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

42 Pages·2015·2.91 MB·English

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