Bacterial Secretion Systems

 

Type I Secretion System: Proteins cross both membranes in a single step and do not form periplasmic intermediates.

 

ABC proteins are involved in the uptake of various substrates like maltose, histidine, siderophores, etc in gram negative bacteria.

 

They are also involved in protein secretion.

 

Most are usually specific to one substrate and are involved in either export or import, although there are exceptions.

 

Consists of 3 proteins

 

1. pore-forming outer membrane protein (usually a trimer?)

2. Membrane fusion protein

3. inner-membrane ATP-binding cassette (ABC) protein

 

The process:

 

-the secreted effector molecule has a secretion signal located on its C-terminal end that interacts with the ABC transporter protein

 

-This binding causes the interaction between the membrane fusion protein and the pore forming protein to be triggered, allowing the secretion of the effector molecule.

 

-ATP hydrolysis by the ABC transporter drives secretion of the molecule across the cell membrane

 

For a review see Buchanan 2001 Trends in Biomedical Sciences or look up ABC transporters in a micro text book.

 

Type II Secretion System: Occurs in 2 membrane translocation steps.

 

1. Proteins contain N-terminal signal peptides, which allows Sec-dependent translocation across the cytoplasmic membrane. The signal peptide is removed and the protein folds and is released into the periplasmic space.

-In the periplasm the protein may undergo further modifications like disulphide bond formation or subunit assembly.

 

2. Translocation across the outer membrane via the Type II secretion apparatus.

           

 

Secretion Apparatus: highly specific

Depending on the species 12-15 genes are thought to be involved (gene products A-O).

 

Protein D: Pore of the apparatus

-large oligomer of 12-14 subunits in the OM.

-belongs to the family of Secretins (components are required for type IV pili)

 

Protein S: small OM protein

-helps stabilize protein D

 

Protein B: Interacts with Protein D

-only been identified in a few species

 

Protein N: Cytoplasmic membrane protein

-interacts with protein D

-Maybe species specific

 

Protein C: interacts with integral cytoplasmic proteins L and M (form together as a stable complex)

 

Protein E: (with L and M) regulate secretion by phosphorylation or ATP hydrolysis between the OM and CM pore.

 

Proteins G,H,I,J,K: processed by protein O and form the pilus structure

-pilus may push the protein out of the pore by extension (polymerization) and retraction (depolymerization)

 

Good Summary fig. Taken from Mol Micro review: Sandkvist 2001.

 

Fig. 1. Assembly and secretion of cholera toxin via the type II secretion pathway Eps in V. cholerae. The cholera toxin A and B subunits are translocated as monomeric precursors across the cytoplasmic membrane (CM) via the Sec pathway. The subunits fold and assemble with the assistance of DsbA into the AB₅ toxin complex. The AB₅ then engages the type II secretion apparatus Eps via specific recognition of B₅, which carries the putative secretion signal. The complex is targeted to the secretion pore in the outer membrane (OM) followed by extracellular release. In this model, EpsD forms the secretion pore, and EpsE, EpsL and EpsM regulate extracellular secretion by communication of critical information through phosphorylation or ATP hydrolysis between the CM and the OM pore, possibly via EpsC. Proteins G, H, I, J and K are processed by protein O and are likely to form a pilus-like structure, with protein G as the major component. The pilus may act as a piston to push the toxin out through the OM pore by repeated extension (polymerization) and retraction (depolymerization), as represented by the white double-headed arrow. The EpsD pore also supports extrusion of the filamentous phage CTXφ. EpsD may shuttle between these two pathways (indicated by the black double-headed arrow) or may be present in excess of the other Eps components. Although not present in V. cholerae, protein S (symbol with dashed outline) supports outer membrane insertion of protein D. Protein A forms a complex in the cytoplasmic membrane with protein B, which in turn appears to interact with protein D. Protein N can be immunoprecipitated with protein D. Proteins A, B, N and S are shown in this model; however, they are not present in all organisms with a type II pathway and may not be required for secretion in every case. Protein F is not shown, as no protein–protein interaction has yet been documented

 

 

Type III Secretion System: “Effectors” are translocated  from bacteria to eukaryotic hosts to hijack host cell signaling to benefit the bacteria (pathogenesis) or both organisms (symbiosis).

