Frédéric Taieb

Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. 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.

Bacterial cyclomodulin Cif blocks the host cell cycle by stabilizing the cyclin-dependent kinase inhibitors p21waf1 and p27kip1

The cycle inhibiting factor (Cif) is a cyclomodulin produced by enteropathogenic and enterohemorrhagic Escherichia coli. Upon injection into the host cell by the bacterial type III secretion system, Cif inhibits the G2/M transition via sustained inhibition of the mitosis inducer CDK1 independently of the DNA damage response. In this study, we show that Cif induces not only G2, but also G1 cell cycle arrest depending on the stage of cells in the cell cycle during the infection. In various cell lines including differentiated and untransformed enterocytes, the cell cycle arrests are correlated with the accumulation of the cyclin‐dependent kinase inhibitors p21waf1/cip1 and p27kip1. Cif‐induced cyclin‐dependent kinase inhibitor accumulation is independent of the p53 pathway but occurs through inhibition of their proteasome‐mediated degradation. Our results provide a direct link between the mode of action of Cif and the host cell cycle control.

Structure of the Cyclomodulin Cif from Pathogenic Escherichia coli

Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.

Cycle Inhibiting Factors (CIFs) Are a Growing Family of Functional Cyclomodulins Present in Invertebrate and Mammal Bacterial Pathogens

The cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery. Cif provokes cytopathic effects characterized by G1 and G2 cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21waf1/cip1 and p27kip1 and formation of actin stress fibres. The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad. Here we report the discovery and characterization of four Cif homologs encoded by different pathogenic or symbiotic bacteria isolated from vertebrates or invertebrates. Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the β-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli. Although these Cif homologs are remarkably divergent in primary sequence, the catalytic triad is strictly conserved and was shown to be crucial for cell cycle arrest, cytoskeleton reorganization and CKIs accumulation. These results reveal that Cif proteins form a growing family of cyclomodulins in bacteria that interact with very distinct hosts including insects, nematodes and humans.

The Enteropathogenic Escherichia coli Effector Cif Induces Delayed Apoptosis in Epithelial Cells

ABSTRACT The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins, the cyclomodulins, which modulate the host cell cycle. Upon injection into the host cell by the type III secretion system of enteropathogenic Escherichia coli (EPEC), Cif induces both G2 and G1 cell cycle arrests. The cell cycle arrests correlate with the accumulation of p21waf1 and p27kip1 proteins that inhibit CDK-cyclin complexes, whose activation is required for G1/S and G2/M transitions. Increases of p21 and p27 levels are independent of p53 transcriptional induction and result from protein stabilization through inhibition of the ubiquitin/proteasome degradation pathway. In this study, we show that Cif not only induces cell cycle arrest but also eventually provokes a delayed cell death. Indeed, 48 h after infection with EPEC expressing Cif, cultured IEC-6 intestinal cells were positive for extracellular binding of annexin V and exhibited high levels of cleaved caspase-3 and lactate dehydrogenase release, indicating evidence of apoptosis. Cif was necessary and sufficient for inducing this late apoptosis, and the cysteine residue of the catalytic site was required for Cif activity. These results highlight a more complex role of Cif than previously thought, as a cyclomodulin but also as an apoptosis inducer.

The molecular basis of ubiquitin-like protein NEDD8 deamidation by the bacterial effector protein Cif

The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The “occluding loop” in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.

Cycle Inhibiting Factors (Cifs): Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

The Enterobacterial Genotoxins: Cytolethal Distending Toxin and Colibactin

While the DNA damage induced by ionizing radiation and by many chemical compounds and drugs is well characterized, the genotoxic insults inflicted by bacteria are only scarcely documented. However, accumulating evidence indicates that we are exposed to bacterial genotoxins. The prototypes of such bacterial genotoxins are the Cytolethal Distending Toxins (CDTs) produced by Escherichia coli and Salmonella enterica serovar Typhi. CDTs display the DNase structure fold and activity, and induce DNA strand breaks in the intoxicated host cell nuclei. E. coli and certain other Enterobacteriaceae species synthesize another genotoxin, colibactin. Colibactin is a secondary metabolite, a hybrid polyketide/nonribosomal peptide compound synthesized by a complex biosynthetic machinery. In this review, we summarize the current knowledge on CDT and colibactin produced by E. coli and/or Salmonella Typhi. We describe their prevalence, genetic determinants, modes of action, and impact in infectious diseases or gut colonization, and discuss the possible involvement of these genotoxigenic bacteria in cancer.

Enterohaemorrhagic Escherichia coli serogroup O111 inhibits NF-kB-dependent innate responses in a manner independent of a type III secreted OspG orthologue

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) inject a repertoire of effector proteins into host cells via a type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). OspG is an effector protein initially identified in Shigella that was shown to inhibit the host innate immune response. In this study, we found ospG homologues in EHEC (mainly of serogroup O111) and in Yersinia enterocolitica. The T3SS encoded by the LEE was able to inject these different OspG homologues into host cells. Infection of HeLa cells with EHEC O111 inhibited the NF-kappaB-dependent innate immune response via a T3SS-dependent mechanism. However, an EHEC O111 ospG mutant was still able to inhibit NF-kappaB p65 transfer to the nucleus in infected cells stimulated by tumour necrosis factor alpha (TNF-alpha). In addition, no difference in the inflammatory response was observed between wild-type EHEC O111 and the isogenic ospG mutant in the rabbit ligated intestinal loop model. These results suggest that OspG is not the sole effector protein involved in the inactivation of the host innate immune system during EHEC O111 infection.

The cyclomodulin Cif of Photorhabdus luminescens inhibits insect cell proliferation and triggers host cell death by apoptosis

Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G(2)/M phase transition in human cell lines. We report here the first direct functional analysis of Cif(Pl), from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cif(Pl) gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cif(Pl) was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. Cif(Pl) inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G(2)/M phase transition. It also triggered host cell death by apoptosis. The integrity of the Cif(Pl) catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.