Type II Secretion in Escherichia coli.

DOMAIN 4 SYNTHESIS AND PROCESSING OF MACROMOLECULES

Type II Secretion in Escherichia coli MARCELLA PATRICK, MIRANDA D. GRAY, MARIA SANDKVIST AND TANYA L. JOHNSON Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109–0620

Received: 16 December 2009 Accepted: 4 March 2010 Posted: 19 October 2010 Supercedes previous posting at EcoSal.org. Editors: James M. Slauch, The School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL and Harris Bernstein, National Institutes of Health, Bethesda, MD Citation: EcoSal Plus 2013; doi:10.1128/ ecosalplus.4.3.4. Correspondence: Tanya L. Johnson: tanyalj@ umich.edu Copyright: © 2013 American Society for Microbiology. All rights reserved. doi:10.1128/ecosalplus.4.3.4

ABSTRACT The type II secretion system (T2SS) is used by Escherichia coli and other gram-negative bacteria to translocate many proteins, including toxins and proteases, across the outer membrane of the cell and into the extracellular space. Depending on the bacterial species, between 12 and 15 genes have been identified that make up a T2SS operon. T2SSs are widespread among gram-negative bacteria, and most E. coli appear to possess one or two complete T2SS operons. Once expressed, the multiple protein components that form the T2S system are localized in both the inner and outer membranes, where they assemble into an apparatus that spans the cell envelope. This apparatus supports the secretion of numerous virulence factors; and therefore secretion via this pathway is regarded in many organisms as a major virulence mechanism. Here, we review several of the known E. coli T2S substrates that have proven to be critical for the survival and pathogenicity of these bacteria. Recent structural and biochemical information is also reviewed that has improved our current understanding of how the T2S apparatus functions; also reviewed is the role that individual proteins play in this complex system.

INTRODUCTION Type II Secretion Systems The double membrane of the gram-negative cell envelope adds a level of complexity for secreted proteins as they must cross two physical barriers to reach the exterior of the cell. Gram-negative bacteria have therefore evolved several proteinaceous systems to secrete proteins into their surroundings. Some of these pathways transport proteins across the bacterial cell envelope in a single step, including the signal sequence-independent type I, III, and IV secretion pathways. On the other hand, the signal peptide-dependent secretion systems (type II secretion [T2S] and autotransporter pathways) involve a two-step process. In these latter pathways, proteins containing an N-terminal signal peptide are first translocated across the inner membrane via the Sec or Tat systems. After removal of their signal peptides and release into the periplasm, the mature proteins cross the outer membrane (for reviews, see references 1, 2, and 3). In the case of the type II secretion system (T2SS),

ASMScience.org/EcoSalPlus

1 Downloaded from www.asmscience.org by IP: 132.239.1.231 On: Fri, 06 Jan 2017 10:29:45

Patrick et al.

proteins fold into their final or near-final tertiary structure before outer membrane translocation (3). The T2SS supports the secretion of many virulence factors, including toxins and proteases, and therefore secretion via this pathway is regarded as a major virulence mechanism. Bacteria that utilize a T2SS include human, animal, and plant pathogens (4). In humans, these bacteria are often the cause of serious diseases such as cholera, other severe diarrheal diseases, and pneumonia. In addition to its role in pathogenesis, the T2S pathway has been implicated in transporting proteins that have a role in symbiosis or other associations between bacteria and eukaryotic hosts. Depending on the bacterial species, 12 to 15 genes have been identified as essential for T2S, and the genes and gene products have been designated by the letters A to O and S (Fig. 1) for most species. The organization of the T2S gene cluster is relatively well conserved, with most of the genes

appearing in a single operon. A large amount of research studying model organisms such as Klebsiella oxytoca, Pseudomonas aeruginosa, Dickeya sp. (formerly Erwinia chrysanthemi), and Vibrio cholerae has given us a reasonably good picture of the individual components that make up the T2S machinery (1, 2, 3). The precise mechanism of the secretion process is not as well understood, however. One thing that has become apparent is that the T2S apparatus shares an evolutionary history with type IV pili (1, 5). While this chapter will focus principally on the T2S transport process, it should be mentioned that much of what has been learned about the T2SS complex can also be applied to type IV pilus biogenesis.

Escherichia coli and T2S The T2SS is widespread among gram-negative bacteria such as V. cholerae, K. oxytoca, P. aeruginosa, Legionella pneumophila, and E. coli. E. coli is an extremely diverse group of bacteria that contribute to normal intestinal

Figure 1 Type II secretion system (T2SS) gene alignments. The genes comprising the T2SSs from individual species are shown schematically. Homologous components are colored similarly; arrowheads show orientation. Dashed lines indicate a deleted region in the operon and do not represent linkage relationships. 2

ASMScience.org/EcoSalPlus Downloaded from www.asmscience.org by IP: 132.239.1.231 On: Fri, 06 Jan 2017 10:29:45

Type II Secretion in Escherichia coli

microbiota, but also include intestinal pathogens of global significance such as enteropathogenic E. coli (EPEC, associated with infant diarrhea), enterohemorrhagic E. coli (EHEC, an important food-borne pathogen), enterotoxigenic E. coli (ETEC, associated with traveler’s diarrhea), enteroinvasive E. coli (EIEC, associated with bacillary dysentery), enteroaggregative E. coli (EAEC, associated with persistent diarrhea in children), and DAEC (diffusely adhering E. coli, also associated with diarrhea in children). Pathogenic E. coli are also important causes of extraintestinal infections. Uropathogenic E. coli (UPEC) is the major cause of community-acquired urinary tract infections, while the meningitis-associated E. coli (MNEC) is the most common cause of gramnegative meningitis in newborns. Most E. coli appear to possess one or two complete T2S operons encoded chromosomally; however, there are exceptions to this rule. In EHEC, for instance, the T2S genes are present on the large virulence plasmid, pO157 (6). The T2SS is expressed by EPEC strain E2348/69 at 37°C and is required for virulence (7). In EHEC strain O157: H7, the T2SS has been shown to promote the bacterium’s adherence and intestinal colonization. Mutants not expressing the T2SS were more than fivefold less able to colonize infant rabbits (8). In UPEC, T2SSs have been demonstrated to be important in spreading between sites in the urinary tract and in avoiding efflux in the mouse bladder (9). Finally, in ETEC, T2S proteins are responsible for secretion of one of the major virulence factors leading to diarrhea, heat-labile enterotoxin (LT). Expression of the etxAB (eltAB) operon, which encodes LT, is repressed by the nucleoid-structuring protein (H-NS) when cells are grown at low temperatures (10), i.e., temperatures outside the human body. The expression of LT may also be regulated by osmolarity (11, 12) and pH (11). The T2S genes of ETEC have been shown to constitute one transcriptional unit, with a σ70 promoter identified at the start, and are also transcriptionally controlled by H-NS in response to low temperatures (7). Regulation in ETEC therefore seems to occur with both expression of the secretion apparatus and LT, thus ensuring the coordinated production and secretion of LT at the appropriate time and place. E. coli K-12 also contains genes encoding a T2SS, but while some of the predicted proteins of the K-12 T2SS share a high degree of similarity with the T2SS proteins of ETEC, others have no significant homology. The E. coli K-12 T2S operon is not expressed under standard labo-

ratory conditions. When expressed on a plasmid in an HNS mutant background, however, the E. coli K-12 T2SS was shown to secrete a protein encoded by a gene directly downstream of its genomic location, a chitinase (ChiA) (13). The T2SS in other E. coli may also be under regulatory control, but not necessarily with the same mechanisms as those controlling the ETEC T2S genes. Analysis of other gram-negative bacteria has revealed that regulation of T2S pathways varies greatly from species to species. In P. aeruginosa, for example, one T2S pathway has been found to be regulated by quorum sensing (14), while another T2SS is only expressed under phosphate-limiting conditions (15). A quorum-sensing system has also been identified for Erwinia carotovora, a plant pathogen that secretes cell wall-degrading enzymes via a T2SS. When a strain with a mutation in a quorum-sensing gene was examined, both the production of T2S-dependent exoenzymes and the ability of the mutant strain to propagate and cause disease were reduced (16). In addition, expression of the T2S genes was induced in early stationary phase, suggesting that these genes may also be under quorum-sensing control (17). In V. cholerae, the T2S genes appear to be regulated by RpoE, an alternative sigma factor that responds to extracytoplasmic stress (18). In contrast to regulated expression of the T2S genes in many species, expression of the T2S genes in Aeromonas hydrophila may be constitutive. Reverse transcriptasePCR analysis of mRNA production in A. hydrophila has shown that at least one gene in the T2S operon is constitutively expressed (19). The expression of the A. hydrophila T2S-dependent serine protease AspA, however, does appear to be regulated by quorum sensing (20). It is possible that constitutive expression of the secretion apparatus is necessary for species that secrete substrates in several different environments.

