Daniel Humphreys

Salmonella takes control: effector-driven manipulation of the host.

Salmonella pathogenesis relies upon the delivery of over thirty specialised effector proteins into the host cell via two distinct type III secretion systems. These effectors act in concert to subvert the host cell cytoskeleton, signal transduction pathways, membrane trafficking and pro-inflammatory responses. This allows Salmonella to invade non-phagocytic epithelial cells, establish and maintain an intracellular replicative niche and, in some cases, disseminate to cause systemic disease. This review focuses on the actions of the effectors on their host cell targets during each stage of Salmonella infection.

Transition to the open state of the TolC periplasmic tunnel entrance

The TolC channel-tunnel spans the bacterial outer membrane and periplasm, providing a large exit duct for protein export and multidrug efflux when recruited by substrate-engaged inner membrane complexes. The sole constriction in the single pore of the homotrimeric TolC is the periplasmic tunnel entrance, which in its resting configuration is closed by dense packing of the 12 tunnel-forming α-helices. Recruitment of TolC must trigger opening for substrate transit to occur, but the mechanism underlying transition from the closed to the open state is not known. The high resolution structure of TolC indicates that the tunnel helices are constrained at the entrance by a circular network of intra- and intermonomer hydrogen bonds and salt bridges. To assess how opening is achieved, we disrupted these connections and monitored changes in the aperture size by measuring the single channel conductance of TolC derivatives in black lipid bilayers. Elimination of individual connections caused incremental weakening of the circular network, accompanied by gradual relaxation from the closed state and increased flexibility of the entrance. Simultaneous abolition of the key links caused a substantial increase in conductance, generating an aperture that corresponds to the modeled open state, with the capacity to allow access and passage of diverse substrates. The results support a model in which transition to the open state of TolC is achieved by an iris-like realignment of the tunnel entrance helices.

WAVE regulatory complex activation by cooperating GTPases Arf and Rac1

The WAVE regulatory complex (WRC) is a critical element in the control of actin polymerization at the eukaryotic cell membrane, but how WRC is activated remains uncertain. While Rho GTPase Rac1 can bind and activate WRC in vitro, this interaction is of low affinity, suggesting other factors may be important. By reconstituting WAVE-dependent actin assembly on membrane-coated beads in mammalian cell extracts, we found that Rac1 was not sufficient to engender bead motility, and we uncovered a key requirement for Arf GTPases. In vitro, Rac1 and Arf1 were individually able to bind weakly to recombinant WRC and activate it, but when both GTPases were bound at the membrane, recruitment and concomitant activation of WRC were dramatically enhanced. This cooperativity between the two GTPases was sufficient to induce WAVE-dependent bead motility in cell extracts. Our findings suggest that Arf GTPases may be central components in WAVE signalling, acting directly, alongside Rac1.

The Salmonella Effector SptP Dephosphorylates Host AAA+ ATPase VCP to Promote Development of its Intracellular Replicative Niche

Summary Virulence effectors delivered into intestinal epithelial cells by Salmonella trigger actin remodeling to direct pathogen internalization and intracellular replication in Salmonella-containing vacuoles (SCVs). One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake. SptP also harbors a C-terminal protein tyrosine phosphatase (PTPase) domain with no clear host substrates. Investigating SptPs longevity in infected cells, we uncover a late function of SptP, showing that it associates with SCVs, and its PTPase activity increases pathogen replication. Direct SptP binding and specific dephosphorylation of the AAA+ ATPase valosin-containing protein (VCP/p97), a facilitator of cellular membrane fusion and protein degradation, enhanced pathogen replication in SCVs. VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase. Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

Salmonella virulence effector SopE and Host GEF ARNO cooperate to recruit and activate WAVE to trigger bacterial invasion.

Summary Salmonella virulence effectors elicit host cell membrane ruffling to facilitate pathogen invasion. The WAVE regulatory complex (WRC) governs the underlying membrane-localized actin polymerization, but how Salmonella manipulates WRC is unknown. We show that Rho GTPase activation by the Salmonella guanine nucleotide exchange factor (GEF) SopE efficiently triggered WRC recruitment but not its activation, which required host Arf GTPase activity. Invading Salmonella recruited and activated Arf1 to facilitate ruffling and uptake. Arf3 and Arf6 could also enhance invasion. RNAi screening of host Arf-family GEFs revealed a key role for ARNO in pathogen invasion and generation of pathogen-containing macropinosomes enriched in Arf1 and WRC. Salmonella recruited ARNO via Arf6 and the phosphoinositide phosphatase effector SopB-induced PIP3 generation. ARNO in turn triggered WRC recruitment and activation, which was dramatically enhanced when SopE and ARNO cooperated. Thus, we uncover a mechanism by which pathogen and host GEFs synergize to regulate WRC and trigger Salmonella invasion.

Enteropathogenic Escherichia coli Recruits the Cellular Inositol Phosphatase SHIP2 to Regulate Actin-Pedestal Formation

Adhesion of enteropathogenic Escherichia coli to epithelial cells triggers actin-rich pedestal formation beneath the bacteria. Pedestal formation requires delivery and insertion of the bacterial translocated intimin receptor (Tir) into the host plasma membrane. The C-terminal regions in Tir, encompassing Y483 and Y511, share sequence similarity with cellular immunoreceptor tyrosine-based inhibition motifs (ITIMs), which are critical regulators of eukaryotic signaling pathways. We demonstrate that Y483 and Y511 within tandem ITIM-like sequences are essential for recruiting SHIP2, a host inositol phosphatase. SHIP2 controls condensed F-actin-pedestal formation by engaging the adaptor SHC and by generating a PI(3,4)P(2)-enriched lipid platform for recruitment of the cytoskeletal regulator lamellipodin. Therefore, mimicry of eukaryotic receptor motifs by Tir controls both the lipid and protein composition of the signaling platform necessary for pedestal formation. Further, the dual action of SHIP2s scaffolding and phosphatase functions ensures tight compartmentalization and coordination of actin dynamics during pedestal formation.

