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The broad spectrum antiviral compound ST-669 affects vesicular trafficking in Chlamydia-infected ... PDF

42 Pages·2015·2.91 MB·English
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The broad spectrum antiviral compound ST-669 affects vesicular trafficking in Chlamydia-infected cells by William G. Valiant A PROJECT submitted to Oregon State University University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science Microbiology (Honors Scholar) Presented May 13, 2015 Commencement June 2015 AN ABSTRACT OF THE THESIS OF William Valiant for the degree of Honors Baccalauriate of Science in Microbiology presented on May 13, 2015. Title: The broad spectrum antiviral compound ST-669 affects vesicular trafficking in Chlamydia-infected cells. Abstract approved: __________________________________________________________________   Daniel Rockey   Our  laboratory  is  exploring  the  mechanism  of  action  of  a  novel  broad   spectrum  antiviral,  ST-­‐669.  This  compound  has  activity  against  a  variety  of   different  viruses  and  the  obligate  intracellular  bacterium,  Chlamydia.  In  this   study,  we  explored  the  effects  of  ST-­‐669  when  the  cell  cycle  of  the  host  cells   was  altered.  Chlamydia  spp.  were  grown  in  Vero  cells  in  the  presence  of   various  compounds  that  inhibit  the  eukaryotic  cell  cycle,  and  examined  for   inclusion  structure  and  production  of  bacteria.  ST-­‐669  was  used  to  examine   the  inclusion  structure  of  C.  caviae:  when  treated  with  ST-­‐669,  C.  caviae   appears  to  fuse  vacuoles  to  form  an  inclusion  with  a  single  lobe.  Most  of  the   cell  cycle  inhibitors  did  not  alter  the  anti-­‐chlamydial  effects  of  ST-­‐669.     However,  treatment  of  infected  cells  with  vincristine  led  to  an  increase  in   bacteria  production  and  change  in  inclusion  morphology  in  the  presence  of   ST-­‐669.  It  is  hypothesized  that  a  protein  that  is  a  target  of  ST-­‐669  is   differently  present  or  activated  when  Vero  cells  were  treated  with vincristine.    These  results  open  the  door  for  future  proteomic  studies  that   might  elucidate  ST-­‐669’s  mechanism  of  action.       Key  Words:  Chlamydia,  Inclusion,  ST-­‐669,  Anti-­‐chlamydial   Corresponding  email  address:  [email protected] ©Copyright by William Valiant May 13, 2015 All Rights Reserved The broad spectrum antiviral compound ST-669 affects vesicular trafficking in Chlamydia-infected cells by William G. Valiant A PROJECT submitted to Oregon State University University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science Microbiology (Honors Scholar) Presented May 13, 2015 Commencement June 2015 Honors Baccalaureate of Science in Microbiology project of William Valiant presented on May 13, 2015. APPROVED: Daniel Rockey, Mentor, representing Biomedical Sciences Ling Jin, Committee Member, representing Biomedical Sciences Brian Dolan, Committee Member, representing Biomedical Sciences Toni Doolen, Dean, University Honors College I understand that my project will become part of the permanent collection of Oregon State University, University Honors College. My signature below authorizes release of my project to any reader upon request. William Valiant, Author Acknowledgements Most importantly I would like to thank Dr. Rockey for all the assistance and mentorship he has provided over the last four years. Thanks to him I will continue to participate in research for many more years. I would also like to thank the entirety of the Rockey lab for their continued assistance as well as Dr. Hruby and Dr. Allen at Siga technologies for their technical assistance. Table of Contents Page Introduction ......................................................................................................................... 1 Chlamydia ....................................................................................................................... 1 ST-669 ............................................................................................................................. 3 The Eukaryotic Cell Cycle .............................................................................................. 5 Thesis Statement ................................................................................................................. 8 Methods ............................................................................................................................... 9 Chlamydial Strains .......................................................................................................... 9 Cell Culture and Chlamydial Infection ........................................................................... 9 Genome quantification .................................................................................................. 10 Fluorescence Microscopy ............................................................................................. 11 Results ............................................................................................................................... 13 ST-669 treatment affects C. caviae inclusion structure ................................................ 13 Anti-chlamydial effects of ST-669 in the presence of Cell Cycle Inhibitors ............... 13 Discussion ......................................................................................................................... 27 References ......................................................................................................................... 29 List of Figures and Tables Page Figure 1: The eukaryotic cell cycle……………………………………………………….5 Figure 2: Description of chlamydial growth method……………………………………12 Table 1: Contents of Media……………………………………………………………...12   Figure 3: The morphology of ST-669 treated C. caviae through an infection…….……17 Figure 4: Lobed inclusions in the presence of ST-669……………………………….…18 Figure 5: qPCR to determine the effects of colchicine and ST-669………………….…19 Figure 6: Visualization of the effects of colchicine and ST-669…………………..……20   Figure 7: qPCR to determine the effects of thymidine and ST-669…………………….21   Figure 8: Visualization of the effects of thymidine and ST-669………………………..22 Figure 9: qPCR to determine the effects of low serum and ST-669……….……………23 Figure 10: Visualization of the effects of low serum and ST-669………………………24 Figure 11: qPCR to determine the effects of vincristine and ST-669……..….…………25 Figure 12: Visualization of the effects of vincristine and ST-669………………………26

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compound ST-669 affects vesicular trafficking in Chlamydia-infected cells. Abstract approved: .. Applied Biosystems. TaqMan universal PCR Master
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