3.A.6 The Type III (Virulence-related) Secretory Pathway (IIISP) Family

Proteins of the IIISP family are found in Gram-negative bacteria and allow secretion of cytoplasmically synthesized proteins across both membranes of the cell envelope (Saier, 2007). They are often concerned with secretion of virulence factors in pathogenic Gram-negative bacteria. These genes are sometimes chromosomally-encoded, in which case they are often found within 'pathogenicity islands' which are inserted DNA segments derived from foreign organismal sources. They may also be plasmid or phage-encoded. Most of these proteins are homologous to proteins concerned with bacterial (both Gram-positive and Gram-negative) flagellar protein export, and the flagellar export machinery has been shown to be capable of secreting virulence factors. They are thus functionally and structurally equivalent, although the constituents generally cluster seperately (Nguyen et al., 2000). Type III systems may transport fully or partially folded substrate proteins (Lee and Schneewind, 2002). The targeting sequence may be in the N-termini of the substrate protein (Hirano et al., 1993; Sorg et al., 2007), but other evidence has led to the suggestions that the system may recognize tertiary structure, or even elements in the mRNA (Petnicki-Ocwieja et al., 2002). The topologies of several of the inner membrane core proteins are known (Berger et al., 2010). Membrane targeting and pore formation by the type III secretion system translocon are discussed by Mattei et al. (2011). Translocation of cell surface-localized effectors can occur via type III secretion systems (Akopyan et al., 2011).

Several new findings have illuminated the evolution of flagella. Cut-down versions of flagella in Buchnera and a dispensable ATPase indicate the occurance of simpler versions of the flagellum. Morever, structural evidence for homology between FliG (a component of the flagellar motor) and MgtE (a magnesium transporter) have come to light (Snyder et al., 2009). Examination of the phylogenetic distribution of flagellar genes warns against a simplistic model of early flagellar evolution. The organization and coordinated assembly of the type III secretion export apparatus has been studied in detail (Wagner et al., 2010).

The biochemical functions of most of the individual constituents of type IIISP systems are not known. However, one constituent is an ATPase (EC 3.6.1.34) that is believed to allow the coupling of ATP hydrolysis to protein export, and six proteins are found in the inner membrane where they may form a complex that provides the transport pathway. The best characterized systems are derived from Yersinia species. These export Yersinia proteins (YOPS). [N-terminal and internal protein secretion signals as well as mRNA signals have been proposed to target proteins to the secretory apparatus.] The ATPase in this system is YscN while the integral inner membrane proteins are LcrD and YscD, R, S, T and U. They exhibit the following numbers of putative transmembrane spanners: LcrD, 8; YscD, 1; YscR, 4; YscS, 2; YscT, 6; YscU, 4. The YscC protein, an outer membrane protein that may form an oligomeric pore (outer diameter of ~200 Å; inner pore diameter of ~50 Å), belongs to the secretin family (TC #1.B.22). As many as 20 proteins may comprise the secretion apparatus.

As noted above, type III, virulence-related, secretion systems are related to the flagellar secretion apparatus, and the latter may have given rise to the former. Phylogenetic analyses have shown that the flagellar (Fla) and type III secretion systems have evolved with little or no shuffling of protein constituents between systems although lateral transfer of type III systems (but not of Fla systems) between various Gram-negative bacterial species has occurred frequently (Nguyen et al., 2000). The bacterial Fla systems use ATP which is hydrolyzed by FliI to drive export of well over a dozen proteins. These include structural constituents of the basal body, the hook, the hook capping and scaffolding protein, the two hook-filament junctional proteins, a hook-length control protein, flagellin and the flagellin capping protein. Exported regulatory proteins include the antiflagellar σ factor, FlgM, and, a muramidase, FlgJ, that allows penetration of the peptidoglycan layer by the nascent rod. FliI forms a hexameric ring-like structure in the presence of ATP with a central cavity of 2.5-3.0 nm, the same size as the flagellar export channel. The enzyme exhibits cooperativity, and cooperativity is enhanced by the presence of E. coli phospholipids (Claret et al., 2003). The FliM, N, H, I and G proteins form a physical complex, the c-ring. The chaperone protein, FliJ, binds to FliM. This complex serves to energize and regulate secretion, but has several other functions as well (Gonzalez-Pedrajo et al., 2006).