SECRETED PROTEINS A wide variety of proteins are secreted via the T2SS. Many of these proteins are associated with degradation of macromolecules or destruction of various tissues. T2S substrates include chitinases, cellulases, pectin lyases, toxins, proteases, lipases, phospholipases, and nucleases, to name a few (Table 1). Secretome projects are only now beginning to uncover the number of proteins that depend on T2SSs for their transport into the extracellular



Patrick et al.

Table 1 Examples of T2S-dependent substrates and their known activities Type II secretion substrate

Target/activity

Bacteria (reference[s])

RNase, nuclease

Nucleic acids

A. hydrophila (22), L. pneumophila (23)

Cellulase, pectinase, pullulanase, amylase

Polysaccharides, cell walls

Dickeya sp. (17), Xanthomonas campestris (24), Klebsiella pneumophila (25), A. hydrophila (26)

Chitinase, chitin-binding protein

Chitin, epithelial cells

E. coli (K-12) (27), V. cholerae (28), L. pneumophila (21)

YodA/ZinT, MtrC, OmcA

Metal or electron transport

E. coli (EHEC) (8), Shewanella oneidensis (29, 30)

Lipase, phospholipase

Lipids/phospholipids

P. aeruginosa (31), L. pneumophila (32)

Protease (including serine and metalloproteases)

Proteins/glycoproteins, extracellular matrix

E. coli (EHEC) (33), P. aeruginosa (34), V. cholerae (35)

Cholera toxin, heat-labile enterotoxin, exotoxin A, aerolysin

Eukaryotic signaling, protein synthesis pathway, pore formation

V. cholerae (36), E. coli (ETEC) (37), P. aeruginosa (34), A. hydrophila (26)

Keywords: T2SS, substrates, activities, Type II secretion substrate, target, bacteria, RNase, nuclease, cellulase, pectinase, pullulanase, amylase, chitinase, chitinbinding protein, YodA/ZinT, MtrC, OmcA, lipase, phospholipase, protease, serine, metalloproteases, cholera toxin, heat-labile enterotoxin, exotoxin A, aerolysin, nucleic acids, polysaccharides, cell walls, chitin, epithelial cells, metal or electron transport, lipids/phospholipids, proteins/glycoproteins, extracellular matrix, eukaryotic signaling, protein synthesis pathway, pore formation, A. hydrophila, L. pneumophila, Dickeya sp., Xanthomonas campestris, Klebsiella pneumophila, E. coli (K-12), V. cholerae, Shewanella oneidensis, P. aeruginosa.

environment (21). While only a few substrates are known in E. coli, the proteins discovered so far have proven to be critical for the pathogenicity of these bacteria.

Heat-Labile Enterotoxins Perhaps the best-characterized substrates of the T2SS in E. coli are the heat-labile enterotoxins (LTs) of ETEC. LT proteins are highly homologous to the cholera toxin of V. cholerae, particularly in structure (Fig. 2) (38). Like cholera toxin, LT is a member of the AB5 family of enterotoxins, where the mature toxin is constructed of a symmetric ring of five identical B subunits joined together with a single A subunit. Although the cholera toxin genes of V. cholerae are phage encoded (39), the etxAB operon is most often located on a plasmid (40) in ETEC. LT genes, however, can also be found within lysogenic phage (41) or on the chromosome (42, 43), suggesting that the genes encoding LTs may have originated from ancestral cholera toxin genes (44). After synthesis in the cytoplasm, the individual A and B polypeptides are transported as precursor proteins across the inner membrane and into the periplasm by the Sec apparatus. Once in the periplasm, the N-terminal signal sequences are removed from the polypeptides and the subunits assemble into a mature AB5 holotoxin. The folding and assembly of the subunits is catalyzed by DsbA, a periplasmic isomerase. Loss of DsbA by gene inactivation greatly reduces the amount of LT holotoxin formed (45).

Once fully assembled, the LT holotoxin is transported across the outer membrane via the T2SS. Inactivation of the T2S pathway by mutagenesis of the outer membrane protein GspD completely abolished LT secretion across the outer membrane and resulted in an accumulation of the toxin in the periplasmic space. Release of LT from the periplasm could then be restored when the gspD mutant was complemented with a plasmid copy of the wild-type gspD gene (37). In contrast to cholera toxin, which is freely secreted away from the cell; most ETEC strains do not release LT into the extracellular environment, but rather the toxin largely remains associated with the bacterial cell surface (Fig. 3) (46, 47, 48). Following secretion through the T2SS, LT binds to lipopolysaccharide (LPS). Extracellular LT is only detected in association with outer membrane vesicles released from the cell. Interestingly, the ability of AB5 toxins to bind to the bacterial cell surface, rather than be released into the extracellular environment in a soluble form, is species specific rather than toxin specific. Both LT and cholera toxin can bind to E. coli LPS and remain associated with the cell. These interactions have been shown to occur via the inner core sugar 3-deoxy-Dmanno-octulosonic acid of LPS. Neither LT nor cholera toxin is able to bind to V. cholerae LPS, however, because the sugar of V. cholerae LPS is phosphorylated (49). It has been proposed that the reduced toxicity of ETEC compared with V. cholerae could be explained, in part, by this difference in locations of the AB5 toxins (50). Because V. cholerae appears to secrete its toxin away from the cell

4



Figure 2 Comparison of heat-labile enterotoxin (LT-IIb) and cholera toxin structures. The structures of LT-IIb (left, PDB ID code 1TII) and cholera toxin (right, PDB ID code 1XTC), portrayed such that their A subunits (brown) are in a similar orientation. The individual B subunits are in red, magenta, green, blue, and cyan.

rather than tightly binding toxin to the surface of the bacteria, more cholera toxin may come into contact with host receptors. During a mutational screen designed to find ETEC genes involved in pathogenesis, a second operon was discovered and shown to be required for maximal LT secretion (51). Fleckenstein et al. (51) demonstrated that deletion of leoA, a gene within this second operon that encodes a protein homologous to bacterial and eukaryotic GTPases, greatly reduced LT secretion. Exactly how this operon contributes to LT release from the cell is unclear, but it appears to play some role in the formation and protein content of outer membrane vesicles produced by ETEC. The host cell surface ganglioside GM1 receptor recognized by the LT is dictated by the B subunits of the holotoxin (52). Once the B subunits bind to this membrane receptor, they facilitate entry of the toxin into the host cell (50). Following endocytosis, LT is transported to the endoplasmic reticulum where the A subunit is reduced and the resulting catalytic A1 subunit retrotranslocates to the cytosol (53, 54). Here, A1 ribosylates

the stimulatory G proteins leading to activation of adenylate cyclase and elevated production of cAMP. Elevation of cAMP results in activation of cAMPdependent protein kinase A and phosphorylation of chloride channels; stimulating Cl− secretion and reducing the absorption of Na+ followed by massive efflux of water (50). Indeed, LT directly contributes to the severity of diarrhea seen in the host (55). Results from piglet infection studies also suggest that LT can lead to enhanced colonization of the intestinal epithelium by ETEC strains by affecting bacterial attachment to the epithelium, as well as causing a general increase in the concentration of bacteria in the small intestine (56).

ChiA The only known T2S substrate in E. coli K-12 is the chitinase, ChiA (13). Chitinases degrade chitin, a polymer of N-acetylglucosamine (GlcNAc) that is a major component of the cell walls of fungi, crustaceans, and other organisms. Chitin is one of the most abundant biopolymers in nature and is used by many microorganisms as a source of carbon and nitrogen. Use of



Patrick et al.

Figure 3 Schematic model of LT secretion. LT toxin subunits are translocated across the inner membrane via the Sec translocon and fold into an AB5 structure (shown in ribbon structure) in the periplasm of the ETEC cell. (1) After holotoxin formation, the assembled LT is targeted to the T2SS by an unknown mechanism. (2) Once engaged with the T2SS, LT is transported across the outer membrane. (3) Upon exposure to the extracellular space, LT binds to LPS on the bacterial cell surface (4), and as outer membrane vesicles are budding off the cell surface, LT is released. (5) B subunits bind to gangliosides on the host cell and facilitate the entry of the toxin by an unknown mechanism, initiating a series of cellular events leading to diarrhea.

chitin as a nutrient, however, requires degradation of the GlcNAc polymer into monomers by the action of chitinases and chitobioses. In other gram-negative bacteria such as V. cholerae and L. pneumophila, chitinase secretion has been well documented (21, 57, 58), and the enzyme has been shown to be a substrate of the T2SS (21, 28, 35). Indeed, when the T2SS of E. coli K-12 was kept under H-NS suppression, ChiA was not secreted, and the vast majority of the protein accumulated in the periplasm (13). As reported for other bacterial chitinases (28, 59), E. coli ChiA was able to degrade ethylene-glycol chitin, a lysozyme substrate (27). Bacteria expressing chitinases with broad specificity could be important for additional aspects of competition and survival, such as promoting pathogenicity or other interactions with host organisms. Along these lines, there has been a renewed interest in examining the effect of chitin, and presumably the action of chitinases and other chitin-binding proteins, on the pathogenicity of Vibrio and Legionella spp. other than

their role in carbon acquisition. In particular, chitin has been shown to induce proteins involved in colonization (21, 60), induce natural competence (61), and promote the survival of the bacteria during exposure to stomach acid (62) and low temperatures (63). Interestingly, the remnants of a second T2SS are present in the E. coli K-12 strain, MG1655, at a second chromosomal location, suggesting that K-12 strains may have originally possessed another T2S pathway, which has since been lost. One gene from the pathway that still remains in K-12 strains is pppA, encoding a prepilin peptidase (64). Francetic et al. (64) cloned the pppA gene from E. coli K-12 and demonstrated that it is functional.