 

Components of the TTSS:

 

Injectisome: a “nanomachine” which allows secretion of the effectors from the bacterial cytoplasm across the IM, periplasm, and OM to the host cytoplasm where is crosses the plasma membrane.

-Consists of 2 parts:

            1. cylindrical base: similar to the flagellar basal body

-Made up of 2 rings that span the IM and OM connected by a rod.

2. needle: hollow, elongated structure

                  -structure for proteins to travel through

                  -made by polymerization of a major subunit

                              -varies in length from 45-80 nm

 

Translocators: set of proteins that are required to translocate the protein to the eukaryotic cell.  2 are hydrophobic proteins and 1 is hydrophilic.

-The hydrophilic protein is thought to be a chaperone, which helps the hydrophobic transporters to insert into the membrane to form a pore.

 

TTSS Regulation: Regulated by different transcriptional regulators in response to the environment.

            -Signal from environment causes the injectisome to be assembled and a certain expression effectors.

            - A specific signal then triggers the proteins to be secreted.

            -If there is no contact with the host cell then protein secretion is blocked by a complex of regulators.

            -When there is contact with the eukaryotic cell plasma membrane the blocking complex is dissembled and secretion starts.

 

Fig. 2. The T3S translocation pore. A model for formation of the T3S translocation pore is schematically shown. T3S is triggered upon contact with the eukaryotic cell lipid membrane. The translocators (two hydrophobic proteins and one hydrophilic protein) should have secretion priority, but this has never been experimentally demonstrated. Then, the two hydrophobic translocators (in orange) insert and form a pore in the host cell lipid membrane in a process assisted by the hydrophilic translocator (in red). The structure and stoichiometry of the pore are unknown. The hetero-heptameric pore structure shown is purely hypothetical. It is also unknown if the hydrophilic translocator binds to the assembled pore and/or needle.

 

 

Fig. 3. Regulation of T3S. A general model on the mechanisms that were described to control T3S is shown. The model summarizes information from different T3S systems; it does not mean that all mechanisms are operative in a single bacterium. In T3S systems, a first response to specific host environmental conditions ensures injectisome assembly and a certain expression level of T3S effectors. However, secretion only occurs when a specific signal triggers T3S. In the absence of contact, secretion is prevented by a multiprotein complex of T3S regulators that somehow blocks the access of T3S substrates to the injectisome. In non-secreting conditions, transcription from T3S substrates promoters is limited. This has been shown to be due to the indirect and uncharacterized action of a negative regulator of T3S transcription and to the inability of an AraC-like transcription activator to have high-affinity access to the T3S promoters. Upon contact with the eukaryotic host cell plasma membrane, the complex blocking the access to the injectisome is disassembled and secretion starts. This leads not only to the secretion of T3S effectors but also of some T3S regulators. In addition, the T3S chaperones are now free in the cytoplasm. The consequences are that the negative regulator of T3S transcription cannot exert its action any more (because is secreted) and the AraC-like transcription activator forms a high-affinity complex for T3S promoters with the now free T3S translocator chaperones, boosting transcription and expression of T3S effector genes. The function of T3S chaperones is far more complex (see text for a discussion) than what is depicted in this model on T3S control. The injectisome and translocation pore are represented as in Fig. 1 and Fig. 2. OM, outer membrane; PG, peptidoglycan; IM, inner membrane.

 

Type IV Secretion: Used for genetic exchange and the delivery of effector molecules to eukaryotic target cells.

The best described Type IV secretion system is the conjugation system of the F plasmid.

-This system allows contact-dependent delivery of DNA to bacterial systems and involved the conjugal pilus.

