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Category: Bacterial Pathogenesis
Protein Secretion in Bacteria is now available on Wiley.comMembers, use the code ASM20 at check out to receive your 20% discount.
Protein transport into and across membranes is a fundamental process in bacteria that touches upon and unites many areas of microbiology, including bacterial cell physiology, adhesion and motility, nutrient scavenging, intrabacterial signaling and social behavior, toxin deployment, interbacterial antagonism and collaboration, host invasion and disruption, and immune evasion. A broad repertoire of mechanisms and macromolecular machines are required to deliver protein substrates across bacterial cell membranes for intended effects. Some machines are common to most, if not all bacteria, whereas others are specific to Gram-negative or Gram-positive species or species with unique cell envelope properties such as members of Actinobacteria and Spirochetes.
Protein Secretion in Bacteria, authored and edited by an international team of experts, draws together the many distinct functions and mechanisms involved in protein translocation in one concise tome. This comprehensive book presents updated information on all aspects of bacterial protein secretion encompassing:
Protein Secretion in Bacteria serves as both an introductory guide for students and postdocs and a ready reference for seasoned researchers whose work touches on protein export and secretion. This volume synthesizes the diversity of mechanisms of bacterial secretion across the microbial world into a digestible resource to stimulate new research, inspire continued identification and characterization of novel systems, and bring about new ways to manipulate these systems for biotechnological, preventative, and therapeutic applications.
Hardcover, 410 pages, full-color illustrations, index.
The envelope of bacterial cells consists of at least one—and often two—membranes, a cell wall, and possibly a surface layer. This envelope allows cells to differentiate themselves from their environment and handle the resulting osmotic pressure, but it also presents a significant obstacle. Anything a cell wishes to export, from a motility appendage to a plasmid, needs to be ushered across this barrier. To accomplish this, bacteria have evolved a battery of secretion systems. Secretion systems are often constructed from dozens of protein building blocks embedded in the cell’s envelope. The size, complexity, and location of these machines make them a particular challenge for structural characterization. High-resolution structure determination techniques such as X-ray crystallography and transmission electron microscopy (TEM)-based single-particle reconstruction (SPR) require objects to be purified from their cellular environment. This is problematic for membrane-associated proteins, which embed in the lipid bilayer by means of exposed hydrophobic patches. These patches can be protected during purification by adding detergents to the solvent, but often structural alterations still occur. Secretion systems are also unusually large targets and frequently lose peripheral or loosely-associated components during purification. In addition, they often cross two membranes and are avidly linked to the cell wall.
Protein transport occurs in all domains of life ( 1 ). Proteins that function outside the cytosol are translocated across membranes. The general system for protein translocation is formed by the Sec translocase at its core the translocon: SecYEG in bacteria ( 2 ), SecYEβ in archaea ( 3 ), and Sec61αβγ in the endoplasmic reticulum of eukaryotes ( 4 , 5 ). The translocon forms a protein conducting channel in the membrane for unfolded preproteins ( 6 ) but also mediates cotranslational insertion of nascent membrane proteins into the membrane ( Fig. 1 ).
The protein export systems of bacteria deliver proteins from the cytoplasm to the cell envelope or extracellular environment, and in doing so, they play critical roles in bacterial physiology and pathogenesis. In bacteria, the majority of protein export is carried out by the general Sec system ( 1 , 2 ). The core components of the Sec system are the integral membrane proteins SecY, SecE, and SecG, which form the SecYEG channel through which unfolded proteins traverse the membrane, and the SecA ATPase, which provides energy for export ( Fig. 1A ). SecA shuttles between the cytoplasm and SecYEG in its role in export. SecDFYajC are auxiliary components that enhance export efficiency. Proteins exported by the Sec pathway are synthesized as preproteins with N-terminal signal peptides that are recognized by the Sec machinery and removed during export to produce the mature protein. Some Gram-positive bacteria, including high-GC Gram-positive actinobacteria such as mycobacteria, possess two SecA proteins. In these cases, SecA (sometimes called SecA1) is the canonical SecA of the Sec pathway, while SecA2 functions in a specialized pathway that exports one or a few proteins. There are at least two evolutionarily and mechanistically distinct types of SecA2 systems: the accessory Sec (aSec) system, which has also been referred to as the SecA2/SecY2 system, and the multisubstrate SecA2 system, which was initially called the SecA2-only system.
