To ensure that the absence of needles was indeed the result of the lesion inyopRand not due to a second-site mutation, bacteria complemented with a plasmid-borne copy ofyopRwere examined for needle polymerization; in this strain, needles were once again observed (Figure 5)

To ensure that the absence of needles was indeed the result of the lesion inyopRand not due to a second-site mutation, bacteria complemented with a plasmid-borne copy ofyopRwere examined for needle polymerization; in this strain, needles were once again observed (Figure 5). == Fig. Keywords:type III secretion, needle polymerization, YopR,Yersinia == Introduction == ThreeYersiniaspecies –Yersinia enterocolitica, Yersinia pestisandYersinia pseudotuberculosis– are pathogenic to animals as well as humans (Butler, 1995) and employ a virulence plasmid-encoded type III secretion system to evade the host immune system (Corneliset al., 1998). Type III secretion machines provide for the transport of Yop effectors from the bacterial cytoplasm into the cytosol of host L-Lysine thioctate target cells (Marketonet al., 2005), thereby disabling phagocytes and other innate immune responses to bacterial infections (Rosqvistet al., 1994,Galn & Wolf-Watz, 2006). Common features of type III machines are their assembly from many different proteins in the cytoplasm, inner and outer membranes as well as the extension of the machine via a hollow needle filament (Kuboriet al., 1998,Hoiczyk & Blobel, 2001,Cornelis, 2006). Needles are formed via the polymerization of YscF and decorated with a cap-like tip comprised of LcrV (Hoiczyk & Blobel, 2001,Muelleret al., 2005). Two ring Rabbit Polyclonal to CPB2 structures, one in the bacterial plasma membrane and one in the outer membrane, are joined via a central rod that is formed from YscI, which is also thought to connect with the needle L-Lysine thioctate protein at the level of the outer YscC ring (Marlovitset al., 2004,Marlovitset al., 2006). Needle polymerization is controlled by YscP, a secreted machine component (Journetet al., 2003,Payne & Straley, 1999). WithoutyscP, Yersiniapolymerize extended needles and are unable to inject effectors into host cells (Agrainet al., 2005). LcrV, the tip protein, as well as YopB and YopD are essential for type III injection, but dispensable for polymerization of needles (Hkanssonet al., 1993,Leeet al., 2000,Muelleret al., 2008). All three proteins are thought to gate type III needles by forming a translocation structure in the plasma membrane of host cells (Mueller et al., 2008). The structural attributes of such membrane translocation complexes (injectisomes) remain however unknown. Pseudomonas aeruginosaPscF, a homolog of YscF, binds two putative chaperones, PscE and PscG, that are conserved in yersiniae (YscE and YscG) (Quinaudet al., 2005,Quinaudet al., 2007).yscH, located downstream of the genes for needle protein/chaperones (yscEFG), encodes a secreted product, named YopR (Allaouiet al., 1995).Y. enterocoliticaW22703yopRmutants are still able to catalyze type III injection of L-Lysine thioctate host cells (Lee & Schneewind, 1999). However, as investigated in a mouse model of intraperitoneal challenge,yopRmutants display a 34 fold reduction in virulence (Lee & Schneewind, 1999). YopR is secreted into the extracellular medium early during development, while needle complexes assemble, and the protein has been investigated as a model substrate of type III machines (Sorget al., 2006,Blaylocket al., 2008). The X-ray structure of YopR revealed the unexpected similarity with YopN (Schubotet al., 2005), another secreted protein that blocks recognition of effector substrates by type III machines until host cell contact has been established (Yother & Goguen, 1985,Chenget al., 2001,Torruellaset al., 2005). YscF has been proposed to bind calcium and may respond to changes in the abundance of this ion by altering the needle structure (Ferracciet al., 2005,Davis & Mecsas, 2007). Such a mechanism may trigger YopN injection into host cells and enable the subsequent transport of effectors along the conduit of type III needles (Ramamurthi & Schneewind, 2002). Regulation ofYersiniatype III L-Lysine thioctate machines can be monitored on agar plates as the low calcium response (LCR); chelation of calcium ions prevents growth of the organisms at 37C due to the massive release of effectors (Goguenet al., 1984,Michielset al., 1990) and mutations in type III.