StcE StcE (secreted protease of C1-esterase inhibitor) is a metalloprotease encoded by the large virulence plasmid pO157 of EHEC O157:H7 (33). Immediately downstream

6



of stcE is the T2SS operon etpC-O. In view of this genetic linkage and the presence of a candidate cleavable Nterminal signal peptide in the StcE protein sequence, Lathem et al. (33) investigated whether StcE was, in fact, secreted by the etp T2SS. Upon disruption of etpD, the gene encoding the putative T2SS outer membrane secretin StcE was no longer detectable in culture supernatants by immunoblot analysis. Complementation in trans with etpD restored the ability of the etpD mutant strain to secrete StcE, thus confirming that the defect in T2S was responsible for the absence of extracellular StcE (33). Expression of the stcE gene is upregulated by the global regulator Ler (LEE-encoded regulator). By inducing Ler expression in O157:H7, the level of extracellular StcE was increased nearly 12-fold over uninduced conditions (33). Ler also plays an important role in the expression of other secretion systems in E. coli. The LEE pathogenicity island is common to enteropathogens that cause disease through attaching and effacing lesions, and it encodes many proteins that are required for virulence, including the proteins required for type III secretion. Upon Ler binding, expression of many genes in the LEE pathogenicity island is permitted (65). To date, several substrates have been identified for StcE. C1-esterase inhibitor is a member of the serine protease inhibitor family in humans, while the others, glycoprotein 340 and mucin 7 and CD43 and CD45 are salivary and neutrophil glycoproteins, respectively (33, 66, 67). StcE can bind these substrates and change the glycoprotein’s activity in both cleavage-dependent and noncleavage-dependent ways. Additionally, purified StcE has been shown to significantly reduce the viscosity of human saliva (66). Because the known StcE substrates are glycosylated proteins with defensive roles in the body, and because the expression of StcE’s mucinase activity has been shown to be coregulated with proteins of the LEE pathogenicity island elements, it has been hypothesized that StcE contributes to the pathogenesis of E. coli O157:H7 by degrading the protective layer of mucins and glycoproteins on host immune and intestinal cells (66). Other enteropathogens also secrete proteins with mucinase activity via the T2SS. For instance, the HA/ protease of V. cholerae has been shown to degrade fibronectin, ovomucin, and lactoferrin and is highly homologous to the P. aeruginosa elastase protease (68, 69).

Both proteases depend on the T2SS for exit from the cell (35, 70). Using the T2SS to export proteins involved in degradation of the mucosal layer appears to be a common theme seen in bacteria that require access to epithelial cells for successful colonization and/or invasion.

YodA (ZinT) Besides the protease StcE, the genome of O157:H7 contains at least one other T2S-dependent protein. This second T2S substrate, YodA (ZinT), has been shown to be essential for adherence of EHEC to cultured HeLa cell monolayers and to aid in the colonization of the infant rabbit intestines (8). The authors demonstrated YodA’s (ZinT’s) dependence on the T2SS by replacing etpC, the first gene in the pO157-encoded etp operon, with an antibiotic cassette. The etpC mutant showed almost no detectable YodA (ZinT) in the supernatant, whereas this protein was readily identified in the supernatant from the wild-type strain (8). These results strongly suggest that YodA (ZinT) is a substrate of the EHEC T2SS. YodA (ZinT) was originally identified in E. coli K-12 as a cadmium-induced protein that is translocated from the cytoplasm to the periplasm following exposure to cadmium stress (71). Besides transcriptional regulation by cadmium, YodA (ZinT) has been demonstrated to bind a variety of metal ions (including cadmium, mercury, nickel, and zinc) via a lipocalin-like domain (72, 73). Lipocalins are a family of small, usually secreted, proteins that are often involved in transport (74). Because the growth of the E. coli K-12 yodA (zinT) mutant was found to be impaired in zinc-limited conditions, and because zinc was one of the metals that bound to purified YodA (ZinT), it has been proposed that YodA (ZinT) may have a function in zinc homeostasis (73). In E. coli K-12, YodA (ZinT) is localized first to the cytoplasm and then to the periplasm, but its secretion to the extracellular environment has not been observed, probably because the genes encoding the E. coli K-12 T2SS are usually transcriptionally silenced (13). In EHEC, not all YodA (ZinT) is extracellularly secreted, and it is not yet clear whether, like LT, the protein becomes associated with the outer membrane and/or becomes incorporated into vesicles. Also like LT, another protein has been implicated in the release of YodA (ZinT) in addition to the EHEC T2SS. Mutation of the protein AdfO (adherence factor encoded on CP-933O phage) reduced secretion of YodA (ZinT) and three other proteins in EHEC



Patrick et al.

O157:H7 by at least 2-fold (8). As with the LT and LeoA relationship, it is unclear exactly what role AdfO is playing in YodA (ZinT) secretion.

DraD In addition to the ability of pathogenic E. coli to secrete mucins and other degradative enzymes via the T2SS to adhere and invade mucosal epithelial layers, other proteins termed adhesins and invasins are also used in promoting infection. These proteins are often located on extended surface appendages known as fimbriae. Although each family of adhesin proteins is generally associated with a specific human disease, the Dr family of fimbriae from E. coli is a notable exception, as its members are associated with both diarrheal (DAEC) and urinary tract infections (UPEC) (75). Dr fimbriae are so named because of their ability to bind the Dr blood group erythrocytes through the decay-accelerating factor (CD55) (76). Dr fimbriae are polymeric structures built from repeating monomers of the DraE fimbrial subunit and may have a minor fimbrial subunit, DraD, serving as the tip adhesin (77). Fimbrial subunits like DraE cross the inner membrane in an unfolded form via the Sec system. After interacting with a periplasmic chaperone, the subunits must be targeted to the outer membrane for the assembly into the fimbriae fiber, followed by transport of the fiber across the outer membrane to the cell surface via the chaperon/usher pathway. Assembly of Dr fimbriae does not require the tip adhesion DraD. In fact, examination of isolated Dr fimbriae suggests that most Dr fimbriae are made up of only DraE subunits, and DraD is surface located in structures independent from the Dr fimbriae (78). When the chaperon/usher pathway was deleted in UPEC, DraD, unlike DraE, was still exported to the cell surface (79); suggesting that this adhesin uses a different system to translocate the outer membrane. With the exception of DraD, all fimbriae subunits have a highly conserved N-terminal extension that participates in subunit-subunit interactions. In contrast, DraD is synthesized as a protein precursor with an N-terminal cleavable signal sequence. In accordance with this observation, DraD, but not the other proteins encoded by the Dr fimbriae operon, has recently been shown to be secreted via the T2SS in UPEC. Inactivation of the T2SS by mutagenesis of the gspD gene blocked DraD transport to the cell surface (79).

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE T2S APPARATUS The characterization of the E. coli T2SS has been quite limited; however, their similarities with more wellcharacterized T2SS in other organisms are relatively high and thus much of the research done on other bacterial T2SS can translate into those of E. coli. Therefore, in this section we will review what is generally known about the workings of the T2S complex based on studies performed with other bacteria, including V. cholerae, K. oxytoca, and P. aeruginosa. The T2S machinery is composed of at least 12 gene products (T2S:C-N, T2S:O, and any combination of T2S: A, B, and S) (Fig. 1), which probably assemble into a large protein complex spanning the entire cell envelope (Fig. 4). Once transported across the cytoplasmic membrane and folded in the periplasm, proteins to be secreted into the extracellular environment are believed to be pushed through a pore in the outer membrane termed the secretin (T2S:D). This is thought to be facilitated by the polymerization of a pseudopilus (consisting of T2S: G-K) using the energy provided by a cytoplasmic ATPase (T2S:E), which associates with the inner membrane protein complex (T2S:C, L, M, and F) (1, 4). The following sections will break down the individual components of the T2SS and review the current knowledge of how the components interact to form a functional apparatus.

