A typical bacterial proteome contains about 8000 proteins with nearly half of these functioning outside of the cytoplasm (1). This implies that export of proteins to extracytoplasmic sites is a crucial step in their biosynthesis. In Gram-negative bacteria, protein export poses a logistical challenge because proteins must, depending on their final destination, traverse different layers of the cell envelope. Therefore, these bacteria have evolved distinct secretion systems (figure 1 adapted from 2), which are numbered type I through type VI and each system is dedicated to the export of specific substrate proteins (2). To reach the periplasm, proteins are translocated across the inner membrane, while transport across the inner membrane, periplasm and outer membrane is required to get at the cell surface. The vast majority of proteins is transported to the periplasm across the inner membrane via the Sec pathway in an unfolded fashion, while a subset of folded proteins is exported through the twin arginine translocation (Tat) system. Following Sec or Tat-dependent periplasmic transport, proteins can either stay in this compartment or may be exported outside of the cell by utilizing the type 2 secretion system (T2SS). This export channel is located in the outer membrane and is able to transport folded proteins, which are either secreted through the Tat system or fold in the periplasm following Sec-dependent transport (2). Unlike the T2SS, the T5SS is only able to export unfolded proteins from the periplasm. In fact, type V secretion includes a variety of systems of which the substrate proteins are translocated across the inner membrane Sec-dependently, while transport through the outer membrane is seemingly autonomous (3). In contrast to T2SS and T5SS, substrate proteins of T1SS are translocated in a single step across the inner and outer membrane through a specialized export system made up of an ABC transporter located in the inner membrane, a periplasmic membrane fusion protein and an outer membrane porin.
These components assemble into a membrane-to-membrane complex which serves to translocate unfolded proteins (2). T4SSs are related to bacterial DNA conjugation systems and represent one of the most versatile export systems as evidenced by its ability to transport monomeric proteins, multi-subunit protein complexes as well as DNA-protein complexes. Structurally, these systems comprise a channel that spans the periplasm and associates with additional subunits, including cytosolic ATPases that probably energize protein export (2,4). The T6SS is a proteinaceous nanomachine that is present in many Gram-negative bacteria and is used for the transport of proteins directly into a variety of target cells. A typical T6SS is made up of three components, namely: the membrane complex, the baseplate complex and the injection apparatus. These substructures assemble into an envelope-spanning secretion apparatus (5). Structurally and mechanistically the T6SS is related to contractile bacteriophage tails (6). Similar to T6SS, the T3SS represents another secreting nanomachine that used to deliver proteins (effector proteins) directly from the bacterial cytosol into a eukaryotic host cell. Although these effectors are functionally and structurally diverse, they are exported in an unfolded state by a common single step mechanism that is ATP and proton motive force (PMF) dependent. It is mainly found in pathogenic Gram-negatives and is critical for virulence in causative agents of plague, typhoid fever and whooping cough (7). Not surprisingly, the T3SS represents an important target for the development of novel vaccines. The fully assembled T3SS represents a syringe-shaped “injectisome”, a cell envelope-spanning complex that is related to flagella. It comprises three major structural components: a basal body, the needle component and the translocon (2,7). Recent high-resolution structures of some of these subcomponents have provided profound atomic insight into injectisome assembly and mechanism (8,9). Here I will discuss these structures as well as the current functional T3SS understanding.
Biochemical features of the bacterial type III system
General architecture of the injectisome
Many important human pathogens, including the causative agents of plague, typhoid fever, whooping cough and dysentery, directly transport numerous effector proteins (toxins and enzymes) into the cytosol of the host cell. These effectors target cellular processes within the host, such as cytoskeletal dynamics, vesicle transport and signal transduction pathways, to promote bacterial spread, survival (prevention of phagocytosis, killing of macrophages) and evasion of the immune response (1,10). To enable the transfer of effector proteins, these bacteria are equipped with a T3SS that represents a syringe-shaped nanomachine. Hence, these are known as injectisomes. The injectisome is a 6 MDa-sized oligomeric complex made up of more than 20 subunits with one to several hundred copies each that spans the bacterial cell envelope and serves as a conduit for the passage of effector proteins into the host cytosol (11). Overall, injectisomes are related to flagella both in sequence and structure (1,10). Important insight into the general architecture and composition of the Salmonella typhimurium injectisome was provided by different structural techniques, including cryo-EM of isolated complexes at subnanometer resolution (12). This revealed its major structural components - the needle and the basal body- as well as its substructures (figure 2 left panel adopted from 12). The basal body is made up three highly oligomerized concentric rings, namely: the outer rings, neck and inner rings. These span both the inner and outer membrane with a 24-fold symmetry for the inner rings and a 15-fold symmetry for the outer rings, respectively (11).
