Publications

2022
Ramachandran RP, I, Nandi , N, Haritan , E, Zlotkin-Rivkin , Y, Keren , T, Danieli , M, Lebendiker , N, Melamed-Book , W, Breuer , D, Reichmann , and B, Aroeti . 10/11/2022. “Esph Interacts With The Host Active Bcr Related (Abr) Protein To Suppress Rhogtpases”. Gut Microbes, 14, 1. https://www.tandfonline.com/doi/full/10.1080/19490976.10/11/2022. 2130657. Abstract
Enteropathogenic Escherichia coli are bacterial pathogens that colonize the gut and cause severe diarrhea in humans. Upon intimate attachment to the intestinal epithelium, these pathogens translocate via a type III secretion system virulent proteins, termed effectors, into the host cells. These effectors manipulate diverse host cell organelles and functions for the pathogen’s benefit. However, the precise mechanisms underlying their activities are not fully understood despite intensive research. EspH, a critical effector protein, has been previously reported to disrupt the host cell actin cytoskeleton by suppressing RhoGTPase guanine exchange factors. However, native host proteins targeted by EspH to mediate these activities remained unknown. Here, we identified the active Bcr related (ABR), a protein previously characterized to possess dual Rho guanine nucleotide exchange factor and GTPase activating protein (GAP) domains, as a native EspH interacting partner. These interactions are mediated by the effector protein’s C-terminal 38 amino acid segment. The effector primarily targets the GAP domain of ABR to suppress Rac1 and Cdc42, host cell cytotoxicity, bacterial invasion, and filopodium formation at infection sites. Knockdown of ABR expression abolished the ability of EspH to suppress Rac1, Cdc42. Our studies unravel a novel mechanism by which host RhoGTPases are hijacked by bacterial effectors.
2021
Nandi I, L, Aroeti , RP, Ramachandran , EG, Kassa , E, Zlotkin-Rivkin , and B, Aroeti . 5/2021. “Type Iii Secreted Effectors That Target Mitochondria.”. Cell Microbiol., Pp. e13352. https://pubmed.ncbi.nlm.nih.gov/33960116/. Abstract
A type III secretion system (T3SS) is used by Gram-negative bacterial pathogens to secrete and translocate a battery of proteins, termed effectors, from the bacteria directly into the host cells. These effectors, which are thought to play a key role in bacterial virulence, hijack and modify the activity of diverse host cell organelles, including mitochondria. Mitochondria-the energy powerhouse of the cell-are important cell organelles that play role in numerous critical cellular processes, including the initiation of apoptosis and the induction of innate immunity. Therefore, it is not surprising that pathogenic bacteria use mitochondrially targeted effectors to control host cell death and immunity pathways. Surprisingly, however, we found that despite their importance, only a limited number of type III secreted effectors have been characterised to target host mitochondria, and the mechanisms underlying their mitochondrial activity have not been sufficiently analysed. These include effectors secreted by the enteric attaching and effacing (A/E), Salmonella and Shigella bacterial pathogens. Here we give an overview of key findings, present gaps in knowledge and hypotheses concerning the mode by which these type III secreted effectors control the host and the bacterial cell life (and death) through targeting mitochondria.
2020
PR Ramachandran, C, Spiegel , Keren, Y, Danieli, T, Melamed-Book, N, Pal, RR , Zlotkin-Rivkin, E, Rosenshine, I, and Aroeti, B. 9/15/2020. “Mitochondrial Targeting Of The Enteropathogenic Escherichia Coli Map Triggers Calcium Mobilization, Adam10-Map Kinase Signaling, And Host Cell Apoptosis”. Mbio, 11, 5, Pp. e01397-20. https://pubmed.ncbi.nlm.nih.gov/32934081/. Abstract

Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.

