Cytoskeletal Alteration Is an Early Cellular Response in Pulmonary Epithelium Infected with Aspergillus fumigatus Rather than Scedosporium apiospermum.

Invasive aspergillosis and scedosporiosis are life-threatening fungal infections with similar clinical manifestations in immunocompromised patients. Contrarily, Scedosporium apiospermum is susceptible to some azole derivative but often resistant to amphotericin B. Histopathological examination alone cannot diagnose these two fungal species. Pathogenesis studies could contribute to explore candidate protein markers for new diagnosis and treatment methods leading to a decrease in mortality. In the present study, proteomics was conducted to identify significantly altered proteins in A549 cells infected with or without Aspergillus fumigatus and S. apiospermum as measured at initial invasion. Protein validation was performed with immunogold labelling alongside immunohistochemical techniques in infected A549 cells and lungs from murine models. Further, cytokine production was measured, using the Bio-Plex-Multiplex immunoassay. The cytoskeletal proteins HSPA9, PA2G4, VAT1, PSMA2, PEX1, PTGES3, KRT1, KRT9, CLIP1 and CLEC20A were mainly changed during A. fumigatus infection, while the immunologically activated proteins WNT7A, GAPDH and ANXA2 were principally altered during S. apiospermum infection. These proteins are involved in fungal internalisation and structural destruction leading to pulmonary disorders. Interleukin (IL)-21, IL-1α, IL-22, IL-2, IL-8, IL-12, IL-17A, interferon-γ and tumour necrosis factor-α were upregulated in both aspergillosis and scedosporiosis, although more predominately in the latter, in accordance with chitin synthase-1 and matrix metalloproteinase levels. Our results demonstrated that during invasion, A. fumigatus primarily altered host cellular integrity, whereas S. apiospermum chiefly induced and extensively modulated host immune responses.

Cross Talk between ARF1 and RhoA Coordinates the Formation of Cytoskeletal Scaffolds during Chlamydia Infection.

Chlamydia trachomatis is an obligate intracellular bacterium that has developed sophisticated mechanisms to survive inside its infectious compartment, the inclusion. Notably, Chlamydia weaves an extensive network of microtubules (MTs) and actin filaments to enable interactions with host organelles and enhance its stability. Despite the global health and economic burden caused by this sexually transmitted pathogen, little is known about how actin and MT scaffolds are integrated into an increasingly complex virulence system. Previously, we established that the chlamydial effector InaC interacts with ARF1 to stabilize MTs. We now demonstrate that InaC regulates RhoA to control actin scaffolds. InaC relies on cross talk between ARF1 and RhoA to coordinate MTs and actin, where the presence of RhoA downregulates stable MT scaffolds and ARF1 activation inhibits actin scaffolds. Understanding how Chlamydia hijacks complex networks will help elucidate how this clinically significant pathogen parasitizes its host and reveal novel cellular signaling pathways. IMPORTANCE Chlamydia trachomatis is a major cause of human disease worldwide. The ability of Chlamydia to establish infection and cause disease depends on the maintenance of its parasitic niche, called the inclusion. To accomplish this feat, Chlamydia reorganizes host actin and microtubules around the inclusion membrane. How Chlamydia orchestrates these complex processes, however, is largely unknown. Here, we discovered that the chlamydial effector InaC activates Ras homolog family member A (RhoA) to control the formation of actin scaffolds around the inclusion, an event that is critical for inclusion stability. Furthermore, InaC directs the kinetics of actin and posttranslationally modified microtubule scaffolds by mediating cross talk between the GTPases that control these cytoskeletal elements, RhoA and ADP-ribosylation factor 1 (ARF1). The precise timing of these events is essential for the maintenance of the inclusion. Overall, this study provides the first evidence of ARF1-RhoA-mediated cross talk by a bacterial pathogen to coopt the host cytoskeleton.

Chlamydia trachomatis TmeA Directly Activates N-WASP To Promote Actin Polymerization and Functions Synergistically with TarP during Invasion.

Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis, such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process.IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.

Biological activity of Nod factors.

Chemically, the Nod factors (NFs) are lipochitooligosaccharides, produced mainly by bacteria of the Rhizobium genus. They are the main signaling molecules involved in the initiation of symbiosis between rhizobia and legume plants. Nod factors affect plant tissues at very low concentrations, even as low as 10-12 mol/L. They induce root hair deformation, cortical cell division, and root nodules' formation in the host plant. At the molecular level, the cytoskeleton is reorganized and expression of genes encoding proteins called nodulins is induced in response to Nod factors in the cell. Action of Nod factors is highly specific because it depends on the structure of a particular Nod factor involved, as well as the plant receptor reacting with it.

