Cycle inhibiting factors (CIFs) are a growing family of functional cyclomodulins present in invertebrate and mammal bacterial pathogens

Abstract

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.

Figure 1. Phylogenetic relationship between Cif_Ec, Cif_Yp, Cif_Bp, Cif_Pl and Cif_Pa.

A multiple alignment of the protein sequences (see Fig. 3A) was used to obtain the unrooted tree with Phylip's DrawTree software.

Figure 2. Genetic organization of the cif-like genes loci from E. coli strain B171, P. luminescens strain TT01, P. asymbiotica strain ATCC43949, B. pseudomallei strains K96243, S13, 9 and 1106a and Y. pseudotuberculosis strains YPIII, AH and 9314/74.

Open reading frames (ORFs) are represented by horizontal arrows and designation of first and last ORF from each schematic are indicated. Vertical arrows indicate position of the yrs sequence (Yersinia recombination site).

Figure 3. The three residues of the Cif_Ec catalytic triad are conserved among members of the Cif protein family.

(A) ClustalW alignment between Cif_Ec, Cif_Yp, Cif_Bp, Cif_Pl, Cif_Pa and GOS_5485515. Fully conserved residues are indicated by a red background and amino acids conserved more than 60 or 80% are indicated by a yellow or an orange background respectively. The cysteine, histidine and glutamine residues that form the catalytic triad of Cif_Ec are indicated with blue stars. (B) Position of the fully conserved residues in the three dimensional structure of Cif_Ec. Side chain carbon atoms of residues comprising the catalytic triad are coloured cyan. The remaining fully conserved residues cluster in three regions, as described in the text. Residues coloured yellow, including glycine positions indicated by spheres, are P107, G110, A113, N159, L163-G164, S186-G189, G191, D200-W201; in green are D170, D172, E264-D266; in purple are K118-L119 and N273.

Figure 4. Cif_Bp is injected by the EPEC T3SS and induces cell cycle arrest and stress fibre formation in HeLa cells.

(A) Translocation of Cif_Ec-TEM, Cif_Bp-TEM, Cif_Pl-TEM, Cif_Pa-TEM and Cif_Yp-TEM fusions by the T3SS of EPEC strain E22. Hela cells were loaded with CCF2/AM substrate and were infected for 2 and a half h with E22Δcif hosting plasmids expressing TEM alone or the different Cif-TEM fusions. Upper panel: intracellular β-lactamase activity detected by measuring cleavage of the CCF2/AM, as described in Material and Methods. This ratio represents the relative translocation efficiency . Experiments were performed in triplicate and error bars represent standard errors of the mean. Lower panel: synthesis of TEM fusions proteins were quantified in bacteria just before the translocation assays by western blot with anti-TEM antibodies. (B) G₁/S synchronized HeLa cells were exposed for 90 min to E22Δcif hosting either empty vector or the plasmids expressing Cif_Ec or Cif_Bp, washed and incubated with antibiotic for 20 or 72 h. Upper panels: F-actin was labelled with phalloidin-rhodamine (red) and DNA with DAPI (blue) 72 h post-infection. Bars represent 20 µm. Lower panels: cell cycle distribution was analysed by flow cytometry 20 h post-infection. 2N and 4N populations are indicated.

Figure 5. Lipofection of purified Cif_Bp, Cif_Pl and Cif_Pa proteins into HeLa cells induce cell cycle arrest and stress fibres formation akin to Cif_Ec.

G₁/S synchronized HeLa cells were treated with purified proteins or PBS in combination with a lipidic delivery agent (BioPORTER). Upper panels: F-actin was stained with phalloidin-rhodamine (red) and DNA with DAPI (blue) 72 h post-treatment. Bars represent 20 µm. Lower panels: cell cycle distribution was analysed by flow cytometry 20 h post-treatment. Percentages of cells with 4N DNA content are indicated.

Figure 6. Transfection of Cif_Yp induces cell cycle arrest in HeLa cells.

HeLa cells were transfected with plasmid expressing GFP, GFP-Cif_Ec or GFP-Cif_Yp (wild-type (WT) or Cys variants (C/A)) fusion proteins. GFP expression and DNA content were analysed by flow cytometry 48 h post-transfection. Data are represented on two dimensional contour plot graphics with DNA content on the X-axis and GFP signal on the Y-axis. Gates corresponding to the GFP negative and GFP positive populations are indicated. Among the GFP positive populations, percentages of cells with 2N or 4N DNA content are indicated within the corresponding quadrants.

Figure 7. Cysteine residues from predicted catalytic triads are essential for Cif homologs activity.

(A) HeLa cells were treated with purified Cif homologs proteins (wild-type (WT) or cysteine variants (C/S)) in combination with a lipidic delivery agent (BioPORTER). F-actin was stained with phalloidin-rhodamine (red) and DNA with DAPI (blue) 72 h post-treatment. Bars represent 20 µm. (B) G₁/S synchronized HeLa cells were treated with PBS or purified Cif proteins (as indicated), in combination with BioPORTER. The percentages of the populations containing 4N DNA content were determined by flow cytometry 20 h post-treatment.

Figure 8. Cif homologs induce p21 and p27 accumulation in cells.

HeLa cells were treated with PBS or purified Cif proteins (wild-type (WT) or cysteine variants (C/S) as indicated), in combination with BioPORTER. Cell extracts were probed with anti-p21, anti-p27 and anti-actin antibodies 24 h post-treatment.