Poster  Print  Size:   This  poster  template  is  36”  high  by   36”  wide.  It  can  be  used  to  print  any   poster  with  a  1:1  aspect  ra@o.   Placeholders:   The  various  elements  included  in   this  poster  are  ones  we  oBen  see  in   medical,  research,  and  scien@fic   posters.  Feel  free  to  edit,  move,     add,  and  delete  items,  or  change   the  layout  to  suit  your  needs.   Always  check  with  your  conference   organizer  for  specific  requirements.   Image  Quality:   You  can  place  digital  photos  or  logo   art  in  your  poster  file  by  selec@ng   the  Insert,  Picture  command,  or  by   using  standard  copy  &  paste.  For   best  results,  all  graphic  elements   should  be  at  least  150-­‐200  pixels   per  inch  in  their  final  printed  size.   For  instance,  a  1600  x  1200  pixel   photo  will  usually  look  fine  up  to   8“-­‐10”  wide  on  your  printed  poster.   To  preview  the  print  quality  of   images,  select  a  magnifica@on  of   100%  when  previewing  your  poster.   This  will  give  you  a  good  idea  of   what  it  will  look  like  in  print.  If  you   are  laying  out  a  large  poster  and   using  half-­‐scale  dimensions,  be  sure   to  preview  your  graphics  at  200%  to   see  them  at  their  final  printed  size.   Please  note  that  graphics  from   websites  (such  as  the  logo  on  your   hospital's  or  university's  home  page)   will  only  be  72dpi  and  not  suitable   for  prin@ng.     [This  sidebar  area  does  not  print.]   Change  Color  Theme:   This  template  is  designed  to  use  the   built-­‐in  color  themes  in  the  newer   versions  of  PowerPoint.   To  change  the  color  theme,  select   the  Design  tab,  then  select  the   Colors  drop-­‐down  list.                     The  default  color  theme  for  this   template  is  “Office”,  so  you  can   always  return  to  that  aBer  trying   some  of  the  alterna@ves.   Prin@ng  Your  Poster:   Once  your  poster  file  is  ready,  visit   www.genigraphics.com  to  order  a   high-­‐quality,  affordable  poster   print.  Every  order  receives  a  free   design  review  and  we  can  deliver  as   fast  as  next  business  day  within  the   US  and  Canada.     Genigraphics®  has  been  producing   output  from  PowerPoint®  longer   than  anyone  in  the  industry;  da@ng   back  to  when  we  helped  MicrosoB®   design  the  PowerPoint®  soBware.       US  and  Canada:    1-­‐800-­‐790-­‐4001   Email:  info@genigraphics.com     [This  sidebar  area  does  not  print.]   Developing a Cancer Therapy: Engineering Salmonella enterica Typhimurium to Express and Secrete Interleukin-21 Kelsey L. Simmons1, Michael J. Mertensotto1, Jeremy J. Drees1, Lance B. Augustin1, Janet L. Schottel2, Arnold S. Leonard1, Daniel A. Saltzman1 1Department of Surgery, University of Minnesota Medical School, Minneapolis, MN 2Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN When most people hear of Salmonella enterica Typhimurium, they think of a pathogen that causes the dreaded foodborne illness, gastroenteritis. When most people hear that you are trying to use S. enterica as a cancer therapeutic, they think you are a little crazy. S. enterica Typhimurium is remarkable in that it can invade and replicate within tumor cells. Tumor to normal tissue ratios of approximately 1000:1 have been observed when measuring the number of S. enterica cells in mouse tissues.1 Cancer immunotherapy is designed to elicit an immune response to inhibit tumor growth and destroy cancerous cells. One major obstacle to using immunotherapies in the clinic is the severe toxicities and negative side effects associated with systemically administering purified immune effector molecules, such as cytokines, to a patient.2 One approach to limiting these toxicities is tumor-targeted bacterial cancer therapy. Attenuated strains of bacteria, such as S. enterica Typhimurium can be used to selectively target tumors and may be engineered to express various immune- stimulating molecules.3,4 Previous studies have demonstrated that IL-21 is a promising cytokine for cancer immunotherapy due to its