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( David Quintero ),( Jamie Carrafa ),( Lena Vincent ),( Hee Jong Kim ),( James Wohlschlegel ),( David Bermudes ) 한국미생물 · 생명공학회 2018 Journal of microbiology and biotechnology Vol.28 No.12
Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culturebased inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.
Asselin, Jo Ann E.,Lin, Jinshan,Perez-Quintero, Alvaro L.,Gentzel, Irene,Majerczak, Doris,Opiyo, Stephen O.,Zhao, Wanying,Paek, Seung-Mann,Kim, Min Gab,Coplin, David L.,Blakeslee, Joshua J.,Mackey, Da American Society of Plant Biologists 2015 Plant Physiology Vol.167 No.3
<P><I>The virulence activity of an effector protein belonging to the widely conserved AvrE family is linked to its ability to cause system-wide reprogramming of phenylpropanoid metabolism in susceptible maize seedlings.</I></P><P>AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of <I>Pantoea stewartii</I> ssp. <I>stewartii</I> (<I>Pnss</I>) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (<I>Zea mays</I>) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by <I>Pnss</I>. Thus, WtsE is a pathogenicity and virulence factor in maize, and an <I>Escherichia coli</I> heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by <I>Pnss</I>. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence.</P>