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Toronto, Canada

Fattouh R.,Cell Biology Program | Czuczman M.A.,Cell Biology Program | Copeland J.W.,University of Ottawa | Pelletier L.,Samuel Lunenfeld Research Institute | And 8 more authors.
Journal of Infectious Diseases

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen whose virulence depends on its ability to spread from cell to cell within an infected host. Although the actin-related protein 2/3 (Arp2/3) complex is necessary and sufficient for Listeria actin tail assembly, previous studies suggest that other actin polymerization factors, such as formins, may participate in protrusion formation. Here, we show that Arp2/3 localized to only a minor portion of the protrusion. Moreover, treatment of L. monocytogenes-infected HeLa cells with a formin FH2-domain inhibitor significantly reduced protrusion length. In addition, the Diaphanous-related formins 1-3 (mDia1-3) localized to protrusions, and knockdown of mDia1, mDia2, and mDia3 substantially decreased cell-to-cell spread of L. monocytogenes. Rho GTPases are known to be involved in formin activation. Our studies also show that knockdown of several Rho family members significantly influenced bacterial cell-to-cell spread. Collectively, these findings identify a Rho GTPase-formin network that is critically involved in the cell-to-cell spread of L. monocytogenes. © 2014 The Author. All rights reserved. Source

Truong D.,Cell Biology Program | Truong D.,University of Toronto | Copeland J.W.,University of Ottawa | Brumell J.H.,Cell Biology Program | And 2 more authors.

The host actin nucleation machinery is subverted by many bacterial pathogens to facilitate their entry, motility, replication, and survival. The majority of research conducted in the past primarily focused on exploitation of a host actin nucleator, the Arp2/3 complex, by bacterial pathogens. Recently, new studies have begun to explore the role of formins, another family of host actin nucleators, in bacterial pathogenesis. This review provides an overview of recent advances in the study of the exploitation of the Arp2/3 complex and formins by bacterial pathogens. Secreted bacterial effector proteins seem to manipulate the regulation of these actin nucleators or functionally mimic them to drive bacterial entry, motility and survival within host cells. An enhanced understanding of how formins are exploited will provide us with greater insight into how a fundamental eurkaryotic cellular process is utilized by bacteria and will also advance our knowledge of host-pathogen interactions. © 2014 WILEY Periodicals, Inc. Source

Roppenser B.,Cell Biology Program | Kwon H.,University of Toronto | Canadien V.,Cell Biology Program | Xu R.,Johns Hopkins Hospital | And 6 more authors.

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host. © 2013 Roppenser et al. Source

D'Costa V.M.,Hospital for Sick Children | Braun V.,Hospital for Sick Children | Landekic M.,University of Toronto | Shi R.,Laval University | And 9 more authors.
Cell Reports

Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target. © 2015 The Authors. Source

Fattouh R.,Cell Biology Program | Guo C.-H.,Cell Biology Program | Lam G.Y.,Cell Biology Program | Gareau M.G.,Cell Biology Program | And 8 more authors.

Recent genetic-based studies have implicated a number of immune-related genes in the pathogenesis of inflammatory bowel disease (IBD). Our recent genetic studies showed that RAC2 is associated with human IBD; however, its role in disease pathogenesis is unclear. Given Rac2's importance in various fundamental immune cell processes, we investigated whether a defect in Rac2 may impair host immune responses in the intestine and promote disease in the context of an infection-based (Citrobacter rodentium) model of colitis. In response to infection, Rac2-/- mice showed i) worsened clinical symptoms (days 13-18), ii) increased crypt hyperplasia at days 11 and 22 (a time when crypt hyperplasia was largely resolved in wild-type mice; WT), and iii) marked mononuclear cell infiltration characterized by higher numbers of T (CD3+) cells (day 22), compared to WT-infected mice. Moreover, splenocytes harvested from infected Rac2-/- mice and stimulated in vitro with C. rodentium lysate produced considerably higher levels of interferon-γ and interleukin-17A. The augmented responses observed in Rac2-/- mice did not appear to stem from Rac2's role in NADPH oxidase-driven reactive oxygen species production as no differences in crypt hyperplasia, nor inflammation, were observed in infected NOX2-/- mice compared to WT. Collectively, our findings demonstrate that Rac2-/- mice develop more severe disease when subjected to a C. rodentium-induced model of infectious colitis, and suggest that impaired Rac2 function may promote the development of IBD in humans. © 2013 Fattouh et al. Source

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