Abstract
Gram-negative bacteria possess diverse transmembrane appendages that allow them to colonize different ecological niches. An essential prokaryotic strategy used for adaptation to various environments is the secretion of several molecules such as proteins and DNA, which is accomplished by specialized secretion systems that enable substrate transport across the cell envelope. Moreover, bacterial motility and adherence, driven by filamentous surface structures as flagella, pili, and curli, also play a crucial role in bacterial colonization. The construction of these macromolecular complexes faces a physical barrier since they must traverse two distinct lipid membrane bilayers and a peptidoglycan cell wall. Therefore, proteins that display special physicochemical characteristics to assemble within the hydrophobic lipid environment serve as building blocks for the biogenesis of the different transmembrane appendages. Here, we review the architecture and function of the main appendages of diderm bacteria and discuss how the interplay between membrane lipids and proteins influences their assembly and activity.
1 Introduction
Gram-negative bacteria are diderm microorganisms enclosed by two structurally and biochemically distinct lipid bilayers, separated by a periplasmic compartment containing the peptidoglycan cell wall (Fig. 1). The inner membrane (IM) is a phospholipid bilayer whose composition varies among different bacterial species. In Escherichia coli it is mainly composed of the amphiphilic lipids, phosphatidylethanolamine and phosphatidylglycerol, and small amounts of cardiolipin. The outer membrane (OM) is highly asymmetric and consists of two distinct layers; the outer leaflet is composed primarily of lipopolysaccharides (LPS) projecting outside and the inner leaflet is formed by glycerophospholipids. The differences between the IM and OM are also reflected in the structure of the integral membrane proteins assembled in each bilayer. While most of the IM proteins are formed by α-helical bundles, OM proteins (OMPs) are β-barrel proteins consisting of multiple β-sheets wrapped into cylinders, which assume a β-barrel conformation. In addition, lipoproteins can be anchored either to the periplasmic leaflet of the IM or to the OM through an N-terminal lipid moiety. These proteinaceous building blocks associate together within the hydrophobic lipid environment of the membranes to create different macromolecular complexes designed to carry out a varied set of cellular tasks, as will be outlined below (Silhavy et al. 2010; Dalbey et al. 2011; Sohlenkamp and Geiger 2016).
2 Transmembrane Appendages
Gram-negative bacteria assemble diverse transenvelope machines such as flagella, pili, curli, and secretion systems. These molecular devices serve critical functions as attachment and invasion, biofilm formation, cell motility, horizontal gene transfer, and transport of small molecules, proteins, or DNA (Dalbey and Kuhn 2012). Surface appendages in Gram-positive bacteria and archaea will not be covered in this review, but readers are referred to extensive reviews on these topics (Jarrell et al. 2013; Anne et al. 2017; Pansegrau and Bagnoli 2017).
2.1 Secretion Systems
To date, six secretion systems have been extensively studied in Gram-negative bacteria, named type I to type VI secretion systems (abbreviated as TXSS) (Fig. 2). These macromolecular complexes are employed for the transport of different cytoplasmic substrates either to other cell compartments, the extracellular space, or into a target cell. Secreted proteins have multiple functions and play a crucial role in the interplay of bacteria with the environment, e.g., protein translocation into eukaryotic cells is indispensable for the virulence of many bacterial pathogens. Secretion systems can be classified into two different categories: those performing the secretion process in a one-step mechanism (T1SS, T3SS, T4SS, and T6SS) and those whose substrates undergo a two-step secretion process in which they are first transported across the IM through the general secretion (Sec) or Twin-arginine translocation (Tat) pathways and then secreted (T2SS and T5SS) (Costa et al. 2015; Green and Mecsas 2016). The Sec pathway allows the insertion into the IM or translocation into the periplasm of unfolded proteins, in a co- or posttranslational manner (Fig. 1). In the co-translational pathway, the signal recognition particle binds to the N-terminal signal sequence of the nascent protein in complex with the ribosome and targets it to its IM receptor FtsY. In the posttranslational pathway, the preprotein, in complex with the SecB chaperone, is targeted to the SecYEG-SecA complex (Fig. 3, SecYEG-SecA). Preproteins can also be targeted in a chaperone-independent manner either alone or directly through SecA, which binds to the ribosome. Translocation across the IM is energized by the proton-motive force and the ATP hydrolysis of SecA. Protein insertion into the IM is carried out either by the SecYEG translocon alone or together with the YidC insertase (Fig. 3, YidC). The Tat pathway catalyzes the export of fully folded proteins. The signal peptide of Tat substrates contains a twin-arginine motif that is recognized by TatC in the membrane-embedded Tat machinery (TatA, TatB, and TatC). The TatB-TatC heteroligomer serves as the primary substrate binding site and is postulated to induce TatA oligomerization and pore formation. In the periplasm, the signal peptidase I (SPase I) cleaves the signal sequence of Sec and Tat substrates to release the mature proteins (Fig. 1) (Patel et al. 2014; Tsirigotaki et al. 2016).