The ATPase T2S is an energy-dependent process. Only one protein encoded by the T2S operon has been characterized to have ATPase activity and that is the cytoplasmic protein, T2S:E. T2S:E is related to a larger family of secretion ATPases, which are known to be involved in many activities including protein secretion, type IV pilus biogenesis, DNA uptake, and archeal flagellar assembly (80). The V. cholerae ATPase was one of the earliest T2S proteins to be crystallized, and from that work it was hypothesized to form a hexameric ring structure based on homologies to other characterized RecA-like ATPases (81). Later functional work supported this hypothesis by demonstrating that a fraction of T2S:E corresponding to the size of a hexamer is capable of hydrolyzing ATP (82). The ATPase activity of T2S:E was stimulated even further by the association of its N-terminal domain with the N-terminal cytoplasmic domain of the inner membrane protein, T2S:L (83); an interaction which is necessary

8



Figure 4 Model of T2SS with LT. The T2SS comprises an ATPase (T2S:E), an inner membrane complex (T2S:C, F, L, and M), pseudopilins (T2S: G, H, I, J, and K), and an outer membrane secretin (T2S:D), which assemble into a complex spanning the entire cell envelope. Fully assembled LT (shown in ribbon structure) engages the T2S apparatus in the periplasm to allow for its extracellular translocation.

to link T2S:E to the greater T2S complex (25, 84, 85, 86). T2S:E and the cytoplasmic domain of T2S:L themselves are monomeric when purified, but they cocrystalize as heterodimers (84). Although T2S:E and cytoplasmic domain of T2S:L copurify in a 1:1 ratio, the final stoichiometry of the T2S:EL complex in the functional T2SS remains to be determined.

comparing the periplasmic T2S:L structure, it was found that its closest structural homolog is the periplasmic domain of another T2S inner membrane protein, T2S:M (94). Interestingly, periplasmic domain of T2S:M also forms a dimer (95, 96); however, the periplasmic domains of T2S:L and T2S:M are not homologous at the primary sequence level and their dimer arrangements are not the same (94).

The Inner Membrane Complex

T2S:L and T2S:M are known to form a stable complex that protects them from degradation (25, 87, 90), and it has been demonstrated that residues 84 to 99 in the periplasmic domain of T2S:M are necessary to interact with T2S:L residues 216 to 296 (92, 96). Unfortunately, structural information is not available for these regions in T2S:M and T2S:L; thus, it is still unknown how these proteins form a complex and the relative stoichiometry of said complex. When the periplasmic domain of T2S: M dimerizes, a hydrophobic cleft with a hydrophilic rim is formed, which may bind a yet undefined ligand or peptide. Because the cleft residues in T2S:M’s are

Although proteins secreted through the T2SS engage the apparatus in the periplasm, the majority of the T2S components are found in the inner membrane. T2S:L is a bitopic membrane protein with a large cytoplasmic domain, a short membrane-spanning helix, and a smaller periplasmic domain, and it is believed to dimerize in the membrane (87). Aside from linking the cytoplasmic T2S: E to the membrane, it has been shown to interact with the other inner membrane proteins, T2S:C, M, F, and J (88, 89, 90, 91, 92, 93). The C-terminal periplasmic domain of T2S:L was recently crystallized as a dimer (94). When ASMScience.org/EcoSalPlus


Patrick et al.

relatively well conserved, the cleft is likely important for secretion (95). A third inner membrane protein, T2S:F, has also been shown to make up part of this inner membrane complex. T2S:F spans the membrane three times with its Nterminal domain in the cytoplasm and its C-terminal domain in the periplasm (97). Only the larger, first cytoplasmic domain has been crystallized and it was found to form dimers. The first 55 residues of T2S:F were too flexible to crystallize, and are therefore thought to potentially interact with other T2S components (97). Using two-yeast hybrids and coimmunoprecipitations, Py et al. (90) demonstrated the formation of an T2S:ELF complex in E. chrysanthemi, but this was conditional on T2S:E and L forming a complex first. Similarly, Arts et al. (98) demonstrated that the T2S:F homolog in P. aeruginosa is unstable unless in the presence of the T2S:E and L proteins. The authors determined the second cytoplasmic domain to be important for this stabilization, which is in contrast to the E. chrysanthemi studies; however, it is feasible that both cytoplasmic domains play a role in interaction with other T2S components (90, 98). The structure of the first cytoplasmic domain of T2S:F was recently solved, and a careful sequence comparison between the first and second cytoplasmic domains shows many similarities between the two that may translate into structural similarities as well (97). Interestingly, T2S:E binds in a pocket within the T2S:L cytoplasmic domain, as demonstrated by cocrystallizing the two proteins. T2S:E fills only a portion of this binding cleft, making it tempting to speculate that T2S:F may also bind within this same pocket in T2S:L (84). Future work is necessary to understand the precise interactions between the various domains of these proteins and other components of the inner membrane complex.

Connecting the Inner and Outer Membranes At least one other T2S protein is associated with the inner membrane complex, T2S:C. T2S:C is a bitopic inner membrane protein with a periplasmic domain that is divided into two subdomains: the HR domain and either a PDZ or coiled-coil domain. T2S:C has been shown to interact and stabilize the inner membrane T2S:LM complex (89, 93, 99). The interaction of T2S:C’s N-terminal 46 residues with the inner membrane proteins T2S: L and M protect them from degradation (89), and similarly, the presence of the T2S:LM complex seems to be necessary to protect T2S:C from degradation and may

play a further role in stabilizing T2S:C in its proper conformation to support secretion (100). T2S:C homologs are known to be degraded in the absence of outer membrane protein T2S:D in K. oxytoca and P. aeruginosa (101, 102), and it was recently demonstrated that the periplasmic subdomains of T2S:C and D could be copurified, indicating a direct interaction between these two T2S components (103). Korotkov et al. (103) further determined that the HR domain is primarily responsible for interaction with T2S:D. As T2S:C spans the periplasm, it is a suitable candidate for substrate recognition and/or gating of the T2S:D pore. The second periplasmic subdomain, the PDZ domain, is the most likely candidate to interact with the secreted proteins, another T2S protein, or possibly both (103). Further research into this region of the protein is ongoing; however, it has been shown that the PDZ domain is required for the secretion of most proteins in E. chrysanthemi (104).

The Secretin T2SSs have only one outer membrane protein, T2S:D, also termed the secretin, which is thought to oligomerize into a pore used in the export of proteins out of the bacterium (105). It is part of a larger secretin superfamily that consists of outer membrane proteins required for T2S, type IV pilus biogenesis, type III secretion, and filamentous phage extrusion. Most secretins require the use of a small lipoprotein termed the pilotin (T2S:S), which binds the C-terminal end of T2S:D and is required for proper positioning of T2S:D in the outer membrane (106, 107). Although many of the secretins that make up the larger family require a pilotin for proper insertion, not all T2SSs have an identified pilotin protein. In fact, only K. oxytoca and E. chrysanthemi have shown their respective T2S:S pilotin to be important for secretin insertion in the outer membrane; however, putative t2s:S genes in other T2S operons (including the E. coli pO157 plasmid-encoded T2SS) have been identified (4, 6). It is possible that the pilotin has not been identified because of limited sequence homology in other T2SSs, or there may be an alternative pathway by which the secretin is inserted in the outer membrane. In support of this latter point, Viarre et al. (108) showed that the secretin for the T2SS in P. aeruginosa, HxcQ, contains a lipid anchor, which is sufficient to target this protein to the outer membrane and allow for correct insertion. Regardless of the mechanism, once properly inserted, secretins are characterized by their ability to form large heat- and detergent-resistant oligomers. They are thought to form

10



rings of 12 to 14 subunits containing a central channel ranging from 50 to 100 Å in diameter that is occluded by a central plug (109, 110, 111). The T2S:D protein has a well conserved C-terminal domain that is protease resistant and thought to be anchored in the outer membrane by amphipathic transmembrane β-strands (109, 112) as well as a more variable N-terminal domain which extends into the periplasm. Recently, Korotkov et al. (113) crystallized the N-terminal domain of the periplasmic domain of T2S:D from ETEC, and showed a region termed the N0:N1 lobe to be easily accessible and thus a good candidate for interacting with other proteins in the periplasm. These interactions may include other T2S components, such as T2S:C and/or T2S:J (88, 103), as well as proteins targeted to, and ultimately secreted by, the T2SS (104, 114, 115).

