Recent insights into type 3 secretion system injectisome structure and mechanism of human enteric pathogens

Abstract

Type 3 secretion system injectisomes are multiprotein complexes that translocate bacterial effector proteins from the bacterial cytoplasm directly into the cytosol of eukaryotic host cells. These systems are present in more than 30 gram-negative bacterial species, including numerous human, animal, and plant pathogens. We review recent discoveries of structural and molecular mechanisms of effector protein translocation through the injectisomes and recent advances in the understanding of mechanisms of activation of effector protein secretion.

Introduction

Type 3 secretion system injectisomes of bacterial pathogens translocate into host cells effector proteins that facilitate bacterial entry and/or modulate cell processes such that bacterial infection is promoted. Injectisomes are large (>3.6 MDa) multi-protein needle-like machines that dock on host membrane (plasma membrane or vacuole)-embedded translocons. Injectisomes consist of a cytoplasmic export apparatus that engages a membrane-embedded needle complex, which, together with the translocon, provides a continuous channel across the bacterial inner membrane, periplasm, outer membrane, extracellular gap between bacterium and host cell, and the host membrane. Effectors, synthesized in the bacterial cytoplasm, are translocated via the channel directly into the host cell cytosol. The translocon proteins, or translocases, are actively involved in effector protein translocation. Effector translocation is tightly regulated; in response to signals generated at host membranes, the export apparatus coordinates the passage of effectors into the injectisome channel. Injectisomes are evolutionarily and structurally similar to flagellar apparatuses, on which much key work has been performed [1-3]. This review uses the unified nomenclature of injectisome and flagellar structural components [4]. Mechanisms of transcriptional regulation of injectisome components are described elsewhere [5-7] and thus not reviewed.

The needle complex

The injectisome needle complex consists of a conserved basal body and helical needle filament, the structures of which are remarkably similar among various species (Fig. 1) [8-12]. Basal body components are multimers that adopt rotational symmetry [9,13-17]. The multiprotein export apparatus in the bacterial cytoplasm docks onto and extends into the proximal channel of the bacterial membrane-embedded needle complex [10,18]. Assembly of these structures is associated with remodeling of the bacterial inner membrane and peptidoglycan [18,19].

Figure 1.

Schematic of the main components of the injectisome. Indicated is where the SctRST complex sits, although the proteins are not depicted. Red lines and lettering indicate the approximate location of the M-gate and Q-1 belt. Adapted with permission from [12].

The export apparatus

The cytoplasmic-most portion of the export apparatus is a sorting platform that participates in the ordered selection of effectors for delivery to the secretion machinery and provides energy for their secretion. Unlike other needle complex components, the shape of the sorting platform varies among injectisomes and differs from those of flagellar basal bodies [8,10-12,20,21], the biological significance of these variations is unclear. Recent work elucidated the structure-function relationships of several components of injectisomes apparatuses, including intact secretion complexes in contact with host membranes [2,3,12,18,22-27]. The export gate protein SctV engages a central stalk protein (SctO) that interacts with an ATPase (SctN) that is peripheral in the apparatus and regulates substrate entry (Fig. 1) [3,24,28]. The ATPase facilitates movement of needle, translocase, and effector proteins through the export apparatus during injectisome assembly and function [25].

Positioned at the opening of the inner rod atrium, just outside the inner membrane, is a complex of SctR₅S₄T₁ that serves as a molecular gasket [1]. A hydrophilic constriction containing 13 conserved glutamines, three within each SctS molecule and one within SctT, sits at the substrate entry site (Q1-belt/Q latch, Fig. 1), enabling side-chain independent effector loading [27]. Substitution of all three glutamines alters function in S. Typhimurium SPI-1 effector recruitment to the needle [27], but single or double substitutions in enteropathogenic E. coli do not [29], indicating the importance of the glutamines and their functional redundancy. Each SctR molecule contains three methionine residues that together with a phenylalanine residue in SctT form a dynamic hydrophobic gasket (M-gate, Fig. 1). Absent effectors, the M-gate is closed [27,30]; upon effector engagement, with a subtle conformational switch, it opens, allowing effector secretion [1,27,31]. Mutational analysis indicates the importance of the closed M-gate in maintaining a tight seal, membrane potential, and consequent bacterial fitness [32,33].

Substrate transit through the apparatus occurs at remarkable speed, with flagellin translocation through the flagellar apparatus estimated at tens of thousands of amino acids per second [34,35]. S. Typhimurium SPI-1 effector SptP enters the Q1-belt unfolded and N-terminus first, proceeds through the M-gate unfolded, then enters the adjacent Q2-belt formed by the SctRST complex [27]. The lumen of the basal body and the needle is sufficiently wide to accommodate alpha helices [27], suggesting that substrates may partially refold during transit through these structures.

