The type 2 secretion (T2S) and the type 4 pilus (T4P) systems are two broadly conserved systems that play important roles in the virulence of pathogenic Escherichia coli and many other bacteria. These systems have been identified in enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and non-pathogenic E. coli strains. The T2S system exports exotoxins and important cell-surface anchored lipoprotein enzymes that digest components of the host cell, including proteins, lipids, and sugars (Economou et al., 2006). The T4P system produces pili that are involved in adherence, motility, and aggregation. Both systems consist of multicomponent machines that are broadly distributed but play distinct roles in bacterial pathogenesis. Components of the T2S and T4P machines are homologous in sequence, structure, and function. Only a few key differences account for the ability of T4P systems to anchor polymerized substrates in the inner membrane (IM) as opposed to the secretion of individual, folded substrates across the outer membrane (OM) by T2S systems. Under artificial conditions, T4P systems can assemble T2S pseudopilins into pili (Sauvonnet et al., 2000).

T2S represents one of at least six secretion systems in Gram-negative bacteria that function in the export of proteins to the extracellular environment (Economou et al., 2006). T2S has been referred to as the main terminal branch of the general secretory pathway (Gsp) and the second part of a two-step process. The first step is transport of the exoprotein across the IM by the Sec or Tat pathway (Cianciotto, 2005). The substrate precursor contains an N-terminal signal peptide that targets the protein to be transported through the IM. The signal peptide is cleaved off and the protein is released into the periplasm where it folds into its functional confirmation. The protein is then transported by the T2S across the OM into the extracellular environment through a pore formed by the secretin protein (Genin and Boucher, 1994).

The T2S system in E. coli consists of 12 core proteins found in all T2S systems and two accessory proteins (Sandkvist, 2001). Mutations in genes encoding most of these proteins result in defects in the secretion process and accumulation of exoproteins in the periplasm (Filloux, 2004). There are chromosomal genes in the non-pathogenic E. coli K-12 and most other strains that encode a T2S system; however, under standard conditions secretion does not occur. The operon encoding this system is silenced by the nucleoid-structuring protein H-NS, and mutants deficient for H-NS can express the T2S system (Francetic et al., 2000). In E. coli pathogens, proteins secreted by accessory T2S systems play multiple roles, including adherence to eukaryotic cells and damage to host tissues. Examples include the EHEC StcE metalloprotease, which promotes adherence to host eukaryotic cells (Grys et al., 2005) and the ETEC heat-labile toxin (LT), a multimeric AB₅ enterotoxin functionally and structurally similar to cholera toxin of Vibrio cholerae (Sixma et al., 1993) that induces fluid secretion and promotes colonization of intestinal epithelia (Tauschek et al., 2002).

T4P are filamentous surface appendages required for adherence, motility, aggregation, and transformation in a wide array of bacterial and archaeal species. T4P were first identified in Pseudomonas aeruginosa and Myxococcus xanthus (Ottow, 1975). Further studies revealed that all T4P have a conserved set of core proteins (Pelicic, 2008). T4P are long, thin, flexible, polymeric, three-start helical filaments approximately 85 Å in diameter (Craig et al., 2004; Ramboarina et al., 2005). The pili are composed primarily of the major pilin protein (Craig et al., 2004), and are assembled by a complex biogenesis machine consisting of 10–18 proteins that spans both bacterial membranes (Sohel et al., 1996; Stone et al., 1996; Hwang et al., 2003; Peabody et al., 2003; Pelicic, 2008). Major pilins are first synthesized as precursor prepilins and then processed by a prepilin peptidase to create mature pilin proteins incorporated into the pilus (Strom et al., 1993).

