Substrate selection and specificity in Yersinia type III secretion machines

Of the many species of Yersiniae, only three, Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis , are pathogenic to humans. They are capable of causing disease ranging in severity from mild gastroenteritis to bubonic and pneumonic plague. What unites these three pathogenic Yersinia species is the presence of a type III secretion system (TTSS) located on an extra-chromosomal virulence plasmid. When confronted with host immune defenses, Yersiniae use the TTSS to construct a needle-like protrusion on the bacterial surface. This needle punctures host immune cells allowing for the deposition of bacterial toxins directly in the host cell cytoplasm resulting in host cell death.;Construction of this apparatus, also known as the injectisome, requires the coordination of over 20 proteins, known as Yersinia secretion proteins (ysc). In this work, we have studied several different machinery genes and examined the diverse roles that they play in apparatus assembly, and substrate specificity, selection, and translocation into eukaryotic cells.;We first explored the ATPase that fuels translocation, YscN, and its negative regulator YscL. YscN is thought to consume ATP in order to unfold secretion substrates in preparation for their secretion through the narrow needle. Next, we characterized the function of YscU, an inner membrane protein. YscU undergoes auto-cleavage in its cytoplasmic domain that plays a role in substrate selection. YscU works in concert with YscP, an early secretion substrate, to coordinate a substrate specificity switch. YscP hybrids were constructed and found to block TTS in a manner dependent on the capture of YscN, the ATPase. Finally, we examined a previously uncharacterized protein, YscO. Work here suggests that YscO plays a role in recruiting YscP to the needle apparatus and ensuring that the substrate specificity switch occurs at the proper time during TTS apparatus assembly.;By studying TTS machinery proteins, we have developed a deeper understanding into the nature of TTS apparatus assembly and a greater insight into the pathogenic mechanisms of Yersinia species.