The needle

The needle, needle tip, and translocases fall into four families (Fig. 2). The needle is a helical polymer of a single subunit protein (SctF), point mutations of which lead to altered effector secretion phenotypes. In S. Typhimurium SPI-1, the needle lumen displays alternating positive and negative charge that are critical to effector secretion [36].

Figure 2.

Classification and relatedness of injectisome needle and translocator proteins. (A) Classification and examples of injectisome needle, needle tip complex, and translocon proteins. n/a, no homologous protein known. (B) Comparison of major and minor translocases with phylogenetic tree to show divergence. Cladogram view of tree with branch distance labeled.

Substrate specificity switching

Proper assembly and function of the injectisome depends on two critical substrate specificity switches: first, from secretion of needle subunits and inner rod proteins to secretion of tip protein and translocases, and second, from secretion of tip protein and translocases to translocation of effectors. As shown for S. Typhimurium SPI-2, initially, a gatekeeper (SctW) interacts with the export gate protein SctV_ST2; subsequent dissociation of SctW_ST2 from SctV_ST2 causes the second specificity switch [37]. An additional role of SctW in the first switch is suggested by P. aeruginosa SctW_PA modulation of affinity of SctN_PA for substrates [25] and regulation of sctW complex expression in Y. pseudotuberculosis by temperature [38,39].

The needle tip complex and translocon

Needle tip complex

At the needle tip is a helical pentameric complex of a single protein, SctA [40-45]. A report that the Shigella tip complex contains 4 subunits of SctA_Sh and one subunit of the translocase SctE_Sh [46] is an outlier; our interpretation of the aggregated data is that, upon its secretion, a single molecule of SctE_Sh may associate, at least transiently, with pentameric SctA_Sh [47,48]. Structural analysis of the S. Typhimurium SPI-1 tip complex shows a conserved negatively charged channel that is 20 Å in diameter at the junction with the needle filament, matching the needle filament channel, and narrows to ~10 Å at the distal end [43]; the lining of the enteropathogenic E. coli tip complex, which forms a short filament, contains positively charged residues [49]. In both cases, electrostatic interactions likely are critical to effector translocation.

The translocon

The translocases (SctB and SctE) are integral membrane proteins that form a heterooligomeric translocon pore in the host membrane onto which the needle complex docks. Within intact translocons, despite divergence among the translocases (Fig. 2), the major translocase, SctE, has two transmembrane domains, with both N and C-termini in the extracellular space [50,51]. Evidence on the topology of the minor translocase, SctB, is conflicting. In silico analyses show either one [52] or two transmembrane domains [53,54], whereas experimental analyses show a single membrane-spanning domain [55], an intra-membrane hairpin [56], or a stable single membrane-spanning domain with an additional pH-sensitive transmembrane domain [54,57].

Prior to secretion, the translocases are maintained in soluble form in the bacterial cytoplasm by a chaperone, with which the major Shigella translocase is in 1:1 stoichiometry [58]. Although not widely conserved at the amino acid level, the translocases display similar secondary structure composition, with alpha-helical transmembrane domains and cytosolic coiled-coil domains. By cryo-electron tomography, natively delivered membrane-embedded S. Typhimurium SPI-1 translocons have a thickness of 8.1 nm and extend beyond the face of the cytosolic side of the membrane, with an external diameter of 13.5 nm [45], substantially narrower than the previous estimate of 55-65 nm for the E. coli translocon, obtained using purified components [59]. The internal diameter of natively delivered translocon pores is 1.2-2.6 nm.

Contact of the needle tip complex with the host membrane triggers secretion, and tip complex-dependent insertion into host membranes, of the two translocases. It remains unclear how the translocon assembles and whether assembly occurs extracellularly at the needle tip or within the host membrane. In P. aeruginosa, the interaction of SctB_Pa with the needle tip is also required for sensing the host cell [57]. The stoichiometry of fully assembled functional P. aeruginosa translocons is predicted by single molecule fluorescence photobleaching to be hexadecameric, with eight molecules each SctB_Pa and SctE_Pa [60]. Membrane insertion of Salmonella and Shigella SctE homologs depends on acylation of a single cysteine by an acyl carrier protein that is genetically associated with injectisomes [50,61,62], suggesting acylation may be common to SctE proteins. The hydrophobic sequences of the transmembrane domains direct targeting to the host membrane rather than the bacterial membrane [63]. Owing to the hydrophobic nature of the intact translocon, its structure still eludes researchers. The nature of the stable interaction that occurs between the needle tip and the translocon upon bacterial docking is poorly understood.