Regulation and Functions of ROP GTPases in Plant-Microbe Interactions.

Rho proteins of plants (ROPs) form a specific clade of Rho GTPases, which are involved in either plant immunity or susceptibility to diseases. They are intensively studied in grass host plants, in which ROPs are signaling hubs downstream of both cell surface immune receptor kinases and intracellular nucleotide-binding leucine-rich repeat receptors, which activate major branches of plant immune signaling. Additionally, invasive fungal pathogens may co-opt the function of ROPs for manipulation of the cytoskeleton, cell invasion and host cell developmental reprogramming, which promote pathogenic colonization. Strikingly, mammalian bacterial pathogens also initiate both effector-triggered susceptibility for cell invasion and effector-triggered immunity via Rho GTPases. In this review, we summarize central concepts of Rho signaling in disease and immunity of plants and briefly compare them to important findings in the mammalian research field. We focus on Rho activation, downstream signaling and cellular reorganization under control of Rho proteins involved in disease progression and pathogen resistance.

Modulation of Immune Responses by Particle Size and Shape.

The immune system has to cope with a wide range of irregularly shaped pathogens that can actively move (e.g., by flagella) and also dynamically remodel their shape (e.g., transition from yeast-shaped to hyphal fungi). The goal of this review is to draw general conclusions of how the size and geometry of a pathogen affect its uptake and processing by phagocytes of the immune system. We compared both theoretical and experimental studies with different cells, model particles, and pathogenic microbes (particularly fungi) showing that particle size, shape, rigidity, and surface roughness are important parameters for cellular uptake and subsequent immune responses, particularly inflammasome activation and T cell activation. Understanding how the physical properties of particles affect immune responses can aid the design of better vaccines.

Transcriptomic analysis of clam extrapallial fluids reveals immunity and cytoskeleton alterations in the first week of Brown Ring Disease development.

The Brown Ring Disease is an infection caused by the bacterium Vibrio tapetis on the Manila clam Ruditapes philippinarum. The process of infection, in the extrapallial fluids (EPFs) of clams, involves alteration of immune functions, in particular on hemocytes which are the cells responsible of phagocytosis. Disorganization of the actin-cytoskeleton in infected clams is a part of what leads to this alteration. This study is the first transcriptomic approach based on collection of extrapallial fluids on living animals experimentally infected by V. tapetis. We performed differential gene expression analysis of EPFs in two experimental treatments (healthy-against infected-clams by V. tapetis), and showed the deregulation of 135 genes. In infected clams, a downregulation of transcripts implied in immune functions (lysosomal activity and complement- and lectin-dependent PRR pathways) was observed during infection. We also showed a deregulation of transcripts encoding proteins involved in the actin cytoskeleton organization such as an overexpression of ß12-Thymosin (which is an actin sequestration protein) or a downregulation of proteins that closely interact with capping proteins such as Coactosin, that counteract action of capping proteins, or Profilin. We validated these transcriptomic results by cellular physiological analyses that showed a decrease of the lysosome amounts and the disorganization of actin cytoskeleton in infected hemocytes.

Role of septins in microbial infection.

Septins are widely recognized as a component of the cytoskeleton that is essential for cell division, and new work has shown that septins can recognise cell shape by assembling into filaments on membrane regions that display micrometer-scale curvature (e.g. at the cytokinetic furrow). Moreover, infection biology studies have illuminated important roles for septins in mediating the outcome of host-microbe interactions. In this Review, we discuss a selection of mechanistic insights recently gained from studying three infection paradigms: the rice blast fungus Magnaporthe oryzae, the poxvirus family member vaccinia virus and the Gram-negative bacterium Shigella flexneri These studies have respectively discovered that higher-order septin assemblies enable fungal invasion into plant cells, entrap viral particles at the plasma membrane and recognize dividing bacterial cells for delivery to lysosomes. Collectively, these insights illustrate how studying septin biology during microbial infection can provide fundamental advances in both cell and infection biology, and suggest new concepts underlying infection control.