2019
Ephrem G Kassa, Zlotkin-Rivkin, Efrat , Friedman, Gil , Ramachandran, Rachana P, Melamed-Book, Naomi , Weiss, Aryeh M, Belenky, Michael , Reichmann, Dana , Breuer, William , Pal, Ritesh Ranjan , Rosenshine, Ilan , Lapierre, Lynne A, Goldenring, James R, and Aroeti, Benjamin . 2019. “Enteropathogenic Escherichia Coli Remodels Host Endosomes To Promote Endocytic Turnover And Breakdown Of Surface Polarity”. Plos Pathog, 15, 6, Pp. e1007851. doi:10.1371/journal.ppat.1007851. Abstract
Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.
2018
Rachana Pattani Ramachandran, Vences-Catalán, Felipe , Wiseman, Dan , Zlotkin-Rivkin, Efrat , Shteyer, Eyal , Melamed-Book, Naomi , Rosenshine, Ilan , Levy, Shoshana , and Aroeti, Benjamin . 2018. “Esph Suppresses Erk By Spatial Segregation From Cd81 Tetraspanin Microdomains”. Infect Immun, 86, 10. doi:10.1128/IAI.00303-18. Abstract
Enteropathogenic (EPEC) belongs to a group of enteric human pathogens known as attaching-and-effacing (A/E) pathogens, which utilize a type III secretion system (T3SS) to translocate a battery of effector proteins from their own cytoplasm into host intestinal epithelial cells. Here we identified EspH to be an effector that prompts the recruitment of the tetraspanin CD81 to infection sites. EspH was also shown to be an effector that suppresses the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) signaling pathway at longer infection times. The inhibitory effect was abrogated upon deletion of the last 38 amino acids located at the C terminus of the protein. The efficacy of EspH-dependent Erk suppression was higher in CD81-deficient cells, suggesting that CD81 may act as a positive regulator of Erk, counteracting Erk suppression by EspH. EspH was found within CD81 microdomains soon after infection but was largely excluded from these domains at a later time. Based on our results, we propose a mechanism whereby CD81 is initially recruited to infection sites in response to EspH translocation. At a later stage, EspH moves out of the CD81 clusters to facilitate effective Erk inhibition. Moreover, EspH selectively inhibits the tumor necrosis factor alpha (TNF-α)-induced Erk signaling pathway. Since Erk and TNF-α have been implicated in innate immunity and cell survival, our studies suggest a novel mechanism by which EPEC suppresses these processes to promote its own colonization and survival in the infected gut.
Benjamin Aroeti and Kassa, Ephrem G. 2018. “Stinging Tight Junctions With Wasps”. Cell Mol Gastroenterol Hepatol, 5, 3, Pp. 420-421. doi:10.1016/j.jcmgh.2017.12.007.
2017
Aaron Brahami, Levy, Hadas , Zlotkin-Rivkin, Efrat , Melamed-Book, Naomi , Tal, Nataly , Lev, Dmitry , Yeshua, Talia , Fedosyeyev, Oleg , Aroeti, Benjamin , and Lewis, Aaron . 2017. “Live Cell Near-Field Optical Imaging And Voltage Sensing With Ultrasensitive Force Control”. Opt Express, 25, 11, Pp. 12131-12143. doi:10.1364/OE.25.012131. Abstract
Force controlled optical imaging of membranes of living cells is demonstrated. Such imaging has been extended to image membrane potential changes to demonstrate that live cell imaging has been achieved. To accomplish this advance, limitations inherent in atomic force microscopy (AFM) since its inception in 1986 [G. Binnig, C. F. Quate, and C. Gerber, "Atomic Force Microscope," Phys. Rev. Lett. 56, 930-933 (1986).] had to be overcome. The advances allow for live cell imaging of a whole genre of functional biological imaging with stiff (1-10N/m) scanned probe imaging cantilevers. Even topographic imaging of fine cell protrusions, such as microvilli, has been accomplished with such cantilevers. Similar topographic imaging has only recently been demonstrated with the standard soft (0.05N/m) cantilevers that are generally required for live cell imaging. The progress reported here demonstrates both ultrasensitive AFM (~100pN), capable of topographic imaging of even microvilli protruding from cell membranes and new functional applications that should have a significant impact on optical and other approaches in biological imaging of living systems and ultrasoft materials.