2.2 Flagella, Pili, and Other Fimbrial Adhesins
Polymerization of multiple protein subunits promotes the assembly of flagella, pili, and curli fibers (Fig. 4). Flagellar rotation, powered by the influx of protons or sodium ions, drives bacterial motility, allowing bacteria to swim through liquid environments or to swarm on solid surfaces. In addition, flagella are also required for adhesion and biofilm formation in certain bacterial species. Structurally, flagella are thicker and longer than pili, although they are less numerous. Pili are long (1–4 μm) flexible filaments composed of thousands of pilin subunits, essential for various bacterial processes such as adherence to both biotic and abiotic surfaces, auto-aggregation, biofilm formation, cellular invasion, bacteriophage infection, electron transfer, motility, horizontal gene transfer, and DNA uptake during natural transformation. Furthermore, bacteria belonging to the Enterobacteriaceae family assemble highly aggregative and flexible amyloid fibers of 4–7 nm named curli in E. coli or thin aggregative fimbriae (Tafi) in Salmonella spp. These recently identified adhesins are important for bacteria-bacteria and bacteria-host interactions and have been shown to be involved in biofilm formation and the colonization of inert surfaces such as teflon and stainless steel (Fronzes et al. 2008; Van Gerven et al. 2011; Hospenthal et al. 2017).
3 Building Blocks for the Construction of Transmembrane Appendages
The ordered association of numerous proteins leads to the construction of the bacterial appendages discussed above. Since these nanomachines traverse the cell envelope, specialized integral membrane proteins serve as essential building blocks for their assembly. In the next section we will describe the architecture and function of the main appendages, emphasizing on the structure of integral membrane proteins and lipoproteins that help build these macromolecular complexes within the hydrophobic lipid bilayers.
3.1 T1SS
The type I secretion system is a tripartite complex composed of two IM components, an ATP-binding cassette (ABC) transporter and a membrane fusion protein (MFP) which functions as a periplasmic adaptor, and an OM protein (OMP) that forms a channel (Fig. 2, T1SS). Together, these pieces assemble a double-membrane spanning and transperiplasmic tunnel-like structure, through which unfolded proteins are secreted in a single ATP hydrolysis-dependent step (Green and Mecsas 2016). The proteins exported by the T1SS greatly vary in size (from the 20 kDa iron scavenger HasA from Serratia marcescens to the 900 kDa large adhesion protein LapA from Pseudomonas fluorescens) (Thomas et al. 2014) and in function (lipases, proteases, adhesins, toxins, etc.), some of which contribute to the virulence of many plant and animal bacterial pathogens. The prototypical T1SS is the hemolysin (HlyA) export machinery of uropathogenic E. coli, formed by HlyB (ABC transporter), HlyD (MFP), and TolC (OMP) (Holland et al. 2005). Most ABC transporters are homodimers, each monomer consisting of two transmembrane domains (TMDs), which form the translocation pathway for substrates across the IM, and two nucleotide-binding domains (Fig. 3, ABC transporter). However, HlyB monomers are formed by only one TMD composed of six predicted transmembrane segments and one ATP-binding domain. Substrates are recognized by this component through a C-terminal uncleavable signal sequence. The MFP HlyD is a trimeric protein predicted to possess an IM single spanning α-helical region and a large periplasmic domain that interacts with TolC. This component also associates with the ABC transporter and participates in substrate recognition. TolC is a homotrimer that forms a short OM β-barrel of 12 strands and a long α-helical domain (four α-helices), which protrudes into the periplasm (10 nm) to form a hydrophilic conduit that narrows toward its periplasmic end to prevent the nonselective passage of folded proteins (Fig. 3, TolC) (Koronakis et al. 2000). Upon substrate binding, HlyB and HlyD associate with TolC, triggering the opening of its α-helical barrel to allow substrate export (Thomas et al. 2014).
3.2 T2SS
The type II secretion system, or general secretory pathway (“Gsp” for a unified nomenclature), exports proteins that are first translocated across the IM by the Sec translocase or the Tat transporter and, in a second step, promotes transport of folded periplasmic proteins through the OM and into the extracellular milieu, by a piston-driven mechanism energized by ATP hydrolysis (Korotkov et al. 2012). A functional T2SS is present in many important human pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa, Legionella pneumophila, and Vibrio cholerae (e.g., cholera toxin is secreted via the T2SS) but is also present in environmental bacteria, some of which promote symbiosis, among other processes. This molecular complex is composed of 12 to 15 distinct proteins that constitute 4 major substructures: an OM channel-forming protein called secretin, a periplasmic pseudopilus, an IM platform, and a homohexameric cytoplasmic ATPase (Fig. 2, T2SS). The OM secretin is a pentadecamer of the GspD protein that forms a gated pore. Each secretin subunit has a conserved membrane-embedded β-barrel C-terminal domain followed by a periplasmic S-domain involved in secretin localization and membrane insertion, and an N-terminal region with four subdomains (N0 to N3), which form a cylindrical structure in the periplasm (Yan et al. 2017; Filloux and Voulhoux 2018). Proteins that belong to the secretin family serve as common building blocks for the biogenesis of different bacterial appendages such as the T3SS and the type IV pilus. In some cases, the assembly of the secretin relies on the assistance of a small lipoprotein known as the pilotin (GspS), which binds to the S-domain. The short pseudopilus in the periplasm is composed of multiple copies of a major pseudopilin, GspG, and four minor pseudopilins (GspH, I, J, and K), and, in contrast to extracellular pilus, this structure remains in the periplasmic space. The IM platform subcomplex, which consists of one polytopic and three bitopic proteins, plays an important role in contacting the OM secretin, the pseudopilus, and is also thought to recruit the oligomeric ATPase (GspE). The latter interaction stimulates the enzymatic activity of GspE, thereby transmitting conformational changes to the IM platform and pseudopilus, which functions as a piston that extends to push secreted substrates through the secretin channel (Thomassin et al. 2017).
