Molecular insights into type I secretion systems.

Type 1 secretion systems are one of the main machineries in Gram-negative bacteria involved in the secretion of a wide range of substrates from the cytoplasm across the inner and outer membrane in one step to the extracellular space. The range of substrates varies from small proteins up to large surface layer proteins of about 900 kDa. Most of the substrates have a non-cleavable C-terminal secretion signal and so-called GG repeats that are able to bind calcium ions. The translocator complex is composed of a trimeric outer membrane protein that provides a pore in the outer membrane. A multimeric membrane fusion protein spans the periplasm and forms a continuous channel connecting the outer membrane protein with a dimeric ATP-binding cassette transporter in the inner membrane. The ATP-binding cassette-transporter is thought to form a channel through the inner membrane and energizes the transport process. This review will provide a detailed view of the components of the translocator and will summarize structural as well as functional data.

Identity Determinants of the Translocation Signal for a Type 1 Secretion System.

The toxin hemolysin A was first identified in uropathogenic E. coli strains and shown to be secreted in a one-step mechanism by a dedicated secretion machinery. This machinery, which belongs to the Type I secretion system family of the Gram-negative bacteria, is composed of the outer membrane protein TolC, the membrane fusion protein HlyD and the ABC transporter HlyB. The N-terminal domain of HlyA represents the toxin which is followed by a RTX (Repeats in Toxins) domain harboring nonapeptide repeat sequences and the secretion signal at the extreme C-terminus. This secretion signal, which is necessary and sufficient for secretion, does not appear to require a defined sequence, and the nature of the encoded signal remains unknown. Here, we have combined structure prediction based on the AlphaFold algorithm together with functional and in silico data to examine the role of secondary structure in secretion. Based on the presented data, a C-terminal, amphipathic helix is proposed between residues 975 and 987 that plays an essential role in the early steps of the secretion process.

Corrigendum: Directionality of substrate translocation of the hemolysin A Type I secretion system.

This corrects the article DOI: 10.1038/srep12470.

In vivo quantification of the secretion rates of the hemolysin A Type I secretion system.

Type 1 secretion systems (T1SS) of Gram-negative bacteria secrete a broad range of substrates into the extracellular space. Common to all substrates is a C-terminal secretion sequence and nonapeptide repeats in the C-terminal part that bind Ca(2+) in the extracellular space, to trigger protein folding. Like all T1SS, the hemolysin A (HlyA) T1SS of Escherichia coli consists of an ABC transporter, a membrane fusion protein and an outer membrane protein allowing the one step translocation of the substrate across both membranes. Here, we analyzed the secretion rate of the HlyA T1SS. Our results demonstrate that the rate is independent of substrate-size and operates at a speed of approximately 16 amino acids per transporter per second. We also demonstrate that the rate is independent of the extracellular Ca(2+) concentration raising the question of the driving force of substrate secretion by T1SS in general.

Directionality of substrate translocation of the hemolysin A Type I secretion system.

Type 1 secretion systems (T1SS) of Gram-negative bacteria are responsible for the secretion of various proteases, lipases, S-layer proteins or toxins into the extracellular space. The paradigm of these systems is the hemolysin A (HlyA) T1SS of Escherichia coli. This multiple membrane protein complex is able to secrete the toxin HlyA in one step across both E. coli membranes. Common to all secreted T1SS substrates is a C-terminal secretion sequence being necessary as well as sufficient for secretion. However, it is not known whether transport occurs directionally, i.e. the N- or the C-terminus of T1SS substrates is secreted first. We have addressed this question by constructing HlyA fusions with the rapidly folding eGFP resulting in a stalled T1SS. Differential labeling and subsequent fluorescence microscopic detection of C- and N-terminal parts of the fusions allowed us to demonstrate vectorial transport of HlyA through the T1SS with the C-terminus appearing first outside the bacterial cells.

Secretion of slow-folding proteins by a Type 1 secretion system.

Protein production through dedicated secretion systems might offer an potential alternative to the conventional cytoplasmical expression. The application of Type 1 secretion systems of Gram-negative bacteria, however, where often not successful in the past for a wide range of proteins. Recently, two studies using the E. coli maltose binding protein (MalE) and the rat intestinal fatty acid binding protein (IFABP) revealed a rational to circumvent these limitations. Here, wild-type passenger proteins were not secreted, while folding mutants with decreased folding kinetics were efficiently exported to the extracellular space. Subsequently, an one-step purification protocol yielded homogeneous and active protein. Taken together, theses two studies suggest that the introduction of slow-folding mutations into a protein sequence might be the key to use Type 1 secretion systems for the biotechnological production of proteins.

Using an E. coli Type 1 secretion system to secrete the mammalian, intracellular protein IFABP in its active form.

A biotechnological production of proteins through protein secretion systems might be superior to the conventional cytoplasmic production, because of the absence of large amounts of proteases present in the extracellular space and the ease of purification or downstream processing. However, secretion of proteins is still a trial-and-error approach and many proteins fail to be secreted. Recently, a study of a Type 1 secretion system revealed that the folding rate of the passenger protein dictates secretion efficiency. Here, the well-known MalE failed to be secreted when fused to a C-terminal fragment of the natural substrate haemolysin A. In contrast, slow-folding mutants of MalE were secreted in high yields. However, MalE is a bacterial protein that is targeted to the periplasmic space of E. coli and possesses the intrinsic capability to cross a membrane. Therefore, we applied the same approach for another eukaryotic protein that resides in the cytoplasm. As an example, we chose the intestinal fatty acid binding protein (IFABP) and highlight the universal potential of this Type 1 secretion system to secrete proteins with slow-folding kinetics (here the G121V mutant). Finally, a one-step purification protocol was established yielding 1mg of pure IFABP G121V per liter culture supernatant. Moreover, secreted IFABP G121V was shown to reach a folded state, which is biologically active.