Investigating Novel Antimicrobial Strategies in vitro and in Murine Models of Infection by Lauren R

236 Pages·2015·4.41 MB·English

by  Rae Brooks

Investigating Novel Antimicrobial Strategies in vitro and in Murine Models of Infection

by
Lauren R. Brooks

Submitted in Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy

Supervised by Professor Stephen Dewhurst
Department of Microbiology and Immunology
School of Medicine and Dentistry

University of Rochester
Rochester, New York

2015
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Dedication

The work in this thesis is dedicated to my husband, my parents, and most importantly to
my sons Kiwi and Quark Brooks.
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Biographical Sketch

The author was born in Baltimore, Maryland. She attended Virginia Polytechnic
Institute and State University from 2004 to 2009 for her undergraduate education where
she received a Bachelor of Science degree in Biochemistry, a Bachelor of Science degree
in Biological Sciences, and a Bachelor of Arts degree in Chemistry. As a student at
Virginia Tech, she completed undergraduate research in the labs of Dr. Liwu Li and Dr.
Lisa Belden, and a Summer Undergraduate Research Internship Program at the Uniformed
Services University of the Health Sciences with Dr. Xin Xiang. She began doctoral studies
in the Department of Microbiology and Immunology at the University of Rochester in the
Fall of 2009. She was a trainee on the T32 Training Grant in Cellular, Biochemical, and
Molecular Sciences in 2011- 2012 and she received a Master of Science degree from the
University of Rochester in 2011.

The following publications were a result of work conducted during doctoral study:

*Blanchard, C., *Brooks, L.R., Beckley, A., Colquhoun, J., Dewhurst, S., and
Dunman, P.M. Neomycin sulfate improves the antimicrobial activity of
mupirocin-based antibacterial ointments. Accepted pending revisions
August 2015. Antimicob Agents Chemother. *Authors contributed
equally.

*Sheik, D., *Brooks, L.R., Frantzen, K., Dewhurst, S., Yang, J. Inhibition of the
enhancement of infection of human immunodeficiency virus by the semen-
derived enhancer of viral infection using amyloid targeting polymeric
nanoparticles. ACS Nano (2015) 9 (2): 1829-36. *Authors contributed
equally. PMID: 25619867
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*Ontiveros, F., *Easterhoff, D., Brooks, L.R., Ross, B., Olsen, J.T., Hardy, D.,
Dunman, P.M., Dewhurst, S. The semen-derived enhancer of virus
infection (SEVI) binds bacterial pathogens enhancing phagocytosis and
pro-inflammatory cytokine secretion. Antimicrob Agents Chemother
(2013) 57 (6): 2443 – 50.*Authors contributed equally. PMID: 23507280
Sullivan, M., Brooks, L.R., Weidenborner, P.J., Domm, W., Mattiacio, J.L., Xu,
Q., Tiberio, M., Wentworth, T.J., Kobie, J.J., Bryk, P., Zheng, B., Sanz, I.,
Dewhurst, S. Anti-idiotypic monobodies derived from a fibronectin
scaffold. Biochemistry (2013) 52 (10): 1802 – 13. PMID 23394681
Domm, W., Brooks, L., Chung, H.-L., Feng, C., Bowers, W.J., Watson, G.E.,
McGrath, J.L., Dewhurst, S. Robust antigen-specific humoral immune
responses to sublingually delivered viral vectors encoding HIV-1 Env
depend on mucoadhesion and efficient penetration of the sublingual
barrier. Vaccine (2011) 29 (40): 7080-9. PMID 21801777
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Acknowledgments

First and foremost, I would like to thank Dr. Stephen Dewhurst for accepting me
into his lab for my tenure as a graduate student. Through his mentorship and support he
has helped me grow from a timid and unsure student into a more confident and hard-
working individual.
Many, many thanks to all of the current and past members of the Dewhurst lab,
especially Matt Brewer, Will Domm, Jeff Chamberlain, Megan Granger, Caitlin Brown,
David Easterhoff, Colleen O’Dell, and Kristen Frantzen. Without all of you, I would never
be where I am today.
To the class of 2009 Cohort of PhD Students: Sara and Zack Knowlden, Yin Yin
Wang, Dan Martinelli, Alison Bilroth-Maclurg, Steve Baker, Katie Lannan, and Liz Boule
– we have been through every step of this together, from the first day of classes to hanging
out every month during our first year, to qual club, paella and movie nights, and writing
parties to finish our dissertations. You have been my greatest friends and I wish you all
the best of luck.
Thank you to my committee, Drs. Baek Kim, Minsoo Kim, and James McGrath.
Thank you for your input and help over the years. To Dr. Brad Nilsson, thank you for
becoming a member of my dissertation committee. To John DiMiao, your help and
expertise in chemistry has been indispensable to the work in this thesis. To Dr. Jerry Yang
and Daniel Sheik, I will miss our monthly conference calls to discuss the progress of our
work. To Catlyn Blanchard, thank you for being with me every 12 hours (if not every 8
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hours) for 8 months of the past year completing animal work. And finally, to Dr. Paul
Dunman. Thank you for pushing me, for supporting me always, and for being a sounding
board for both personal and professional decisions. Without your support I would not have
graduated; thank you.
I would like to thank the Department of Microbiology and Immunology, especially
Brenda Knorr for always taking time to talk with me and answer my questions. To Dr.
Alexandra Livingstone, thank you for always having an open door and a willing ear every
time I needed to talk.
Lastly and most importantly, I would like to thank my husband and my mom. Your
daily support, encouragement, love, and confidence in me has bolstered me to this point.
Thank you.
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Abstract

