1.C.36 The Bacterial Type III-Target Cell Pore (IIITCP) Family

The Type III secretory pathway (IIISP, TC #3.A.6) allows bacteria to pass proteins directly from the bacterial cytoplasm into the host animal or plant cell cytoplasm. In order for this to occur, the bacteria construct a complex secretory apparatus allowing a direct pathway for the passage of proteins. One bacterial protein of each type IIISP system is secreted directly into the host cell membrane where it oligomerizes to form a transmembrane translocation pore at the site of bacterial contact. These homologous proteins comprise the IIITCP family. They are very divergent in sequence but have a uniform topology with two conserved transmembrane spanners (TMSs) and one conserved coiled coil region. The best characterized members of the IIITCP family include EspD for enteropathogenic E. coli, YopB for Yersinia species, PopB and PepB for Pseudomonas species, IpaB for Shigella species and SipB for Salmonella species. The three clusters of proteins indicated in the table (1, EspD; 2, YopB, PopB and PepB; and 3, IpaA and SipB) are very divergent in sequence so that PSI-BLAST reveals only limited sequence similarity between clusters. Proteins of the third cluster, including IpaB and SipB, are also much larger than the proteins in clusters 1 and 2 due in part to a 150 amino acyl residue N-terminal extension.

YopB and YopD of Yersinia species form a complex in the bacterial cytoplasm with their chaperone, SycD, suggesting that they are secreted as a heterocomplex. They may act together in forming the pore structure in the host cell membrane. YopB contains two central hydrophobic domains and is predicted to be an integral membrane protein, while YopD has a single central hydrophobic domain. In Bordetella bronchiseptica, BopB and BopD are also complexed to form a pore in the host cell plasma membrane (Nogawa et al., 2004). It seems likely that these two proteins are required for pore formation in all of these homologous (but very distantly related) systems.

YopB and YopD of Yersinia enterocolitica have been reported to both be required for pore formation in macrophage plasma membranes. The estimated inner pore diameter is about 2.0 nm. There is disagreement as to the requirement for YopD; some suggest that YopB alone forms the pore while others suggest that the YopB-YopD complex comprise the structural components of the pore. Studies in artificial membranes suggest that YopB alone forms channels with no ionic selectivity and that YopD alters the channel (Tardy et al., 1999). Thus, YopB is probably the channel while YopD is an accessory protein. The P. aeruginosa homologues, PopB/PopD with the PcrV protein form channels (2.8-3.5 nm inner diameter) both in red blood cells and in macrophage. In the former, they induce haemolysis. By electron microscopy PopB and PopD form 80 Å wide rings that encircle a ~40 Å cavity (Schoehn et al., 2003).

The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. The tip of the TTSS needle interacts with the translocon. Mutations in the needle protein, YscF (87 aas; TC# 3.A.6.1.1) separate its function in secretion from its role in translocation (Davis and Mecsas, 2006). Two yscF mutants formed needles and secreted Yops normally displayed distinct translocation defects. One showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. The second formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions.

The Salmonella SipB protein has been studied topologically in model bilayers (McGhie et al., 2002). The protein can exist in soluble and membrane-inserted forms, a property typical of bacterial exotoxins. SipB inserts into the host cell membrane and has heterotypic membrane fusion activity in vitro. The membrane integrated protein (593 aas) is predominantly α-helical as is true for the soluble form. Two hydrophobic α-helices (residues 320-353 and 409-427) integrate into the membrane obliquely, allowing residues 354-408 to cross the bilayer while a C-terminal region associates with the bilayer surface. The homologous IpAB of Shigella assembles into a homotrimer via an N-terminal coiled-coil domain (Hume et al., 2003). SipB and IpaB integrate with the same membrane topology.

The proteins in this family appear to be related to the putative pore-forming bacterial proteins in TC sub-family 1.C.127.2 which appear to be distantly related to pore-forming lipoproteins of animals in TC sub-family 1.C.127.1 However, the relationships between these subfamilies and 1.C.36 is not fully established. 

