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). The process involving the delivery of bacterial effector proteins into eukaryltic host cells has been reviewed (Wagner et al. 2018). T#SS substrate proteins contain an N-terminal signal sequence and often a chaerone-binding domain for T#SS chaperones. These proteins are unfolded before entering the secretion channel and exiting using the pmf (Wagner et al. 2018).
Several 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). At the sequence level, a low degree of identity (22%) was observed (residues 87 to 286 in FliG align with residues 2 - 202 in MgtE). This is a hydrophilic domain. 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).
A high-resolution in situ structure of the intact machine from Shigella was revealed by high-throughput cryoelectron tomography (cryo- ET) showing a cytoplasmic sorting platform consisting of a central hub and six spokes, with a pod-like structure at the terminus of each spoke (Hu et al. 2015). Molecular modeling allowed proposal of a structure of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. ATP-hydrolysis energy may be able to drive protein transport in the absence of a proton-motive force (PMF) (Terashima and Imada 2018) although the PMF may be able to drive transport as well. Thus, the protein export apparatus consists of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. In addition, the basal body C ring acts as a sorting platform for the cytoplasmic ATPase complex that efficiently brings export substrates and type III export chaperone-substrate complexes from the cytoplasm to the export gate complex (Minamino et al. 2019). A cryo-EM structure of the isolated Shigella T3SS needle complex has been pulished (Lunelli et al. 2020). The inner membrane (IM) region of the basal body adopts 24-fold rotational symmetry and forms a channel system that connects the bacterial periplasm with the export apparatus cage. The secretin oligomer adopts a heterogeneous architecture with 16- and 15-fold cyclic symmetry in the periplasmic N-terminal connector and C-terminal outer membrane ring, respectively. Two out of three IM subunits bind the secretin connector via a β-sheet augmentation. The cryo-EM map also reveals the helical architecture of the export apparatus core, the inner rod, the needle and their intervening interfaces (Lunelli et al. 2020).
The biochemical functions of most of the individual constituents of type IIISP systems are not known. However, one constituent is an ATPase (EC 220.127.116.11) 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). FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly (Minamino et al. 2016).
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 flagellar type III export apparatus utilizes ATP and the proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Minamino et al. 2016 reported that the export gate complex can use the sodium motive force (SMF) in addition to the PMF to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM external NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. Minamino et al. 2016 proposed that the export gate by itself is a dual fuel engine that uses both the PMF and the SMF for protein export, and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.
(Gaytán et al. 2016). The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial envelope, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. Two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows E. coli pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform (Gaytán et al. 2016). Type III small hydrophobic export apparatus components SpaP and SpaR nucleate assembly of the needle complex and form the central 'cup' substructure of a Salmonella Typhimurium secretion system. Gaytán et al. 2016 presented evidence that a SpaP pentamer forms a 15 Å wide pore. They provided a map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. They also refined the current view of export apparatus assembly, consolidated transmembrane topology models for SpaP and SpaR, and suggested interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ. Their results indicated how the export apparatus and needle filament are connected to create a continuous conduit for substrate translocation.
Protein export via the T3SS is energized by the proton gradient. Erhardt et al. 2017 used a mutational approach to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identified residues R147, R154 and D158 of FlhA. These lie in a small, well-conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5, and this domain forms a multimeric array. A mutation that mimiced protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results suggest a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA (Erhardt et al. 2017).
As noted above, the flagellar type III export apparatus utilizes ATP and the PMF as energy sources, and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing flagellar structure. The apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, the PMF is the primary fuel for protein unfolding and translocation (Minamino et al. 2017).
Virulence-associated type III secretion systems inject a great diversity of bacterial effector proteins into eukaryotic host cells and can sense host cell contact. T3SS substrates contain an N-terminal signal sequence and often also a chaperone-binding domain for cognate T3SS chaperones. These signals guide the substrates to the machine where substrates are unfolded and handed over to the secretion channel formed by the transmembrane domains of the export apparatus including the needle filament. Secretion itself is driven by the pmf. The needle filament measures 20-150 nm in length and is crowned by a needle tip that mediates host cell sensing. Secretion through T3SS is a highly regulated process with early, intermediate, and late substrates. A strict secretion hierarchy is required to build an injectisome capable of reaching, sensing, and penetrating the host cell membrane, before host cell acting effector proteins are deployed (Wagner et al. 2018).
The generalized reaction catalyzed by type IIISP systems is:
Protein (bacterial cytoplasm) + ATP + pmf (or smf) + H+ or Na+ (out) → Protein (out or in the host cell cytoplasm) + ADP + Pi + H+ or Na+ (in)