Secretion systems 34

Secretion in gram-negative bacteria requires transport across two hydrophobic membranes separated by a peptidoglycan-containing aqueous periplasm. The movement of large macromolecules across this cellular envelope poses

challenges in terms of maintaining the integrity of the membrane, as well as regulation of cross-membrane movement. Estimates place at least 25-30% of the typical gram-negative total protein content in the cell envelope or secretome; as such, gram negative bacteria have developed six known large multi-gene secretion systems – types I through VI – including both one-step “tunnels” and two-step processes separated by a periplasmic stage (198). A type VII secretion system has also been identified in gram-positive bacteria (2). Secretion is

particularly important in intercellular signalling for cooperation or competition, and in virulence effector delivery to eukaryotic cells, and thus some secretion systems are contact-dependent (188).

1.2.1

The Sec and Tat systems

The general secretory (Sec) pathway and the twin-arginine translocation (Tat) pathway both function to move proteins across the inner membrane (IM); the former transports unfolded proteins, while the latter translocates folded proteins (261). Protein substrates are targeted for export via an amino (N)-terminal signal sequence called a signal peptide (SP), which is cleaved when the protein

reaches the periplasm. The composition of the Tat sytem is varied, minimally consisting of a TatABC substrate-binding receptor complex which associates with a separate TatA complex to form an active translocon, through which tranport is driven by the proton motive force (385). Proteins targeted to the Sec system bind to cytosolic motor protein SecA, an ATPase that drives translocation through a SecYEG channel; in the periplasm, the SP is cleaved by signal peptidase, allowing folding of the mature protein (135).

1.2.2

Type I

The type I secretion system (T1SS) is a simple trimeric complex spanning the entire cell envelope (199). The translocon consists of an IM ATP-binding cassette (ABC) transporter, with a nucleotide binding domain (NBD) fused to a transmembrane domain, and an outer membrane (OM) pore linked by an adaptor protein, or membrane fusion protein. Unfolded substrates are targeted to the T1SS, where a non-cleavable carboxy (C)-terminal signal sequence binds to the NBD of the ABC transporter to induce translocation.

1.2.3

Type II

The type II secretion system (T2SS), often referred to as the general secretory pathway, mediates a two-step process. First, exoproteins with an N-terminal SP are transported to the periplasm by either the Sec or Tat systems; following

cleavage of the SP, fully folded proteins are translocated across the OM by the secreton (217). The secreton is a large complex composed of an OM pore called a secretin, and an IM pilus-like structure postulated to act as a piston to force substrates through the secretin (90). The T2SS is closely related to the type IV pili (217), and secretes a wide variety of exoenzymes (90).

1.2.4

Type III

The type III secretion system (T3SS) is a generally contact-dependent system found in pathogenic bacteria for the delivery of effectors to the cytosol of eukaryotic cells. It is characterized by an injectisome, or needle-like structure capable of injecting substrates directly into a eukaryotic cell through a cell-

envelope-spanning channel; outside the bacterial OM, the T3SS is capped with a needle, a tip complex, and a translocation pore forming a channel in the

eukaryotic membrane (188). In structure and assembly, the T3SS is homologous to the flagellar export system (143), though secreted effectors are varied.

1.2.5

Type IV

The type IV secretion system (T4SS) is a mostly contact-dependent system able to transport substrates directly into both prokaryotic and eukaryotic cells.

Homologous to bacterial conjugation machinery, T4SSs are able to translocate both DNA and protein substrates; the former contribute to rapid inter-bacterial spread of resistance genes and fitness traits, while the latter contribute to virulence against eukaryotic cells (86). The main structures of the T4SS comprise a cell surface adhesin or pili for intercellular contact, a transenvelope channel, and a Type IV coupling protein (T4CP), which mediates substrate entrance into the channel (188). There are two general lineages of T4SS, one

homologous to the Dot/Icm system of L. pneumophila, although there are T4SSs

that fit well into neither lineage (86).

1.2.6

Type V

The simplest of the secretion systems, Type V secretion systems (T5SS) also use a two-step secretion mechanism with a periplasmic intermediate for the secretion of very large proteins. Unfolded proteins are transported to the periplasm through the Sec system, where a -barrel translocator domain is needed for transport across the OM (189). T5SS can be divided into

autotransporters (AT) and two-partner secretion systems (TPS). ATs consist of a signal domain and a -barrel domain linked by a passenger domain; the signal domain directs the protein to the periplasm where the -barrel domain forms a pore in the OM for secretion of the passenger domain. The passenger domain may remain associated with the OM or be released into the extracellular milieu by proteolysis. AT proteins often contribute to cellular adhesion, aggregation, biofilm formation, invasion, and toxicity (519). TPSs are very similar to ATs, but the passenger domain, or exoprotein, and the pore-forming -barrel domain, or transporter, are translated as two separate proteins (189). TPSs are implicated in contact-dependent growth inhibition (188).

1.2.7

Type VI

The type VI secretion system (T6SS) is the most recently discovered of the large multi-gene secretion systems, and as such, has not been as well characterized. Some proteins of the T6SS are homologous to bacteriophage proteins, including tube-like hexameric ring protein Hcp, and tailspike-like protein VgrG, and the T6SS can participate in contact-dependent effector delivery (215). However, the purpose of the T6SS is a hotly debated topic, with phenotypes ranging from

restriction of H. hepaticus colonization and downregulation of the host immune

response in infected epithelial cells (83), to P. aeruginosa toxin-mediated killing of

neighbouring prokaryotic cells (204), B. thailandensis resistance to cell contact-

induced growth inhibition (427), B. mallei virulence in hamsters (421), and

translocation of an actin cross-linking effector into host cells by intracellular Vibrio cholerae (284).