ability to induce the proliferation of T cells and natural killer cells.5,6,7 Consequently, we are interested in engineering S. enterica to express and secrete mouse IL-21 via bacterial expression plasmids. My research has focused on the addition of secretion tags to the mIL-21 protein in order for mIL-21 to be secreted from S. enterica into the tumor microenvironment. Once expression of the mIL-21 protein is confirmed and shown to be biologically active, a strain of virulence-attenuated S. enterica that secretes mIL-21 will be administered to mice to test its efficacy as a tumor-targeted immunotherapy. If the secreted mIL-21 shows promising results in preliminary experiments in mice, the same secretion tags could be applied to other molecules known to stimulate the immune system. Background Materials and Methods Four strains of S. enterica Typhimurium designed to express mouse IL-21 (pBla-mIL21, pBla-OmpA-mIL21, pLacUV5-mIL21-Hly , & pBla-mIL21-Hly) were successfully engineered. Protein expression was assessed in the media, periplasmic, soluble, and insoluble fractions via Western blot. Addition of the Hly tag to the pBla-mIL21 and pLacUV5-mIL21 constructs, which was designed to cause extracellular secretion of mIL-21, resulted in loss of detectable protein expression. Addition of the OmpA tag, which was designed to cause protein secretion into the periplasm, showed mIL-21 protein in only the soluble and insoluble cytoplasmic fractions. Stronger promoters such as pTrc have been used previously in our laboratory, and a similar lack of mIL-21 expression was seen. These results suggest that the OmpA tag does not effectively direct secretion of mIL-21 into the periplasm, and the Hly tag is deleterious to production of mIL-21. Next steps include testing additional secretion tags such as YebF and SopE in an attempt to construct S. enterica Typhimurium strains that secrete mIL-21. Additionally, the mIL-21 cDNA may be codon-optimized for expression in S. enterica to help remove mRNA structures or less frequently used codons to improve overall expression of mIL-21. Conclusions Figure 2. Western Blot of Protein Extracts from S. enterica Expressing OmpA-mIL-21. Overnight cultures of S. enterica χ4550 producing OmpA-mIL21 (17.1 kDa) were grown, and the secreted, soluble, and insoluble protein fractions were prepared. Proteins were subjected to SDS-PAGE, transferred to a PVDF membrane, and analyzed by immunoblotting using antibodies against mIL-21 (15.0 kDa) and DnaK (70 kDa). mIL-21 was found in both the soluble and insoluble cytoplasmic protein fractions, which is shown within the purple box on the gel picture above. mIL-21 was not found in the periplasmic or medium fractions. DnaK protein was found mainly in the soluble cell protein fraction as expected, although it was also detected in the insoluble protein fraction and in the culture medium, indicating the presence of unsonicated cells in the insoluble extract and cell lysis in the overnight culture. Figure 3. Western Blot of Protein Extracts from S. enterica Expressing mIL-21-Hly. Overnight cultures of S. enterica χ4550 carrying either the pBla-mIL21-Hly, pLacUV5- mIL21-Hly, or pBla-mIL2 constructs were grown. Soluble, insoluble, and secreted protein fractions were prepared. Proteins were subjected to SDS-PAGE, transferred to a PVDF membrane, and analyzed by immonoblotting using antibodies against mIL-21 (15.0 kDa) and DnaK (70 kDa). Recombinant mIL-21 (rmIL-21) protein is labeled green and DnaK protein is labeled red on the blot. mIL-21 was not found in the soluble, insoluble, or medium protein samples. DnaK protein was only found in the soluble cytoplasmic protein fraction, indicating no cell lysis in the overnight cultures and no unsonicated cells in the insoluble protein sample. Results 1.  Wall DM, Srikanth CV, McCormick BA. Targeting Tumors with Salmonella Typhimurium- Potential for Therapy. Oncotarget, 1(8): 721-728, 2010. 2.  Vial T, Descotes J. Immune-Mediated Side-Effects of Cytokines in Humans. Toxicology, 105(1): 31-57, 1995. 3.  