2014
Merav Cohen, Kitsberg, Daniel , Tsytkin, Sabina , Shulman, Maria , Aroeti, Benjamin , and Nahmias, Yaakov . 2014. “Live Imaging Of Glut2 Glucose-Dependent Trafficking And Its Inhibition In Polarized Epithelial Cysts”. Open Biol, 4, 7. doi:10.1098/rsob.140091. Abstract
GLUT2 is a facilitative glucose transporter, expressed in polarized epithelial cells of the liver, intestine, kidney and pancreas, where it plays a critical role in glucose homeostasis. Together with SGLT1/2, it mediates glucose absorption in metabolic epithelial tissues, where it can be translocated apically upon high glucose exposure. To track the subcellular localization and dynamics of GLUT2, we created an mCherry-hGLUT2 fusion protein and expressed it in multicellular kidney cysts, a major site of glucose reabsorption. Live imaging of GLUT2 enabled us to avoid the artefactual localization of GLUT2 in fixed cells and to confirm the apical GLUT2 model. Live cell imaging showed a rapid 15 ± 3 min PKC-dependent basal-to-apical translocation of GLUT2 in response to glucose stimulation and a fourfold slower basolateral translocation under starvation. These results mark the physiological importance of responding quickly to rising glucose levels. Importantly, we show that phloretin, an apple polyphenol, inhibits GLUT2 translocation in both directions, suggesting that it exerts its effect by PKC inhibition. Subcellular localization studies demonstrated that GLUT2 is endocytosed through a caveolae-dependent mechanism, and that it is at least partly recovered in Rab11A-positive recycling endosome. Our work illuminates GLUT2 dynamics, providing a platform for drug development for diabetes and hyperglycaemia.
2013
Victor Yashunsky, Kharilker, Leorah , Zlotkin-Rivkin, Efrat , Rund, David , Melamed-Book, Naomi , Zahavi, Eitan Erez , Perlson, Eran , Mercone, Silvana , Golosovsky, Michael , Davidov, Dan , and Aroeti, Benjamin . 2013. “Real-Time Sensing Of Enteropathogenic E. Coli-Induced Effects On Epithelial Host Cell Height, Cell-Substrate Interactions, And Endocytic Processes By Infrared Surface Plasmon Spectroscopy”. Plos One, 8, 10, Pp. e78431. doi:10.1371/journal.pone.0078431. Abstract
Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.
2012
Victor Yashunsky, Marciano, Tal , Lirtsman, Vladislav , Golosovsky, Michael , Davidov, Dan , and Aroeti, Benjamin . 2012. “Real-Time Sensing Of Cell Morphology By Infrared Waveguide Spectroscopy”. Plos One, 7, 10, Pp. e48454. doi:10.1371/journal.pone.0048454. Abstract
We demonstrate that a live epithelial cell monolayer can act as a planar waveguide. Our infrared reflectivity measurements show that highly differentiated simple epithelial cells, which maintain tight intercellular connectivity, support efficient waveguiding of the infrared light in the spectral region of 1.4-2.5 µm and 3.5-4 µm. The wavelength and the magnitude of the waveguide mode resonances disclose quantitative dynamic information on cell height and cell-cell connectivity. To demonstrate this we show two experiments. In the first one we trace in real-time the kinetics of the disruption of cell-cell contacts induced by calcium depletion. In the second one we show that cell treatment with the PI3-kinase inhibitor LY294002 results in a progressive decrease in cell height without affecting intercellular connectivity. Our data suggest that infrared waveguide spectroscopy can be used as a novel bio-sensing approach for studying the morphology of epithelial cell sheets in real-time, label-free manner and with high spatial-temporal resolution.
