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

Jubelin G, Chavez CV, Taieb F, Banfield MJ, Samba-Louaka A, Nobe R, Nougayrède JP, Zumbihl R, Givaudan A, Escoubas JM, Oswald E - PLoS ONE (2009)

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.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.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1225, Toulouse, France.

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.

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CifBp is injected by the EPEC T3SS and induces cell cycle arrest and stress fibre formation in HeLa cells.(A) Translocation of CifEc-TEM, CifBp-TEM, CifPl-TEM, CifPa-TEM and CifYp-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 [9]. 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 CifEc or CifBp, 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.
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pone-0004855-g004: CifBp is injected by the EPEC T3SS and induces cell cycle arrest and stress fibre formation in HeLa cells.(A) Translocation of CifEc-TEM, CifBp-TEM, CifPl-TEM, CifPa-TEM and CifYp-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 [9]. 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 CifEc or CifBp, 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.

Mentions: An EPEC strain deleted for its chromosomal cifEc gene (E22Δcif) has previously been described [3]. To test whether the Cif-like proteins are functional homologs of CifEc, the E22Δcif strain was complemented with a plasmid encoding each of the cifEc-like genes, and these bacteria were used to infect cultured HeLa cells. Since the whole amino acid sequence of the putative protein derived from the marine metagenome is not available, this truncated protein was not included in these assays. Before phenotypic characterization of cells infected with EPEC producing the Cif homologs, the translocation efficiency of the proteins by the EPEC T3SS was monitored using the TEM/CCF2 assay [9]. As expected, the CifEc-TEM fusion protein was properly translocated, as demonstrated by detection of intracellular β-lactamase activity (Fig. 4A). TEM activity was also detected in cells infected with E22Δcif producing CifBp-TEM, but levels of β-lactamase activity for CifPl-TEM, CifPa-TEM and CifYp-TEM were similar to the basal level detected with the negative control (TEM alone, Fig. 4A). Since TEM fusion proteins were produced to similar levels in the bacteria (Fig. 4A), absence of intracellular TEM activity likely results from inefficient recognition and/or injection of CifPl-TEM, CifPa-TEM and CifYp-TEM by the T3SS of EPEC. The lower translocation level of CifBp-TEM compared to CifEc-TEM probably also reflects a poor recognition of the secretion/translocation signal (STS) of CifBp by the T3SS from EPEC. Indeed, when this fusion protein was expressed in an escN mutant (T3SS ATPase defective mutant), β-lactamase activity was no longer detected in infected cells, confirming that translocation of CifBp-TEM by E22 strain is T3SS-dependent (data not shown).


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

Jubelin G, Chavez CV, Taieb F, Banfield MJ, Samba-Louaka A, Nobe R, Nougayrède JP, Zumbihl R, Givaudan A, Escoubas JM, Oswald E - PLoS ONE (2009)

CifBp is injected by the EPEC T3SS and induces cell cycle arrest and stress fibre formation in HeLa cells.(A) Translocation of CifEc-TEM, CifBp-TEM, CifPl-TEM, CifPa-TEM and CifYp-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 [9]. 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 CifEc or CifBp, 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654923&req=5

pone-0004855-g004: CifBp is injected by the EPEC T3SS and induces cell cycle arrest and stress fibre formation in HeLa cells.(A) Translocation of CifEc-TEM, CifBp-TEM, CifPl-TEM, CifPa-TEM and CifYp-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 [9]. 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 CifEc or CifBp, 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.
Mentions: An EPEC strain deleted for its chromosomal cifEc gene (E22Δcif) has previously been described [3]. To test whether the Cif-like proteins are functional homologs of CifEc, the E22Δcif strain was complemented with a plasmid encoding each of the cifEc-like genes, and these bacteria were used to infect cultured HeLa cells. Since the whole amino acid sequence of the putative protein derived from the marine metagenome is not available, this truncated protein was not included in these assays. Before phenotypic characterization of cells infected with EPEC producing the Cif homologs, the translocation efficiency of the proteins by the EPEC T3SS was monitored using the TEM/CCF2 assay [9]. As expected, the CifEc-TEM fusion protein was properly translocated, as demonstrated by detection of intracellular β-lactamase activity (Fig. 4A). TEM activity was also detected in cells infected with E22Δcif producing CifBp-TEM, but levels of β-lactamase activity for CifPl-TEM, CifPa-TEM and CifYp-TEM were similar to the basal level detected with the negative control (TEM alone, Fig. 4A). Since TEM fusion proteins were produced to similar levels in the bacteria (Fig. 4A), absence of intracellular TEM activity likely results from inefficient recognition and/or injection of CifPl-TEM, CifPa-TEM and CifYp-TEM by the T3SS of EPEC. The lower translocation level of CifBp-TEM compared to CifEc-TEM probably also reflects a poor recognition of the secretion/translocation signal (STS) of CifBp by the T3SS from EPEC. Indeed, when this fusion protein was expressed in an escN mutant (T3SS ATPase defective mutant), β-lactamase activity was no longer detected in infected cells, confirming that translocation of CifBp-TEM by E22 strain is T3SS-dependent (data not shown).

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.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.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1225, Toulouse, France.

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.

Show MeSH
Related in: MedlinePlus