Typhoid toxin hijacks Wnt5a to establish host senescence and Salmonella infection.

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a senescence-associated secretory phenotype (SASP) that propagates the stress response to bystander cells. Thus, acute senescence is a powerful tumor suppressor. Salmonella enterica hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (TxSCs) and proteomics of their secretome identify the factors as Wnt5a, INHBA, and GDF15. Wnt5a establishes a positive feedback loop, driving INHBA and GDF15 expression. In fibroblasts, Wnt5a and INHBA mediate autocrine senescence in TxSCs and paracrine senescence in naive cells. Wnt5a synergizes with GDF15 to increase Salmonella invasion. Intestinal TxSCs undergo apoptosis without Wnt5a, which is required for establishing intestinal TxSCs. The study reveals how an innate defense against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections.

The great host-pathogen war: U.K. Cellular microbiology meeting 2020.

In 2019 we started a new annual meeting, aimed at bringing together researchers from across the United Kingdom studying cellular microbiology and the cell biology of host-pathogen interactions. In contrast to large glamourous meetings, featuring the great and the good from across the world, we wanted to create a forum for early career researchers to present their work and enjoy lively discussion. In particular, we hope that focussing on making the meeting accessible, affordable, and informal would help integrate and build the U.K. community working on this exciting topic.

Senescence and Host-Pathogen Interactions.

Damage to our genomes triggers cellular senescence characterised by stable cell cycle arrest and a pro-inflammatory secretome that prevents the unrestricted growth of cells with pathological potential. In this way, senescence can be considered a powerful innate defence against cancer and viral infection. However, damage accumulated during ageing increases the number of senescent cells and this contributes to the chronic inflammation and deregulation of the immune function, which increases susceptibility to infectious disease in ageing organisms. Bacterial and viral pathogens are masters of exploiting weak points to establish infection and cause devastating diseases. This review considers the emerging importance of senescence in the host-pathogen interaction: we discuss the pathogen exploitation of ageing cells and senescence as a novel hijack target of bacterial pathogens that deploys senescence-inducing toxins to promote infection. The persistent induction of senescence by pathogens, mediated directly through virulence determinants or indirectly through inflammation and chronic infection, also contributes to age-related pathologies such as cancer. This review highlights the dichotomous role of senescence in infection: an innate defence that is exploited by pathogens to cause disease.

Typhoid toxin exhausts the RPA response to DNA replication stress driving senescence and Salmonella infection.

Salmonella Typhi activates the host DNA damage response through the typhoid toxin, facilitating typhoid symptoms and chronic infections. Here we reveal a non-canonical DNA damage response, which we call RING (response induced by a genotoxin), characterized by accumulation of phosphorylated histone H2AX (γH2AX) at the nuclear periphery. RING is the result of persistent DNA damage mediated by toxin nuclease activity and is characterized by hyperphosphorylation of RPA, a sensor of single-stranded DNA (ssDNA) and DNA replication stress. The toxin overloads the RPA pathway with ssDNA substrate, causing RPA exhaustion and senescence. Senescence is also induced by canonical γΗ2ΑΧ foci revealing distinct mechanisms. Senescence is transmitted to non-intoxicated bystander cells by an unidentified senescence-associated secreted factor that enhances Salmonella infections. Thus, our work uncovers a mechanism by which genotoxic Salmonella exhausts the RPA response by inducing ssDNA formation, driving host cell senescence and facilitating infection.

Arf GTPase interplay with Rho GTPases in regulation of the actin cytoskeleton.

The Arf and Rho subfamilies of small GTPases are nucleotide-dependent molecular switches that act as master regulators of vesicular trafficking and the actin cytoskeleton organization. Small GTPases control cell processes with high fidelity by acting through distinct repertoires of binding partners called effectors. While we understand a great deal about how these GTPases act individually, relatively little is known about how they cooperate, especially in the control of effectors. This review highlights how Arf GTPases collaborate with Rac1 to regulate actin cytoskeleton dynamics at the membrane via recruiting and activating the Wave Regulatory Complex (WRC), a Rho effector that underpins lamellipodia formation and macropinocytosis. This provides insight into Arf regulation of the actin cytoskeleton, while putting the spotlight on small GTPase cooperation with emerging evidence of its importance in fundamental cell biology and interactions with pathogenic bacteria.

MYO6 is targeted by Salmonella virulence effectors to trigger PI3-kinase signaling and pathogen invasion into host cells.

To establish infections, Salmonella injects virulence effectors that hijack the host actin cytoskeleton and phosphoinositide signaling to drive pathogen invasion. How effectors reprogram the cytoskeleton network remains unclear. By reconstituting the activities of the Salmonella effector SopE, we recapitulated Rho GTPase-driven actin polymerization at model phospholipid membrane bilayers in cell-free extracts and identified the network of Rho-recruited cytoskeleton proteins. Knockdown of network components revealed a key role for myosin VI (MYO6) in Salmonella invasion. SopE triggered MYO6 localization to invasion foci, and SopE-mediated activation of PAK recruited MYO6 to actin-rich membranes. We show that the virulence effector SopB requires MYO6 to regulate the localization of PIP3 and PI(3)P phosphoinositides and Akt activation. SopE and SopB target MYO6 to coordinate phosphoinositide production at invasion foci, facilitating the recruitment of cytoskeleton adaptor proteins to mediate pathogen uptake.