A cartoon representation of a fully assembled injectisome is presented in the right panel of figure 2 (adopted from 1). This shows that the basal body is associated with additional components, including a membrane embedded export apparatus located between inner membrane rings of the basal body. The export apparatus comprises five hydrophobic proteins that probably form a narrow pore that acts as size-restricting filter, providing selectivity for substrate proteins on their way from the cytoplasm across the inner membrane (10). Moreover, the cytoplasmic side of the basal body is attached to several soluble factors such as an ATPase and a cytoplasmic ring (C-ring). The C-ring serves as attachment site for the ATPase (1,10). Specific subunits of the ATPase complex and C-ring comprise the sorting platform that facilitates the capture and unfolding of type III-dependent substrate proteins. Hence, these additional components - export apparatus and sorting platform-regulate the secretion of substrate proteins through controlling their selection, unfolding and subsequently feeding them to the injectisome. The extracellular segment ensures attachment to the host plasma membrane and comprises the needle filament and the translocator pore. The needle filament is anchored to the basal body and is made up of different subunits that polymerize on the periplasmic face of the injectisome, thereby forming the rod and the needle. The needle filament measures 20-150 with an inner diameter of 20 Å. This is compatible with the export of unfolded substrate proteins, while its inner surface is mostly polar. The needle ends with a tip that mediates host cell sensing and upon contact with the host cell the translocator pore is formed. Its subunits penetrate the host cell membrane and ultimately establish a conducting channel for direct delivery of effector proteins from the bacterial cytoplasm to the host cytoplasm.
Structural features of the bacterial type III system
Structure of the basal body
The basal body represents the structural foundation of the injectisome and is made up of mainly three proteins, which oligomerize into ring structures that span the inner and outer membrane. Recently, important insight into the architecture of the basal body from the injectisome of Salmonella enterica was provided by determining its cryo-EM structure with a resolution of 6.3 Å (8). This structure is shown in figure 3 both in surface and ribbon representation. The overall structure reveals the concentric IM ring and the cylindrical OM ring as well as the periplasmic neck between the two ring assemblies. Moreover, the basal body is in the closed conformation because the periplasmic gate of the OM ring (indicated by as asterisks in the right panel) is closed.
To probe the structure of the membrane-spanning rings in more detail, these were isolated after which their individual structure was assessed by cryo-EM at 4.3 Å for the IM ring and 3.6 Å for the OM ring, respectively (8). These structures are presented in figure 4 and show that the IM ring (bottom panel) is made up of two different membrane proteins, namely: PrgH and PrgK. Within the IM ring, PrgH and PrgK form extensive interactions that stabilize the ring. Both proteins are assembled into two concentric rings comprising 24 PrgH and 24 PrgK subunits with PrgH on the outside and PrgK on the inside. PrgH represents an integral membrane protein with one transmembrane domain (TMD), while PrgK is a lipoprotein that is anchored to the inner membrane by virtue of its lipid-modified N-terminus and C-terminal TMD. In addition to its TMD, PrgH contains a small N-terminal cytoplasmic domain and a large periplasmic domain. The bottom panel of figure 4 shows the structure of this periplasmic domain, revealing that is composed of three topologically similar α/β domains (helices are in red and β sheets in yellow). Of note, to obtain the high resolution of the IM ring, a PrgH variant lacking its cytoplasmic domain was used. The subdomains of PrgH attain a boot-shaped conformation and probably function as ring-building motif for the assembly ring structures (14). The periplasmic domain of PrgK is displayed in the bottom panel of figure 4 and shows that it is made up of two α/β domains with similar topology that are also thought to function as ring-building motif (15).