Benjamin Aroeti, Friedman, Gil , Zlotkin-Rivkin, Efrat , and Donnenberg, Michael S. 2012. “Retraction Of Enteropathogenic E. Coli Type Iv Pili Promotes Efficient Host Cell Colonization, Effector Translocation And Tight Junction Disruption”. Gut Microbes, 3, 3, Pp. 267-71. doi:10.4161/gmic.19814. Abstract
Type IV pili (Tfp) play a primary role in mediating the adherence of pathogenic bacteria to their hosts. The pilus filament can retract with an immense force. However, the role of this activity in microbial pathogenesis has not been rigorously explored. Experiments performed on volunteers suggested that the retraction capacity of enteropathogenic Escherichia coli (EPEC) Tfp is required for full virulence. Here we review our recent study(1) in which we showed that the retraction capacity of the EPEC Tfp facilitates tight-junction disruption and actin-rich pedestal formation by promoting efficient bacterial protein effector translocation into epithelial host cells. We also present new data using live imaging confocal microscopy suggesting that EPEC adheres to monolayers in microcolonies and that Tfp retraction facilitates significant changes in the microcolony shape, which may be critical for efficient effector delivery. Our studies hence suggest novel insights into the role of pili retraction in EPEC pathogenesis.
Victor Yashunsky, Lirtsman, Vladislav , Zilbershtein, Alexander , Bein, Amir , Schwartz, Betty , Aroeti, Benjamin , Golosovsky, Michael , and Davidov, Dan . 2012. “Surface Plasmon-Based Infrared Spectroscopy For Cell Biosensing”. J Biomed Opt, 17, 8, Pp. 081409-1. doi:10.1117/1.JBO.17.8.081409. Abstract
Cell morphology is often used as a valuable indicator of the physical condition and general status of living cells. We demonstrate a noninvasive method for morphological characterization of adherent cells. We measure infrared reflectivity spectrum at oblique angle from living cells cultured on thin Au film, and utilize the unique properties of the confined infrared waves (i.e., surface plasmon and guided modes) traveling inside the cell layer. The propagation of these waves strongly depends on cell morphology and connectivity. By tracking the resonant wavelength and attenuation of the surface plasmon and guided modes we measure the kinetics of various cellular processes such as (i) cell attachment and spreading on different substrata, (ii) modulation of the outer cell membrane with chlorpromazine, and (iii) formation of intercellular junctions associated with progressive cell polarization. Our method enables monitoring of submicron variations in cell layer morphology in real-time, and in the label-free manner.
2011
Eitan E Zahavi, Lieberman, Joshua A, Donnenberg, Michael S, Nitzan, Mor , Baruch, Kobi , Rosenshine, Ilan , Turner, Jerrold R, Melamed-Book, Naomi , Feinstein, Naomi , Zlotkin-Rivkin, Efrat , and Aroeti, Benjamin . 2011. “Bundle-Forming Pilus Retraction Enhances Enteropathogenic Escherichia Coli Infectivity”. Mol Biol Cell, 22, 14, Pp. 2436-47. doi:10.1091/mbc.E11-01-0001. Abstract
Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.
Meirav Matto, Sklan, Ella H, David, Naama , Melamed-Book, Naomi , Casanova, James E, Glenn, Jeffrey S, and Aroeti, Benjamin . 2011. “Role For Adp Ribosylation Factor 1 In The Regulation Of Hepatitis C Virus Replication”. J Virol, 85, 2, Pp. 946-56. doi:10.1128/JVI.00753-10. Abstract
We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.