The Pseudopilins On the basis of sequence homology between several T2S proteins and type IV pilus biogenesis components, it is believed that the T2S pathway may function similarly to the type IV pilus machinery, which supports the assembly of the pilus, as well as its extension and retraction (5). In particular, the T2S:G-K proteins have been termed the “pseudopilins” because of their sequence similarity to the type IV pilins, with both T2S pseudopilins and the type IV pilins containing a conserved N-terminal leader peptide that is required for transport across the cytoplasmic membrane. Following insertion into the cytoplasmic membrane, the N terminus of the pseudopilins is cleaved and methylated by the prepilin peptidase, which also shows a high degree of homology to the type IV prepilin peptidase (116). The relative abundance of T2S:G protein compared with the other pseudopilins has led to it being termed the major pseudopilin, whereas T2S:H-K proteins have been termed the minor pseudopilins (116). Reports in several organisms have shown that the major pseudopilin component, T2S:G, can assemble into multimers, suggesting that T2S:G may be able to form a pilus-like structure or “pseudopilus” (117). Studies in K. oxytoca and P. aeruginosa have demonstrated that overexpression of the major pseudopilin T2S:G resulted in the presence of bundled surface pseudopili, further illustrating that the T2S pathway is able to assemble a piluslike structure (118, 119, 120). In addition, the overexpression of T2S:G inhibited secretion through the T2S pathway (118). This result indicates that the pseudopilus may extend through the secretin in the outer membrane

when overexpressed and that the formation of the surface pseudopili occludes the pore and prevents secreted proteins from passing through the outer membrane. These findings have led to a model where the pseudopilin components normally polymerize into a shorter periplasmic pseudopilus, which either acts as a piston to push secreted proteins through the outer membrane pore, or as a plug within the secretin that could depolymerize to allow proteins to be secreted. The exact role of the minor pseudopilins is not known. The minor pseudopilins were found to be required for secretion; however, their overexpression did not inhibit secretion as shown with T2S:G in K. oxytoca and P. aeruginosa (25, 121). T2S:I was the only minor pseudopilin found to be required for the production of surface pseudopili when T2S:G was overexpressed (121). Conversely, deletion of T2S:K increased the length and number of the surface pseudopili observed following overexpression of T2S:G, and overexpression of T2S:K abolished the presence of the surface pseudopili (120, 122). These results suggest that T2S:I may be an initiation factor required for the pseudopilus to form, and that T2S:K may be a termination factor that stops polymerization of the pseudopilus. Deletion of T2S:H and J had no effect on the production of surface pseudopili, indicating that one or both of these proteins may act as a chaperone for the other pseudopilins. In recent years, the structures for all five pseudopilins have been determined, allowing a better view into what role these proteins may be playing in T2S (119, 123, 124, 125). The structures for the pseudopilins further confirmed the homology with the type IV pilins, with both groups containing an N-terminal α-helix, a variable domain, and a C-terminal antiparallel β-sheet that completes the pilin fold. However, despite this core homology, a great degree of dissimilarity was found within the variable domain between the pseudopilins. Both T2S: H and J have large, elaborate variable domains (124, 125), whereas T2S:I and K have smaller variable regions (123). T2S:K is the largest of the pseudopilins and contains a unique α-domain that is absent in all other pseudopilins. This unique α-domain was found to contain a dinuclear metal binding site and a pair of cysteine residues that form a disulfide bridge (123). The structure for T2S:G has been resolved for K. oxytoca, P. aeruginosa, V. cholerae, Vibrio vulnificus, and EHEC; however, slight variation occurred among the structures



Patrick et al.

from these species (113, 119, 126). K. oxytoca illustrated a C-terminal β-strand that was observed in a helical conformation in the other species (119). This difference is believed to occur because of a β-strand swap occurring during the K. oxytoca crystallization process. Interestingly, Korotkov et al. (9) found a calcium-binding loop within the C terminus of T2S:G in three organisms: V. cholerae, V. vulnificus, and EHEC. Furthermore, the sequence of the calcium-coordinating side chains was highly conserved among these T2S:G homologs. Disruption of the amino acids coordinating the calcium ion inhibited T2S in V. cholerae, illustrating that calcium is playing an important role during secretion, perhaps acting as a stabilizing factor for T2S:G or within the assembled pseudopilus (113). The structures for the minor pseudopilins also gave insight into their interactions with each other. T2S:I and J initially crystallized as a heterodimer and were subsequently shown to form a ternary complex with T2S:K (123, 125, 127). It has been suggested that the T2S:IJK complex may be positioned at the tip of the pseudopilus and therefore serves as a cap to the pseudopilus. This hypothesis further supports the finding that T2S:K may act as a termination factor for pseudopilus growth, because the bulky α-domain of T2S:K would prevent the pseudopilus from being able to pass through the secretin pore. In addition to the structural data, both genetic and biochemical approaches have been utilized to uncover interactions between the pseudopilins. T2S:G was found in heterodimers with T2S:H, I, and J. T2S:H was found to be associated with T2S:G, I, and J, and the absence of T2S:H disrupted the observed T2S:GJ and T2S:IJ interactions. In the absence of T2S:I, T2S:G and H were no longer associated (128). In addition to the tertiary complex of T2S: IJK seen in 3D structures, the existence of a quaternary complex of all four minor pseudopilins has also been shown (127). Furthermore, yeast two-hybrid assays showed that both T2S:H and J can form homodimers and also pointed to a T2S:IJ interaction (88). These findings suggest that the interactions between the pseudopilins are complex and that each individual pseudopilin is playing an important role in the formation of the pseudopilus. Less is known about the interactions between the pseudopilins and the other components of the T2S complex. Yeast two-hybrid data indicated an interaction between T2S:J and the secretin, T2S:D. This same assay also indicated an interaction between T2S:J and the inner

membrane protein T2S:L (88). These data support the hypothesis that the pseudopilins are polymerized in the inner membrane as they build into the pseudopilus, and that T2S:J is part of a complex with T2S:I, H, and K at the pseudopilus tip, which could then interact with the outer membrane.

Stoichiometry of the T2S Complex Although much is known about the interactions between the various proteins of the T2S apparatus, the relative stoichiometry of the individual components remains elusive. It is probable that the cytoplasmic component, T2S:E exists as a hexamer, while T2S:D is found in an oligomer of 12 or more proteins. Most of the inner membrane components analyzed to date appear to form dimers, although whether they remain so in a functional apparatus has yet to be determined. As the inner membrane proteins are likely interacting with each other (either transiently or in a stable complex), it is anticipated that heterocomplexes form in the membrane. The relative combinations of each protein in those complexes remain in question, however. Likewise, if T2S:C is able to interact with proteins both in the inner membrane and the outer membrane, it seems likely that it would need to conformationally stretch between a 6-fold symmetry in the inner membrane and a larger 12+-fold symmetry in the outer membrane. Finally, some of the interactions between the T2S proteins are probably transient and it is unknown whether the complex actually exists as a stable entity at any given time. One important question to address is how the various components come together across the entire gram-negative cell envelope to form a functional apparatus. It is known that T2S:C is a key protein in bridging the inner membrane complex to the outer membrane secretin. In addition, Lybarger et al. (100) determined that T2S:D is required for proper localization and focal assembly of the T2S complex in the membrane. These data, in combination with results from the K. oxytoca T2S:D homolog, demonstrate that the secretin can focally localize in the outer membrane in the absence of other T2S components (129) and suggests that T2S:D may be the driving force behind T2S apparatus assembly. Furthermore, T2S:C appears to be able to interact with T2S: D in the absence of any other T2S components and therefore could direct the T2S:L and M proteins (and presumably the rest of the inner membrane proteins) to where the secretin is located (100).

12



FUTURE DIRECTIONS Secretion Signal The interaction between the secretion system and its substrate is an obvious important step in the secretion process. How are proteins that are to be secreted distinguished from periplasmic proteins? Does a specific member of the T2S apparatus recognize substrates? Examination of many different substrates and secretion machineries reveal that there is no simple answer to these questions. A specific secretion signal or motif has not yet been discovered for T2S substrates. Because T2S substrates are likely folded into tertiary or quaternary structures when recognized by the secretion apparatus, it is currently thought that the targeting information probably resides in the final or near-final conformation of the protein. It is only after the folding of the substrate occurs in the periplasm that the secretion system is thought to recognize proteins designated for export into the extracellular environment. A possible result of this is that the secretion signal need not be a linear string of amino acids in the primary sequence of the secreted protein. Alternatively, the sequence may be linear, but only can be recognized by the apparatus when folded into a final conformation. Some success for identifying the minimum sequence needed for secretion has been made, however. For LT of ETEC, all of the information for secretion is included in the B pentamer portion of the holotoxin. In addition to secretion of B pentamers of cholera toxin and the close homolog LT (130), V. cholerae is capable of secreting the B pentamer of the LT variant, LTIIb, which is less than 10% homologous to CT (131). When the 3D structures of the pentamers are examined, they appear structurally very similar, further substantiating the idea that the secretion signal is not found at the primary sequence level alone (Fig. 2). The 3D structures of several T2S substrates are now available. Even with this information, however, a clear structural motif that is shared between T2S substrates has not been visualized. Clearly, much more work is needed before any conclusions can be made about T2S signals.