Membrane proteins constitute between 20–30% of the cellular proteome ( 1 ) and perform critical functions like signal transduction, molecular transport, and cell adhesion. The molecular machineries that catalyze their targeting, insertion, and assembly in the different cellular and subcellular membranes are remarkably conserved. The Sec translocon is responsible for moving the majority of the proteins across/into the bacterial, archaeal, thylakoidal, and endoplasmic reticulum (ER) membranes in an unfolded state ( 2 ). In bacteria, it is proposed to form a holocomplex composed of the heterotrimeric protein channel SecYEG and the accessory elements SecDFYajC, SecA ATPase, and YidC ( 3 ).
About 20 to 30% of proteins synthesized in the bacterial cytoplasm are destined for extracytoplasmic locations ( 1 ). They pass the cytoplasmic membrane using specialized transport systems, involving gated pores, energy, and signal peptides to direct protein export. Two major protein export systems are known, namely, the general secretory (Sec) pathway and the twin-arginine translocation (Tat) pathway ( Fig. 1 ). Most proteins use the Sec pathway, common to all domains of life. The Tat pathway, the focus of this review, is more exclusive. For example, it has only ∼30 native substrates in the Gram-negative bacterium Escherichia coli, and it is not universally conserved ( 2 ).
Lipoproteins are a family of secreted proteins that are acylated after their translocation across the plasma membrane ( 1 – 3 ). Acylation spatially confines lipoproteins by anchoring them into membranes. Lipoproteins are bioinformatically identifiable by the highly conserved lipobox motif in their short signal peptides ( 4 ). Within the lipobox is a cleavage site for signal peptidase II (SPII; Lsp). Immediately adjacent is an invariant Cys residue which is the target of acylation reactions. Most lipoproteins are secreted from the cytosol via the SecYEG translocon ( 5 – 7 ), though secretion via the twin-arginine transport (Tat) system has also been identified ( 8 – 11 ). Following translocation, the inner membrane (IM) enzyme Lgt attaches a diacyl moiety to the lipobox Cys of prolipoproteins via a thioester linkage ( Fig. 1 ) ( 12 – 14 ). The diacylated product is a substrate for Lsp, which releases the apolipoprotein from its signal peptide ( Fig. 1 ) ( 15 – 17 ). The diacylated Cys residue then becomes the first amino acid of the lipoprotein (Cys+1). In Gram-negative bacteria, a third acyl group is attached by the enzyme Lnt to the Cys+1 amino group (which was made available following Lsp cleavage) ( Fig. 1 ) ( 18 – 21 ). The acyl chain donors in Lgt and Lnt reactions are plasma membrane phospholipids ( Fig. 1 ). Gram-negative bacteria produce triacylated lipoproteins; lnt , lsp , and lgt are therefore conserved and essential in the majority of these organisms. Low-GC Gram-positive bacteria lack lnt homologs and generate considerable diversity in lipoprotein acylation; in addition to the triacyl form, these bacteria can variously generate diacyl, lyso, peptidyl, and N-acetyl lipoprotein forms ( 22 , 23 ) ( Fig. 1 ). How such diversity is generated largely awaits discovery, although recent progress has identified the enzyme, Lit, that is responsible for producing lyso-form lipoproteins in Enterococcus faecalis and Bacillus cereus ( 24 ).
Spirochetes form a distinct bacterial phylum of slender, diderm (dual-membrane) bacteria that exhibit either a coiled “corkscrew” or flat-wave “serpentine” morphology. These distinct phenotypes are at least partly due to various numbers of periplasmic flagella that are inserted subterminally at both poles of the bacteria, wrapping around the protoplasmic cylinder and often overlapping in the middle of the cell. Coordinated rotation of the flagellar bands or bundles, sometimes referred to as axial filaments, leads to rotation of the cell cylinder and cellular motility that is particularly prominent in viscous environments.
The presence in Gram-negative bacteria of an extracytoplasmic outer membrane (OM), which is distinct from the inner membrane (IM) both in constitution and in function, presents a complex topological problem, as all proteinaceous and lipidic OM components are synthesized cytoplasmically ( 1 ). In order to reach their destination in the growing OM, these components must translocate across the IM and traverse the aqueous, crowded periplasmic space. This problem is solved through a series of semi-independent and highly conserved transport pathways that coordinate the efficient delivery and integration of all OM constituents.