3.3 T3SS
The type III secretion system, or injectisome, is a 3.5 MDa complex composed of approximately 25 proteins that assemble a nanosyringe capable of traversing three membranes (Fig. 2, T3SS). This protein export machinery is used by symbiotic and pathogenic Gram-negative bacteria to translocate a myriad of effector proteins directly from the bacterial cytoplasm into a eukaryotic target cell, in an ATP- and proton-motive force-dependent manner (Deng et al. 2017). The injectisome is evolutionarily related to the bacterial flagellum and it has been suggested that it originated from an exaptation process, i.e., the recruitment of flagellar components for a new protein transport function. The T3SS can be divided into three substructures: (i) the extracellular appendages, which consist of a filamentous needle-like structure that serves as a scaffold for the assembly of a tip complex made up of a hydrophilic protein and a translocation pore formed by two hydrophobic proteins that insert into the host cell membrane. In some bacteria, such as enteropathogenic and enterohemorrhagic E. coli, instead of a small tip, a large filament is assembled on top of the needle; (ii) the basal body, which comprises three membrane rings connected through a periplasmic rod and which houses the export apparatus, an IM embedded protein complex that controls access of cytosolic proteins to the T3SS inner channel, (iii) the cytoplasmic components, some of which associate with the IM, as the sorting platform (cytoplasmic C-ring) and the ATPase complex that help in the recruitment and classification of substrates, as well as in energizing the secretion process promoting chaperone-effector dissociation and initial substrate engagement. T3SS substrates are transported in a partially unfolded conformation through a continuous 2.5 nm narrow conduit that extends from the basal body and needle to the host cell. The translocated effectors subvert specific host cell processes to enable bacterial colonization (Gaytan et al. 2016; Deng et al. 2017; Galan and Waksman 2018).
The membrane-embedded protein rings in the basal body are composed of 15 subunits of the OM secretin SctC (homologous to the T2SS-associated secretin) and 24 subunits each of the SctD and SctJ proteins in the IM. The structure of the OM ring, defined at 3.6 Å resolution by single-particle cryoelectron microscopy, showed that it forms a double-layered β-barrel (Fig. 3, InvG) (Worrall et al. 2016). The IM-concentric rings house the export apparatus, formed by the association of five polytopic proteins in the following stoichiometry, 5 SctR, 1 SctS, 1 SctT, 1 SctU, and 9 SctV, which could give a total of 104 TM segments in a membrane patch at the base of the injectisome (Zilkenat et al. 2016). These proteins are highly conserved among different T3SSs (including the flagellar T3SS) and they are essential for protein secretion, although little is known about the structure of their membrane-spanning domains.
3.4 T4SS
The type IV secretion system is an envelope-spanning versatile nanomachine capable of delivering DNA, proteins, or nucleo-protein complexes to the extracellular milieu or into a wide range of target cells (bacterial and eukaryotic cells). The evolutionary origin of this system is the ancestral bacterial conjugation machinery. There are three functional types of T4SSs: one used for transferring DNA through conjugation, which is the main mechanism of antibiotic resistance gene dissemination, one that mediates DNA uptake (transformation) and release, and one engaged in effector protein translocation into target cells. Protein-exporting T4SSs are usually employed by human pathogenic bacteria such as Helicobacter pylori, which delivers one effector protein, and Legionella pneumophila, which injects over 300 effectors into its eukaryotic target cell. In addition, it has been reported that Xanthomonas citri employs its T4SS to kill other Gram-negative bacterial species (Galan and Waksman 2018; Grohmann et al. 2018).
Agrobacterium tumefaciens transfers oncogenic DNA into plant cells via a conjugative T4SS and has been the prototypical bacterium for studying this macromolecular complex, which is composed of 12 proteins named VirB1 to VirB11 and VirD4. The architecture of this secretory system consists of an OM core complex connected by a narrow stalk to an IM complex (Fig. 2, T4SS) (Low et al. 2014). The OM complex is a ring structure formed by 14 subunits each of the proteins VirB7, VirB9, and VirB10. This complex spans both bacterial membranes and is in turn subdivided into an outer (O-layer) and an inner layer (I-layer), inserting in the OM and IM, respectively (Rivera-Calzada et al. 2013). The O-layer is formed by the protein VirB7 and the C-terminal domains of VirB9 and VirB10, the latter protein forms the inner side of the core complex while VirB7 and VirB9 bind around VirB10. The 14 VirB10 subunits project a double-helical TM region that together form the OM channel. This helical barrel arrangement is infrequent in OM proteins (Fig. 3, O-layer) (Chandran et al. 2009). The I-layer is formed by the N-terminal domains of the VirB9 and VirB10 subunits (Costa et al. 2015). The IM complex consists of a large membrane-embedded platform composed of 12 copies each of VirB3, VirB5, VirB6, and VirB8 and two barrel structures protruding into the cytoplasm, each made up of six subunits of the VirB4 ATPase (Low et al. 2014). The composition of the stalk connecting the OM and IM complexes is still unknown (Galan and Waksman 2018). Furthermore, the pilus of the T4SS is composed of the major pilin subunit VirB2 and the minor pilin VirB5, which is localized at the tip of the pilus and is believed to function as an adhesion protein for cell contact. Protein translocation by the T4SS is powered by the ATP hydrolysis of three energizing ATPases, VirB4, VirB11, and VirD4 (Costa et al. 2015; Grohmann et al. 2018).
