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Identification of new bacterial effector(s) involved in the cytopathic

Certain AEEC strains inhibit host cell proliferation and trigger irreversible alterations of the cytoskeleton, characterised by the recruitment of vinculin and the assembly of actin stress fibres. This phenotype called CythoPathic Effect (CPE), is dependent on a functional type III secretion machinery but is Tir and intimin independent (Tir and intimin mutants are FAS negative but CPE positive). We used two approaches to identify the bacterial effector(s) responsible for CPE:
- Construction of a mutant strain that show an enhanced secretion of all protein secreted through the type III secretion system and characterisation of new secretion products.

- Development of random mutagenesis and screening for failure to induce CPE.

Major finding during the project:

We have identified a new TTSS translocated effector molecule responsible of the CPE that we called Cif (Cycle inhibiting factor), which blocks cell cycle G2/M transition, and induce the formation of stress fibres through the recruitment of focal adhesions. Cif is the first effector not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface but is defective in the ability to block mitosis. When expressed in TTSS competent LEE positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins, which subvert the host eukaryotic cell cycle.

The use of mutant with enhanced secretion was less efficient than the detection of CPE effectors by random mutagenesis. Therefore, we developped a new strategy to analyze and identify new TTSS effectors using an originalreporter system based on a translational fusion of the effector proteins with mature TEM-1 b-lactamase.

Translocation was detected directly in living host cells by using the fluorescent b-lactamase substrate CCF2/AM. The TEM-1 system is not based on a cellular process but on the differential entry of the TEM-1 substrate in bacterial and eukaryotic cells and the use of the CCF2/AM fluorescent substrate enables the translocation analysis in living cells, without disruption of the host cell.

In addition, this new reporter can be fused to the end of certain effectors without affecting their activity, which means that double activity tests can be carried out with efficiently translocated proteins, making the data more reliable. Because fewer than 100 molecules of TEM-1 can be readily detected within a cell, the system was sensitive enough to detect translocation of a weakly produced chromosomal-encoded Cif-TEM fusion.

In the context of a growing importance of TTSS in bacterial pathogenicity, TEM-1 fusions used in combination with CCF2/AM fluorescent substrate is a new powerful tool for identifying undiscovered bacterial encoded molecules that are delivered into host cells.

Related information

Reported by

INRA-ENVT Microbiologie
23 chemin des Capelles
31000 Toulouse
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