Cycle inhibiting factors (CIFs) are a growing family of functional cyclomodulins present in invertebrate and mammal bacterial pathogens.
Bottom Line: 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.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.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.
Affiliation: INRA, UMR1225, Toulouse, France.
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 G(1) and G(2) cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21(waf1/cip1) and p27(kip1) 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 beta-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.
Related in: MedlinePlus
Mentions: As the EPEC T3SS was not able to translocate CifPl, CifPa and CifYp into infected cells, the cytopathic activity of these CifEc-like proteins was investigated using purified recombinant samples combined with a lipid mediated delivery system (BioPORTER) as previously described . The effects of CifBp delivered with this system were also investigated. CifBp, CifPl and CifPa were all readily overexpressed and purified in a soluble form (see Materials and Methods). However, despite many efforts, it was not possible to obtain a purified soluble form of CifYp at levels necessary for activity assays using the BioPORTER delivery system. As previously reported , treatment of HeLa cells with BioPORTER mixed with purified CifEc leads to cell enlargement and formation of actin stress fibres (Fig. 5), identical to the phenotype observed with the infection model. However, as protein delivery with BioPORTER is not as efficient as bacterial infection , only ∼50% of the treated cells exhibit morphological alterations (not shown). Studies of cell cycle patterns were therefore realized using G1/S synchronized cells to improve visualization of G2 arrest. In contrast to cells incubated with the lipid delivery agent mixed with PBS alone, cells treated with BioPORTER+CifEc accumulated in G2 phase (38% of CifEc-treated cells contained 4N DNA-content against 10% for PBS-treated cells). Lipofection of purified CifBp into HeLa cells led to actin stress fibres and cell accumulation in G2 phase (27%) (Fig. 5), confirming the functionality of CifBp observed with the infection assays. Introduction of purified CifPl or CifPa into HeLa cells with BioPORTER also led to cell enlargement, cytoskeleton alteration and accumulation of cells with 4N DNA content (40 and 25% for CifPl and CifPa respectively versus 10% for PBS treated cells, see Fig. 5). Therefore, CifPl and CifPa are also functional homologs of CifEc. As these phenotypes are observed with purified proteins, the results demonstrate that the proteins alone are sufficient to induce the Cif-associated cytopathic effects.