T4P can be subdivided into two distinct classes, type 4 class A pili (T4aPs) and type 4 class B pili (T4bPs), based on differences in component structure, genetic organization, distribution, and function. T4bPs are characterized by larger major pilins compared to those of T4aPs that begin with an amino acid other than phenylalanine and are encoded in a contiguous operon often found on plasmids (Craig et al., 2004). As with the common T2S system, most strains of E. coli have the genes for a T4aP that has been called, somewhat confusingly, the hemorrhagic coli pilus (HCP) (Xicohtencatl-Cortes et al., 2007). In addition, some E. coli pathotypes express accessory T4bPs that play important roles in infection.

History

The T2S system was initially discovered in Klebsiella oxytoca and Pseudomonas aeruginosa and is widely conserved among Gram-negative bacteria (Filloux et al., 1990). Sequencing of plasmid pO157 from EHEC identified a T2S system conserved among all EHEC O157 and some non-O157 strains (Schmidt et al., 1997). A silenced T2S operon was also found in an E. coli K-12 strain (Blattner et al., 1997). This system can secrete endochitinase ChiA when the silencing protein is inactivated (Francetic et al., 2000). In ETEC a T2S system highly similar to that responsible for cholera toxin secretion in Vibrio cholerae was discovered to secrete LT (Tauschek et al., 2002). This gene cluster is absent from E. coli K12, however the K12 genome appears to have once contained this sequence.

Although discovered years earlier in other bacteria, T4P where first identified in E. coli with the discovery of the EPEC bundle-forming pilus (BFP) (Girón et al., 1991). Subsequent identification of the gene encoding the major structural subunit confirmed its similarity to other T4P pilin genes and suggested the presence of a large operon encoding genes required for pilus processing and biogenesis (Donnenberg et al., 1992). Additional E. coli T4P were discovered subsequently.

Distribution

Both T2S and T4P are produced by a diverse number of Gram-negative bacteria including many plant, animal, and human pathogens. Within E. coli, one T2S system and the HCP are ubiquitous, while additional T2S and T4P systems are found on specific subsets of pathotypes (Girón et al., 1993, 1994; Schmidt et al., 1997; Tauschek et al., 2002).

The components of T4P systems share significant sequence similarity and structural homology with components of T2S systems, DNA uptake systems (Averhoff and Friedrich, 2003; Peabody et al., 2003), and filamentous phage assembly systems (Linderoth et al., 1996; Russel et al., 1997). T4P assembly components also are orthologous to proteins involved in archaeal flagellum assembly (Peabody et al., 2003). The sequence and structural similarities across such a wide range of organisms strongly suggest an ancient and shared evolutionary history (Peabody et al., 2003; Pelicic, 2008). T4Ps have also been found in Gram-positive (Varga et al., 2006; Rodgers et al., 2011) and archaeal species (Herdendorf et al., 2002; Bardy and Jarrell, 2003).

Only a few T4bP systems have been identified in E. coli, where some are virulence factors used for adherence to eukaryotic cells, an essential first step for colonization. This is evident in the decreased virulence of EPEC strains deficient for functional pili (Bieber et al., 1998). The best-characterized system is the BFP of EPEC. Through the remainder of this chapter we will refer to the BFP system as the model of T4bP biogenesis, referring to other systems primarily to highlight differences when they occur. In addition to BFP, two related T4Ps have been identified in ETEC, the colonization factor antigen III (CFA/III) and Longus systems. Another system called the R64 pilus is encoded on a plasmid, and a T4aP called the hemorrhagic coli pilus or HCP has been described in EHEC, although the genes encoding HCP are ubiquitous among E. coli strains (Xicohtencatl-Cortes et al., 2007).

