Investigations on the substrate binding sites of hemolysin B, an ABC transporter, of a type 1 secretion system.

The ABC transporter hemolysin B (HlyB) is the key protein of the HlyA secretion system, a paradigm of type 1 secretion systems (T1SS). T1SS catalyze the one-step substrate transport across both membranes of Gram-negative bacteria. The HlyA T1SS is composed of the ABC transporter (HlyB), the membrane fusion protein (HlyD), and the outer membrane protein TolC. HlyA is a member of the RTX (repeats in toxins) family harboring GG repeats that bind Ca2+ in the C-terminus upstream of the secretion signal. Beside the GG repeats, the presence of an amphipathic helix (AH) in the C-terminus of HlyA is essential for secretion. Here, we propose that a consensus length between the GG repeats and the AH affects the secretion efficiency of the heterologous RTX secreted by the HlyA T1SS. Our in silico studies along with mutagenesis and biochemical analysis demonstrate that there are two binding pockets in the nucleotide binding domain of HlyB for HlyA. The distances between the domains of HlyB implied to interact with HlyA indicated that simultaneous binding of the substrate to both cytosolic domains of HlyB, the NBD and CLD, is possible and required for efficient substrate secretion.

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region.

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

Biotechnological applications of type 1 secretion systems.

Bacteria have evolved a diverse range of secretion systems to export different substrates across their cell envelope. Although secretion of proteins into the extracellular space could offer advantages for recombinant protein production, the low secretion titers of the secretion systems for some heterologous proteins remain a clear drawback of their utility at commercial scales. Therefore, a potential use of most of secretion systems as production platforms at large scales are still limited. To overcome this limitation, remarkable efforts have been made toward improving the secretion efficiency of different bacterial secretion systems in recent years. Here, we review the progress with respect to biotechnological applications of type I secretion system (T1SS) of Gram-negative bacteria. We will also focus on the applicability of T1SS for the secretion of heterologous proteins as well as vaccine development. Last but not least, we explore the employed engineering strategies that have enhanced the secretion efficiencies of T1SS. Attention is also paid to directed evolution approaches that may offer a more versatile approach to optimize secretion efficiency of T1SS.