FlhB, with 4 putative TMSs, appears to gate the flagellar export pathway and determines the substrates transported (Fraser et al., 2003). An oligomeric form of it may comprise all or part of the channel for protein export. After translocation across the cytoplasmic membrane, the proteins diffuse sequentially down the channel formed by the secretion apparatus at the nascent flagellum, and they assemble at its distal end. The export apparatus includes three cytoplasmic proteins (FliH, I and J) and six integral cytoplasmic membrane proteins (FlhA and B; FlhO, P, Q and R), but a secretin, present in type III secretion systems, is absent. Early and late flagellar subunits dock at the ATPase, FliI, and are probably distinguished (Stafford et al., 2007) not by late chaperones but by N-terminal export signals of the subunits themselves. FlhA and FlhB form a docking platform for the 'soluble' components of the export apparatus FliH, FliI and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhAC) is required for protein export. Minamino et al. (2010) showed that FlhAC not only forms a part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of export substrates into the central channel of the growing flagellar structure.

Type IIISP systems often secrete their products, or some of their products directly into the host cell cytoplasm without an intermediary stage in the extracellular millieu. This fact implies the existence of a pore complex that spans the host cell cytoplasmic membrane and is contiguous with the bacterial secretion apparatus. The Yersinia protein that is believed to provide this function is YopB. YopB (401 aas; spP37131) exhibits two hydrophobic domains (residues 168-208 and 224-258) that may span the host cell cytoplasmic membrane. The protein probably forms an oligomeric pore complex. This protein is homologous to PepB of Pseudomonas aeruginosa (gbAF035922), which may serve the same function. It may show some similarity with bacterial toxins such as IpaB of S. flexneri (580 aas; spP18011) and SipB of S. typhimurium (593 aas; gbU25631) as well as with a diverse group of eukaryotic proteins. These proteins comprise the Bacterial Type III-Target Cell Pore (IIITCP) family (TC #1.C.36).

The pathway for protein transport from the bacterial cell surface is not well defined. One report (Jin and He, 2001) suggests that the pilus of a Pseudomonas syringae type III protein secretion system functions as a conduit for protein delivery. Another report (Sekiya et al., 2001) suggests that in the enteropathogenic E. coli type III secretion system, the EspA protein forms a filamentous structure that assembles as a physical bridge between the bacterial surface and the host cell surface, where the EspB/EspD proteins form the pore in the host membrane. EspA may thus provide the pathway for protein transfer between bacterial and animal cells. A hydrophilic protein forms a complex on the distal end of the injectisome needle, the tip complex, and serves as an assembly platform for the two hydrophobic translocators, EspB/D (Mueller et al., 2008). The structure of the needle protein is divergent from the flagellar filament protein (Galkin et al., 2010).

The 'injectisome', consisting of more than 20 different proteins, has been viewed as a result of T3SS crystal structures of the major oligomeric inner membrane ring, the helical needle filament, the needle tip protein, the associated ATPase, and outer membrane pilotin (Moraes et al., 2008). Two reports have demonstrated that the FliH-FliI complex facilitates only the initial entry of export substrates into the gate, with the energy of ATP hydrolysis being used to disassemble and release the FliH-FliI complex from the protein about to be exported. The rest of the successive unfolding/translocation process of the substrates is driven by the proton motive force (Minamino and Namba, 2008; Paul et al., 2008).

During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Zarivach et al. (2008) showed that a surface type II beta-turn in the stabliized Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. Deane et al. (2008) have determined the crystal structure of the cytoplasmic complex of a homologue, Spa40 of Shigella flexneri. Riordan & Schneewind (2008) suggest that auto-cleavage of YscU in Yersinia promotes interaction with YschU and recruitment of ATPase complexes that initiate secretin.

The assembly of a type III secretion injectisome culminates in the formation of the needle. In Yersinia, this step requires not only the needle subunit (YscF), but also the small components YscI, YscO, YscX and YscY. Diepold et al. (2012) found that these elements act after the completion of the transmembrane export apparatus. YscX and YscY co-purified with the export apparatus protein YscV. YscY is probably present in multiple copies. YscO and YscX are required for export of the early substrates, YscF, YscI and YscP, but they were only exported after the substrate specificity switch had occurred. Unlike its flagellar homologue FliJ, YscO was not required for assembly of the ATPase, YscN.  No export of the reporter substrate, YscP(1-137)-PhoA, into the periplasm was observed in absence of YscI, YscO or YscX, confirming that these proteins are required for export of the first substrates. In contrast, YscP(1-137)-PhoA accumulated in the periplasm in the absence of YscF, suggesting that YscF is not required for the function of the export apparatus, but that its polymerization opens the secretin YscC channel (Diepold et al. 2012).

The generalized reaction catalyzed by type IIISP systems is:

Protein (bacterial cytoplasm) + ATP + pmf (?) → Protein (out, or in the host cell cytoplasm) + ADP + P_i.