Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Redefining the bacterial Type I protein secretion system.

Type I secretion systems (T1SS) are versatile molecular machines for protein transport across the Gram-negative cell envelope. The archetypal Type I system mediates secretion of the Escherichia coli hemolysin, HlyA. This system has remained the pre-eminent model of T1SS research since its discovery. The classic description of a T1SS is composed of three proteins: an inner membrane ABC transporter, a periplasmic adaptor protein and an outer membrane factor. According to this model, these components assemble to form a continuous channel across the cell envelope, an unfolded substrate molecule is then transported in a one-step mechanism, directly from the cytosol to the extracellular milieu. However, this model does not encapsulate the diversity of T1SS that have been characterized to date. In this review, we provide an updated definition of a T1SS, and propose the subdivision of this system into five subgroups. These subgroups are categorized as T1SSa for RTX proteins, T1SSb for non-RTX Ca2+-binding proteins, T1SSc for non-RTX proteins, T1SSd for class II microcins, and T1SSe for lipoprotein secretion. Although often overlooked in the literature, these alternative mechanisms of Type I protein secretion offer many avenues for biotechnological discovery and application.

Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system.

Protein acylation is critical for many cellular functions across all domains of life. In bacteria, lipoproteins have important roles in virulence and are targets for the development of antimicrobials and vaccines. Bacterial lipoproteins are secreted from the cytosol via the Sec pathway and acylated on an N-terminal cysteine residue through the action of three enzymes. In Gram-negative bacteria, the Lol pathway transports lipoproteins to the outer membrane. Here, we demonstrate that the Aat secretion system is a composite system sharing similarity with elements of a type I secretion systems and the Lol pathway. During secretion, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue. Mutations disrupting glycine acylation interfere with membrane incorporation and trafficking. Our data reveal CexE as the first member of a new class of glycine-acylated lipoprotein, while Aat represents a new secretion system that displays the substrate lipoprotein on the cell surface.