2010
Victor Yashunsky, Lirtsman, Vladislav , Golosovsky, Michael , Davidov, Dan , and Aroeti, Benjamin . 2010. “Real-Time Monitoring Of Epithelial Cell-Cell And Cell-Substrate Interactions By Infrared Surface Plasmon Spectroscopy”. Biophys J, 99, 12, Pp. 4028-36. doi:10.1016/j.bpj.2010.10.017. Abstract
The development of novel technologies capable of monitoring the dynamics of cell-cell and cell-substrate interactions in real time and a label-free manner is vital for gaining deeper insights into these most fundamental cellular processes. However, the label-free technologies available today provide only limited information on these processes. Here, we report a new (to our knowledge) infrared surface plasmon resonance (SPR)-based methodology that can resolve distinct phases of cell-cell and cell-substrate adhesion of polarized Madin Darby canine kidney epithelial cells. Due to the extended penetration depth of the infrared SP wave, the dynamics of cell adhesion can be detected with high accuracy and high temporal resolution. Analysis of the temporal variation of the SPR reflectivity spectrum revealed the existence of multiple phases in epithelial cell adhesion: initial contact of the cells with the substrate (cell deposition), cell spreading, formation of intercellular contacts, and subsequent generation of cell clusters. The final formation of a continuous cell monolayer could also be sensed. The SPR measurements were validated by optical microscopy imaging. However, in contrast to the SPR method, the optical analyses were laborious and less quantitative, and hence provided only limited information on the dynamics and phases of cell adhesion.
Michal Simovitch, Sason, Hagit , Cohen, Shulamit , Zahavi, Eitan Erez , Melamed-Book, Naomi , Weiss, Aryeh , Aroeti, Benjamin , and Rosenshine, Ilan . 2010. “Espm Inhibits Pedestal Formation By Enterohaemorrhagic Escherichia Coli And Enteropathogenic E. Coli And Disrupts The Architecture Of A Polarized Epithelial Monolayer”. Cell Microbiol, 12, 4, Pp. 489-505. doi:10.1111/j.1462-5822.2009.01410.x. Abstract
Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and 'bulging out' morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.
2009
Hagit Sason, Milgrom, Michal , Weiss, Aryeh M, Melamed-Book, Naomi , Balla, Tamas , Grinstein, Sergio , Backert, Steffen , Rosenshine, Ilan , and Aroeti, Benjamin . 2009. “Enteropathogenic Escherichia Coli Subverts Phosphatidylinositol 4,5-Bisphosphate And Phosphatidylinositol 3,4,5-Trisphosphate Upon Epithelial Cell Infection”. Mol Biol Cell, 20, 1, Pp. 544-55. doi:10.1091/mbc.E08-05-0516. Abstract
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection. However, the role of PI(4,5)P(2) and PI(3,4,5)P(3) in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P(2) accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P(2) depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P(2) contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P(3) clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P(3) beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.
Victor Yashunsky, Shimron, Simcha , Lirtsman, Vladislav , Weiss, Aryeh M, Melamed-Book, Naomi , Golosovsky, Michael , Davidov, Dan , and Aroeti, Benjamin . 2009. “Real-Time Monitoring Of Transferrin-Induced Endocytic Vesicle Formation By Mid-Infrared Surface Plasmon Resonance”. Biophys J, 97, 4, Pp. 1003-12. doi:10.1016/j.bpj.2009.05.052. Abstract
We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.
2007
Julieta Leyt, Melamed-Book, Naomi , Vaerman, Jean-Pierre , Cohen, Shulamit , Weiss, Aryeh M, and Aroeti, Benjamin . 2007. “Cholesterol-Sensitive Modulation Of Transcytosis”. Mol Biol Cell, 18, 6, Pp. 2057-71. doi:10.1091/mbc.e06-08-0735. Abstract
Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-beta-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia.
Ella H Sklan, Staschke, Kirk , Oakes, Tina M, Elazar, Menashe , Winters, Mark , Aroeti, Benjamin , Danieli, Tsafi , and Glenn, Jeffrey S. 2007. “A Rab-Gap Tbc Domain Protein Binds Hepatitis C Virus Ns5A And Mediates Viral Replication”. J Virol, 81, 20, Pp. 11096-105. doi:10.1128/JVI.01249-07. Abstract
Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population.

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