Proteases of Sporothrix schenckii: cytopathological effects on a host-cell model / Proteasas de Sporothrix schenckii: efectos citopatológicos en un modelo de células huésped

Background. Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. Aims. To evaluate the proteolytic activity of S. schenckii on epithelial cells. Methods. The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. Results. The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. Conclusions. Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors (AU)

Antecedentes. La esporotricosis es una infección fúngica causada por el complejo Sporothrix schenckii. La adhesión del hongo al tejido hospedero se ha considerado un paso clave en la colonización e invasión, sin embargo poco se conoce de los eventos tempranos en la interacción hospedero-parasito. Objetivos. Evaluar la actividad proteolítica de S. schenckii en células epiteliales. Métodos. El sistema proteolítico (bajo los valores pH 5 y 7) fue evaluado mediante azocoll y zimogramas. Además, la interacción hospedero-parasito y la respuesta celular fueron analizadas con el examen de los microfilamentos del citoesqueleto mediante faloidina-FITC y microscopia electrónica de transmisión. Finalmente, la actividad metabólica (viabilidad celular) fue determinada por un ensayo de XTT. Resultados. Los zimogramas de S. schenckii muestran que posee una alta actividad proteolítica intracelular y extracelular (Mr≥200, 116, 97 y 70kD) dependientes de pH e inhibidas por PMSF y E64, que actúan sobre serin- y cistein proteasas. Durante la interacción de las células epiteliales-proteasas, las células mostraron alteraciones en la distribución de los microfilamentos y la estructura de la membrana plasmática. Además, la actividad metabólica (viabilidad celular) de las células epiteliales disminuyó un 60% sin inhibidores de proteasas. Conclusiones. Nuestros datos demuestran la complejidad de la respuesta celular durante el proceso de infección, proceso que puede ser en parte contrarrestado por la acción de los inhibidores de proteasas. Además, los resultados proporcionan información crítica para el entendimiento de la naturaleza en la interacción hospedero-hongo y para una nueva terapia antifúngica eficaz que incluya inhibidores de proteasas (AU)

Manipulation of the Host Cell Cytoskeleton by Chlamydia.

Chlamydiae are obligate intracellular pathogens. They undergo a biphasic developmental cycle differentiating between the infectious but metabolically quiescent elementary body and the vegetative, but non-infectious reticulate body. Chlamydia spends a significant portion of its development in the non-infectious stage, demanding an effective strategy of manipulating the host cells to ensure its intracellular survival and replication. A common target of all Chlamydia species studied so far is the host cell cytoskeleton, with past and recent findings revealing crucial roles in invasion, inclusion maintenance, nutrient acquisition, and egress. The molecular details of how Chlamydia co-opts the cytoskeleton is becoming clearer, with bacterial factors and their corresponding host cell targets identified.

Remodeling of tick cytoskeleton in response to infection with Anaplasma phagocytophilum.

The obligate intracellular pathogen Anaplasma phagocytophilum infects vertebrate and tick hosts. In this study, a genome-wide search for cytoskeleton components was performed in the tick vector, Ixodes scapularis. The available transcriptomics and proteomics data was then used to characterize the mRNA and protein levels of I. scapularis cytoskeleton components in response to A. phagocytophilum infection. The results showed that cytoskeleton components described in other model organisms were present in the I. scapularis genome. One type of intermediate filaments (lamin), a family of septins that was recently implicated in the cellular response to intracellular pathogens, and several members of motor proteins (kinesins and dyneins) that could be implicated in the cytoplasmic movements of A. phagocytophilum were found. The results showed that levels of tubulin, actin, septin, actin-related proteins and motor proteins were affected by A. phagocytophilum, probably to facilitate infection in I. scapularis. Functional studies demonstrated a role for selected cytoskeleton components in pathogen infection. These results provided a more comprehensive view of the cytoskeletal components involved in the response to A. phagocytophilum infection in ticks.

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.

The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant.

