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

Abstract

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 recombinant proteins from E. coli

“…The secreted protein is characterized by a C-terminal signal sequence that is not cleaved during translocation. A lot of effort has been made in order to establish T1SS for recombinant protein production, especially the use of HlyA T1SS [35][36][37][38][39][40]. Interestingly, there is a broad range of different substrates naturally secreted by T1SS including heme-binding proteins, toxins, proteases, lipases, adhesins, and S-layer proteins, with molecular weights between 19 kDa up to 900 kDa [32].…”

“…A lot of effort has been made in order to establish T1SS for recombinant protein production, especially the use of HlyA T1SS [35][36][37][38][39][40]. The system consists of the inner membrane ABC translocator protein HlyB, the OMP TolC as well as the MFP HlyD, that serves as a linker between HlyB and TolC [35,40,41]. The HlyA T1SS derived from uropathogenic E. coli strains is well-characterized, reliable and can be used in nonpathogenic laboratory strains [35].…”

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

“…The unfolded protein is transported in a C to N direction, leading to initial exposure of the C-terminus and thus the first RTX repeat segment. [77] In contrast to the Ca 2þ -depleted prokaryotic cytoplasm, the Ca 2þ concentration of extracellular space allows for the folding of the capping structure in front of the first RTX repeat segment. [86,92,93] The C-terminal capping structure provides the required entropic stabilization for Ca 2þ -dependent folding of the first RTX repeat segment, preventing its backwards diffusion into the cytosol.…”

“…At the cytoplasmic side, a non-cleavable, N-terminal signal sequence of the cargo protein interacts with the nucleotide binding domain (NBD) of the ABC transporter [77] ( Figure 4A). This interaction triggers the assembly of the T1SS complex.…”

“…[84] A non-cleavable C-terminal signal sequence mediates the initiation of transport by inserting the C-terminus in the T1SS duct. [77] The remainder of the RTX domain consists of tandem repeats of Ca 2þ -binding, negatively charged nonapeptides. [85] The RTX repeats are organized in blocks that fold into Ca 2þ -loaded structures termed parallel ß-rolls.…”