BFP fibers are produced from an operon of 14 genes encoded on the large (∼90 kb) adherence factor plasmid (pEAF) (Nataro et al., 1987; Stone et al., 1996). The BFP is a confirmed virulence factor (Bieber et al., 1998) that mediates the initial stages of adherence to the host intestinal epithelium (Cleary et al., 2004; Hyland et al., 2006a; Zahavi et al., 2011). CFA/III fibers are peritrichous pili that are 5–10 µm long and are often encoded on a large plasmid (Honda et al., 1984; Shinagawa et al., 1993). Some ETEC strains produce the Longus pilus, so named because it can grow over 20 µm in length, which is essentially allelic to CFA/III (Girón et al., 1994). The Longus T4P display a polar distribution on the cell surface (Girón et al., 1994). The N-termini of the major pilin proteins in each system, CofA and LngA, and of all accessory genes are nearly identical (Taniguchi et al., 1995; Gomez-Duarte et al., 2007). Despite their similarity, these pilus systems are expressed under different conditions and by different serogroups of ETEC (McConnell et al., 1989; Girón et al., 1995). The R64 pilus is encoded on a large conjugative plasmid. Unlike its counterparts in EPEC and ETEC, the R64 pilus has not been shown to have a role in adherence but rather is specialized for bacterial conjugation in liquid media (Yoshida et al., 1998). The HCP can grow to over 10 µm in length and is involved in adherence of EHEC to host cells (Xicohtencatl-Cortes et al., 2007).

Introduction

The T2S and T4P machines are structurally and functionally homologous (Craig et al., 2004; Ayers et al., 2009; Korotkov et al., 2012). These complexes can be conceptualized as consisting of three distinct subcomplexes: the inner membrane (IM) subassembly associated with at least one cytoplasmic ATPase; the OM subassembly which consists of a transmembrane pore; and the translocated substrate. Remarkably few differences between the T2S and T4P machines account for their capacity to process very different substrates: the T2S extrudes fully folded proteins likely driven through the pore by a pseudopilus while the T4P system substrate is itself a pilus. Variations and accessory proteins do exist and are discussed after the conserved components. All components are listed using the General Secretory Pathway (Gsp) nomenclature used for ETEC (Table 13.1).

Structure and function of type 2 secretion systems in E. coli

The T2S pseudopilus is believed to interact with substrate exoproteins in the periplasmic vestibule of the secretin and force the exoprotein into the extracellular space through an as yet undocumented piston mechanism (Reichow et al., 2011). The pseudopilus consists of the major pseudopilin, GspG, and minor pseudopilins GspH, I, J, and K, all of which are translocated across the IM via the Sec-dependent pathway (Francetic et al., 2007). A short, positively charged segment at the N-terminal region of each pseudopilin is cleaved off by the prepilin peptidase GspO (Bally et al., 1992; Dupuy et al., 1992). The peptidase is an aspartic acid protease with eight putative transmembrane helices and its catalytic residues in cytoplasmic loops (LaPointe and Taylor, 2000). The enzyme is bifunctional and also N-methylates the cleavage product (Strom et al., 1993). The pseudopilins GspI, J, and K appear to be incorporated as a trimer at the tip of the pseudopilus (Korotkov and Hol, 2008; Douzi et al., 2009) with GspK at the top and GspI/J forming the base. How monomers of GspG and the pseudopilin GspH are added to the pseudopilus remains unknown. All five pseudopilins have markedly different globular structures but share common features: an N-terminal alpha helix (the first half of which is hydrophobic) followed by a variable region and a conserved beta-sheet that varies in length with the various proteins.

T2S biogenesis requires the insertion of the IM proteins, GspC, F, L, and M, into the IM. These proteins create a complex together and appear to protect each other from proteolysis (Sandkvist et al., 1999). GspC contains a short cytoplasmic segment, a transmembrane helix, and two periplasmic domains: the homology region (HR) and PDZ domains (Bleves et al., 1999) although the PDZ domain is not universal across all T2S systems (Korotkov et al., 2006). The HR domain of GspC interacts with the periplasmic N-terminal regions of the secretin, GspD (Korotkov et al., 2011). The HR domain may also influence formation of the IM complex (Lybarger et al., 2009). GspF is the only polytopic protein in the complex, consisting of two cytoplasmic domains sharing some sequence homology and three transmembrane helices (Abendroth et al., 2009a,b). As discussed above, this polytopic transmembrane protein is one of the most highly conserved members of the T2S system and related machines (Peabody et al., 2003).