The vast majority of integral membrane proteins residing within the outer membrane of Gram-negative bacteria adopt a β-barrel architecture. Mechanistically, how these proteins fold remains uncertain, but the process requires assistance from at least two nanomachines: the translocation and assembly module (TAM) and the β-barrel assembly machinery (BAM) complex ( 1 – 3 ). But whether the TAM and the BAM complex collaborate or act independently on each nascent membrane protein substrate arriving at the outer membrane has yet to be determined. The TAM is comprised of two subunits: TamA, an integral outer membrane protein ( 2 , 4 , 5 ), and TamB, an inner membrane-anchored protein ( 2 ). The BAM complex is variable in composition between genera, and it is comprised of 2 to 5 accessory lipoproteins attached to an integral outer membrane protein, BamA ( 1 , 6 , 7 ).
The fundamental type IVa pilus (T4aP)-like architecture includes a retractable pilus fiber, a motor, an alignment subcomplex, and—in Gram-negative bacteria—an outer membrane secretin pore ( Fig. 1 ). The pilus is an extracellular polymer of pilins. Pilins subunits are stored in the inner membrane and the motor powers their polymerization (extension) and depolymerization (retraction) at the pilus base. The alignment subcomplex connects the secretin with the motor and controls pilus dynamics. Finally, the secretin pore allows the pilus to extend through the outer membrane. Since publication of previous T4aP reviews ( 1 – 4 ), discoveries made using cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), X-ray crystallography, and nuclear magnetic resonance (NMR) have dramatically reshaped our understanding of T4P-like systems. Here we put these discoveries in context with the structure and function of the T4aP, using the Pseudomonas aeruginosa T4aP system nomenclature.
In recent years, numerous complete bacterial genome sequences became available and led to the identification of surprisingly diverse type IV pili (T4P) across a broad range of Gram-positive bacteria. The genes encoding T4P components cluster together in distinct loci, and three subsets of T4P loci present in Gram-positive bacteria have been described: (i)pil (pilin) loci, (ii)com (competence) loci, and (iii)tad (tight adherence) loci ( 1 ). Interestingly, they are not mutually exclusive. In fact, many Gram-positive bacteria harbor a combination of pil, com, and tad loci, suggesting diverse functional roles for T4P in Gram-positive bacteria ( 1 ). pil loci are commonly found in Clostridium spp., and clostridial T4P are best studied for Clostridium perfringens and Clostridium difficile ( 2 , 3 ). com loci are widespread in Firmicutes, among Bacillales and Lactobacillales. tad loci are present in archaea and Gram-negative and Gram-positive bacteria. Proteins of the tad system assemble adhesive fimbrial low-molecular-weight protein (Flp) pili that are largely unexplored in Gram-positive bacteria ( 4 ). pil and tad loci are not extensively discussed here. Instead we focus on the com operon present in the human respiratory pathogen Streptococcus pneumoniae and containing the genes involved in the formation of the pneumococcal type IV pilus, also referred to as competence pilus or transformation pilus.
Chaperone-usher (CU) pili are virulence factors displayed on a wide variety of Gram-negative bacterial pathogens ( 1 ), mediating bacterial attachment and biofilm formation ( 2 ). The two best-studied examples of CU pili are the type 1 and P pili of uropathogenic Escherichia coli (UPEC), which is the most important causative agent of urinary tract infections ( 3 ). We here summarize the steps of CU pilus biogenesis and highlight the most recent structural advances relating to type 1 pili that allow UPEC to thrive in the urinary tract.
The chaperone-usher (CU) pathway is dedicated to the biogenesis of surface structures termed pili or fimbriae that play indispensable roles in the pathogenesis of a wide range of bacteria ( 1 – 4 ). Pili are hair-like fibers composed of multiple different subunit proteins. They are typically involved in adhesion, allowing bacteria to establish a foothold within the host. Following attachment, pili modulate host cell signaling pathways, promote or inhibit host cell invasion, and mediate bacterium-bacterium interactions leading to formation of community structures such as biofilms ( 5 , 6 ). Gram-negative bacteria express multiple CU pili that contribute to their ability to colonize diverse environmental niches ( 1 , 7 – 10 ). Pili thus function at the host-pathogen interface to both initiate and sustain infection and represent attractive therapeutic targets.