In contrast to the IM ring, the OM ring consists of only one protein, InvG. Fifteen copies of this subunit are assembled into a double-layered β-barrel-shaped OM ring (figure 4 top panel). InvG belongs to the group of secretins. These proteins form large portals in the outer membrane and are an essential part of different bacterial secretion systems. The structure of an individual InvG protomer is shown in the top panel of figure 4, revealing that it is made up of three different regions, namely: an N-terminal domain consisting of three subdomains (N0, N1 and N3) that protrude into the periplasm, a secretin domain comprising an inner and outer β-sheet, a membrane association region as well as a C-terminal pilotin-binding S-domain (8). The outer secretin β-sheet forms the outer wall of the barrel, while the inner β-sheet comprises the periplasmic gate. The S-domain adopts a helix-turn-helix conformation and wraps around the midsection of barrel, extending laterally across neighboring subunits. This further stabilizes the secretin barrel. The oligomerization of InvG is mainly mediated by its secretin domain, although its smaller domains also play a role in this process. The S-domain serves as binding site for cognate chaperones known as pilotins. These are small outer membrane localized proteins that assist in secretin sorting, assembly and insertion into the outer membrane. It is thought that pilotins act as molecular stapler, mediating the folding of the S-domain and thereby enabling additional stabilizing interactions between tow InvG subunits that proceed through folded S-domains. Secretins probably insert spontaneously into the outer membrane. Interestingly, InvG contains a small amphipatic helical loop (secretin flip) with a hydrophobic face suitable for membrane association. Conceivably, this loop facilitates the first stages of OM insertion, serving as an anchor to localize InvG to the OM, and thereby allowing further assembly and oligomerization. The periplasmic gate (right panel of figure 3) is a crucial feature of the secretin barrel and is formed by inner projections of the inner β-sheet of the secretin domain. The gate has a width of 15 Å, which is consistent with the export of unfolded substrates, and is lined by differently charged residues (e.g. glutamine and arginine). This creates a ring of opposing charge on the extracellular and periplasmic face of the gate. These charged residues appear to support the closed state.
Structure of the needle complex
The structure of the basal body discussed above lacks the needle filament. However, the overall structure of the needle complex, comprising the basal body and helical needle of the Salmonella typhimurium injectisome was recently reported at a resolution of 7.4 Å (9). To this end, needle complexes were isolated and subjected to cryo-EM analysis. The molecular architecture of this complex is shown in the left panel of figure 5, revealing the IM and OM rings of the basal body as well as attached substructures that include the needle filament. One prominent feature at the base of the needle filament within the lumen of the basal body comprises the so-called socket and cup. This substructure is made up of components of the export apparatus. The export apparatus provides a structural foundation for the assembly of the needle filament and this basically forms a continuation from the export apparatus with a diameter that is similar to that of the export apparatus. Moreover, the needle is buried deeply within the lumen of the secretin barrel of the OM ring. To probe the structural details of the needle, native filaments were isolated and also studied by cryo-EM. This resulted in a detailed reconstruction with a resolution of 3.3 Å (9). Structurally, the needle filament comprises the rod and the needle. The latter is a homo oligomer made up of more than one hundred copies of a single subunit known as PrgI. The structure of an individual protomer is presented in the right panel of figure 5 and shows that it adopts a helix-turn-helix conformation. PrgI polymerizes with neighboring subunits into a helical structure with 11 subunits per two turns, which results in a right-handed spiral groove. The polymer is shown in the center panel of figure 5 both in surface (with helices in blue and loops in magenta) as well as ribbon representation. A top and bottom view of the needle is also shown in the center panel, detailing its central pore. The interior of the needle is highly conserved and mainly polar with an internal diameter of about 15Å. This sufficient to accommodate unfolded or partially unfolded substrate proteins.
Injectisome assembly and type III-dependent protein export
The assembly of the injectisome is a highly coordinated process that occurs in three distinct steps (figure 6 adopted from 7). Initially, the components of the export apparatus are localized to the inner membrane. This step requires the Sec system that is used for most exported and membrane proteins and comprises therefore the first Sec-dependent step (1,10). Subsequently, the subunits of the basal body’s inner and outer membrane rings are inserted into the membrane to form the basal body’s outer shell which also occurs Sec dependently (second Sec-dependent step). Once the ring structures are in place, they are connected through stabilizing interactions. Interestingly, assembly of the C-ring occurs independently of other basal body components, suggesting that a pre-assembled C-ring can dock onto the cytoplasmic side of the basal body (10). The final step, however, proceeds Sec-independently (T3SS-dependent step). After assembly of the complete basal body, the ATPase complex associates with the C-ring and export apparatus, resulting in a secretion competent machine, which finalizes the injectisome via T3SS-dependent assembly of the needle and translocator pore subunits (1,10). This process comprises the initial secretion of rod and needle subunits (early substrates) that polymerize onto the periplasmic side of the export apparatus forming the needle filament that extends extracellularly. Following contact with the host cell, the subunits of the translocator pore are secreted (mid substrates). This establishes a conducting channel between the bacterial and host cytoplasm for the direct transfer of effector proteins (late substrates).