Future Challenges The gene products that assemble into the T2S apparatus function both in pathogenic and nonpathogenic bacteria, where they play a crucial role in the survival and persistence of many gram-negative bacteria. A challenge for the future is to uncover how these products create a large,

highly organized, complex capable of secreting a variety of fully folded proteins across the outer membrane of gram-negative bacteria. To do this, numerous interactions between T2S proteins still need to be better understood. Recent structural characterizations along with classical protein-protein interaction experiments are important steps toward completing our picture of how the T2SS assembles and functions in vivo. Further investigation of the stoichiometry and the attempt to define the ordered assembly of the T2S complex should also lead to insights into the mechanism of secretion used by these exported proteins. While only a small number of T2S-dependent substrates are currently known in E. coli, it is likely that, as has been discovered in other gram-negative bacteria, many more are encoded in the genomes of these organisms. The ability to sequence entire genomes of bacteria inhabiting diverse environments around the globe will also lead to the ability for researchers to explore the role of the T2SS in both environmental, as well as pathogenic, bacteria. Investigation of the T2S secretomes of gram-negative bacteria, including E. coli, will likely increase not only the number of T2S-dependent proteins, but also the types of proteins exported through the system. It is also likely that secreted proteins that do not share any homology to known proteins, and that therefore could be novel substrates secreted by the T2SS, will also be discovered. Another exciting possibility that can be addressed by genomic sequencing is the existence of variations of the T2SS. One such system, Stt (for second type II), has already been reported in the plant pathogen Dickeya dadantii. Ferrandez and Condemine (132) identified a cluster of genes in this bacterium encoding a second T2SS that contained homologs of all the T2S proteins except T2S:B, H, and O. pnlH, a gene encoding a homolog of the E. carotovora pectinase, was located immediately downstream of the cluster and was therefore considered a candidate for secretion via this second T2SS. In wild-type cells, PnlH was translocated across the inner membrane via the Tat system and apparently targeted to the outer membrane by its uncleaved N-terminal signal peptide. When examined in a stt mutant or in E. coli, PnlH was localized to the outer membrane but could not be detected on the outside of the cell (132). Thus, this alternative T2SS appears to be responsible for translocating PnlH to the extracellular surface of the outer membrane, although it is not responsible for targeting the protein to the outer membrane per se.



Patrick et al.

ACKNOWLEDGMENTS No potential conflicts of interest relevant to this review were reported.

REFERENCES 1. Filloux A. 2004. The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694:163–179. 2. Johnson TL, Abendroth J, Hol WGJ, Sandkvist M. 2006. Type II secretion: from structure to function. FEMS Microbiol Lett 255:175– 186. 3. Sandkvist M. 2001. Biology of type II secretion. Mol Microbiol 40:271–283. 4. Sandkvist M. 2001. Type II secretion and pathogenesis. Infect Immun 69:3523–3535. 5. Nunn D. 1999. Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol. 9:402–408. 6. Schmidt H, Henkel B, Karch H. 1997. A gene cluster closely related to type II secretion pathway operons of Gram-negative bacteria is located on the large plasmid of enterohemorrhagic Escherichia coli O157 strains. FEMS Microbiol Lett 148:265–272. 7. Yang J, Baldi DL, Tauschek M, Strugnell RA, Robins-Browne RM. 2007. Transcriptional regulation of the yghJ-pppA-yghGgspCDEFGHIJKLM cluster, encoding the type II secretion pathway in enterotoxigenic Escherichia coli. J Bacteriol 189:142–150. 8. Ho TD, Davis BM, Ritchie JM, Waldor MK. 2008. Type 2 secretion promotes enterohemorrhagic Escherichia coli adherence and intestinal colonization. Infect Immun 76:1858–1865. 9. Kulkarni R, Dhakal BK, Slechta ES, Kurtz Z, Mulvey MA, Thanass DG. 2009. Roles of putative type II secretion and type IV pilus systems in the virulence of uropathogenic Escherichia coli. PLoS ONE 4:e4752. 10. Trachman JD, Maas WK. 1998. Temperature regulation of heatlabile enterotoxin (LT) synthesis in Escherichia coli is mediated by an interaction of H-NS protein with the LT A-subunit DNA. J Bacteriol 180:3715–3718. 11. Kunkel SL, Robertson DC.. 1979. Factors affecting release of heat-labile enterotoxin by enterotoxigenic Escherichia coli. Infect Immun 23:652–659. 12. Trachman JD, Yasmin M. 2004. Thermo-osmoregulation of heatlabile enterotoxin expression by Escherichia coli. Curr Microbiol 49:353–360. 13. Francetic O, Belin D, Badaut C, Pugsley AP. 2000. Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. EMBO J 19:6697–6703. 14. Chapon-Herve V, Akrim M, Latifi A, Williams P, Lazdunski A, Bally M. 1997. Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa. Mol Microbiol 24:1169–1178. 15. Ball G, Durand E, Lazdunski A, Filloux A. 2002. A novel type II secretion system in Pseudomonas aeruginosa. Mol Microbiol 43:475– 485. 16. Jones S, Yu B, Bainton NJ, Birdsall M, Bycroft BW, Chhabra SR, Cox AJR, Golby P, Reeves PJ, Stephens S, Winson MK, Salmond GPC, Stewart GSAB, Williams P. 1993. The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. EMBO J 12:2477–2482. 17. Lindeberg M, Collmer A. 1992. Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria. J Bacteriol 174:7385–7397.

18. Ding Y, Davis BM, Waldor MK. 2004. Hfq is essential for Vibrio cholerae virulence and downregulates sigma E expression. Mol Microbiol. 53:345–354. 19. Swift S, Karlyshev AV, Fish L, Durant EL, Winson MK, Chhabra SR, Williams P, MacIntyre S, Stewart GSAB. 1997. Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate Nacylhomoserine lactone signal molecules. J Bacteriol 179:5271–5281. 20. Swift S, Lynch MJ, Fish L, Kirke DF, Tomas JM, Stewart GSAB, Williams P. 1999. Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect Immun 67:5192–5199. 21. DebRoy S, Dao J, Soderberg M, Rossier O, Cianciotto NP. 2006. Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung. Proc Natl Acad Sci USA 103:19146–19151. 22. Dodd HN, Pemberton JM. 1996. Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636. J Bacteriol 178:3926–3933. 23. Rossier O, Dao J, Cianciotto NP. 2009. A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis. Microbiology 155:882–890. 24. Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, Wu CY. 1992. Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol 174:2679–2687. 25. Possot OM, Vignon G, Bomchil N, Ebel F, Pugsley AP. 2000. Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE. J Bacteriol 182:2142–2152. 26. Jiang B, Howard SP. 1992. The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer-membrane biogenesis, is a member of a general secretion pathway. Mol Microbiol 6:1351–1361. 27. Francetic O, Badaut C, Rimsky S, Pugsley AP. 2000. The ChiA (YheB) protein of Escherichia coli K-12 is an endochitinase whose gene is negatively controlled by the nucleoid-structuring protein H-NS. Mol Microbiol 35:1506–1517. 28. Connell TD, Metzger DJ, Lynch J, Folster JP. 1998. Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae. J Bacteriol 180:5591–5600. 29. Donald JW, Hicks MG, Richardson DJ, Palmer T. 2008. The c-type cytochrome OmcA localizes to the outer membrane upon heterologous expression in Escherichia coli. J Bacteriol 190:5127–5131. 30. Shi L, Deng S, Marshall MJ, Wang ZM, Kennedy DW, Dohnalkova AC, Mottaz HM, Hill EA, Gorby YA, Beliaev AS, Richardson DJ, Zachara JM, Fredrickson JK. 2008. Direct involvement of type II secretion system in extracellular translocation of Shewanella oneidensis outer membrane cytochromes MtrC and OmcA. J Bacteriol 190:5512–5516. 31. Filloux A, Bally M, Murgier M, Wretlind B, Lazdunski A. 1989. Cloning of xcp genes located at the 55-min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa. Mol Microbiol 3:261–265. 32. Aragon V, Rossier O, Cianciotto NP. 2002. Legionella pneumophila genes that encode lipase and phospholipase C activities. Microbiology 148:2223–2231. 33. Lathem WW, Grys TE, Witowski SE, Torres AG, Kaper JB, Tarr PI, Welch RA. 2002. StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor. Mol Microbiol 45:277–288. ASMScience.org/EcoSalPlus



34. Lindgren V, Wretlind B. 1987. Characterization of a Pseudomonas aeruginosa transposon insertion mutant with defective release of exoenzymes. J Gen Microbiol 133:675–681. 35. Overbye LJ, Sandkvist M, Bagdasarian M. 1993. Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae. Gene 132:101–106. 36. Sandkvist M, Morales V, Bagdasarian M. 1993. A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae. Gene 123:81–86. 37. Tauschek M, Gorrell RJ, Strugnell RA, Robins-Browne RM. 2002. Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli. Proc Natl Acad Sci USA 99:7066–7071. 38. Sixma TK, Pronk SE, Kalk KH, Wartna ES, Vanzanten BAM, Witholt B, Hol WGJ. 1991. Crystal-structure of a cholera toxinrelated heat-labile enterotoxin from Escherichia coli. Nature 351:371– 377. 39. Waldor MK, Mekalanos JJ. 1996. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910–1914. 40. Gyles CL, Palchaudhuri S, Maas WK. 1977. Naturally occurring plasmid carrying genes for enterotoxin production and drug-resistance. Science 198:198–199. 41. Takeda Y, Murphy JR. 1978. Bacteriophage conversion of heatlabile enterotoxin in Escherichia coli. J Bacteriol 133:172–177. 42. Imamura S, Kido N, Kato M, Kawase H, Miyama A, Tsuji T. 1997. A unique DNA sequence of human enterotoxigenic Escherichia coli enterotoxin encoded by chromosomal DNA. FEMS Microbiol Lett 146:241–245. 43. Pickett CL, Weinstein DL, Holmes RK. 1987. Genetics of type-IIa heat-labile enterotoxin of Escherichia coli—operon fusions, nucleotide sequence, and hybridization studies. J Bacteriol 169:5180–5187. 44. Yamamoto, Yokota TT. 1983. Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans. J Bacteriol 155:728–733. 45. Yu J, Webb H, Hirst TR. 1992. A homolog of the Escherichia coli DsbA protein involved in disulfide bond formation is required for enterotoxin biogenesis in Vibrio cholerae. Mol Microbiol 6:1949– 1958. 46. Horstman AL, Kuehn MJ. 2000. Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol Chem 275:12489–12496. 47. Lariviere S, Gyles CL, Barnum DA. 1973. Preliminary characterization of heat-labile enterotoxin of Escherichia coli F11(P155). J Infect Dis 128:312–320. 48. Wensink J, Gankema H, Jansen WH, Guinee PAM, Witholt B. 1978. Isolation of membranes of an enterotoxigenic strain of Escherichia coli and distribution of enterotoxin activity in different subcellular fractions. Biochim Biophys Acta 514:128–136. 49. Horstman AL, Bauman SJ, Kuehn MJ. 2004. Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins. J Biol Chem 279:8070–8075. 50. Spangler BD. 1992. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 56:622–647. 51. Fleckenstein JM, Lindler LE, Elsinghorst EA, Dale JB. 2000. Identification of a gene within a pathogenicity island of enterotoxigenic Escherichia coli H10407 required for maximal secretion of the heat-labile enterotoxin. Infect Immun 68:2766–2774. 52. Fukuta S, Magnani JL, Twiddy EM, Holmes RK, Ginsburg V. 1988. Comparison of the carbohydrate binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb. Infect Immun 56:1748–1753.