Curli are extracellular proteinaceous fibers made by Gram-negative bacteria. Curli-specific genes (csg) are primarily found in Proteobacteria and Bacteroidetes ( 1 – 3 ). The main function of curli fibers is associated with a sedimentary lifestyle and multicellular behavior in biofilms, as they form scaffolds that provide adhesive and structural support to the community ( 4 – 8 ). In certain pathogenic bacteria, curli have also been implicated in host colonization, innate response activation, and cell invasion ( 9 – 13 ).
Prior to bacterial genome sequencing and the genetic analysis of pathogenesis, microbiologists identified molecules on microbial surfaces and studied their role in disease processes ( 1 ). The ultimate goal of this research was the identification of molecular formulations inciting antibody responses in vaccine recipients that prevented disease yet would otherwise not cause harm ( 2 ). Oswald Avery’s discovery of the pneumococcus capsule and the demonstration that capsular polysaccharide vaccine protects against pneumococcal pneumonia represent an important paradigm ( 3 , 4 ). Another was Rebecca Lancefield’s characterization of M protein as the determinant of type-specific immunity against Streptococcus pyogenes, the causative agent of streptococcal pharyngitis and rheumatic fever ( 2 ). Lancefield and Sjöquist required proteases or peptidoglycan (murein) hydrolases, but not membrane detergents, to solubilize surface proteins of Gram-positive bacteria ( 2 , 5 , 6 ). The underlying reason for this biochemical phenomenon is that surface proteins are covalently linked to peptidoglycan at their C-terminal ends ( 7 , 8 ).
The flagellum is a major organelle for motility in many bacterial species. It confers locomotion and is often associated with virulence of bacterial pathogens. Flagella from different species share a conserved core but also exhibit profound variations in flagellar structure, flagellar number, and placement ( 1 , 2 ), resulting in distinct flagella that appear to be adapted to the specific environments that the bacteria encounter. While many bacteria possess multiple peritrichous flagella, such as those found in Escherichia coli and Salmonella enterica, other bacteria, such as Vibrio spp. and Pseudomonas aeruginosa, normally have a single flagellum at one cell pole ( Fig. 1 ). Spirochetes uniquely assemble flagella that are embedded in periplasmic space between their inner and outer membranes, thus called periplasmic flagella ( 3 ). Although the flagella of E. coli and Salmonella have been extensively studied for several decades, periplasmic flagella are less understood, despite their profound impact on the distinctive morphology and motility of spirochetes. In this chapter, many aspects of periplasmic flagella are discussed, with particular focus on their structure and assembly.
Outer membrane vesicles (OMVs) are nanosized, spherical proteoliposomes. They are secreted via vesiculation of the outer membrane by Gram-negative bacteria as part of the normal growth process ( 1 ). OMVs play diverse roles in intracellular communication, microbial virulence, and modulation of the host immune response ( 2 ).
Gram-negative bacteria are equipped with at least seven dedicated secretion systems that mediate the export of proteins beyond the outer membrane ( 1 , 2 ). These are called type 1 to 6 and type 9 secretion systems (T1SS to T6SS and T9SS). Among those, T3SS, T4SS, and T6SS are even capable of delivering their cargo directly into the cytosol of the host cell. In this minireview, we place the major emphasis on the hemolysin A (HlyA) secretion system in Escherichia coli. This is by far the most studied and illustrates very well the largely conserved, essential features of T1SS. Interestingly, however, an important mechanistic variation in the translocation of some of the unusually extended giant RTX proteins—adhesins—was discovered recently ( 3 ) and is also discussed.
The type II secretion system (T2SS) is one of several extracellular secretion systems in Gram-negative bacteria. While highly prevalent in gamma- and betaproteobacteria, the T2SS is also recognized to a lesser extent in members of the delta and alpha classes ( 1 , 2 ). It is known for its prolific protease secretion activity. In addition, the T2SS mediates extracellular delivery of a variety of toxins, lipases, and enzymes that break down complex carbohydrates, thus conferring a survival advantage to pathogenic as well as environmental species ( 2 – 4 ). The T2SS is not restricted to extracellular pathogens, such as Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae; it is also present and contributes to growth of intracellular pathogens, including Legionella pneumophila, which replicates in aquatic amoebae, alveolar macrophages, and epithelial cells ( 5 – 7 ). The obligate intracellular pathogen Chlamydia trachomatis also depends on T2SS components for extracellular secretion; however, its T2SS is atypical, as some components are missing or are too different from homologs in other species to be identified using BLAST algorithms ( 8 , 9 ).