Three types of type 4 dependent mechanisms of secretion:

1. Conjugation: deliver DNA to recipient bacteria and other cell types by cell-cell contact. These cell types can be other bacterial species, plants, animals, and fungi. The mobile elements can be antibiotic resistance genes and can cause multiple drug resistance.

2. DNA uptake and release: exchange DNA with the surrounding environment independently of contact between cells. These systems promote genetic exchange and can also acquire antibiotic resistance genes.

3. Effector translocators: deliver DNA and proteins to eukaryotic cells during infection. They differ depending on the organism, and are reminiscent of T3SS causing the translocation of effectors directly to the recipient cell through cell contact.

 

Conjugation - 3 substructures: work together to recruit cognate DNA and protein substrates to the transfer machine, the transfer of substrates across the cell envelope, and the delivery of substrates to target cells.

1. coupling protein (CP) homomultimer

2. transenvelope protein complex

3. conjugative pilus

 

Overview of Type I, II, III, and IV Secretion Systems:

FIG. 1. Schematic representation of the type I, II, III, and IV protein secretion systems. The type I pathway is exemplified by hemolysin A

(HlyA) secretion in E. coli, the type III system is exemplified by Yop secretion in Yersinia, the type II system is exemplified by pullulanase secretion

in Klebsiella oxytoca, and the type IV system is exemplified by the VirB system in A. tumefaciens. ATP hydrolysis by HlyB, YscN, SecA, and VirB11

is indicated. Secreted effector molecules are depicted as grey ovals. The type II and in some cases the type IV secretion systems utilize the

cytoplasmic chaperone SecB, although the Tat export pathway has recently been implicated in the secretion of molecules via the type II pathway.

Type III secretion also involves cytoplasmic chaperones (SycE); however, they do not interact with the Sec inner membrane translocon. The major

structural proteins of each system are depicted in relation to their known or deduced position in the cell envelope. EM, extracellular milieu; OM,

outer membrane; Peri, periplasm; IM, inner membrane; Cyto, cytoplasm.

694 HENDERSON ET AL. MICROBIOL. MOL. BIOL. REV.

 

Type V Secretion Systems:

Va: Autotransporter secretion pathway

-These proteins consist of 3 domains

            1. signal sequence: present at the N-terminal end and directs the protein to the periplasm (like in type 2 secretion)

            2. Passenger domain: this confers the diverse effector function of the various proteins.

            3. translocation unit: located at the C-terminal end and contains a short linker region with an α-helical secondary structure and a β-core that adopts a β-barrel tertiary structure when embedded in the outer membrane facilitating the translocation of the passenger domain through the outer membrane.

 

Vb: two-partner secretion pathway

-contains an N-terminal signal sequence like Va that directs its translocation to the periplasm.

-The passenger domain then inserts itself into a pore in the outer membrane formed by a β-barrel.

-When the passenger domain is at the surface of the bacterial cell it can undergo further proteolytic cleavage to achieve its physiological function.

-What makes this different than Va is the passenger domain (termed the exoprotein) and the β-domain (termed the transporter domain) are translated as 2 separate proteins.

FIG. 3. Schematic overview of the type V secretion systems. The secretion pathway of the autotransporter proteins (type Va) is depicted at the

bottom left of the diagram, the two-partner system (type Vb) is depicted in the center of the diagram, and the type Vc or AT-2 family is depicted

on the right. The four functional domains of the proteins are shown: the signal sequence, the passenger domain, the linker region, and the

_-domain. The autotransporter polyproteins are synthesized and generally exported through the cytoplasmic membrane via the Sec machinery.

Interestingly, effector proteins with an unusual extended signal sequence, which purportedly mediates Srp-dependent export, are found in all three

categories of type V secretion. Once through the inner membrane, the signal sequence is cleaved and the _-domain inserts into the outer

membrane in a biophysically favored _-barrel structure that forms a pore in the outer membrane. After formation of the _-barrel, the passenger

domain inserts into the pore and is translocated to the bacterial cell surface, where it may or may not undergo further processing.

 A review for type V secretion see Henderson et al. 2005 Micro and Molecular biology reviews.