Saltzman DA, Katsanis E, Hasz DE, Vigdorovich V, Curtiss III RE, Kelly SM, Anderson PM, & Leonard AS. Anti-tumor Mechanisms of Attenuated Salmonella typhimurium Containing the Gene for Human Interleukin-2. Journal of Pediatric Surgery, 32(2): 301-306, 1997. 4.  Saltzman DA, Katsanis E, Heise CP, Hasz DE, Kelly SM, Curtiss R, Anderson PM, and Leonard AS. Hepatic and Splenic Colonization for the Attenuated Salmonella Typhimurium Containing the Gene for Human Interleukin-2: A Novel Anti-Tumor Agent. Cancer Biotherapy and Radiopharmaceuticals, 12910:37-45, 1997. 5.  Pan X, Li L, Juan-Juan, M, Yeo W, Zheng J, Liu M, & Fu J. Synergistic Effects of Soluble PD-1 and IL-21 on Antitumor Immunity Against H22 Murine Hepatocellular Carcinoma. Oncology Letters, 5(1): 90-96, 2013. 6.  Schulze KS, Kim HS, Fan Q, Kim DW, Kaufman HL. Local IL-21 Promotes the Therapetuic Activity of Effector T Cells by Decreasing Regulatory T Cells Within the Tumor Microenvironment. Molecular Therapy, 17(2): 380-388, 2009. 7.  Spolski R, Leonard WJ. Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity. Annual Review of Immunology, 26:57-79, 2008. Plasmid Construction Attenuated χ4550 (Δcrp-1 Δcya-1 ΔasdA1) S. enterica Typhimurium and plasmid pYA292 were obtained from Roy Curtiss III (Arizona State University). Plasmid pYA292 contains the aspartate semialdehyde dehydrogenase (asd) gene, which is used as a conditional-lethal selection for plasmid maintenance to avoid engineering antibiotic resistance into the bacteria. pYA292 was modified to contain the mouse IL-21 (mIL-21) cDNA under control of the lacUV5 promoter (Figure 1A). Using polymerase chain reaction and restriction enzymes, plasmids were constructed with a secretion sequence fused to the mIL-21 cDNA, resulting in either OmpA-mIL21 or mIL21-Hly, under control of the lacUV5 or bla promoters (Figure 1B). Once the mIL-21 fusions were constructed, they were used to transform S. enterica with standard electroporation methodology. Transformants were chosen via selective plating. Plasmid Isolation & Identification Transformants were grown in liquid culture, and the plasmids were isolated using Qiagen’s Qiaprep Miniprep kit. The plasmids were analyzed by restriction enzyme digest and gel electrophoresis to check the presence and directionality of the cloned DNA. Plasmids that contained the appropriate mIL-21 fusion constructs in the proper orientation were sequenced by the University’s Biomedical Genomics Center.  Western Blotting To determine if the S. enterica transformants were producing and secreting the mIL-21 fusion proteins, cultures were grown in LB broth. To detect secreted proteins, the culture medium was concentrated using Millipore Amicon Ultra Filters with a 10 kDa cutoff. Periplasmic proteins were isolated by subjecting cells to osmotic shock. Cytoplasmic proteins were isolated from cells by sonication and centrifugation to separate soluble cytoplasmic protein in the supernatant from insoluble protein in the pellet. The protein samples were boiled in loading dye and separated by SDS- PAGE. The proteins were transferred to a PVDF membrane for immunoblotting using antibodies targeting mIL-21 and E. coli DnaK. Materials and Methods Results References rmIL21   rmIL21   80  kDa   80kDa   Figure 1. IL-21 Constructs A. Attenuated χ4550 S. enterica Typhimurium was transformed with pLacUV5-mIL21 originating from plasmid pYA292 (see Materials and Methods). pYA292 was modified to contain the mouse IL-21 (mIL-21) cDNA under control of the lacUV5 promoter; this construct was used to make pBla- mIL21, pBla-OmpA-mIL21, pBla- mIL21-Hly, and pLacUV5-mIL21- Hly. B. The pBla-OmpA-mIL21 construct was made by fusing the mIL-21 protein to the N-terminal 21 amino acids of the E. coli OmpA protein secretion signal to export mIL-21 into the periplasm. The pBla-mIL21-Hly and pLacUV5- mIL21-Hly constructs were made by fusing the mIL-21 protein to the C-terminal 60 amino acids of the E. coli hemolysin A protein to export the mIL-21 fusion protein to the extracellular space. Transcription of the mIL-21 gene was controlled by either the β-lactamase promoter (PBla) or the lac-derived LacUV5 promoter (PLacUV5).   A   B   15  kDa   20  kDa   25  kDa   44  kDa   60  kDa   37  kDa   60  kDa   44  kDa   37  kDa   25  kDa   20  kDa   15  kDa