Type III secretion signals for YplA, a secreted phospholipase of Yersinia enterocolitica

Many Gram-negative bacteria that interact with eukaryotic hosts utilize type III secretion (TTS) systems to deliver bacterial derived proteins into targeted host cells. The human pathogen Y. enterocolitica utilizes three TTS systems for pathogenesis: the Ysa and Ysc contact-dependant TTS systems and the contact-independent flagellar TTS system. The phospholipase YplA is a virulence factor exported by the flagellar TTS system. In addition, when ectopically expressed, YplA is secreted by each of the Ysc and Ysa TTS systems. Based on this intriguing property, this study utilizes YplA as a model TTS substrate to understand how proteins are recognized and exported by distinct TTS systems. Through utilization of chimeras containing various amino-terminal regions of YplA fused to the carboxy-terminal activity domain of PhoA from E. coli, a secretion signal for YplA was mapped. The analysis revealed that the first 20 amino acids of YplA were sufficient for export of YplA-PhoA chimeras by each TTS system. In addition, export of native YplA by each TTS system was sensitive to alterations that removed or changed the primary structure of the amino-terminal 20 residues, but alteration of other regions was tolerated. I also provide evidence that YplB is a TTS chaperone for YplA. I show that YplA export is abolished by a chromosomal insertion mutation in yplB. Similarly, when YplA is constitutely expressed, the addition of I in cis or in trans can enhance YplA export. YplB enhancement of YplA export occurs only when the amino-terminal secretion signal is present. Intriguingly, the degree by which YplA export is enhanced by yplB differs in each of the three TTS systems of Y. enterocolitica. Through yeast two-hybrid analysis and affinity blotting, I show that YplA and YplB can specifically interact. Further, this interaction appears to occur at the carboxy-terminus of YplA. These data indicate that YplB plays a role in YplA secretion and is likely a TTS chaperone. Overall, this work supports a model in which TTS substrates contain an amino-terminal secretion signal that is recognized by distinct TTS systems. Export is then enhanced through the use of an additional protein, the TTS chaperone.