Research

 

The Disease: Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are human pathogens that infect the epithelial cells of the small and large intestines, respectively, causing severe intestinal diseases. EPEC causes gastroenteritis in infants, while EHEC can cause bloody diarrhea and kidney failure in children and the elderly. Diarrhea remains the second leading cause of death in children younger than five years old, globally, accounting for ~1.3 million deaths every year (1). Moreover, E. coli infections have been linked to inflammatory bowel diseases, such as Crohn's disease (2). Exploration of the mechanisms by which pathogenic E. coli interact with their enteric host cells in the human body may pave the way to the development of novel therapeutic and diagnostic strategies to combat a wide range of severe human illnesses. Such developments are of particular importance in a world that rapidly moves to a post-antibiotic era, where bacteria that cause common human infections become resistant to antibiotic treatments. 
 

The Domain: We hypothesize that upon contacting the apical plasma membrane, EPEC and EHEC generate a unique membrane domain, reminiscent of a lipid raft domain, at their attachment site to the plasma membrane. This bacteria-made membrane domain is enriched with distinct lipids and proteins that transmit harmful signals to the host cells. However, the host cell may respond by launching counteracting signals, such as innate immune danger signals, aimed at exterminating the domain and the pathogen. Therefore, studying the biochemical composition of this ‘pathogenic module’ is crucial for understanding the EPEC and EHEC diseases. 

 

Open Questions:
•    How do bacteria generate and modulate the membrane domain?
•    What is the biochemical composition of the domain?
•    Which host signaling pathways are hijacked by the domain, and how do they affect the pathogen and the host?

Our Contribution: PMID: 18987340; PMID: 30037792; PMID: 31242273

The Injection: Upon contacting the apical plasma membrane of intestinal epithelial cells, a specialized syringe-like structure called the type III secretion system (T3SS) is formed. This syringe-like machinery enforces the injection of  ~21 different bacterial proteins, also called effector proteins, from the bacterial cytoplasm directly into the mammalian host cell. The coordinated action of these secreted effectors in space and time is thought to play a pivotal role in inducing the EPEC/EHEC infectivity and disease (3). The main changes induced by the T3SS include: i) the loss of absorptive microvilli and the production of filamentous actin-rich structures (pedestals) underneath the adherent bacterium; ii) inhibition of nutrient/water transporter localization and functions; iii)  remodeling of membrane trafficking; iv) targeting mitochondrial functions; v) weakening inflammatory responses; vi) disruption of epithelial barrier functions. However, the precise mode by which these effectors act on their host targets is largely unclear. 

 

Open Questions:
•    Which host proteins and lipids are targeted by the effectors, and by which mechanims?
•    How do the translocated effectors affect the host cell physiology and shape? 
•    How do the effector proteins contribute to bacterial infection and survival?

Our Contribution:  PMID: 18987340; PMID: 21613538; PMID: 22572833; PMID: 24194932; PMID: 30037792; PMID: 31242273

The Pedestal: Actin-rich pedestals are raised structures formed by actin polymerization of the host. The effector protein that plays a central role in pedestal formation by EPEC and EHEC is the 'translocated intimin receptor' (Tir) (4-6). Tir adopts a loop-like structure when inserted into the host cell plasma membrane, with the N and C termini of the protein facing the host cell cytoplasm. Following translocation, a bacterial adhesion protein, intimin, binds the loop of Tir that projects to the extracellular space, anchoring the bacterium firmly onto the apical surface of the host cell. The Tir-intimin interactions cause actin polymerization through different signaling mechanisms in EPEC and EHEC. In EPEC, the tyrosine residue at position 474 (Y474) located in the C-terminus of Tir is phosphorylated by the host kinases Fyn and Abl, resulting in the recruitment of the host adaptor protein NcK that binds directly to Tir. NcK then recruits and activates the host cell actin-promoting nucleating factor N-WASP, which in turn recruits and activates the Arp2/3 actin polymerizing complex (7).
In EHEC, the Tir tyrosine phosphorylation step is circumvented by the EHEC effector EspFu. EspFu interacts with IRTKS and IRSp53, two remodeling proteins in the host plasma membrane. Signaling is then initiated from the Asn-Pro-Tyr motif at the C-terminus of Tir, which recruits N-WASP and Arp2/3, leading to actin polymerization. 
Apart from actin and actin-binding proteins, host cell adhesion proteins (e.g., vinculin, talin, alpha-actinin) are also localized at the actin-rich pedestals, raising the hypothesis that the pedestal is similar to adhesion plaques, like focal adhesions, podosomes or filopodia in mammals. Even more surprising, multiple components of the clathrin-mediated endocytic pathway have been found at actin pedestals (8-10). Nonetheless, despite extensive research, neither the precise mechanisms involved in pedestal biogenesis nor the functional significance of pedestals in the bacterium life is fully understood.  One possibility is that the actin-rich pedestal is generated as an anti-phagocytic mechanism that positions the microbe mostly extracellularly.  Another option is that the actin-rich pedestal functions as an adhesive platform that modulates bacterial adherence and dissemination during infection.

Open Questions:
•    How does cytoskeletal remodeling contribute to pedestal formation and shape?
•    How is Tir translocated into the host cell plasma membrane?
•    What role does the pedestal play in the microbe lifestyle?

Our Contribution: PMID:18987340; PMID:19912240; PMID:21613538; PMID:22572833

References:
1.    Kaper JB, Nataro JP, Mobley HL. 2004. Pathogenic Escherichia coli. Nat Rev Microbiol 2:123-40.
2.    Mirsepasi-Lauridsen HC, Vallance BA, Krogfelt KA, Petersen AM. 2019. Escherichia coli Pathobionts Associated with Inflammatory Bowel Disease. Clinical microbiology reviews 32.
3.    Dean P, Kenny B. 2009. The effector repertoire of enteropathogenic E. coli: ganging up on the host cell. Curr Opin Microbiol 12:101-9.
4.    Ross NT, Miller BL. 2007. Characterization of the binding surface of the translocated intimin receptor, an essential protein for EPEC and EHEC cell adhesion. Protein science : a publication of the Protein Society 16:2677-83.
5.    Luo Y, Frey EA, Pfuetzner RA, Creagh AL, Knoechel DG, Haynes CA, Finlay BB, Strynadka NC. 2000. Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex. Nature 405:1073-7.
6.    Lai Y, Rosenshine I, Leong JM, Frankel G. 2013. Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli. Cellular microbiology 15:1796-808.
7.    Phillips N, Hayward RD, Koronakis V. 2004. Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation. Nat Cell Biol 6:618-25.
8.    Alto NM, Weflen AW, Rardin MJ, Yarar D, Lazar CS, Tonikian R, Koller A, Taylor SS, Boone C, Sidhu SS, Schmid SL, Hecht GA, Dixon JE. 2007. The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways. J Cell Biol 178:1265-78.
9.    Cossart P, Veiga E. 2008. Non-classical use of clathrin during bacterial infections. J Microsc 231:524-8.
10.    Kassa EG, Zlotkin-Rivkin E, Friedman G, Ramachandran RP, Melamed-Book N, Weiss AM, Belenky M, Reichmann D, Breuer W, Pal RR, Rosenshine I, Lapierre LA, Goldenring JR, Aroeti B. 2019. Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity. PLOS Pathogens 15:e1007851.