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J. Bacteriol. doi:10.1128/JB.01602-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Regulation of expression and secretion of NleH, a new non-LEE-encoded effector in Citrobacter rodentium

Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada and Department of Microbiology and Immunology/Medicine, Dalhousie University, Halifax, Nova Scotia, Canada

^* To whom correspondence should be addressed. Email: puente{at}ibt.unam.mx.

	Abstract

Together with enterohemorrhagic (EHEC) and enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium is a member of the "attaching and effacing" (A/E) family of bacterial pathogens. A/E pathogens use a type III secretion system (T3SS) to translocate an assortment of effector proteins, encoded both within and outside the locus of enterocyte effacement (LEE), into the colonized host cell leading to the formation of A/E lesions and disease. Here we report the identification and characterization of a new non-LEE encoded effector NleH in C. rodentium. NleH is conserved among A/E pathogens and shares identity with OspG, a type III secreted effector protein in Shigella flexneri. Downstream of nleH, genes encoding homologues of the non-LEE-encoded effectors EspJ and NleG/NleI are found. NleH secretion and translocation into Caco-2 cells requires a functional T3SS and signals located at its amino terminal domain. Transcription of nleH is not significantly reduced in mutants lacking the LEE-encoded regulators Ler and GrlA; however, NleH protein levels are highly reduced in these strains, as well as in escN and cesT mutants. Inactivation of Lon, but not of ClpP, protease restores NleH levels even in the absence of CesT. Our results indicate that the efficient engagement of NleH in active secretion is needed for its stability, thus establishing a post-translational regulatory mechanism that co-regulates NleH levels with the expression of LEE-encoded proteins. A C. rodentium nleH mutant shows a moderate defect during the colonization of C57BL/6 mice at early stages of infection.