3.5 T5SS
The type V secretion system, also known as the autotransporter (AT) system, is one of the simplest pathways for protein secretion because the information required for transport through the OM is present in the secreted substrate polypeptide (or two polypeptides in the two-partner secretion system). Classical ATs consist of an N-terminal signal sequence for export to the periplasm by the Sec apparatus, followed by an N-terminal (secreted or extracellularly exposed) passenger domain and a C-terminal translocation domain that inserts into the OM as a β-barrel, to allow secretion of the passenger domain through the barrel pore (Fig. 2, T5SS). The β-barrel is formed by 12 antiparallel β-strands and its interior is occupied by a linker region that connects it to the passenger domain (Fig. 3, EstA). The energy for transport has been postulated to originate from the passenger domain folding when exiting the pore (Dalbey and Kuhn 2012; Costa et al. 2015; Fan et al. 2016). After transport across the IM, the AT interacts with periplasmic chaperones that prevent its folding during the targeting to the OM β-barrel assembly machinery or Bam complex (formed by the OM protein BamA and four lipoproteins BamB, BamC, BamD, and BamE), which promotes membrane insertion of β-barrel proteins. There is increasing evidence indicating that the Bam complex and, for some ATs, the translocation and assembly module (TAM) complex play a direct role in facilitating passenger domain secretion, and so the autonomous secretion of the AT system has been recently re-evaluated. It has been reported that BamA actively participates in AT secretion through its interaction with the passenger domain (Albenne and Ieva 2017). In many cases, after translocation, the passenger domain is cleaved by an autocatalytic process and released into the extracellular space. In contrast, non-processed ATs remain associated to the cell envelope via the translocation domain (Fan et al. 2016).
3.6 T6SS
The type VI secretion system is the most recently characterized macromolecular secretion machine, which translocates effector proteins into both prokaryotic and eukaryotic cells in a single step process. This system injects a wide variety of effectors including nucleases, lipases, glycoside hydrolases, amidases, and esterases that are crucial for pathogenicity toward animal and plant hosts as well as to target competitor bacteria (Cianfanelli et al. 2016; Galan and Waksman 2018). The T6SS is formed by 13 essential core components (TssA to TssM), some of which are homologous to the contractile bacteriophage tails. The overall architecture consists of three main structures: a membrane complex, a cytoplasmic baseplate, and a sheathed inner tube (or effector delivery module) (Fig. 2, T6SS). The membrane complex is composed of 10 heterotrimers containing the lipoprotein TssJ, anchored to the OM, and the IM proteins TssM and TssL. This complex is used as a docking station for assembly of the cytoplasmic components (or tail complex), formed by the baseplate, the inner tube, and the tail sheath. The baseplate-like structure, composed of TssA, TssE, TssF, TssG, TssK, and VgrG proteins, associates with the membrane complex and serves as a platform for tube and sheath polymerization. The extended inner tube (1000 nm) is formed by stacked hexamers of the Hcp protein enclosed by a contractile sheath formed by two proteins, TssB and TssC. VgrG is a trimeric protein localized at the membrane end of the Hcp tube forming the tail spike, which is further sharpened into a conical tip by a PAAR repeat-containing protein that binds to the VgrG trimer (Costa et al. 2015; Alteri and Mobley 2016; Gallique et al. 2017; Galan and Waksman 2018). This bacterial weapon delivers effectors using a cell-puncturing mechanism that resembles an inverted phage contractile tail, where, during contraction of the sheath, the inner tube, which is loaded with effectors, VgrG and PAAR, is ejected toward the target cell (Gallique et al. 2017). Finally, the T6SS is disassembled by the cytoplasmic ClpV ATPase, completing the assembly, contraction, and depolymerization cycle necessary for sheath recycling during the injection process (Cianfanelli et al. 2016).
3.7 Surface Appendages for Motility and Adhesion
Many of the building blocks required for the assembly of flagella and pili are homologous to components of the secretion systems. The bacterial flagellum is a rotating organelle used for motility that is assembled through a T3SS (Fig. 4) (Minamino 2014). Although the flagellum and the injectisome differ in overall structure and function, they have a conserved T3S machinery for protein export with several core components that are highly similar between these macromolecular complexes. These proteins are localized in the flagellar basal body, forming the IM-MS ring (FliF) and the export apparatus (FliP, FliQ, FliR, FlhB, and FlhA) located in a patch of membrane within the MS ring, and also as part of the cytosolic components, the ATPase complex (FliI, FliH, and FliJ) and the cytoplasmic C-ring (FliM and FliN). In contrast, the OM ring structures are different; while in the injectisome a secretin pore is formed, in the flagellum there are two rings, the P ring (FlgI) within the peptidoglycan layer and the L-ring formed by the lipoprotein FlgH in the OM. Once the flagellar core T3SS is assembled, the export of subunits forming the axial structures is initiated, first the periplasmic rod (FliE, FlgB, FlgC, FlgF, and FlgG), then the extracellular hook (FlgE) and hook-associated proteins (FlgK, FlgL, and FliD), and lastly the filament formed by the polymerization of thousands of flagellin monomers (FliC). Substrates are transported through a narrow channel of ~ 2.0 nm and the energy for protein export is provided by both the proton-motive force and ATP hydrolysis. The motor proteins MotA and MotB are assembled in the IM forming the stator complex which is the pathway of proton influx for flagellar rotation (Erhardt et al. 2010; Diepold and Armitage 2015).
