A dominant-negative needle mutant blocks type III secretion of early but not late substrates in Yersinia

Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to deliver effectors into host cells. A key component of the T3SS is the needle, which is a hollow tube on the bacterial surface through which effectors are secreted, composed of the YscF protein. To study needle assembly, we performed a screen for dominant-negative yscF alleles that prevented effector secretion in the presence of wild-type (WT) YscF. One allele, yscF-L54V, prevents WT YscF secretion and needle assembly, although purified YscF-L54V polymerizes in vitro. YscF-L54V binds to its chaperones YscE and YscG, and the YscF-L54V-EG complex targets to the T3SS ATPase, YscN. We propose that YscF-L54V stalls at a binding site in the needle assembly pathway following its release from the chaperones, which blocks the secretion of WT YscF and other early substrates required for building a needle. Interestingly, YscF-L54V does not affect the activity of pre-assembled actively secreting machines, indicating that a factor and/or binding site required for YscF secretion is absent from T3SS machines already engaged in effector secretion. Thus, substrate switching may involve the removal of an early substrate-specific binding site as a mechanism to exclude early substrates from Yop-secreting machines.