53. Lencer WI, Tsai B. 2003. The intracellular voyage of cholera toxin: going retro. Trends Biochem Sci 28:639–645. 54. Tsai B, Rodighiero C, Lencer WI, Rapoport TA. 2001. Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell 104:937–948. 55. Berberov EM, Zhou Y, Francis DH, Scott MA, Kachman SD, Moxey RA. 2004. Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins. Infect Immun 72:3914–3924. 56. Zhang WP, Berberov EM, Freeling J, He D, Moxley RA, Francis DH. 2006. Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies. Infect Immun 74:3107–3114. 57. Li XB, Roseman S. 2004. The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase. Proc Natl Acad Sci USA 101:627–631. 58. Yamano N, Oura N, Wang JY, Fujishima S. 1997. Cloning and sequencing of the genes for N-acetylglucosamine use that construct divergent operons (nagE-nagAC) from Vibrio cholerae non-O1. Biosci Biotechnol Biochem 61:1349–1353. 59. Wang SL, Chang WT. 1997. Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl Environ Microbiol 63:380–386. 60. Reguera G, Kolter R. 2005. Virulence and the environment: a novel role for Vibrio cholerae toxin-coregulated pili in biofilm formation on chitin. J Bacteriol 187:3551–3555. 61. Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK. 2005. Chitin induces natural competence in Vibrio cholerae. Science 310:1824–1827. 62. Nalin DR, Daya V, Reid A, Levine MM, Cisneros L. 1979. Adsorption and growth of Vibrio cholerae on chitin. Infect Immun 25:768–770. 63. Amako K, Shimodori S, Imoto T, Miake S, Umeda A. 1987. Effects of chitin and its soluble derivatives on survival of Vibrio cholerae O1 at low temperature. Appl Environ Microbiol 53:603– 605. 64. Francetic O, Lory S, Pugsley AP. 1998. A second prepilin peptidase gene in Escherichia coli K-12. Mol Microbiol 27:763–775. 65. Elliott SJ, Sperandio V, Giron JA, Shin S, Mellies JL, Wainwright L, Hutcheson SW, McDaniel TK, Kaper JB. 2000. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 68:6115– 6126. 66. Grys TE, Siege MB, Lathem WW, Welch RA. 2005. The StcE protease contributes to intimate adherence of enterohemorrhagic Escherichia coli O157:H7 to host cells. Infect Immun 73:1295–1303. 67. Szabady RL, Lokuta MA, Walters KB, Huttenlocher A, Welch RA. 2009. Modulation of neutrophil function by a secreted mucinase of Escherichia coli O157:H7. Plos Pathog 5:e1000320. 68. Finkelstein RA, Boesman-Finkelstein M, Holt P. 1983. Vibrio cholerae hemagglutinin lectin protease hydrolyzes fibronectin and ovomucin: Burnet, F.M. revisited. Proc Natl Acad Sci USA 80:1092– 1095. 69. Häse CC, Finkelstein RA. 1990. Comparison of the Vibrio cholerae hemagglutinin protease and the Pseudomonas aeruginosa elastase. Infect Immun 58:4011–4015. 70. Bally M, Filloux A, Akrim M, Ball G, Lazdunski A, Tommassen J. 1992. Protein secretion in Pseudomonas aeruginosa—characterization



Patrick et al. of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol 6:1121–1131. 71. Puskárová, A, Ferianc P, Kormanec J, Homerova D, Farewell A, Nyström T. 2002. Regulation of yodA encoding a novel cadmiuminduced protein in Escherichia coli. Microbiology 148:3801–3811. 72. David G, Blondeau K, Schiltz M, Penel S, Lewit-Bentley A. 2003. YodA from Escherichia coli is a metal-binding, lipocalin-like protein. J Biol Chem 278:43728–43735. 73. Kershaw CJ, Brown NL, Hobman JL. 2007. Zinc dependence of zinT (yodA) mutants and binding of zinc, cadmium and mercury by ZinT. Biochem Biophys Res Commun 364:66–71. 74. Flower DR, North ACT, Attwood TK. 1993. Structure and sequence relationships in the lipocalins and related proteins. Protein Sci 2:753–761. 75. Le Bouguenec C, Lalioui L, Du Merle L, Jouve M, Courcoux P, Bouzari S, Selvarangan R, Nowicki BJ, Germani Y, Andremont A, Gounon P, Garcia MI. 2001. Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of afa adhesins that do or do not recognize Dr blood group antigens. J Clin Microbiol 39:1738– 1745. 76. Medof ME, Kinoshita T, Nussenzweig V. 1984. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (Daf) into their membranes. J Exp Med 160:1558–1578. 77. Anderson KL, Billington J, Pettigrew D, Cota E, Simpson P, Roversi P, Chen HA, Urvil P, Du Merle L, Barlow PN, Medof ME, Smith RAG, Nowicki B, Le Bouguenec C, Lea SM, Matthews S. 2004. An atomic resolution model for assembly, architecture, and function of the Dr adhesins. Mol Cell 15:647–657. 78. Zalewska B, Piatek R, Bury K, Samet A, Nowicki B, Nowicki S, Kur J. 2005. A surface-exposed DraD protein of uropathogenic Escherichia coli bearing Dr fimbriae may be expressed and secreted independently from DraC usher and DraE adhesin. Microbiology 151:2477–2486. 79. Zalewska-Piatek B, Bury K, Piatek R, Bruzdziak P, Kur J. 2008. Type II secretory pathway for surface secretion of DraD invasin from the uropathogenic Escherichia coli Dr(+) strain. J Bacteriol 190:5044– 5056. 80. Planet PJ, Kachlany SC, DeSalle R, Figurski DH. 2001. Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci USA 98:2503–2508. 81. Robien MA, Krumm BE, Sandkvst M, Hol WGJ. 2003. Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol 333:657–674. 82. Camberg, Sandkvist JLM. 2005. Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol 187:249– 256. 83. Camberg JL, Johnson TL, Patrick M, Abendroth J, Hol WGJ, Sandkvist M. 2007. Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids. EMBO J. 26:19–27. 84. Abendroth J, Murphy P, Sandkvist M, Bagdasarian M, Hol WGJ. 2005. The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J Mol Biol 348:845–855. 85. Py B, Loiseau L, Barras F. 1999. Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL. J Mol Biol 289:659– 670.