The generalized transport reaction that is facilitated by IIITCP family members is:

proteins (bacterial cytoplasm) → proteins (host cell cytoplasm).

 



This family belongs to the .

 

References:

Barta, M.L., N.E. Dickenson, M. Patil, A. Keightley, G.J. Wyckoff, W.D. Picking, W.L. Picking, and B.V. Geisbrecht. (2012). The structures of coiled-coil domains from type III secretion system translocators reveal homology to pore-forming toxins. J. Mol. Biol. 417: 395-405.

Chatterjee A., Caballero-Franco C., Bakker D., Totten S. and Jardim A. (2015). Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD. J Biol Chem. 290(42):25579-94.

Dacheux, D., J, Goure, J. Chabert, Y. Usson, and I. Attree. (2001). Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages. Mol. Microbiol. 40: 76-85.

Davis A.J., Mecsas J. (2006). Mutations in the Yersinia pseudotuberculosis type III secretion system needle protein, YscF, that specifically abrogate effector translocation into host cells. J Bacteriol. 189: 83-97.

Hueck, C.J. (1998). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62: 379-433.

Hume, P.J., E.J. McGhie, R.D. Hayward, and V. Koronakis. (2003). The purified Shigella IpaB and Salmonella SipB translocators share biochemical properties and membrane topology. Mol. Microbiol. 49: 425-439.

Lee, V.T. and O. Schneewind. (1999). Type III machines of pathogenic yersiniae secrete virulence factors into the extracellular milieu. Mol. Microbiol. 31: 1619-1629.

Luo, W. and M.S. Donnenberg. (2011). Interactions and predicted host membrane topology of the enteropathogenic Escherichia coli translocator protein EspB. J. Bacteriol. 193: 2972-2980.

McGhie, E.J., P.J. Hume, R.D. Hayward, J. Torres, and V. Koronakis. (2002). Topology of the Salmonella invasion protein SipB in a model bilayer. Mol. Microbiol. 44: 1309-1321.

Montagner, C., C. Arquint, and G.R. Cornelis. (2011). Translocators YopB and YopD from Yersinia enterocolitica form a multimeric integral membrane complex in eukaryotic cell membranes. J. Bacteriol. 193: 6923-6928.

Neyt, C. and G.R. Cornelis. (1999). Insertion of a Yop translocation pore into the macrophage plasma membrane byYersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG. Mol. Microbiol. 33: 971-981.

Nguyen, V.S., C. Jobichen, K.W. Tan, Y.W. Tan, S.L. Chan, K. Ramesh, Y. Yuan, Y. Hong, J. Seetharaman, K.Y. Leung, J. Sivaraman, and Y.K. Mok. (2015). Structure of AcrH-AopB Chaperone-Translocator Complex Reveals a Role for Membrane Hairpins in Type III Secretion System Translocon Assembly. Structure 23: 2022-2031.

Nikolaus, T., J. Deiwick, C. Rappl, J.A. Freeman, W. Schröder, S.I. Miller, and M. Hensel. (2001). SseBCD proteins are secreted by the Type III secretion system of Salmonella pathogenicity island 2 and function as a translocon. J. Bacteriol. 183: 6036-6045.

Nogawa, H., A. Kuwae, T. Matsuzawa, and A. Abe. (2004). The Type III secreted protein BopD in Bordetella bronchiseptica is complexed with BopB for pore formation on the host plasma membrane. J. Bacteriol. 186: 3806-3813.

Olsson, J., P.J. Edqvist, J.E. Bröms, A. Forsberg, H. Wolf-Watz, and M.S. Francis. (2004). The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains. J. Bacteriol. 186: 4110-4123.

Romano, F.B., K.C. Rossi, C.G. Savva, A. Holzenburg, E.M. Clerico, and A.P. Heuck. (2011). Efficient Isolation of Pseudomonas aeruginosa Type III Secretion Translocators and Assembly of Heteromeric Transmembrane Pores in Model Membranes. Biochemistry 50: 7117-7131.