Arf6 coordinates actin assembly through the WAVE complex, a mechanism usurped by Salmonella to invade host cells

Significance The small GTPase ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates actin filament polymerization to remodel the membrane-associated cytoskeleton in diverse cell processes, yet how Arf6 directs actin polymerization is unknown. By reconstituting membrane-associated actin assembly, we find that Arf6 assembles actin via the WAVE regulatory complex (WRC). In contrast to other Arf family members, which directly bind and activate WRC, Arf6 signaled to WRC indirectly by recruiting the Arf1 activator ARNO to the membrane. Remarkably, we demonstrate this mechanism is hijacked by the pathogen Salmonella that usurped Arf6 and WRC to invade human host cells and establish intracellular infection. This study describes a mechanism for Arf6-driven actin polymerization. ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1–WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.

Clustering transfers the translocated Escherichia coli receptor into lipid rafts to stimulate reversible activation of c-Fyn.

Enteropathogenic Escherichia coli (EPEC) mimic a ligand–receptor interaction to induce ‘pedestal‐like’ pseudopodia on mammalian cells, providing a tractable system to study tyrosine kinase signalling to the actin cytoskeleton. EPEC delivers its own receptor (Tir), which is engaged by a bacterial surface ligand (intimin). When Tir delivery and activity are uncoupled, intimin‐induced Tir clustering stimulates TirY474 phosphorylation by the Src‐family kinase (SFK) c‐Fyn, triggering actin polymerization and pedestal formation. How c‐Fyn specifically targets Tir and is regulated remains unknown. We show that clustering transfers Tir into cholesterol‐rich detergent‐resistant microdomains (DRMs), a signal prompting transient c‐Fyn accumulation at bacterial adhesion sites. Co‐clustering of TirY474 and c‐Fyn in DRMs rapidly stimulates robust kinase activation both by induced c‐FynY531 dephosphorylation to unlock the inactive state and by reciprocal c‐FynY417 autophosphorylation to promote activity. After signal induction, c‐Fyn dissipates and the resting state restored by Csk‐dependent phosphorylation of c‐FynY531. These data illustrate a sophisticated mechanism evolved by a pathogen effector to reversibly regulate SFKs, and resolve early interactions at a model receptor initiating tyrosine kinase signalling.

The Arf GTPase-Activating Protein Family Is Exploited by Salmonella enterica Serovar Typhimurium To Invade Nonphagocytic Host Cells

ABSTRACT To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections. IMPORTANCE To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP family in Salmonella invasion. Surprisingly, we found that Arf1 and Arf6 GAPs cooperate with their Arf GEF counterparts to facilitate cycles of Arf GTPase activation and inactivation, which direct pathogen invasion. This report illustrates that GAP proteins promote actin-dependent processes and are not necessarily restricted to negatively regulating cellular signaling. It uncovers a remarkable interplay between Arf GEFs and GAPs that is exploited by Salmonella to establish infection and expands our understanding of Arf GTPase-regulated cytoskeleton remodeling. To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP family in Salmonella invasion. Surprisingly, we found that Arf1 and Arf6 GAPs cooperate with their Arf GEF counterparts to facilitate cycles of Arf GTPase activation and inactivation, which direct pathogen invasion. This report illustrates that GAP proteins promote actin-dependent processes and are not necessarily restricted to negatively regulating cellular signaling. It uncovers a remarkable interplay between Arf GEFs and GAPs that is exploited by Salmonella to establish infection and expands our understanding of Arf GTPase-regulated cytoskeleton remodeling.

The Drosophila Arf1 homologue Arf79F is essential for lamellipodium formation

Summary The WAVE regulatory complex (WRC) drives the polymerisation of actin filaments located beneath the plasma membrane to generate lamellipodia that are pivotal to cell architecture and movement. By reconstituting WRC-dependent actin assembly at the membrane, we recently discovered that several classes of Arf family GTPases directly recruit and activate WRC in cell extracts, and that Arf cooperates with Rac1 to trigger actin polymerisation. Here, we demonstrate that the Class 1 Arf1 homologue Arf79F colocalises with the WRC at dynamic lamellipodia. We report that Arf79F is required for lamellipodium formation in Drosophila S2R+ cells, which only express one Arf isoform for each class. Impeding Arf function either by dominant-negative Arf expression or by Arf double-stranded RNA interference (dsRNAi)-mediated knockdown uncovered that Arf-dependent lamellipodium formation was specific to Arf79F, establishing that Class 1 Arfs, but not Class 2 or Class 3 Arfs, are crucial for lamellipodia. Lamellipodium formation in Arf79F-silenced cells was restored by expressing mammalian Arf1, but not by constitutively active Rac1, showing that Arf79F does not act via Rac1. Abolition of lamellipodium formation in Arf79F-silenced cells was not due to Golgi disruption. Blocking Arf79F activation with guanine nucleotide exchange factor inhibitors impaired WRC localisation to the plasma membrane and concomitant generation of lamellipodia. Our data indicate that the Class I Arf GTPase is a central component in WRC-driven lamellipodium formation.