86. Sandkvist M, Bagdasarian M, Howard SP, Dirita VJ. 1995. Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J 14:1664–1673. 87. Sandkvist M, Hough LP, Bagdasarian MM, Bagdasarian M. 1999. Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae. J Bacteriol 181:3129–3135. 88. Douet V, Loiseau L, Barras F, Py B. 2004. Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi. Res Microbiol 155:71–75. 89. Lee HM, Chen JR, Lee HL, Leu WM, Chen LY, Hu NT. 2004. Functional dissection of the XpsN (GspC) protein of the Xanthomonas campestris pv. campestris type II secretion machinery. J Bacteriol 186:2946–2955. 90. Py B, Loiseau L, Barras F. 2001. An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep 2:244–248. 91. Robert V, Filloux A, Michel GP. 2005. Role of XcpP in the functionality of the Pseudomonas aeruginosa secreton. Res Microbiol 156:880–886. 92. Sandkvist M, Keith JM, Bagdasarian M, Howard SP. 2000. Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae. J Bacteriol 182:742–748. 93. Tsai RT, Leu WM, Chen LY, Hu NT. 2002. A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J 367:865–871. 94. Abendroth J, Kreger AC, Hol WGJ. 2009. The dimer formed by the periplasmic domain of EpsL from the type 2 secretion system of Vibrio parahaemolyticus. J Struct Biol 168:313–322. 95. Abendroth J, Rice AE, McLuskey K, Bagdasarian M, Hol WGJ. 2004. The crystal structure of the periplasmic domain of the type II secretion system protein EpsM from Vibrio cholerae: the simplest version of the ferredoxin fold. J Mol Biol 338:585–596. 96. Johnson TL, Scott ME, Sandkvist M. 2007. Mapping critical interactive sites within the periplasmic domain of the Vibrio cholerae type II secretion protein EpsM. J Bacteriol 189:9082–9089. 97. Abendroth J, Mitchell DD, Korotkov KV, Johnson TL, Kreger A, Sandkvist M, Hol WGJ. 2009. The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae. J Struct Biol 166:303–315. 98. Arts J, de Groot A, Ball G, Durand E, El Khattabi M, Filloux A, Tommassen J, Koster M. 2007. Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF). Microbiology 153:1582–1592. 99. Gerard-Vincent M, Robert V, Ball G, Bleves S, Michel GPF, Lazdunski A, Filloux A. 2002. Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus. Mol Microbiol 44:1651–1665. 100. Lybarger SR, Johnson TL, Gray MD, Sikora AE, Sandkvist M. 2009. Docking and assembly of the type II secretion complex of Vibrio cholerae. J Bacteriol 191:3149–3161. 101. Bleves S, Gerard-Vincent M, Lazdunski A, Filloux A. 1999. Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa. J Bacteriol 181:4012–4019. 102. Possot OM, Gerard-Vincent M, Pugsley AP. 1999. Membrane association and multimerization of secreton component PulC. J Bacteriol 181:4004–4011. ASMScience.org/EcoSalPlus



103. Korotkov KV, Krumm B, Bagdasarian M, Hol WG. 2006. Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae. J Mol Biol 363:311–321. 104. Bouley J, Condemine G, Shevchik VE. 2001. The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi. J Mol Biol 308:205–219. 105. Brok R, Van Gelder P, Winterhalter M, Ziese U, Koster AJ, de Cock H, Koster M, Tommassen J, Bitter W. 1999. The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity. J Mol Biol 294:1169–1179. 106. Hardie KR, Lory S, Pugsley AP. 1996. Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein. EMBO J 15:978–988. 107. Shevchik VE, Condemine C. 1998. Functional characterization of the Erwinia chrysanthemi OutS protein, an element of a type II secretion system. Microbiology 144:3219–3228. 108. Viarre V, Cascales E, Ball G, Michel GPF, Filloux A, Voulhoux R. 2009. HxcQ liposecretin is self-piloted to the outer membrane by its N-terminal lipid anchor. J Biol Chem 284:33815–33823. 109. Bitter W, Koster M, Latijnhouwers M, de Cock H, Tommassen J. 1998. Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 27:209–219. 110. Chami M, Guilvout I, Gregorini M, Remigy HW, Muller SA, Valerio M, Engel A, Pugsley AP, Bayan N. 2005. Structural insights into the secretin PulD and its trypsin resistant core. J Biol Chem 280:37732–37741. 111. Nouwen N, Stahlberg H, Pugsley AP, Engel A. 2000. Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy. EMBO J 19:2229–2236. 112. Guilvout I, Hardie KR, Sauvonnet N, Pugsley AP. 1999. Genetic dissection of the outer membrane secretin PulD: are there distinct domains for multimerization and secretion specificity? J Bacteriol 181:7212–7220. 113. Korotkov KV, Gray MD, Kreger A, Turley S, Sandkvist M, Hol WGJ. 2009. Calcium is essential for the major pseudopilin in the type 2 secretion system. J Biol Chem 284:25466–25470. 114. Lindeberg M, Salmond GP, Collmer A. 1996. Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. Mol Microbiol 20:175– 190. 115. Shevchik VE, Robert-Baudouy J, Condemine G. 1997. Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins. EMBO J 16:3007–3016. 116. Nunn DN, Lory S. 1993. Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, XcpU, XcpV, and XcpW. J Bacteriol 175:4375–4382. 117. Hu NT, Leu WM, Lee MS, Chen A, Chen SC, Song YL, Chen LY. 2002. XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J 365:205–211.

118. Durand E, Bernadac A, Ball G, Lazdunski A, Sturgis JN, Filloux A. 2003. Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure. J Bacteriol 185:2749–2758. 119. Köhler R, Schafer K, Muller S, Vignon G, Diederichs K, Philippsen A, Ringler P, Pugsley AP, Engel A, Welte W. 2004. Structure and assembly of the pseudopilin PulG. Mol Microbiol 54:647–664. 120. Vignon G, Kohler R, Larquet E, Giroux S, Prevost MC, Roux P, Pugsley AP. 2003. Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization, and surface presentation of peptides. J Bacteriol 185:3416–3428. 121. Sauvonnet N, Vignon G, Pugsley AP, Gounon P. 2000. Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J 19:2221–2228. 122. Durand E, Michel G, Voulhoux R, Kurner J, Bernadac A, Filloux A. 2005. XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus. J Biol Chem 280:31378– 31389. 123. Korotkov KV, Hol WGJ. 2008. Structure of the GspK-GspIGspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. Nat Struct Mol Biol 15:462–468. 124. Yanez ME, Korotkov KV, Abendroth J, Hol WGJ. 2008. Structure of the minor pseudopilin EpsH from the type 2 secretion system of Vibrio cholerae. J Mol Biol 377:91–103. 125. Yanez ME, Korotkov KV, Abendroth J, Hol WGJ. 2008. The crystal structure of a binary complex of two pseudopilins: EpsI and EpsJ from the type 2 secretion system of Vibrio vulnificus. J Mol Biol 375:471–486. 126. Alphonse S, Durand E, Douzi B, Waegele B, Darbon H, Filloux A, Voulhoux R, Bernard C. 2010. Structure of the Pseudomonas aeruginosa XcpT pseudopilin, a major component of the type II secretion system. J Struct Biol 169:75–80. 127. Douzi B, Durand E, Bernard C, Alphonse S, Cambillau C, Filloux A, Tegoni M, Voulhoux R. 2009. The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus. J Biol Chem 284:34580–34589. 128. Kuo WW, Kuo HW, Cheng CC, Lai HL, Chen LY. 2005. Roles of the minor pseudopilins, XpsH, XpsI and XpsJ, in the formation of XpsG-containing pseudopilus in Xanthomonas campestris pv. campestris. J Biomed Sci 12:587–599. 129. Buddelmeijer N, Krehenbrink M, Pecorari F, Pugsley AP. 2009. Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane. J Bacteriol 191:161–168. 130. Hirst TR, Sanchez J, Kaper JB, Hardy SJ, Holmgren J. 1984. Mechanism of toxin secretion by Vibrio cholerae investigated in strains harboring plasmids that encode heat-labile enterotoxins of Escherichia coli. Proc Natl Acad Sci USA 81:7752–7756. 131. Connell TD, Metzger DJ, Wang ML, Jobling MG, Holmes RK. 1995. Initial Studies of the Structural Signal for Extracellular Transport of Cholera Toxin and Other Proteins Recognized by Vibrio cholerae. Infect Immun 63:4091–4098. 132. Ferrandez Y, Condemine G. 2008. Novel mechanism of outer membrane targeting of proteins in Gram-negative bacteria. Mol Microbiol 69:1349–1357.



Downloaded from www.asmscience.org by IP: 132.239.1.231 On: Fri, 06 Jan 2017 10:29:45

Distribution of non-LEE-encoded type 3 secretion system dependent effectors in enteropathogenic Escherichia coli.

Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli.

Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides.

The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression.

Dynamics of expression and maturation of the type III secretion system of enteropathogenic Escherichia coli.

Escherichia coli type III secretion system 2: a new kind of T3SS?

The type III secretion effector NleF of enteropathogenic Escherichia coli activates NF-κB early during infection.

Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition.

Secretion of active bovine somatotropin in Escherichia coli.

Deciphering the role of the type II glyoxalase isoenzyme YcbL (GlxII-2) in Escherichia coli.

Escherichia coli Type III Secretion System 2 ATPase EivC Is Involved in the Motility and Virulence of Avian Pathogenic Escherichia coli.

Uptake and subcellular distribution of Escherichia coli lipopolysaccharide by isolated rat type II pneumocytes.

Secretion of active truncated CD4 into Escherichia coli periplasm.

Extracellular expression of Thermobifida fusca cutinase with pelB signal peptide depends on more than type II secretion pathway in Escherichia coli.

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Charge requirements of lipid II flippase activity in Escherichia coli.

Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system.

Novel type of murein transglycosylase in Escherichia coli.

Escherichia coli sequence type 131: epidemiology and challenges in treatment.

Expression of fowl adenovirus type 10 antigens in Escherichia coli.

Maleimide II--interaction with L-asparaginase from Escherichia coli.

Purification and characterization of Escherichia coli heat-stable enterotoxin II.

Protease II from Escherichia coli. Substrate specificity and kinetic properties.