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Cycle Inhibiting Factor, Cif

Many pathogenic bacteria of both plants and animals inject virulence factors into host cells through a type III secretion system (T3SS). These translocated proteins possess a diverse array of biochemical activities, reprogramming eukaryotic cell biochemistry to serve the requirements of the pathogen. Examples of translocated effector proteins hijacking essential host functions such as cytoskeleton assembly, vesicular transport, and the process of programmed cell death are numerous. However, until recently, less progress has been made with bacteria that also possess virulence mechanisms that target the host cell-cycle. Such cyclomodulins are a growing family of bacterial toxins and effectors that interfere with the eukaryotic cell-cycle. These effects are thought to aid in bacterial dissemination as well as suppress proliferative-dependent processes in the innate and acquired immune system.

Enteropathogenic (EPEC) and most enterohemorrhagic Escherichia coli (EHEC) possess a T3SS encoded by the locus of enterocyte effacement pathogenicity island. EPEC and EHEC share similar virulence strategies with other mucosal pathogens: colonization of the mucosal site, multiplication, evasion of host defenses, and subversion of the host cell. EPEC is the leading cause of infantile diarrhea in developing countries. EHEC causes bloody diarrhea and, if serious, hemolytic uremic syndrome with possible kidney damage and failure. The locus of enterocyte effacement-encoded T3SS translocates effector proteins that are encoded at different locations in the genome and has an essential role in infection and disease.

One substrate of the EPEC and EHEC T3SS has been shown to inhibit cell-cycle progression. The cycle inhibiting factor (Cif) is an effector protein encoded by a lambdoid prophage that is incorporated into the bacterial genome. Cif is a modular protein composed of an N-terminal secretion and translocation signal and a C-terminal domain with effector activity in the host cell. Upon translocation into the host cell, Cif induces a cytopathic effect characterized by cell-cycle arrest at the G2/M transition associated in HeLa cells with the formation of stress fibers and focal adhesion recruitment.

In order to better understand the function of Cif, we have determined its crystal structure by X-ray crystallography to a resolution of 1.7 Å. We show that Cif is a divergent member of the family of enzymes that includes cysteine proteases, transglutaminases, and acetyltransferases, identifying a putative catalytic triad consisting of cysteine, histidine, and glutamine residues. We show that these residues are essential for the ability of Cif to cause the cytopathic effect when introduced into cells and during infection with pathogenic strains of E. coli.

Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria.

 

Y. Hsu, G. Jubelin, F. Taieb, J.P. Nougayrède, E. Oswald, C.E. Stebbins. (2008) “Structure of the cyclomodulin Cif from pathogenic Escherichia coli.”  J Mol Biol. Dec 12;384(2):465-77.  PMID: 18845161 [Abstract] [pdf] [pdb]

G. Jubelin, F. Taieb, D.M. Duda, Y. Hsu, A. Samba-Louaka, R. Nobe, M. Penary, C. Watrin, J.P. Nougayrède, B.A. Schulman, C.E. Stebbins, E. Oswald. (2010) “Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways” PLoS Pathog. 2010 Sep 30;6(9). PMID: 20941356 [Abstract] [pdf]

 

Dr. Yun Hsu was the lead scientist on the Cif project. Our collaborators included the groups of Eric Oswald and Brenda Schulman.