Type III protein secretion systems (T3SSs) are multiprotein nanomachines present in many Gram-negative bacteria with a close relationship with a eukaryotic host. The primary function of these machines is the delivery of bacterially encoded effector proteins into target eukaryotic cells ( 1 – 4 ), to modulate a myriad of cell biological processes for the benefit of the bacteria that encode them ( 5 , 6 ). T3SSs are widespread in nature, playing a central role in the pathogenic and symbiotic interactions between many bacteria and their hosts. Among the bacteria that encode T3SSs are many important human and plant pathogens. As the field has progressed, so has the amount of information available, precluding a comprehensive review of the literature. Therefore, here we focus on the structural and architectural aspects of the type III system. To reflect current knowledge, and to help the reader better understand the structural organization of this machine, we refer throughout to the complete type III secretion machine as the injectisome, and we describe in detail the different substructures that integrate it (i.e., the needle complex [NC], the export apparatus, and the sorting platform). Readers are referred to other reviews for more specific aspects of the structure and function of these secretion machines ( 1 – 4 ).
Antibiotic resistance is a great and growing threat to public health, motivating scientists to find innovative strategies to cure infections ( 1 – 3 ). An alternative approach to classical antibiotics is to target virulence factors ( 4 ): bacterial factors required for infection or damage but not for growth outside the host ( 2 , 5 , 6 ). An antivirulence factor should render the bacteria nonpathogenic by neutralizing a critical virulence element, thereby allowing clearance of the pathogen by the host immune system ( 5 – 8 ).
The bacterial type IV secretion systems (T4SSs) are a large, versatile family of macromolecular translocation systems functioning in Gram-negative (G−) and Gram-positive (G+) bacteria ( 1 ). These systems mediate the transfer of DNA or monomeric or multimeric protein substrates to a large range of prokaryotic and eukaryotic cell types ( Fig. 1A ). Conjugation systems, the earliest described subfamily of T4SSs ( 2 ), transfer mobile genetic elements (MGEs) between bacteria. They pose an enormous medical problem because MGEs often harbor cargoes of antibiotic resistance genes and fitness traits that endow pathogens with antibiotic resistance and other growth advantages under selective pressures ( 3 – 5 ). Effector translocators, a more recently described T4SS subfamily ( 6 , 7 ), are deployed by pathogenic bacteria to deliver effector proteins to eukaryotic cells during the course of infection ( 8 – 11 ). The conjugation and effector translocator systems, as well as newly discovered interbacterial killing systems, transmit their cargos through direct donor-target cell contact ( 12 – 14 ). A few other T4SSs designated uptake or release systems acquire DNA substrates from the milieu or release DNA or protein substrates into the milieu ( Fig. 1A ) ( 1 , 6 ).
Multiple intracellular pathogens utilize the type III secretion system (T3SS) and type IV secretion system (T4SS) to target functions of the host cell’s endoplasmic reticulum (ER). While pathogens such as Legionella pneumophila and Brucella abortus have long been known to replicate in association with the ER ( 1 , 2 ), the connection of vacuoles containing other intracellular pathogens, such as Coxiella burnetii ( 3 , 4 ), Anaplasma spp. ( 5 , 6 ), and Chlamydia trachomatis and its relatives ( 7 , 8 ), with the ER has been recognized relatively recently. However, manipulation of ER function is not limited to pathogens that replicate within a vacuole, as cytosolic pathogens such as Orientia tsutsugamushi ( 9 , 10 ) and Rickettsia rickettsii ( 11 ) also target ER-based functions via secreted effectors to promote their intracellular growth.
