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Functional characterization of neuroactive toxins using an engineered bacterial type-III secretion system

Final Report Summary - CONOTOX (Functional characterization of neuroactive toxins using an engineered bacterial type-III secretion system)

One of the major challenges of modern neuroscience is to understand the mammalian central nervous system at the molecular level. A general problem is that a wide diversity of ion channel families are expressed, and the functional role of each molecular isoform is extremely challenging to investigate. Thus, novel pharmacological agents are essential to understand what occurs in the central nervous system on a mechanistic level.

The venoms of poisonous animals provide a virtually untapped reservoir of millions of neuroactive peptides that have evolved to block or activate ion channels, often with highly diverse selectivity. The fundamental problem is to identify and purify peptides of interest among the large number of molecules present in animal venoms. Engineered bacteria that export potentially neuroactive peptides into the surrounding media - where the peptides are ready for subsequent characterization - could be used in an innovative approach for the rapid purification of polypeptides of interest.

The proposed project aims at:
(i) adapting and utilizing the powerful flagellar type-III secretion system of Salmonella enterica for the expression and secretion of recombinant polypeptides;
(ii) constructing a Salmonella strain optimized for efficient secretion and expression tests of selected polypeptides;
(iii) creating an arbitrary polypeptide library based on Conus venoms;
(iv) high-throughput expression and affinity purification of potentially neuroactive peptides;
(v) developing electrophysiological, calcium-imaging based assays to screen purified polypeptides for neuroactivity.

The experiments performed so-far in this project have successfully demonstrated that:
(i) the type-III export apparatus can readily be engineered for the secretion of a variety of polypeptides;
(ii) the capacity of the export machinery can be increased by adapting the type-III secretion signal and optimizing flagellar gene expression and
(iii) neuroactive polypeptides secreted via the flagellar type-III secretion apparatus can be purified and used for further activity assays.

We have determined the secretion capability of all early- and late-type flagellar secretion substrates using a dual approach: translocation across the inner membrane and quantification of secreted protein using a beta-lactamase reporter protein fusion and export to the culture supernatant with subsequent detection using immunostaining. We identified FlgD and FlgM/FlgL as the most efficient early- and late-type export signals, respectively. The secretion signal of FlgD and FlgL was further optimized. A manuscript that describes the features of flagellar type-III secretion signals is on track to be prepared.

We further discovered a novel, pronounced effect of the 5'-untranslated region of various flagellar type-III secretion substrates for substrate stability and recognition by the export apparatus. This result will be followed up in detail in further studies.

In a complementary approach, we performed a genetic screen to identify novel factors that positively regulate flagellar gene expression (thereby activating the flagellar type-III export machinery). Two new regulators have been identified that negatively and positively regulate the flagellar master operon and thereby result in an decrease or increase of flagellar type-III export systems, respectively. Flagellar gene expression could be significantly increased by deleting the negative regulator and activating the positive regulator. Two manuscripts that describe the two new regulatory proteins are currently in preparation. We additionally noticed that activity of the negative regulator is dependent on a third factor discovered in the same transposon mutagenesis screen. The two proteins directly interact and the precise mechanism will be followed up in future studies.

We have thus demonstrated that the flagellar type-III secretion system can be manipulated by:
(i) optimizing the secretion signal and
(ii) optimizing flagellar gene expression to efficiently export a reporter protein into the periplasm and culture supernatant.

We therefore utilized these results to engineer a Salmonella strain optimized for efficient secretion and performed expression tests of neuroactive polypeptides. The pore-forming domain of tetanus toxin (TeNT) is is especially difficult to purify because of rapid proteolytic degradation and therefore represents an attractive target for a proof-of-principle expression and purification test using the above-mentioned bacterial secretion strain. We could show that TeNT fused to a type-III secretion substrate is readily secreted into the culture supernatant in an unfolded state through the flagellar secretion channel, thereby circumventing proteolytic degradation. The TeNT protein was purified and will be used for future functional assays.

The work on this project performed so far successfully demonstrates the potential of using bacteria to export polypeptides of interest into the surrounding media, readily available for subsequent characterization. The described, engineered type-III export system of Salmonella will be used in the future for the expression and secretion of recombinant neuropeptides that will be analyzed for neurotoxic activity.

In summary, this project validates the approach of using engineered bacterial secretion system for the production and export of various polypeptides that might be potentially valuable for many researchers and pharmaceutical companies of the European Community.