Novel approaches are needed for the therapeutic intervention of microbial
pathogens. This research describes the use of human immunodeficiency virus (HIV-1) and
Staphylococcus aureus (SA) as model organisms to test three novel antimicrobial
strategies.
First, we designed synthetic nanoparticles intended to sterically interfere with the
ability of a recently described cationic amyloid fibril, the semen derived enhancer of viral
infection (SEVI), to promote HIV-1 infection in vitro. These amyloid-binding
nanoparticles bound SEVI, and potently reduced SEVI mediated HIV-1 infection in a
manner consistent with a steric effect.
Second, we tested the hypothesis that SEVI may possess antibacterial activity, as
has been reported for other amyloidogenic peptides. SEVI did not directly inhibit bacterial
growth, but it did bind bacteria, including SA, and enhanced their phagocytic uptake by
macrophages. We then generated self-assembling synthetic cationic peptides with similar
charge-to-mass ratio to SEVI and investigated their antimicrobial properties – which were
found to be very similar to SEVI. Finally, we examined SEVI’s antimicrobial properties
using a mouse model for Neisseria gonorrhoeae infection of the vaginal tract, and found
that SEVI accelerated bacterial clearance in this model. Collectively, these data suggest
that amyloid cationic fibrils have potential as antimicrobial agents.
Third, we tested the antimicrobial effectiveness of rationally designed drug
combinations that act on bacterial RNA metabolism. To do this, a high-throughput screen
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was conducted to identify anti-staphylococcal small molecules that target tRNA synthesis.
This identified a previously unrecognized activity of the antibiotic, neomycin, which we
then tested for its ability to enhance the activity of mupirocin, which also acts on tRNA
metabolism. In a murine model for nasal infection with SA, mice treated with combination
therapy had improved bacterial decolonization rates and reduced development of
mupirocin resistance.
Collectively, we conducted proof-of-concept studies that validate three new
antimicrobial approaches: (i) nanoparticles that sterically interfere with virus binding, (ii)
cationic amyloid fibrils, and (iii) additive combinations of small molecules that block
bacterial RNA metabolism. This work points to new directions in treating deadly microbial
infections.
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Contributors and Funding Sources

This work was supported by a dissertation committee consisting of Professors
Stephen Dewhurst (advisor), Paul Dunman, and Minsoo Kim of the Department of
Microbiology and Immunology, Professor James McGrath of the Department of
Biomedical Engineering, and Professor Brad Nilsson of the Department of Chemistry.
The materials used in Chapter 2 were created, characterized, and provided by Dr.
Jerry Yang and Daniel Sheik and data was published in 2015 in ACS Nano. The work in
Chapter 3 was published in part in 2013 in AAC and completed in part by Dr. David
Easterhoff and Dr. Fernando Ontiveros. Work in Chapter 4 was completed in part by
Catlyn Blanchard and submitted for publishing in 2015 in AAC. Work in Appendix A was
completed with Dr. Mark Sullivan and published in 2013 in Biochemistry.
Funding sources for the work completed during graduate study and presented in
this thesis were T32 training grant GM068411, NIH grants R21 AI094511/R33 AI094511,
R21 AI087149, and University of Rochester Center for AIDS Research support NIH P30
AI078498.
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Table of Contents

Dedication ii
Biographical Sketch iii
Acknowledgements v
Abstract vii
Contributors and Funding Sources ix
List of Tables xiii
List of Figures xiv
List of Abbreviations xviii
Chapter 1: Introduction 1
1.1 Overview 2
1.2 Mucosal barrier as the first line of defense against infection 2
1.3 Male and female reproductive tracts 3
1.4 Human Immunodeficiency Virus 6
1.5 Cationic amyloid mediated enhancement of HIV-1 infection 8
1.6 Current prevention therapies and treatments for HIV-1 8
1.7 Staphylococcus aureus 9
1.8 Host mechanisms of preventing bacterial infection 11
1.9 Antibiotics classes and mechanisms of action 12
1.10 Hypothesis and Aims 14
1.11 Figures 15