Pili or fimbriae are mainly involved in bacterial adhesion to other bacteria, host tissues, and other surfaces, so these appendages are considered important virulence factors. The type IV pili (T4P) is a very versatile surface structure that participates in bacterial adherence, biofilm establishment, microcolonies formation, electron transfer, natural competence, and twitching motility. The T4P is evolutionarily related to the T2SS, where homologous proteins are named pseudopilins. However, in contrast to the periplasmic T2SS pseudopilus, the T4P is a surface-exposed long filament of 4 μm in length and 4–9 nm in diameter. Nonetheless, the assembly of these appendages is highly similar. Prepilins are inserted into the IM by the Sec translocase and then processed by the prepilin peptidase PilD, which cleaves the N-terminal signal sequence. Mature pilins (PilA and minor pilins) are extracted from the IM and assembled into the base of the T4P, which is then extended from an IM assembly platform into a secretin complex (PilQ) embedded in the OM (Fig. 4). The pore complex PilQ from P. aeruginosa is made of 14 subunits, whereas InvG (T3SS) and GspD (T2SS) are pentadecameric; however, the overall architecture of the T4P OM secretin is similar to the ones of type II and III secretion systems. The T4P is unique in their ability to extend and retract through the action of the cytoplasmic ATPases PilB and PilT (Dalbey and Kuhn 2012; Gold and Kudryashev 2016; Koo et al. 2016; Hospenthal et al. 2017; Filloux and Voulhoux 2018).
Chaperone-usher pili are present on the surface of many bacterial pathogens. Uropathogenic E. coli (UPEC) expresses type 1 and P pili that are crucial for bacterial ascension from the bladder to the upper urinary tract. The type 1 pilus (T1P) of UPEC is assembled through the chaperone-usher pathway and consists of two substructures: a thick helical rod of ~ 2 μm in length composed of the major pilus subunit FimA and a short flexible tip component at the distal end formed by the minor pilus subunits FimF, FimG, and FimH (Fig. 4). Pilin subunits are first transported across the IM through the Sec pathway, and once in the periplasm they are stabilized by the periplasmic chaperone FimC, the chaperone-pilin complex is then recruited to the OM usher FimD, a β-barrel pore that mediates pilus assembly. The pilus subunits possess an incomplete immunoglobulin domain with six β-strands (instead of seven) which makes the proteins unstable; therefore, the assembly process involves a donation of a β-strand by the chaperone FimC (donor strand complementation) or by an N-terminal extension of the incoming pilus subunit during polymerization (donor strand exchange). Pilin subunits polymerize at the usher pore although it is still unknown what energy source powers the translocation process (Dalbey and Kuhn 2012; Costa et al. 2015; Hospenthal et al. 2017).
Curli is another type of fimbrial adhesin present in enteric bacteria, composed of highly aggregative proteinaceous fibers. These functional amyloid fibers mediate adhesion to human matrix proteins as fibronectin and laminin and also interaction with the host immune system having important roles in pathogenicity and biofilm formation. Similarly to pilins in T1P and T4P, curli subunits are transported through the IM by the Sec pathway. Curli is formed by the major (CsgA) and the minor (CsgB) pilin subunits. In the periplasm these subunits interact with the CsgC chaperone to avoid premature amyloid fiber formation. The OM curli subunit secretion machinery comprises three proteins: the lipoprotein CsgG, which spans the OM and forms a nonameric ring structure of 36 β-barrel strands with an inner diameter of 2 to 4 nm; CsgE, an accessory factor which binds CsgG and functions as the OM pore gate, serving also to target curli subunits to the CsgG channel for secretion; and the extracellular protein CsgF, which interacts with CsgG and stabilizes and localizes the minor curli subunit CsgB (Fig. 4). CsgA is translocated through CsgG in an unfolded manner while CsgB functions as the nucleation point for CsgA homopolymer formation in a mechanism known as nucleation-precipitation (Cao et al. 2014; Evans and Chapman 2014; Costa et al. 2015; Van Gerven et al. 2015).
4 Influence of Lipids on the Structure and Function of Bacterial Appendages
Given that bacterial appendages are assembled within the hydrophobic environment of lipidic membranes, it is not surprising that membrane lipids influence their structure and function. Some examples are described below.
In general, protein secretion is an energy-dependent process in which ATP hydrolysis by specific ATPases is coupled to protein secretion. Therefore, bacteria have developed mechanisms to regulate ATPase activity, to ensure that it occurs at the appropriate time and in the right place. For instance, in the Sec pathway, the ATP hydrolytic activity of SecA, and, in turn, protein translocation, is stimulated by the presence of the acidic phospholipids phosphatidylglycerol (PG) and cardiolipin (CL). These anionic phospholipids are not only essential for SecA ATPase activity but also promote its association with the membrane. It has been shown that lipid clusters enriched with acidic phospholipids, via their negatively charged surface, increase the amount of SecA bound to lipid bilayers. Moreover, CL stabilizes the formation of SecYEG dimers, creating a binding surface for SecA and increasing its ATP turnover (Lill et al. 1990; Breukink et al. 1992; Ulbrandt et al. 1992; Ahn and Kim 1998; Gold et al. 2010). A similar phospholipid activation effect has been observed on the activity of the so-called secretion ATPases of the type II, III, IV, and VI secretion systems (Krause et al. 2000). The enzymatic activity of EpsE, the T2SS-associated ATPase of V. cholerae, is synergistically activated by the phospholipids PG and CL together with the cytoplasmic domain of EpsL, which is an assembly platform component that promotes the association of EpsE to the IM. In contrast, the zwitterionic phospholipid phosphatidylethanolamine (PE) had no effect on the ATPase activity of the EpsE/EpsL complex. The stimulation of ATPase activity occurs through a direct binding of acidic phospholipids to the EpsE/EpsL complex. Thus, it is proposed that the cytoplasmic domain of EpsL could regulate the interaction of the ATPase EpsE with phospholipids, which thereby induces its oligomerization and activation (Camberg et al. 2007). Likewise, the activity of the flagellar ATPase FliI has been shown to be stimulated by acidic phospholipid binding, specifically in the presence of PG and CL, but not with the zwitterionic lipid phosphatidylcholine (Auvray et al. 2002).
