Protein Secretion in Bacteria

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 ).

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 ).

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 ).

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 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.

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.

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 ).

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.

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.

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 ).

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 ).

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 ).

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.

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.

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.