Yops and flagellar proteins
The T3SS of the Yersinia proteins causing plague in humans is related to the assembly pathway of bacterial flagellum. T3SS is found in pathogenic and symbiotic bacteria that infect both animal and plant cells.
The T3SS substrates, also called effector proteins, are exported across three membranes: the inner and outer membrane of bacteria and the plasma membrane of the eukaryotic target cell. The T3SS substrate proteins are synthesized with N-terminal uncleaved signals and require export-specific chaperones to prevent tight folding (He et al., 2004). The substrates are exported out of the cell in a 1-step process. The secretion system involves more than 25 proteins that are highly conserved among the pathogenic bacterial species and several show sequence similarities to flagellar assembly genes. The secretion system is usually activated by a prior contact of the bacteria with the host cell (Pettersson et al., 1996).
Yops (for Yersinia outer membrane proteins) are arguably the best-studied T3SS-exported proteins. Indeed, the T3SS was discovered in Yersinia pestis where Yops were shown to be exported from the cytoplasm of the Gram-negative bacteria directly into the cytoplasm of the eukaryotic host rather than into the extracellular medium (Rosqvist et al., 1994; Sory & Cornelis, 1994; Cornelis, 2006). Secretion of Yops requires amino-terminal signals that function as binding sites for the cytoplasmic chaperone SycD (Buttner et al., 2008). The substrate is then believed to move through the export apparatus energized by the cytoplasmic YscN ATPase (Fig. 7) (He et al., 2004). The secretion device used in T3SS is an injectisome that functions as a molecular syringe to inject Yops (or other T3SS substrates) into the eukaryotic host cell (Fig. 7) (Kubori et al., 1998). The injectisome has a basal structure characterized with ring structures in the inner membrane (C ring), in the periplasm, and in the outer membrane. A needle-like extension protrudes from the outer ring.
Figure 7. The flagellum and an archetypal Type 3 secretion system. To assemble the flagellar filament, the flagellin FliC protein engages the flagellar secretion complex and is secreted through the inner cavity of the flagellum to the tip where it assembles (left). The system is related to the Type3SS (shown on the right) that is involved in effector protein export of pathogenic bacteria.
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The T3SS substrates that are transported from the cytoplasm move through the basal body and directly through the needle of the injectisome. The double ring in the inner membrane is formed by the YscJ protein (Yip et al., 2005) where Ysc stands for Yop secretion protein component. In addition, YscR,S,T,U,V and LcrD reside in the inner membrane and join the C ring together with the soluble protein YscQ and the YscN ATPase. In the outer membrane, the secretin YscC forms a double ring structure of the injectisome that forms the channel in the outer membrane in which T3SS substrates are exported across (Fig. 7) (Koster et al., 1997). Recently, ring models of the secretin were constructed and placed within the injectisome (Spreter et al., 2009).
The first secreted proteins (YscC, F, O, P and YscX; not to be confused with the T3SS Yops) make up the external part of the injectisome. For example, YscF is the major protein forming the needle. After the needle reaches a specific length, the export system secretes the T3SS YopB and YopD proteins to localize them to the host membrane. They are required for translocation of the T3SS effector Yops into the host cell and most likely the secreted proteins move through the central channel of’the needle, in a manner similar to that proposed for the flagellins (Fig. 7). The effector Yops are translocated into the eukaryotic cell via a channel formed by YopB and YopD in the plasma membrane of the target cell (Tardy et al., 1999).
The flagellum uses a T3SS for export of the hook-filament junction protein, the filament-capping protein, and flagellin, the major subunit of the filament in flagellum. These secreted proteins become incorporated into the filament (Fig. 7) (see Macnab, 2004, for review). Like the Yop-secreted proteins, the recognition signal of flagellar secreted proteins is located in the amino-terminal region of the T3SS substrates. These flagellum secreted proteins also require substrate-specific chaperones (Fraser et al., 1999; Auvray et al., 2001).
As seen with the T3SS injectisome, the flagellum has a basal body of similar architecture. The components comprising the flagellar export complex, which are essential for the formation of the flagella, are: FliH, I, J, O, P, Q, R and FlhA, B. FlhAB and FliOPQR are inner membrane proteins localized within the central pore of the ring basal body, and FliHIJ are peripheral cytoplasmic proteins (Fig. 7). This export complex first secretes proteins needed to form the external parts of the basal body. Once formed, then the device can secrete type 3-secreted proteins such as flagellin. The FliI ATPase (homologous to the YscN ATPase) drives the export of the individual flagellar components (Fan & Macnab, 1996). ATP hydrolysis is controlled by FliH that interacts with the FliI hexamer. FlhA and FlhB interact with each other and might function as the transmembrane export pore because they are known to interact with the substrate (Minamino & Macnab, 2000).
The energy sources required for flagellar assembly and Type 3 secretion are the pmf and ATP hydrolysis (Galan, 2008). The pmf is believed to drive the movement of the protein substrates through the central channel (Minamino & Namba, 2008; Paul et al., 2008). ATP hydrolysis by the Flil cytosolic ATPase is important for unfolding and presenting the substrate to the secretion machinery (Galan, 2008). However, FliI is not essential and flagellar assembly can occur in a pmf-dependent manner in the absence of the ATPase FliI and its regulatory protein FliH, although inefficiently (Minamino & Namba, 2008).