Romano, F.B., Y. Tang, K.C. Rossi, K.R. Monopoli, J.L. Ross, and A.P. Heuck. (2016). Type 3 Secretion translocators spontaneously assemble a hexadecameric transmembrane complex. J. Biol. Chem. [Epub: Ahead of Print]

Russo, B.C., J.K. Duncan, and M.B. Goldberg. (2019). Topological Analysis of the Type 3 Secretion System Translocon Pore Protein IpaC following Its Native Delivery to the Plasma Membrane during Infection. MBio 10:.

Ryndak, M.B., H. Chung, E. London, and J.B. Bliska. (2005). Role of predicted transmembrane domains for type III translocation, pore formation, and signaling by the Yersinia pseudotuberculosis YopB protein. Infect. Immun. 73: 2433-2443.

Schoehn, G., A.M. Di Guilmi, D. Lemaire, I. Attree, W. Weissenhorn, and A. Dessen. (2003). Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas. EMBO J. 22: 4957-4967.

Solomon, R., W. Zhang, G. McCrann, J.B. Bliska, and G.I. Viboud. (2015). Random Mutagenesis Identifies a C-Terminal Region of YopD Important for Yersinia Type III Secretion Function. PLoS One 10: e0120471.

Tang, Y., F.B. Romano, M. Breña, and A.P. Heuck. (2018). The type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes. J. Biol. Chem. [Epub: Ahead of Print]

Tardy, F., F. Homblè, C. Neyt, R. Wattiez, G.R. Cornelis, J.-M. Ruysschaert, and V. Cabiaux. (1999). Yersinia enterocholitica type III secretion-translocation system: channel formation by secreted Yops. EMBO J. 18: 6793-6799.

Veenendaal, A.K., J.L. Hodgkinson, L. Schwarzer, D. Stabat, S.F. Zenk, and A.J. Blocker. (2007). The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol. Microbiol. 63: 1719-1730.

Wachter, C., C. Beinke, M. Mattes, and M.A. Schmidt. (1999). Insertion of EspD into epithelial target cell membranes by infecting enteropathogenic Escherichia coli. Mol. Microbiol. 31: 1695-1707.

Winans, S.C. (2004). Reciprocal regulation of bioluminescence and type III protein secretion in Vibrio harveyi and Vibrio parahaemolyticus in response to diffusible chemical signals. J. Bacteriol. 186: 3674-3676.

Zurawski, D.V. and M.A. Stein. (2004). The SP12-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD. Microbiology 150: 2055-2068.

Examples:

TC#NameOrganismal TypeExample
1.C.36.1.1

IIITCP protein complex EspB/EspD. The topology of and EspD interaction sites in EspB have been defined (Luo and Donnenberg, 2011).  EspD inserts into the membrane with its two helical hairpins traversing the membrane with the N- and C-termini on the extraluminal surface, forming 2.5 diameter pores (Chatterjee et al. 2015).

Gram-negative bacteria

EspB/EspD of E. coli
EspB (NP_290254)
EspD (NP_290255)

 
Examples:

TC#NameOrganismal TypeExample
1.C.36.2.1

IIITCP protein complex, YopB/YopD (Olsson et al., 2004). TMS2 is essential for function, while TMS1 is partially defective for translocation, pore formation, and signaling (Ryndak et al. 2005). The system forms a multimeric integral membrane complex (Montagner et al., 2011).  Mutants have been isoated which show defects in effector translocation and pore formation, and many of these are in a C-terminal domain (Solomon et al. 2015).

Proteobacteria

YopB/YopD of Yersinia pseudotuberculosis
YopB (Q06114)
YopD (Q06131)

 
1.C.36.2.2

IIITCP protein complex, PopB/PopD. Purified PopB and PopD form pores in model membranes (Romano et al., 2011).  PopB in isolation forms a biimodal distribution of two complexes with 6 and 12 subunits while PopD forms a hexameric complex.  However when present together, they form a hexadecameric transmembrane complex (Romano et al. 2016). PopB assists with the proper insertion of PopD into cell membranes and is required for the formation of a functional translocon and host infection (Tang et al. 2018).

