Study for Expression Mechanism of Shiga-Toxin in Enterohemorrhagic E. Coli and Development of Inhibitor for the Toxin Expression.

The most severe cases with E. coli infection are caused by Shiga-toxin producing E. coli (STEC), induces hemorrhagic colitis and sometimes severe hemorrhagic uremic syndrome. Life-threatening cases also sometimes happen, especially in young children and elderly people. However, antibiotics is not available and non-specific supportive therapy is recommended to treat the bacterial infection. In the STEC genome, Shiga-toxin is encoded in a prophage region, and the toxin is expressed when the phage is produced as in its lytic cycle. An antibiotic treatment induces the phage production through SOS response, therefore the toxin expression is also induced by the antibiotic. Here, I want to establish a specific therapy for STEC infection. Then, I propose to develop an anti-phage agent (APA) to suppress the phage production to treat STEC infection. The APA will inhibit the phage production, consequently, Shiga-toxin will also be not expressed. In addition to decrease the pathogenicity by suppressing the toxin production, an antibiotic is also available to treat the bacterial infection, when the APA suppresses the toxin production. As far as I know, the idea of APA has not been studied in any type of infectious diseases yet. Clinically isolated E. coli O157:H7 EDL933 is a model strain for STEC research, and 933W is the prophage encoding Shiga-toxin. 933W has been transferred to E. coli K-12 strain to target the phage and avoid infection risk during experiments. The toxin gene (stx2AB) will be replaced with gfp gene, then the expression of toxin can be measured by fluorescence. The lytic cycle of lysogenized 933W is induced by low concentration of an antibiotic mitomycin C (500ng/ml). When the test strain is cultured with mitomycin C, OD600nm will be decreased, because the induced phage lyses the host E. coli cell. GFP will be expressed in the same time. However, when a chemical compound inhibits the phage production, OD600nm will be increased and GFP fluorescence will be vanished. By using the principle, APA candidates will be screened from chemical libraries. To identify a target protein of the compound, resistant mutants will be isolated by using flow cytometry and cell sorter through GFP expression, and mutation sites will be determined by genomic sequencing. If the compound has no serious toxicity to mouse, the APA will be applied to treat STEC infection in the animal model. Titer of the phage and expression of Shiga-toxin will be analyzed in cecal and large intestinal luminal contents. If the compound cannot be applied to mouse because of the toxicity, Caenorhabditis elegans will be used as an infectious model.