Mutations in the Y. pseudotuberculosis TTSS needle protein, YscF, that specifically abrogate effector translocation into host cells

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111

^* To whom correspondence should be addressed. Email: joan.mecsas{at}tufts.edu.

	Abstract

Trafficking of effectors, termed Yops, from Yersinia into host cells is a multi-step process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, needle and translocon, which work together to ensure polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein, YscF, that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions of YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores, but showed non-polar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for assembly of the translocon and/or productive interactions with the translocon have allowed us to initiate mapping of the needle-translocon interface.