Gram-negative bacteria

PopB/PopD of Pseudomonas aeruginosa
PopB (AAO91773)
PopD (AAO91774)

 
1.C.36.2.3

Translocator complex AopB/AopD of 347 and 299 aas, respectively.  AopB has been crystalized and the structure determined for this protein in complex with the AcrH chaperone protein (Nguyen et al. 2015). The structure revealed unique interactions between the various interfaces of AopB and AcrH, with the N-terminal "molecular anchor" of AopB crossing into the "N-terminal arm" of AcrH. AopB adopts a novel fold, and its transmembrane regions form two pairs of helical hairpins.

AopBD of Aeromonas hydrophila

 
Examples:

TC#NameOrganismal TypeExample
1.C.36.3.1

IIITCP protein complex, IpaB/IpaC/IpaD. Physical contact with host cells initiates secretion and leads to assembly of a pore, IpaB/IpaC, in the host cell membrane. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and the two pore-forming proteins, IpaB and IpaC. The atomic structures of IpaD and a protease-stable coiled-coil fragment in the N-terminal regions of IpaB from S. flexneri and the homologous SipB from Salmonella enterica have been determined (Barta et al. 2012).  Structural comparisons revealed similarity to the coiled-coil regions of pore-forming proteins such as colicin Ia (TC# 1.C.1.1.1).  Interaction between IpaB and IpaD at the needle tip is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips (Veenendaal et al. 2007). The N-terminus of IpaC is extracellular and the C-terminus is intracellular, and its topology has been studied (Russo et al. 2019).

Proteobacteria

IpaB/IpaD of Shigella dysenteriae
IpaB (P18011)
IpaD (P18013)
IpaC (P18012)

 
1.C.36.3.2IIITCP protein complex, SipB/SipD of pathogenicity island 1 (SPI1)Gram-negative bacteria SipB/SipD of Salmonella typhimurium
SipB (AAL21765)
SipD (AAL21763)
 
1.C.36.3.3

IIITCP complex, BipB/BipD (BipB, 620aas; BipD, 310aas)

Proteobacteria

BipB/BipD of Burkholderia pseudomallei
BipB (Q3JL23)
BipD (Q3JL26)

 
1.C.36.3.4

IIITCP complex, BipB/BipD (Cell invasion protein complex).

Proteobacteria

BipB/D of Protens mirablis
BipB (B4EYC8)
BipD (B4EYC6)

 
1.C.36.3.5

IIITCP complex protein, CopB of 852aas; 4TMSs

Chlamydia

CopB of Parachlamydia acathamoebae (F8KWQ0)

 
1.C.36.3.6

Putative channel-forming system of a bacterial type III secretion system (see family description).  The two proteins included in this system are encoded by genes that are in a gene cluster that includes a type III secretion system and a chaparone protein of 166 aas, SicA (AKM45441).

Pore-forming two component system of Burkholderia contaminans

 
Examples:

TC#NameOrganismal TypeExample
1.C.36.4.1IIITCP protein complex, BopB/BopD (Nogawa et al., 2004)BacteriaBopB/BopD of Bordetella bronchiseptica
BopB (NP_888166)
BopD (NP_888165)
 
Examples:

TC#NameOrganismal TypeExample
1.C.36.5.1

IIICP protein complex SseB/SseC/SseD; SseB: translocon sheath protein; SseC and SseD: translocon pore subunits of the Salmonella pathogenicity island 2 (SPI2)

Gram-negative bacteriaSseB/SseC/SseD of Salmonella typhimurium
SseB (CAA12185)
SseC (CAA12187)
SseD (CAA12188)
 
Examples:

TC#NameOrganismal TypeExample