Type V, or “autotransporter,” secretion is an umbrella term that is often used to refer to a group of distinct but conceptually related protein export pathways that are widely distributed in Gram-negative bacteria. Autotransporters are generally single polypeptides that contain a signal peptide that promotes translocation across the inner membrane (IM) via the Sec pathway, an extracellular (“passenger”) domain, and a domain that anchors the protein to the outer membrane (OM). Passenger domains have a wide variety of functions, but they often promote virulence ( 1 ). In the archetypical, or “classical” (type Va), autotransporter pathway, which was discovered in 1987, the passenger domain is located at the N terminus of the protein adjacent to the signal peptide ( 2 ). Although passenger domains range in size from ∼20 to 300 kDa and are highly diverse in sequence ( 3 ), X-ray crystallographic and in silico studies predict that they usually fold into a repetitive structure known as a β helix ( 4 – 8 ) ( Fig. 1 ). The membrane anchor domains are ∼30 kDa and are also highly diverse in sequence but contain short conserved sequence motifs ( 3 , 9 ). Like most membrane-spanning segments associated with OM proteins (OMPs), these domains fold into a closed, amphipathic β sheet or “β barrel” structure. The C-terminal domains that have been crystallized to date all form nearly superimposable 12-stranded β barrels ( 10 – 15 ). The two domains are connected by a short α-helical “linker” that is embedded inside the β barrel domain ( 10 , 12 , 13 , 16 ). Many passenger domains are released from the cell surface by a proteolytic cleavage following their secretion ( 17 ).
Bacteria use surface molecules to interact with inanimate objects during biofilm development, other bacteria during sociomicrobiological community activities, and host organisms during mutualistic, commensal, and parasitic symbioses. Among the mechanisms for delivering proteins to the surface of Gram-negative bacteria are type V secretion systems (T5SS) ( 1 – 3 ). T5SS comprise a passenger domain and an associated β-barrel transporter domain that, once integrated into the outer membrane via the Bam assembly complex, is sufficient for export of the passenger from the periplasm to the cell surface. Based on domain architecture, T5SS are categorized into five classes, with type Vb or two-partner secretion (TPS) pathway systems being distinct because the passenger domain (referred to generically as a TpsA protein) is synthesized independently from the transporter domain (the TpsB protein). This arrangement requires a mechanism for passenger-transporter recognition in the periplasm and may allow reuse of the transporter for export of multiple copies of the same, or closely related, passenger proteins.
The type VI secretion system (T6SS) is a multiprotein machine that belongs to the versatile family of contractile injection systems (CISs) ( 1 – 4 ). CISs deliver effectors into target cells using a spring-like mechanism ( 4 – 6 ). Briefly, CISs assemble a needle-like structure, loaded with effectors, wrapped into a sheath built in an extended, metastable conformation ( Fig. 1 ). Contraction of the sheath propels the needle toward the competitor cell. Genomes of Gram-negative bacteria usually encode one or several T6SSs, with an overrepresentation in Proteobacteria and Bacteroidetes ( 8 – 10 ; for a review on the role of T6SS in gut-associated Bacteroidales, see the chapter by Coyne and Comstock [ 7 ]). The broad arsenal of effectors delivered by T6SSs includes antibacterial proteins such as peptidoglycan hydrolases, eukaryotic effectors that act on cell cytoskeleton, and toxins that can target all cell types, such as DNases, phospholipases, and NAD+ hydrolases ( 11 – 14 ). Consequently, the T6SS plays a critical role in reshaping bacterial communities and, directly or indirectly, in pathogenesis ( 15 – 19 ). Destroying bacterial competitors also provides exogenous DNA that can be acquired in naturally competent bacteria and that serves as a reservoir for antibiotic resistance gene spread ( 20 ). This chapter lists the major effector families and summarizes the current knowledge on the assembly and mode of action of the T6SS.
