large material and microorganisms, relies on actin polymerization and on the focal delivery of intracellular endocytic compartments. The molecular mechanisms involved in the formation and delivery of the endocytic vesicles that are recruited at sites of phagocytosis are not well characterized. Here we show that adaptor protein (AP)-1 but not AP-2 clathrin adaptor complexes are recruited early below the sites of particle attachment and are required for efficient receptor-mediated phagocytosis in murine macrophages. Clathrin, however, is not recruited with the AP complexes. We further show that the recruitment of AP-1-positive structures at sites of phagocytosis is regulated by the GTP-binding protein ARF1 but is not sensitive to brefeldin A. Furthermore, AP-1 depletion leads to increased surface levels of TNF-alpha, a cargo known to traffic through the endosomes to the plasma membrane upon stimulation of the macrophages. Together, our results support a clathrin-independent role for AP complexes in endosomal dynamics in macrophages by retaining some cargo proteins, a process important for membrane remodeling during phagocytosis.

Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

Bacterial invasion: entry through the exocyst door.

Salmonella entry into host cells involves rearrangements of actin and mobilization of membranes. Here we discuss new findings showing that Salmonella recruits the exocyst complex, which plays a role in vesicle secretion, to the site of invasion to promote its entry.

The phosphoinositide phosphatase SopB manipulates membrane surface charge and trafficking of the Salmonella-containing vacuole.

Shifts in electrostatic surface charge of membranes have recently been highlighted as a significant factor contributing to protein targeting to the plasma membrane and nascent phagosomes. Intracellular, vacuole-adapted pathogens may also regulate surface charge of their vacuoles to establish a replicative niche. Since Salmonella enterica serovar Typhimurium controls trafficking of the Salmonella-containing vacuole (SCV) and inhibits its fusion with lysosomes, we investigated the contribution of surface charge to this process. Using recently developed fluorescent biosensors, we show that the bacterial phosphoinositide phosphatase SopB controls membrane surface charge of nascent SCVs by reducing levels of negatively charged lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine. This SopB activity results in dissociation of a number of host-cell endocytic trafficking proteins from this compartment and inhibits SCV-lysosome fusion. Moreover, inducible reduction of negative charge rescues DeltasopB bacteria-containing SCVs from fusion with lysosomes. These results reveal a membrane-charge-based mechanism used by S. Typhimurium to control SCV maturation.

Linking exocytosis and endocytosis during phagocytosis.

Phagocytosis is used by macrophages, dendritic cells and neutrophils to capture and destroy pathogens and particulate antigens. Although localized assembly of actin filaments is the driving force for particle internalization, exocytosis of intracellular compartments, and in particular endocytic compartments, has been shown recently to be required for the early steps of phagosome formation. Here we report on the different compartments undergoing exocytosis during phagocytosis, with a special focus on late endosomes. We then compare this process with secretion from lysosomes or lysosome-related organelles in specialized cells. Finally, we discuss how some of the molecular mechanisms responsible for lysosome-related organelle secretion could also be implicated in phagosome formation.

CLIPR-59 is a lipid raft-associated protein containing a cytoskeleton-associated protein glycine-rich domain (CAP-Gly) that perturbs microtubule dynamics.

We recently have identified a new cytoplasmic linker protein (CLIP), CLIPR-59, which is involved in the regulation of early endosome/trans-Golgi network dynamics. In contrast with CLIP-170, CLIPR-59 is not localized to microtubules at steady state but is associated with the trans-Golgi network and the plasma membrane. Here we show that the last 30 amino acids (C30) are sufficient for membrane targeting and that two cysteines in the C30 domain are palmitoylated. We demonstrate that CLIPR-59 is associated with lipid rafts via its C-terminal palmitoylated domain. In vitro experiments suggest that CLIPR-59 and its microtubule-binding domain alone have a better affinity for unpolymerized tubulin or small oligomers than for microtubules. In contrast with the CLIP-170 microtubule-binding domain, the CLIPR-59 microtubule-binding domain diminishes microtubule regrowth after nocodazole washout in vivo, showing that this domain can prevent microtubule polymerization. In contrast with the role of linker between membranes and microtubules that was proposed for CLIP function, CLIPR-59 thus may have an "anti-CLIP" function by preventing microtubule-raft interactions.

TI-VAMP/VAMP7 is required for optimal phagocytosis of opsonised particles in macrophages.

Phagocytosis relies on extension of plasmalemmal pseudopods generated by focal actin polymerisation and delivery of membranes from intracellular pools. Here we show that compartments of the late endocytic pathway, bearing the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7), are recruited upon particle binding and undergo exocytosis before phagosome sealing in macrophages during Fc receptor (FcR)-mediated phagocytosis. Expression of the dominant-negative amino-terminal domain of TI-VAMP or depletion of TI-VAMP with small interfering RNAs inhibited phagocytosis mediated by Fc or complement receptors. In addition, inhibition of TI-VAMP activity led to a reduced exocytosis of late endocytic vesicles and this resulted in an early blockade of pseudopod extension, as observed by scanning electron microscopy. Therefore, TI-VAMP defines a new pathway of membrane delivery required for optimal FcR-mediated phagocytosis.