In addition to ATPase activity, membrane lipids influence other steps in the translocation process. Proteins that are transported across the IM through the Sec and Tat pathways possess a positively charged N-terminal signal sequence that allows their targeting and secretion. The positively charged signal peptide interacts with the negatively charged phospholipids in the IM, which favors its insertion into the bilayer, thereby facilitating early steps of the translocation process. The insertion of the signal peptide into the IM induces its conformational change to an α-helix (Phoenix et al. 1993; Brehmer et al. 2012). Furthermore, it has been demonstrated that extracted and purified SecYEG complex from E. coli lipid membranes is greatly enriched in anionic phospholipids, which are essential for protein translocation since the reconstituted SecYEG complex in liposomes without PG and CL is inactive (Prabudiansyah et al. 2015).
Moreover, lipid posttranslational modification of proteins is a relevant process in the assembly of surface appendages. A null mutant in the phosphoethanolamine (pEtN) transferase EptC of Campylobacter jejuni presented a decreased motility, with ~ 95% of the population lacking flagella. This defect is caused by the absence of pEtN modification of the flagellar rod protein FlgG. Thus, the lipid modification of FlgG is proposed to confer structural stability between rod subunits during flagellar assembly (Cullen and Trent 2010; Cullen et al. 2012). EptC belongs to a family of proteins that catalyze the periplasmic decoration of bacterial structures. Another member of this protein family is PptA (pilin phospho-form transferase A), which catalyzes the modification of the major pilin subunit protein, PilE, of the T4P of Neisseria gonorrhoeae, with pEtN and phosphocholine. These modifications confer structural stability to its target and provide antigenic diversity (Aas et al. 2006). A phosphoglycerol modification of PilE has also been reported in N. meningitidis, which affects T4P-dependent interactions between bacteria, promoting its dissemination during invasive infection (Chamot-Rooke et al. 2011). Interestingly, the recent cryoelectron microscopy structure of the conjugative sex F pilus of the T4SS revealed that it is assembled from stoichiometric protein-phospholipid units. The phospholipid PG interacts with the pilin, whereas PE and CL did not. The lipid head groups are directed toward the pilus lumen, creating a moderately negative inner conduit which could facilitate transport of the negatively charged ssDNA (Costa et al. 2016).
Finally, the correct function of the YscU protein, an export apparatus component of the Yersinia pseudotuberculosis T3SS, depends on its association with lipids. YscU is an IM polytopic protein with a large C-terminal cytoplasmic domain. This protein possesses a positively charged linker domain that connects the membrane and the cytoplasmic domains. The linker region associates with the negatively charged IM undergoing a coil-to-helix transition. However, upon alanine substitution of its positively charged amino acids, the membrane binding affinity was attenuated and the T3SS-dependent protein secretion was significantly reduced (Weise et al. 2014).
5 Research Needs
The successful colonization of different ecological niches relies on the ability of bacteria to adapt to diverse environments and to establish associations with other prokaryotic and eukaryotic cells. The secretion systems and extracellular appendages described in this work play a determinant role in this adaptation process and are crucial for bacterial persistence and survival. In many cases, these transmembrane organelles are employed as virulence weapons against host cells, resulting in the development of multiple diseases that have a dramatic impact on human health. This, together with the increasing prevalence of multidrug-resistant bacterial pathogens, requires the urgent design of novel antimicrobial strategies that specifically target virulence mechanisms instead of viability. Therefore, given the central role of the aforedescribed appendages in pathogenesis, it is of utmost importance to further our knowledge on the structural and mechanistic basis of the assembly and function of these molecular machineries.
The molecular assembly of the different bacterial appendages has been extensively studied. Cryoelectron microscopy has transformed the field of structural biology, creating high-resolution models and providing structural insights of complete protein complexes embedded in the bacterial membranes, e.g., the injectisome and the T4SS core complex. This has unraveled some remarkable structural similarities between the systems. However, atomic-resolution structures of various individual components are still not available, in particular those of the membrane-embedded building blocks, which will be a big challenge for the future. In addition, the influence of membrane lipids on the assembly of these protein complexes remains to be determined.
Membrane lipid composition varies within different bacterial species and under diverse environmental conditions, and it is known to affect the structure and function of membrane-embedded components. Nevertheless, there are only few examples in the literature of the role of lipids in the assembly and function of bacterial appendages. The recent unprecedented finding that the F pilus is composed of a 1:1 stoichiometric phospholipid-protein complex opens the field for future studies on the mechanisms of pili biogenesis. However, more efforts are required to understand the operation of these nanomachines within the hydrophobic lipid environment. In addition, protein insertion into bacterial membranes also alters the physical properties of the membrane, causing perturbations that might have an impact in cell physiology. This field is still largely unexplored.
Numerous questions and challenges remain for future investigations, such as the detailed molecular mechanisms underlying the energetics of protein secretion and substrate recognition, to elucidate the structure of membrane components, to determine the influence of lipids in bacterial appendages assembly and function, etc. This information will contribute to current efforts in the development of new anti-infective drugs that interfere with the function of these virulence factors.