Inhibition of WAVE Regulatory Complex Activation by a Bacterial Virulence Effector Counteracts Pathogen Phagocytosis.

To establish pathogenicity, bacteria must evade phagocytosis directed by remodeling of the actin cytoskeleton. We show that macrophages facilitate pathogen phagocytosis through actin polymerization mediated by the WAVE regulatory complex (WRC), small GTPases Arf and Rac1, and the Arf1 activator ARNO. To establish extracellular infections, enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli hijack the actin cytoskeleton by injecting virulence effectors into the host cell. Here, we find that the virulence effector EspG counteracts WRC-dependent phagocytosis, enabling EPEC and EHEC to remain extracellular. By reconstituting membrane-associated actin polymerization, we find that EspG disabled WRC activation through two mechanisms: EspG interaction with Arf6 blocked signaling to ARNO while EspG binding of Arf1 impeded collaboration with Rac1, thereby inhibiting WRC recruitment and activation. Investigating the mode of EspG interference revealed sites in Arf1 required for WRC activation and a mechanism facilitating pathogen evasion of innate host defenses.

The Arf GTPase-activating protein family is exploited by Salmonella enterica serovar Typhimurium to invade nonphagocytic host cells.

UNLABELLED: 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.

WAVE regulatory complex activation.

The WAVE regulatory complex (WRC) is critical to control of actin polymerization at the eukaryotic cell membrane. By reconstituting WAVE-dependent actin assembly on silica microspheres coated with phospholipid bilayers in mammalian brain extracts, we discovered that membrane recruitment and activation of WRC require the cooperative action of two mammalian GTPases, Arf and Rac. Here, we describe detailed methods to generate phospholipid-coated microspheres and porcine brain extract and outline conditions necessary to reconstitute WRC-dependent motility. In addition, we describe how to generate acylated recombinant GTPases, anchor them to lipid-coated microspheres, and reconstitute GTPase activation of WRC.

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

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.

The Drosophila Arf1 homologue Arf79F is essential for lamellipodium formation.

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.

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

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.

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.

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 SHIP2's scaffolding and phosphatase functions ensures tight compartmentalization and coordination of actin dynamics during pedestal formation.

The Salmonella effector SptP dephosphorylates host AAA+ ATPase VCP to promote development of its intracellular replicative niche.

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 SptP's 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 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.

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 Tir(Y474) 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 Tir(Y474) and c-Fyn in DRMs rapidly stimulates robust kinase activation both by induced c-Fyn(Y531) dephosphorylation to unlock the inactive state and by reciprocal c-Fyn(Y417) autophosphorylation to promote activity. After signal induction, c-Fyn dissipates and the resting state restored by Csk-dependent phosphorylation of c-Fyn(Y531). 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.

Antiviral activities and phosphorylation of 5-halo-2'-deoxyuridines and N-methanocarbathymidine in cells infected with vaccinia virus.

BACKGROUND: The antipoxviral activities and phosphorylation of N-methanocarbathymidine ([N]-MCT) and four 5-halo-2'-deoxyuridines, namely 5-fluoro-(FdU), 5-chloro-(CldU), 5-bromo-(BrdU), and 5-iodo-(IdU) derivatives, were explored. METHODS: Antiviral activities and nucleoside metabolism were determined in C127I mouse, LLC-MK2 monkey, and A549 human cells infected with thymidine-kinase-containing and -deficient (TK+ and TK-) vaccinia (WR strain) viruses. RESULTS: The antiviral potencies of CldU, BrdU and IdU were increased 16-26-fold in LLC-MK2 cells infected with TK+ compared with TK- virus infections, but enhancement of activity was much less in the other cell lines. (N)-MCT was nearly equally active against TK+ and TK- viruses in the three cell lines. Antiviral activity of FdU was associated with cytotoxicity. Uninfected and infected cells metabolized compounds to mono-, di- and triphosphates. The thymidine, BrdU and IdU triphosphate levels were higher in C127I and LLC-MK2 cells infected with TK+ than with TK- virus. (N)-MCT monophosphate levels were much higher in TK+ virus-infected cells, but without corresponding increases in (N)-MCT triphosphate. Furthermore, TK+ virus infections did not appreciably alter (N)-MCT triphosphate levels in other mouse (L929), monkey (MA-104 and Vero) and human cell lines (A549). Antiviral potency of the compounds was greater in C127I than in LLC-MK2 cells, yet lower intracellular triphosphate levels were found in C127I cells. CONCLUSION: We conclude that viral TK plays an important role in increasing the antiviral potencies of these compounds in some cell lines, but minimally in others. These findings may have implications in treating infected animals with compounds that are dependent upon poxvirus TK for their activation, because viral TK activity may vary greatly due to cell type.

Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex.

The Burkholderia cepacia complex (BCC) comprises a group of bacteria associated with opportunistic infections, especially in cystic fibrosis patients. B. cenocepacia J2315, of the transmissible ET12 lineage, contains a type III secretion (TTS) gene cluster implicated in pathogenicity. PCR and hybridisation assays indicate that the TTS gene cluster is present in all members of the BCC except B. cepacia (formerly genomovar I). The TTS gene clusters of B. cenocepacia J2315 and B. multivorans are similar in organisation but have variable levels of gene identity. Nucleotide sequence data obtained for the equivalent region of the B. cepacia genome indicate the absence of TTS structural genes due to a rearrangement likely to involve more than one step.