title: A Secretion-Amplification Role for Salmonella enterica Translocon Protein SipD.
abstract: The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins. However, we discovered that complementation of SipD in trans via exogenous addition to T3SS-expressing cultures further improves heterologous protein secretion titers, suggesting a previously unknown but important role for this protein. Building on this knowledge, we have engineered a hyper-secreting strain of S. enterica for a greater than 100-fold improvement in the production of a variety of biotechnologically valuable heterologous proteins that are challenging to produce, such as toxic antimicrobial peptides and proteolysis-prone biopolymer proteins. We determined that transcription by several T3SS promoters is upregulated with the addition of SipD, that the N-terminal domain of SipD is sufficient to observe the increased secretion phenotype, and that the effect is post-transcriptional and post-translational. These results lend support to the use of bacterial secretion as a powerful protein production strategy, and the hypothesis that translocon proteins contribute to type III secretion regulation.
authors.affiliation: UC Berkeley-UCSF Graduate Program in Bioengineering, University of California Berkeley , Berkeley, California 94720, United States.
authors.forename: Anum Azam
authors.identifier: 0000-0002-0938-881X
authors.initials: AA
authors.lastname: Glasgow
authors.affiliation: Department of Plant and Microbial Biology, University of California Berkeley , Berkeley, California 94720, United States.
authors.forename: Han Teng
authors.identifier: 0000-0001-9836-1338
authors.initials: HT
authors.lastname: Wong
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.|Center for Synthetic Biology, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Danielle
authors.identifier: 0000-0002-4555-4803
authors.initials: D
authors.lastname: Tullman-Ercek
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000235:genetics*; D000069296:Type III Secretion Systems / Q000235:genetics*; D014774:Virulence
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C107188:SspD protein, Salmonella typhimurium; D000069296:Type III Secretion Systems
keywords: Salmonella; gene networks; protein engineering; translocon; type III secretion; virulence mechanisms