Exoenzyme Y (ExoY) was identified as a component of the Pseudomonas aeruginosa type 3 secretion system secretome in 1998. It is a common contributor to the arsenal of type 3 secretion system effectors, as it is present in approximately 90% of Pseudomonas isolates. ExoY has adenylyl cyclase activity that is dependent upon its association with a host cell cofactor. However, recent evidence indicates that ExoY is not just an adenylyl cyclase; rather, it is a promiscuous cyclase capable of generating purine and pyrimidine cyclic nucleotide monophosphates. ExoY's enzymatic activity causes a characteristic rounding of mammalian cells, due to microtubule breakdown. In endothelium, this cell rounding disrupts cell-to-cell junctions, leading to loss of barrier integrity and an increase in tissue edema. Microtubule breakdown seems to depend upon tau phosphorylation, where the elevation of cyclic nucleotide monophosphates activates protein kinases A and G and causes phosphorylation of endothelial microtubule associated protein tau. Phosphorylation is a stimulus for tau release from microtubules, leading to microtubule instability. Phosphorylated tau accumulates inside endothelium as a high molecular weight, oligomeric form, and is then released from the cell. Extracellular high molecular weight tau causes a transmissible cytotoxicity that significantly hinders cellular repair following infection. Thus, ExoY may contribute to bacterial virulence in at least two ways; first, by microtubule breakdown leading to loss of endothelial cell barrier integrity, and second, by promoting release of a high molecular weight tau cytotoxin that impairs cellular recovery following infection.

Candida albicans Modifies the Protein Composition and Size Distribution of THP-1 Macrophage-Derived Extracellular Vesicles.

The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, noncoding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus we have undertaken the first quantitative proteomic analysis on the protein composition of THP-1 macrophage-derived EVs during the interaction with Candida albicans. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP-1-derived EVs, both from control and from Candida-infected macrophages, had similar effector functions on other THP-1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP-1 nondifferenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to C. albicans infection and in macrophages communication.

The Roles of RNase-L in Antimicrobial Immunity and the Cytoskeleton-Associated Innate Response.

The interferon (IFN)-regulated endoribonuclease RNase-L is involved in multiple aspects of the antimicrobial innate immune response. It is the terminal component of an RNA cleavage pathway in which dsRNA induces the production of RNase-L-activating 2-5A by the 2'-5'-oligoadenylate synthetase. The active nuclease then cleaves ssRNAs, both cellular and viral, leading to downregulation of their expression and the generation of small RNAs capable of activating retinoic acid-inducible gene-I (RIG-I)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome. This leads to IFNß expression and IL-1ß activation respectively, in addition to broader effects on immune cell function. RNase-L is also one of a growing number of innate immune components that interact with the cell cytoskeleton. It can bind to several cytoskeletal proteins, including filamin A, an actin-binding protein that collaborates with RNase-L to maintain the cellular barrier to viral entry. This antiviral activity is independent of catalytic function, a unique mechanism for RNase-L. We also describe here the interaction of RNase-L with the E3 ubiquitin ligase and scaffolding protein, ligand of nump protein X (LNX), a regulator of tight junction proteins. In order to better understand the significance and context of these novel binding partners in the antimicrobial response, other innate immune protein interactions with the cytoskeleton are also discussed.

Mechanics and morphogenesis of fission yeast cells.

The integration of biochemical and biomechanical elements is at the heart of morphogenesis. While animal cells are relatively soft objects which shape and mechanics is mostly regulated by cytoskeletal networks, walled cells including those of plants, fungi and bacteria are encased in a rigid cell wall which resist high internal turgor pressure. How these particular mechanical properties may influence basic cellular processes, such as growth, shape and division remains poorly understood. Recent work using the model fungal cell fission yeast, Schizosaccharomyces pombe, highlights important contribution of cell mechanics to various morphogenesis processes. We envision this genetically tractable system to serve as a novel standard for the mechanobiology of walled cell.

Interferon-gamma and nitric oxide synthase 2 mediate the aggregation of resident adherent peritoneal exudate cells: implications for the host response to pathogens.

Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2-/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or "group behavior" of APECs are discussed in the context of host resistance to infectious organisms.

Micropatterned macrophage analysis reveals global cytoskeleton constraints induced by Bacillus anthracis edema toxin.

Bacillus anthracis secretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.

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.

Molecular mechanisms of host cytoskeletal rearrangements by Shigella invasins.

Pathogen-induced reorganization of the host cell cytoskeleton is a common strategy utilized in host cell invasion by many facultative intracellular bacteria, such as Shigella, Listeria, enteroinvasive E. coli and Salmonella. Shigella is an enteroinvasive intracellular pathogen that preferentially infects human epithelial cells and causes bacillary dysentery. Invasion of Shigella into intestinal epithelial cells requires extensive remodeling of the actin cytoskeleton with the aid of pathogenic effector proteins injected into the host cell by the activity of the type III secretion system. These so-called Shigella invasins, including IpaA, IpaC, IpgB1, IpgB2 and IpgD, modulate the actin-regulatory system in a concerted manner to guarantee efficient entry of the bacteria into host cells.