The GspL component recruits pseudopilins processed by GspO, the prepilin peptidase, to the IM complex (Sandkvist et al., 1995) and also recruits the ATPase, GspE, to the cytoplasmic face of the IM (Sandkvist et al., 2000; Abendroth et al., 2005). GspE is a member of a large ATPase family (Robien et al., 2003) providing the necessary energy for the secretion apparatus to function through ATP hydrolysis and, unlike most other proteins of its type contains zinc (Camberg and Sandkvist, 2005). The protein contains typical Walker boxes A and B (Planet et al., 2001) and is active as a hexamer (Satyshur et al., 2007). GspM contains a short cytoplasmic sequence, a transmembrane helix, and a ferrodoxin fold in its periplasmic domain that could serve as a binding site for another protein (Abendroth et al., 2004b). GspM interacts directly with GspL through contacts in the transmembrane domain and periplasm (Sandkvist et al., 1999; Py et al., 2001).

The secretin GspD is part of a large protein superfamily of multimeric, pore-forming OM proteins (Linderoth et al., 1996; Collins et al., 2004; Reichow et al., 2010). Although variations exist, some common themes of secretin structure have emerged. Like many of the secretin family members, GspD forms a dodecameric ring structure in the OM approximately 200 Å in height and 150–160 Å in width with a central pore through which exoprotein substrates pass (Korotkov et al., 2009; Reichow et al., 2010). The periplasmic side of the pore consists of a chamber that appears to serve as a docking site for secreted exoproteins (Reichow et al., 2011) with an opening of ∼70 Å, which narrows to ∼50 Å at a periplasmic gate (Korotkov et al., 2009; Reichow et al., 2010, 2011). The periplasmic chamber is formed by concentric rings composed of the N-termini of the monomers. How the secretin accommodates the passage of substrate proteins through the gates and narrow center of the channel, and how it prevents secretion of other molecules that are not substrates, remains unknown. The pore opens into a smaller chamber on the extracellular face that is capped. Since GspD is not a liposecretin, the pilotin GspS is required for secretin stability and transport to the OM via an interaction with the distal C-terminus of the secretin (Daefler et al., 1997; Nickerson et al., 2011). GspS is itself a lipoprotein that is acylated and transported to the OM via the Lol pathway (Collin et al., 2011).

Despite accumulating knowledge regarding the structures of the various T2S components, the actual mechanism of secreting a protein like LT of ETEC through the T2S apparatus has not been well established. However, a theoretical process has been proposed. Initially, the target proteins are synthesized in the cytoplasm and translocated via the Sec or Tat export pathways depending on whether the protein is folded in the periplasm or cytoplasm, respectively (Pugsley, 1993; Berks et al., 2005). Molecular modeling strongly suggests an interaction between the correctly folded substrate protein, the secretin, the pseudopilus tip complex of Gsp I/J/K, and GspC (Reichow et al., 2011), although interactions with additional machine components are possible. The binding of the exoprotein then stimulates the ATPase activity of GspE, leading to the addition of GspG monomers to the pseudopilus (Hobbs and Mattick, 1993; Shevchik et al., 1997). It is not known how the system chooses substrates from the plethora of periplasmic proteins available, or how the ATPase is specifically activated by substrate binding. The exoprotein is pushed upward through the secretin pore with the pseudopilus acting like a piston. However, it is not known how the pseudopilus is retracted. The trimer tip of the pseudopilus may interact with the secretin pore, inducing a conformational change destabilizing the GspG monomers below it, causing the pseudopilus to depolymerize (Korotkov et al., 2012). It notable that the degree of expression of the equivalent of GspK in the Pseudomonas aeruginosa T2S is inversely proportional to the length of the pseudopilus and an interaction between the GspK and GspG equivalent subunits destabilizes GspG (Durand et al., 2005).