Enterohemorrhagic E. coli (EHEC) serotype O157:H7, an important agent of diarrheal disease, triggers the formation of filamentous actin pedestals on intestinal epithelial cells beneath sites of bacterial attachment. The ability to generate actin pedestals promotes late stage intestinal colonization and permits the formation of large aggregates on the epithelial surface. To generate pedestals, EHEC injects two effectors, Tir and EspFU, into mammalian cells via a type III secretion system. Tir is inserted into the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. The C-terminal cytoplasmic domain of Tir is recognized by IRTKS (Insulin Receptor Tyrosine Kinase Substrate), a mammalian adaptor/effector that promotes the formation of F-actin and protrusive membrane structures at the plasma membrane. IRTKS also binds PI(4,5)P2 and deforms membranes, and binds the GTPase Rac, which is also known to stimulate actin assembly. Importantly, a C-terminal IRTKS SH3 domain binds to EspFU,, potentially linking it to Tir. EspFU contains multiple 47-residue proline-rich repeats and activates the actin nucleation promoting factor (NPF) N- WASP by mimicking and displacing an autoinhibitory N-WASP peptide. N-WASP is required for efficient translocation of Tir and EspFU, but if this block is overcome, EspFU can trigger an N-WASP-independent pathway for actin assembly, presumably by interacting with an alternative mammalian actin NPF. These findings suggest a model in which host actin assembly initially promotes translocation of Tir and EspFU, both of which bind IRTKS to assemble a complex at the plasma membrane, clustered by interaction with bacterial intimin, that potently stimulates two pathways of actin assembly. Tir/EspFU-mediated actin assembly may in turn promote more efficient type III translocation, and, by unknown means, epithelial colonization in vivo. IRTKS may play a role in pedestal formation in addition to linking Tir to EspFU, since pilot experiments suggest that the IRTKS binding sequence of EspFU enhances pedestal formation even when EspFU is directly fused to Tir. The following aims will be pursued to investigate both N-WASP- dependent and -independent mechanisms of actin pedestal formation, and to examine potential roles of pedestal formation during mammalian infection: (1) Determine whether Tir-, EspFU-, PI(4,5)P2-, and/or Rac-binding activity is important for IRTKS to promote actin pedestal formation;(2) Identify mutants of EspFU that are defective for the N-WASP-dependent and/or -independent pathways of pedestal formation;(3) Determine the relative importance of the N-WASP-dependent and N-WASP- independent pathways in pedestal formation on polarized intestinal epithelial cells, and (4) Investigate whether pedestal formation promotes stable bacterial attachment, disruption of tight junctions and/or translocation in vitro, and the clonal expansion of microcolonies on intestinal epithelium during infection.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in North America, Europe and Japan (79) that causes both intestinal disease and a life-threatening systemic illness (51, 52) The interaction of intestinal bacteria with the gut wall is a critical step in disease (33), and EHEC triggers the host cell to generate striking pedestal-like structures beneath bound bacteria. These pedestal promote enhanced colonization of the gut later in infection, and these studies investigate EHEC induces pedestals and how this process promotes disease.
|Garber, John J; Mallick, Emily M; Scanlon, Karen M et al. (2018) Attaching-and-Effacing Pathogens Exploit Junction Regulatory Activities of N-WASP and SNX9 to Disrupt the Intestinal Barrier. Cell Mol Gastroenterol Hepatol 5:273-288|
|Jandhyala, Dakshina M; Wong, John; Mantis, Nicholas J et al. (2016) A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response. Toxins (Basel) 8:|
|Jandhyala, Dakshina M; Ahluwalia, Amrita; Schimmel, Jennifer J et al. (2016) Activation of the Classical Mitogen-Activated Protein Kinases Is Part of the Shiga Toxin-Induced Ribotoxic Stress Response and May Contribute to Shiga Toxin-Induced Inflammation. Infect Immun 84:138-48|
|Flowers, Laurice J; Bou Ghanem, Elsa N; Leong, John M (2016) Synchronous Disease Kinetics in a Murine Model for EnterohemorrhagicE. coliInfection Using Food-Borne Inoculation. Front Cell Infect Microbiol 6:138|
|DesRochers, Teresa M; Kimmerling, Erica Palma; Jandhyala, Dakshina M et al. (2015) Effects of Shiga toxin type 2 on a bioengineered three-dimensional model of human renal tissue. Infect Immun 83:28-38|
|Battle, Scott E; Brady, Michael J; Vanaja, Sivapriya Kailasan et al. (2014) Actin pedestal formation by enterohemorrhagic Escherichia coli enhances bacterial host cell attachment and concomitant type III translocation. Infect Immun 82:3713-22|
|Mallick, Emily M; Garber, John J; Vanguri, Vijay K et al. (2014) The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment. Cell Microbiol 16:1405-24|
|Kailasan Vanaja, Sivapriya; Rathinam, Vijay A K; Atianand, Maninjay K et al. (2014) Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome. Proc Natl Acad Sci U S A 111:7765-70|
|Hartland, Elizabeth L; Leong, John M (2013) Enteropathogenic and enterohemorrhagic E. coli: ecology, pathogenesis, and evolution. Front Cell Infect Microbiol 3:15|
|Jandhyala, Dakshina M; Vanguri, Vijay; Boll, Erik J et al. (2013) Shiga toxin-producing Escherichia coli O104:H4: an emerging pathogen with enhanced virulence. Infect Dis Clin North Am 27:631-49|
Showing the most recent 10 out of 39 publications