References
Aas FE, Egge-Jacobsen W, Winther-Larsen HC, Lovold C, Hitchen PG, Dell A, Koomey M (2006) Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases. J Biol Chem 281:27712–27723
Ahn T, Kim H (1998) Effects of nonlamellar-prone lipids on the ATPase activity of SecA bound to model membranes. J Biol Chem 273:21692–21698
Albenne C, Ieva R (2017) Job contenders: roles of the beta-barrel assembly machinery and the translocation and assembly module in autotransporter secretion. Mol Microbiol 106:505–517
Alteri CJ, Mobley HL (2016) The versatile type VI secretion system. Microbiol Spectr 4. https://doi.org/10.1128/microbiolspec.VMBF-0026-2015
Anne J, Economou A, Bernaerts K (2017) Protein secretion in Gram-positive Bacteria: from multiple pathways to biotechnology. Curr Top Microbiol Immunol 404:267–308
Auvray F, Ozin AJ, Claret L, Hughes C (2002) Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH. J Mol Biol 318:941–950
Brehmer T, Kerth A, Graubner W, Malesevic M, Hou B, Bruser T, Blume A (2012) Negatively charged phospholipids trigger the interaction of a bacterial Tat substrate precursor protein with lipid monolayers. Langmuir 28:3534–3541
Breukink E, Demel RA, de Korte-Kool G, de Kruijff B (1992) SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study. Biochemistry 31:1119–1124
Camberg JL, Johnson TL, Patrick M, Abendroth J, Hol WG, Sandkvist M (2007) Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids. EMBO J 26:19–27
Cao B, Zhao Y, Kou Y, Ni D, Zhang XC, Huang Y (2014) Structure of the nonameric bacterial amyloid secretion channel. Proc Natl Acad Sci U S A 111:E5439–E5444
Chamot-Rooke J, Mikaty G, Malosse C, Soyer M, Dumont A, Gault J, Imhaus AF, Martin P, Trellet M, Clary G, Chafey P, Camoin L, Nilges M, Nassif X, Dumenil G (2011) Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination. Science 331:778–782
Chandran V, Fronzes R, Duquerroy S, Cronin N, Navaza J, Waksman G (2009) Structure of the outer membrane complex of a type IV secretion system. Nature 462:1011–1015
Cianfanelli FR, Monlezun L, Coulthurst SJ (2016) Aim, load, fire: the type VI secretion system, a bacterial Nanoweapon. Trends Microbiol 24:51–62
Costa TR, Felisberto-Rodrigues C, Meir A, Prevost MS, Redzej A, Trokter M, Waksman G (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13:343–359
Costa TRD, Ilangovan A, Ukleja M, Redzej A, Santini JM, Smith TK, Egelman EH, Waksman G (2016) Structure of the bacterial sex F pilus reveals an assembly of a stoichiometric protein-phospholipid complex. Cell 166(1436–1444):e1410
Cullen TW, Trent MS (2010) A link between the assembly of flagella and lipooligosaccharide of the Gram-negative bacterium Campylobacter jejuni. Proc Natl Acad Sci U S A 107:5160–5165
Cullen TW, Madsen JA, Ivanov PL, Brodbelt JS, Trent MS (2012) Characterization of unique modification of flagellar rod protein FlgG by Campylobacter jejuni lipid A phosphoethanolamine transferase, linking bacterial locomotion and antimicrobial peptide resistance. J Biol Chem 287:3326–3336
Dalbey RE, Kuhn A (2012) Protein traffic in Gram-negative bacteria – how exported and secreted proteins find their way. FEMS Microbiol Rev 36:1023–1045
Dalbey RE, Wang P, Kuhn A (2011) Assembly of bacterial inner membrane proteins. Annu Rev Biochem 80:161–187
Deng W, Marshall NC, Rowland JL, McCoy JM, Worrall LJ, Santos AS, Strynadka NCJ, Finlay BB (2017) Assembly, structure, function and regulation of type III secretion systems. Nat Rev Microbiol 15:323–337
Diepold A, Armitage JP (2015) Type III secretion systems: the bacterial flagellum and the injectisome. Philos Trans R Soc Lond Ser B Biol Sci 370. https://doi.org/10.1098/rstb.2015.0020
Erhardt M, Namba K, Hughes KT (2010) Bacterial nanomachines: the flagellum and type III injectisome. Cold Spring Harb Perspect Biol 2. https://doi.org/10.1101/cshperspect.a000299
Evans ML, Chapman MR (2014) Curli biogenesis: order out of disorder. Biochim Biophys Acta 1843:1551–1558
Fan E, Chauhan N, Udatha DB, Leo JC, Linke D (2016) Type V secretion systems in bacteria. Microbiol Spectr 4. https://doi.org/10.1128/microbiolspec.VMBF-0009-2015
Filloux A, Voulhoux R (2018) Multiple structures disclose the Secretins’ secrets. J Bacteriol 200. https://doi.org/10.1128/JB.00702-17
Fronzes R, Remaut H, Waksman G (2008) Architectures and biogenesis of non-flagellar protein appendages in Gram-negative bacteria. EMBO J 27:2271–2280
Galan JE, Waksman G (2018) Protein-injection machines in bacteria. Cell 172:1306–1318
Gallique M, Bouteiller M, Merieau A (2017) The type VI secretion system: a dynamic system for bacterial communication? Front Microbiol 8:1454
Gaytan MO, Martinez-Santos VI, Soto E, Gonzalez-Pedrajo B (2016) Type three secretion system in attaching and effacing pathogens. Front Cell Infect Microbiol 6:129
Gold V, Kudryashev M (2016) Recent progress in structure and dynamics of dual-membrane-spanning bacterial nanomachines. Curr Opin Struct Biol 39:1–7
Gold VA, Robson A, Bao H, Romantsov T, Duong F, Collinson I (2010) The action of cardiolipin on the bacterial translocon. Proc Natl Acad Sci U S A 107:10044–10049
Green ER, Mecsas J (2016) Bacterial secretion systems: an overview. Microbiol Spectr 4. https://doi.org/10.1128/microbiolspec.VMBF-0012-2015