The vast majority of bacterial proteins that are exported out of the cytoplasm are synthesized with a cleavable N-terminal signal sequence, which funnels it into either the Sec or Tat pathway. However, substrate proteins of type III dependent protein export lack a cleavable signal sequence, although they contain export information within the first 20 N-terminal residues. These targeting signals are markedly prominent in polar residues, display a highly disordered secondary structure and lack sequence conservation in contrast to Sec or Tat-dependent signal sequences (10). T3SS-dependent export signals are sufficient for translocation through either the flagellar or injectisome system, although secretion via the latter requires the action of molecular chaperones. Typically, substrate proteins of type III-dependent export need cognate chaperones that bind to newly synthesized substrates, thereby keeping them in an unfolded state, and deliver them at the injectisome for subsequent secretion (1,10). Chaperones bind to partially unfolded structures within the first 100 amino acids following the T3SS-dependent export signal. The precise route substrate proteins take through the injectisome is still unclear, although it has been shown that subunits of the ATPase complex, located at the cytoplasmic side of the basal body, recognize export signals of type III-dependent substrate proteins as well as their chaperones (10). The ATPase is responsible for removing chaperones from their substrate proteins and subsequent unfolding of substrates prior to secretion. It has also been established that substrate proteins interact with subunits of the export apparatus. After translocation across the inner membrane, substrates traverse the inner rod, needle filament and the needle tip towards the host cell (13). How type III-dependent protein export is powered is not fully understood, although the limited diameter of the needle suggests that substrate proteins need to be unfolded during secretion. It is therefore thought that substrate unfolding is ATP dependent, while secretion itself requires the proton motive force (PMF). With regards to the latter it should be noted that it was proposed, based on the helical nature of the needle filament, that an as yet uncharacterized rotational force, possibly linked to the PMF, drives the export of substrate proteins (9). Thus, protein export through the injectisome is dependent on two different energy sources – ATP and PMF (1,10).
Gram-negative bacteria utilize a variety of dedicated secretion systems- numbered type I through type VI- to translocate substrate proteins across biological membranes (2). The type III injectisome is employed by a number of pathogens, that include causative agents of plague, typhoid fever and whooping cough, to transport virulence effector proteins directly into the cytoplasm of the host cell. These typically modulate host cell functions at the pathogen’s benefit e.g. prevention of phagocytosis, killing of macrophages and evasion of the immune response. Based on its important role as virulence factor, the injectisome has emerged as potential target for the development of novel vaccines and antimicrobial drugs. The injectisome represents a syringe-shaped nanomachine that is related to the flagellar system for bacterial motility. Structurally, it is an oligomeric complex is made up of more than 20 subunits with one to several hundred copies each, resulting in a mass of 6 MDa. The injectisome spans the bacterial cell envelope and serves as a conduit for the passage of effector proteins into the host cytosol. It comprises several major structural components, namely: the needle, the basal body and associated substructures (7,10). The basal body represents the structural foundation of the injectisome and is made up of mainly three proteins, which oligomerize into ring structures that span the inner and outer membrane. The export apparatus is located between the inner membrane rings of the basal body and probably provides selectivity for substrate proteins on their way from the cytoplasm across the inner membrane (10). At the cytoplasmic side, the basal body is associated with a few soluble factors that include the C-ring and an ATPase. The C-ring serves as attachment site for the ATPase Subunits of the ATPase complex and C-ring comprise the sorting platform that facilitates the capture and unfolding of type III-dependent substrate proteins. The needle filament is anchored to the periplasmic face of the basal body and passes through the outer membrane ring, extending extracellularly. This segment ensures attachment to the host plasma membrane and is crowned by the translocator pore. The latter structure is only formed upon contact with the host cell and penetrates the host plasma membrane. Since its discovery in 1998, important insight into the injectisome’s molecular architecture and mechanism has been obtained in part owing to recent detailed structures of its major components that now include the basal body, needle filament and cytoplasmic ATPase complex (8,9,16). It can be expected that these novel structures will contribute to the design of type III dependent inhibitors or serve as target for the development of new vaccines.
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