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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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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.
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pone-0004855-g007: 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.

Mentions: Most of the conserved residues in Cif proteins are clustered in discrete regions (Fig. 3). The cysteine, histidine and glutamine residues forming the catalytic triad in CifEc were shown to be critical for activity [10]. To determine whether an equivalent functional catalytic site exists in the Cif homologs, the conserved cysteines in CifBp, CifPl and CifPa (C90, C128 and C123 respectively) were substituted with a serine residue, and the corresponding proteins were purified prior to delivery into HeLa cells using the BioPORTER system. In contrast to the wild-type proteins, the cysteine variants did not induce cell enlargement and stress fibre formation (Fig. 7A). Further, analysis of DNA content revealed that accumulation of G2-arrested cells did not occur when cells were treated with the cysteine variants (Fig. 7B). Expression of the cysteine variant from CifYp by transfection in HeLa cells also revealed that the cell cycle was not arrested in contrast to cells producing the wild-type protein (Fig. 6). These results demonstrate that the conserved cysteine residue is critical for Cif activity. Also, as the histidine and glutamine residues that complete the triad are also conserved in the sequences of the Cif homologs, this suggests that catalytic triads also exist in CifBp, CifPl, CifPa and CifYp.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654923&req=5

pone-0004855-g007: 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.
Mentions: Most of the conserved residues in Cif proteins are clustered in discrete regions (Fig. 3). The cysteine, histidine and glutamine residues forming the catalytic triad in CifEc were shown to be critical for activity [10]. To determine whether an equivalent functional catalytic site exists in the Cif homologs, the conserved cysteines in CifBp, CifPl and CifPa (C90, C128 and C123 respectively) were substituted with a serine residue, and the corresponding proteins were purified prior to delivery into HeLa cells using the BioPORTER system. In contrast to the wild-type proteins, the cysteine variants did not induce cell enlargement and stress fibre formation (Fig. 7A). Further, analysis of DNA content revealed that accumulation of G2-arrested cells did not occur when cells were treated with the cysteine variants (Fig. 7B). Expression of the cysteine variant from CifYp by transfection in HeLa cells also revealed that the cell cycle was not arrested in contrast to cells producing the wild-type protein (Fig. 6). These results demonstrate that the conserved cysteine residue is critical for Cif activity. Also, as the histidine and glutamine residues that complete the triad are also conserved in the sequences of the Cif homologs, this suggests that catalytic triads also exist in CifBp, CifPl, CifPa and CifYp.

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