| Type III secretion systems are virulence strategies utilized by Gram-negative bacteria to evade the host's immune system. Yersinia spp. transport ten proteins through a hollow conduit within the type III secretion machine and into the cytoplasm of the eukaryotic cell. These proteins, termed Yops, catalyze events within the host's cytosol that result in depolymerization of the actin cytoskeleton and eventual demise of the target cell. Secretion of substrates occurs in stages. The first substrates secreted by type III secretion are involved in the biogenesis of the type III machine (early substrates), followed by substrates that are involved in the translocation process (middle substrates) and effector Yops (late substrates).; Studies on late substrates, YopE and YopQ, revealed that information required for secretion and injection is encoded within the first 15 codons of yop mRNA. Specifically codon 3, isoleucyl residue, is sensitive to mutagenesis. All but two Yop proteins, YopN and YopH, carry isoleucines within the first 15 codons. Therefore recognition of YopN may occur by a mechanism that is different than YopE and YopQ. Here we describe the secretion signal of YopN. We find that like YopE and YopQ, information required for secretion of YopN lies within the yopN mRNA and may be unique when compared to that of yopE or yopQ.; Fusions of impassable reporter molecules such as DHFR or GST to secretion substrates typically result in jammed secretion machines. We test the hypothesis that initiation of YopE-DHFR into type III pathway results in a blockade of type III secretion and conclude that fusions of late substrates to DHFR or GST do not block type III secretion but decrease the total amount of protein synthesized. We find that fusions of early substrates, YopR and YscP, to impassable reporter molecules lead to a complete block in type III secretion. Initiation of YopR- and YscP-GST into the type III machine results in the co-purification of the type III secretion ATPase, YscN. Further, delayed expression of YopR-DHFR results in a specific block in the secretion of early substrates but not late substrates suggesting that YopR is an early substrate. |