Type VI secretion systems (T6SSs) were first identified and characterized for pathogenic bacteria of the proteobacterial phylum ( 1 , 2 ). The discovery in 2010 that these secretion systems can target and intoxicate not only eukaryotic cells but also other bacteria ( 3 ) revealed that some T6SSs help bacteria compete with other bacteria in a community setting. Indeed, many proteobacterial symbionts, including the plant symbiont Pseudomonas putida, the bumble bee gut symbiont Snodgrassella alvi, and the squid symbiont Vibrio fischeri, all have T6SSs that provide a competitive advantage in their natural ecosystems ( 4 – 6 ). An early in silico analysis using clusters of orthologous groups (COGs) models of proteobacterial T6SS proteins against primary sequence databases suggested that T6SSs are largely confined to proteobacterial species, which are minor members of some human-associated microbial communities such as the gut microbiota ( 7 , 8 )
The different bacterial species within the tree of life ( 1 ) possess a range of secretion systems, which play important roles in the transport of proteins across the various types of bacterial cell envelopes. Classically, Gram staining was used for differentiating Gram-positive and Gram-negative bacteria, but classifications on cell envelope architecture might come closer to the biological reality, and thus, bacteria may also be differentiated according to their cell envelopes into diderm-lipopolysaccharide (archetypal Gram-negative), monoderm (archetypal Gram-positive), and diderm-mycolate (archetypal acid-fast) bacteria ( 2 ). For Gram-negative bacteria a range of at least eight different secretion systems has been described (types I to VI, VIII, and IX) ( 3 – 5 ). While in monoderm bacteria secretion and export are synonymous, in diderm bacteria the secretion is completed only upon translocation of the substrates across the outer membrane ( 2 ). The here-reviewed mycobacterial ESAT-6 secretion (ESX) systems ( 6 , 7 ), which were also named type VII secretion (T7S) systems ( 8 ), represent a particular class of protein export and/or secretion systems, for which at present only the inner-membrane translocation machinery has been explored in more detail ( 9 , 10 ), whereas it remains unknown how ESX/T7S-exported proteins get transported through the mycobacterial outer membrane into the extracellular environment ( 11 ). Indeed, one of the remarkable characteristics of mycobacteria is their complex cell envelope, which is shared to some extent with other members of the Corynebacterineae, a suborder of the phylum Actinobacteria ( 1 , 12 , 13 ). Mycobacteria are surrounded by a diderm cell envelope, consisting of an inner membrane, a peptidoglycan layer, an arabinogalactan layer, an outer membrane, named mycomembrane, which is composed of covalently linked mycolic acids and extractable lipids, and a capsule ( 14 , 15 ). This unusual cell envelope requires complex secretion systems for the export/secretion of proteins, such as those of the SecA and twin-arginine translocation pathways, as well as the specialized ESX/T7S systems ( 7 , 8 , 16 ), which were first discovered almost 20 years ago during in silico analyses of the genome sequence and the proteome of Mycobacterium tuberculosis H37Rv ( 17 , 18 ). Moreover, T7S-like systems that share some core components of mycobacterial ESX/T7S systems exist in various genera of the phylum Firmicutes, representing many classical Gram-positive bacterial species ( 19 ), which, however, are not the subject of the current review.
Members of the phylum Bacteroidetes have many unique features, including novel machinery for protein secretion and gliding motility ( 1 – 3 ). Most members secrete proteins across the outer membrane (OM) using the type IX protein secretion system (T9SS), which is confined to this phylum. Many also crawl rapidly over surfaces by gliding motility. For these gliding bacteria, the motility machinery and T9SS appear to be intertwined. Here we explore gliding motility, the T9SS, and the connections between them.
The cell envelope of Gram-negative bacteria, such as Escherichia coli, is characterized by the presence of two membranes, the inner (IM) and outer (OM) membranes, separated by the periplasm and a thin layer of peptidoglycan (PG). This envelope is a formidable barrier against a myriad of harmful compounds, while simultaneously allowing the entry of nutrients necessary for cell survival. However, this barrier, like the Maginot Line in France during the Second World War, is not completely impenetrable, and exogenous particles, including some toxins and viruses, can pierce it.
Bacteria utilize sophisticated nanomachines to transport proteins, small molecules, and DNA across membranes to the extracellular environment. These transport machineries, also known as secretion systems, are involved in various cellular functions, such as adhesion to surfaces or host cells, cell-cell communication, motility (flagella), virulence effector protein secretion, and, notably, bacterial pathogenesis ( 1 – 5 ). Several of the identified protein secretion systems comprise large complexes that localize and assemble in and around the bacterial membrane(s), forming specialized channels through which the selected substrate(s) is actively delivered ( 6 – 9 ). Although exhibiting significant diversity in structure, substrate, and function, the dedicated type II, III, IV, and IV-pilus secretion systems (T2SS, T3SS, T4SS, and T4PS, respectively) in didermic Gram-negative bacteria each transport a specific subset of proteins to the extracellular milieu via passage through large stacked ring-shaped channels that span the inner membrane (IM) and outer membrane (OM).
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