Grohmann E, Christie PJ, Waksman G, Backert S (2018) Type IV secretion in Gram-negative and Gram-positive bacteria. Mol Microbiol 107:455–471
Holland IB, Schmitt L, Young J (2005) Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review). Mol Membr Biol 22:29–39
Hospenthal MK, Costa TRD, Waksman G (2017) A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat Rev Microbiol 15:365–379
Jarrell KF, Ding Y, Nair DB, Siu S (2013) Surface appendages of archaea: structure, function, genetics and assembly. Life (Basel) 3:86–117
Koo J, Lamers RP, Rubinstein JL, Burrows LL, Howell PL (2016) Structure of the Pseudomonas aeruginosa Type IVa pilus secretin at 7.4 A. Structure 24:1778–1787
Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C (2000) Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405:914–919
Korotkov KV, Sandkvist M, Hol WG (2012) The type II secretion system: biogenesis, molecular architecture and mechanism. Nat Rev Microbiol 10:336–351
Krause S, Pansegrau W, Lurz R, de la Cruz F, Lanka E (2000) Enzymology of type IV macromolecule secretion systems: the conjugative transfer regions of plasmids RP4 and R388 and the cag pathogenicity island of Helicobacter pylori encode structurally and functionally related nucleoside triphosphate hydrolases. J Bacteriol 182:2761–2770
Lill R, Dowhan W, Wickner W (1990) The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell 60:271–280
Low HH, Gubellini F, Rivera-Calzada A, Braun N, Connery S, Dujeancourt A, Lu F, Redzej A, Fronzes R, Orlova EV, Waksman G (2014) Structure of a type IV secretion system. Nature 508:550–553
Minamino T (2014) Protein export through the bacterial flagellar type III export pathway. Biochim Biophys Acta 1843:1642–1648
Pansegrau W, Bagnoli F (2017) Pilus assembly in gram-positive bacteria. Curr Top Microbiol Immunol 404:203–233
Patel R, Smith SM, Robinson C (2014) Protein transport by the bacterial Tat pathway. Biochim Biophys Acta 1843:1620–1628
Phoenix DA, Kusters R, Hikita C, Mizushima S, de Kruijff B (1993) OmpF-Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol-signal sequence interaction during protein translocation across the Escherichia coli inner membrane. J Biol Chem 268:17069–17073
Prabudiansyah I, Kusters I, Caforio A, Driessen AJ (2015) Characterization of the annular lipid shell of the Sec translocon. Biochim Biophys Acta 1848:2050–2056
Rivera-Calzada A, Fronzes R, Savva CG, Chandran V, Lian PW, Laeremans T, Pardon E, Steyaert J, Remaut H, Waksman G, Orlova EV (2013) Structure of a bacterial type IV secretion core complex at subnanometre resolution. EMBO J 32:1195–1204
Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2:a000414
Sohlenkamp C, Geiger O (2016) Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiol Rev 40:133–159
Thomas S, Holland IB, Schmitt L (2014) The Type 1 secretion pathway – the hemolysin system and beyond. Biochim Biophys Acta 1843:1629–1641
Thomassin JL, Santos Moreno J, Guilvout I, Tran Van Nhieu G, Francetic O (2017) The trans-envelope architecture and function of the type 2 secretion system: new insights raising new questions. Mol Microbiol 105:211–226
Tsirigotaki A, De Geyter J, Šoštaric N, Economou A, Karamanou S (2016) Protein export through the bacterial Sec pathway. Nat Rev Microbiol 15:21
Ulbrandt ND, London E, Oliver DB (1992) Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding. J Biol Chem 267:15184–15192
Van Gerven N, Waksman G, Remaut H (2011) Pili and flagella biology, structure, and biotechnological applications. Prog Mol Biol Transl Sci 103:21–72
Van Gerven N, Klein RD, Hultgren SJ, Remaut H (2015) Bacterial amyloid formation: structural insights into curli biogensis. Trends Microbiol 23:693–706
Weise CF, Login FH, Ho O, Grobner G, Wolf-Watz H, Wolf-Watz M (2014) Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein. Biophys J 107:1950–1961
Worrall LJ, Hong C, Vuckovic M, Deng W, Bergeron JRC, Majewski DD, Huang RK, Spreter T, Finlay BB, Yu Z, Strynadka NCJ (2016) Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body. Nature 540:597
Yan Z, Yin M, Xu D, Zhu Y, Li X (2017) Structural insights into the secretin translocation channel in the type II secretion system. Nat Struct Mol Biol 24:177–183
Zilkenat S, Franz-Wachtel M, Stierhof YD, Galan JE, Macek B, Wagner S (2016) Determination of the stoichiometry of the complete bacterial type III secretion needle complex using a combined quantitative proteomic approach. Mol Cell Proteomics 15:1598–1609
Acknowledgments
Work in our laboratory is supported by grants from Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (DGAPA, UNAM; PAPIIT IN209617) and Consejo Nacional de Ciencia y Tecnología (CONACyT 284081). We acknowledge Dra. Norma Espinosa Sánchez for critical reading of the manuscript and excellent technical assistance.
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Díaz-Guerrero, M.Á., Gaytán, M.O., González-Pedrajo, B. (2018). Structure:Function of Transmembrane Appendages in Gram-Negative Bacteria. In: Geiger, O. (eds) Biogenesis of Fatty Acids, Lipids and Membranes. Handbook of Hydrocarbon and Lipid Microbiology . Springer, Cham. https://doi.org/10.1007/978-3-319-43676-0_51-1
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