Efficient translocation of effectors into the host requires that they be secreted only after the needle complex is stably docked onto the translocon. In Shigella, the translocon actively participates in sensing docking, undergoing conformational changes that are associated with the initiation of effector secretion [64], suggesting that conformational changes in the translocon transduce a signal to the gatekeeper that triggers effector secretion. These conformational changes depend on binding of the cytosolic domain of SctB to host intermediate filaments [65] in the cell cortex. As SctB interaction with intermediate filaments is also necessary for effector translocation in S. Typhimurium SPI-1 and Y. pseudotuberculosis, signal transduction arising from conformational changes in the translocon may be a general feature of injectisome secretion activation. Conformational changes are also required to activate effector translocation in P. aeruginosa [57]

Translocon topology

Detailed topology mapping has been performed for the Shigella translocon. The N-terminal transmembrane domain of SctE_Sf makes up the bulk of the interior of the translocon pore channel with polar, hydrophilic residues facing the lumen, whereas the bulk of the second SctE_Sf and single SctB_Sf transmembrane domains face the lipid bilayer (Fig. 3) [50,55]. Towards the cytosolic side, the channel appears to narrow, as cross-linking of sulfhydryl groups between adjacent molecules becomes efficient [50], supporting that the translocon adopts a funnel shape (Fig. 4). Both translocases contain cytosolic domains that interact with host proteins. Sequences within the cytosolic domains of each translocase are accessible from the extracellular side of the plasma membrane to labeling reagents, including membrane and pore impermeant reagents [50,55], suggesting that these cytosolic domain residues loop back into the translocon channel (Fig. 3, asterisks). Further evidence supporting this topology is that when replaced by cysteines, these positions are reactive to copper phenanthroline [50,64], which induces disulfide bond formation only in an oxidizing environment, yet the cell cytosol is reducing.

Figure 3.

Topology of injectisome translocases in the plasma membrane. Depicted for Shigella translocases. Purple, major translocase SctE, which contains two transmembrane domains. Green, minor translocase SctB, which contains a single transmembrane domain. Yellow asterisks, cytosolic domain residues of SctE and SctB that are accessible from the extracellular side of the host membrane.

Figure 4.

Putative overall shape of translocon with docked needle tip complex. Putative funnel shape of the translocon, narrowing toward the cytosolic side of the plasma membrane. Intermediate filaments and actin filaments in the cell cortex.

Whether the cytosolic residues residing in the pore channel participate in the structural integrity or regulation of effector protein translocation remains to be determined. Unlike Shigella, the corresponding region of S. Typhimurium SctB_ST1 is not accessible to extracellular labeling reagents [55]. Whereas this observation does not rule out the possibility that these residues in SctB_ST1 reside in the translocon pore channel, their inaccessibility indicates that at least subtle structural differences exist between the translocons of these closely related injectisomes.

Needle docking and opening of the translocon pore

Docking of the needle tip onto the translocon completes the establishment of a continuous channel for effector translocation from bacterial cytoplasm to host cytosol. In S. flexneri, S. Typhimurium SPI-1, and Y. pseudotuberculosis, stable docking is genetically separable from translocon assembly and depends on interaction of SctB with intermediate filaments, which induces conformational changes in the extracellular face of the translocon that enable stable interaction [64,65].

Also described in S. flexneri, opening of the translocon pore, presumably occurring together with gatekeeper-mediated substrate specificity switching, results from distinct conformational changes of SctB_Sf that are induced by actin polymerization [66]. For pathogens that invade host cells, actin polymerization is also required for formation of membrane ruffles that engulf bacteria during internalization; this actin polymerization process is genetically separable from that which mediates pore opening [66]. Since several injectisomes require actin polymerization for effector translocation, actin polymerization-dependent pore opening may be a general feature of injectisomes.

Feedback inhibition of effector secretion

For certain injectisomes, including P. aeruginosa, Yersinia spp., and enteropathogenic E. coli, translocation of effectors is negatively feedback regulated, i.e., the translocation of a specific effector inhibits further effector translocation. For P. aeruginosa, feedback inhibition is abrogated in phagocytic cells, in a manner that is consistent with the environment of the phagosome stabilizing the translocon and its association with the translocation apparatus [67], potentially enabling the pathogen to specifically damage phagocytic cells. The data also suggest that in all cells, feedback inhibition depends in part on translocon stability.

Concluding remarks

Injectisomes are intricate multiprotein machines that deliver bacterial effector proteins into host cells in a process that is highly regulated to translocate proteins only after the complete translocation channel is established. For bacterial and cell viability, these apparatuses are designed to maintain membrane integrity with a tight seal before, during, and after effector translocation. Activation of effector translocation depends on a gatekeeper-mediated switch in substrate specificity and that likely occurs in response to conformational signal transduction. Recent findings that docking of the needle onto the translocon and opening of the translocon pore each induce conformational changes in the translocon [64,66] and that needle filament point mutants are associated with altered secretion phenotypes together support the hypothesis that conformational changes in the needle filament relay secretion activation signals from the plasma membrane translocon to the export apparatus. Together with the observation that uncapped needle filaments display the same conformation as needle filaments capped with the needle tip complex [43], any conformational signal would likely be induced at the translocon per se and transmitted via the docked needle tip complex into the needle filament. Future structural and mechanistic studies, correlated with function, will elucidate the detailed mechanisms involved in these signaling events.