Divergent evolution of di-lysine ER retention vs. farnesylation motif-mediated anchoring of the ankb

188 Pages·2017·3.35 MB·English

by  John David Perpich

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Electronic Theses and Dissertations
5-2018
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John David Perpich
University of Louisville
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Perpich, John David, "Divergent evolution of di-lysine ER retention vs. farnesylation motif-mediated
anchoring of the ankb virulence effector." (2018). Electronic Theses and Dissertations. Paper 2978.
https://doi.org/10.18297/etd/2978
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DIVERGENT EVOLUTION OF DI-LYSINE ER RETENTION VS.
FARNESYLATION MOTIF-MEDIATED ANCHORING OF THE ANKB
VIRULENCE EFFECTOR

By

John David Perpich
B.S., Michigan State University, 1999
M.S., Michigan State University, 2001
Pharm.D., Medical University of South Carolina, 2007

A Dissertation
Submitted to the Faculty of the
School of Medicine of the University of Louisville
in Partial Fulfillment of the Requirements
for the Degree of

Doctor of Philosophy
in Microbiology and Immunology

Department of Microbiology and Immunology
University of Louisville
Louisville, KY

May 2018
Copyright 2018 by John David Perpich

All rights reserved

DIVERGENT EVOLUTION OF DI-LYSINE ER RETENTION VS.
FARNESYLATION MOTIF-MEDIATED ANCHORING OF THE ANKB
VIRULENCE EFFECTOR

By

John David Perpich
B.S., Michigan State University, 1999
M.S., Michigan State University, 2001
Pharm.D., Medical University of South Carolina, 2007

A Dissertation Approved on

April 12, 2018

by the following Dissertation Committee:

___________________________________
Dissertation Director
Dr. Yousef Abu Kwaik

___________________________________
Dr. Donald Demuth

___________________________________
Dr. Matthew Lawrenz

___________________________________
Dr. Jonathan Warawa

___________________________________
Dr. Michael Merchant

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DEDICATION
To my wife, Amy, for her willingness to support me in this endevour and in
the next phase of my career. For staying by my side during our most difficult
times. To my son, Ethan, for always putting a smile on my face and teaching me
to always be positive. I love them both more than words can express.

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ACKNOWLEDGMENTS
I thank my mentor, Yousef Abu Kwaik, for always being available and
willing to guide me and for helping me find my faults and grow beyond them.
Also for his many pieces of valuable advice. I also thank my committee for their
guidance. In particular, Dr. Michael Merchant for many hours of troubleshooting
as well as career guidance. I am grateful to Dr. Michele Kosiewicz for all her
time and invaluable advice on my career.

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ABSTRACT
DIVERGENT EVOLUTION OF DI-LYSINE ER RETENTION VS.
FARNESYLATION MOTIF-MEDIATED ANCHORING OF THE ANKB
VIRULENCE EFFECTOR
John Perpich

April 12, 2018

Legionella pneumophila is an aquatic organism capable of intracellular
replication within a wide range of protozoan hosts and within human
macrophages where it causes Legionnaires’ Disease. L. pneumophila
manipulates a variety of host cell processes by translocating, into the host cell
cytosol, more than 300 effector proteins via the type IVB secretion system
(T4SS). The AnkB effector from the AA100/130b strain (AnkB-AA100) contains a
C-terminal CaaX motif that rapidly becomes farnesylated upon translocation into
the host cell and anchored to the cytosolic face of the Legionella-containing
vacuole (LCV) membrane, which is essential for intracellular replication. We
show that a homolog of AnkB from the Paris strain (AnkB-Paris) has a frameshift
mutation that truncates the C-terminus eliminating the CaaX motif, but creating a
unique KNKYAP sequence that resembles a eukaryotic di-lysine ER retention
motif (KxKxx). AnkB-Paris localizes to the cytosolic face of the LCV membrane
most likely through the ER retention motif. Phylogenetic analyses indicate that
the AnkB-Paris allele is common among environmental isolates and is positively
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selected specifically for the di-lysine motif. Ectopic expression of AnkB-Paris
results in a perinuclear distribution and trans-rescues the ankB mutant of the
AA100/130b strain for intravacuolar replication. The trans-rescue is dependent
on an intact di-lysine ER-retention motif, which most likely enables anchoring of
AnkB to the ER-derived LCV membrane. AnkB contains a N-terminal eukaryotic
F-box domain that recruits the cellular ubiquitylation machinery by interacting
with the host Skp1 protein resulting in decoration of the LCV with
polyubiquitylated proteins. Proteins targeted for ubiquitylation during infection
are likely bound by ankyrin domains in the C-terminus of AnkB. Based on the
crystal structure, we identified four residues within the ankyrin domains likely
involved in binding specific substrate proteins. Mutation of these residues did not
affect protein folding but resulted in loss of accumulation of ubiquitylated proteins
around the LCV and a severe defect in intracellular replication, similar to the
ankB null mutant. Overall, we identified a di-lysine ER retention motif in the C-
terminus of AnkB-Paris and 4 substrate binding residues within the 3 ankyrin
domains of AnkB-AA100 both of which are essential for biological function.

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TABLE OF CONTENTS
PAGE
DEDICATION…………………………………………………………………………iii
ACKNOWLEDGEMENTS…………………………………………………………...iv
ABSTRACT……………………………………………………………………………v
LIST OF TABLES…………………………………………………………………….ix
LIST OF FIGURES…………………………………………………………………..xi

INTRODUCTION……………………………………………………………………...1
I. History and Etiology of Legionnaires’ Disease………………………...1
II. Epidemiology and Clinical Manifestations……………………………...2
III. Microbial Ecology and Transmission.…………………………………...5
IV. Metabolism…………………………………………………………………6
V. Intracellular Life Cycle…………………………………………………….8
VI. T4SS and Manipulation of Host Cell Processes……………………….8
VII. Genome Plasticity………………………………………………………..10
VIII. Ankyrin Domain-Containing Proteins in L. pneumophila…………….14
IX. Protein Ubiquitylation…………………………………………………….15
X. Manipulation of Host Ubiquitylation Pathways………………………...18
XI. Manipulation of Host Prenylation……………………………………….20
XII. AnkB……………………………………………………………………….22

SPECIFIC AIMS………………………………………………………………………27

MATERIALS AND METHODS………………………………………………………29

CHAPTER 1: Identification of Host Protein Targets of AnkB……………………41

I. Results……………………………………………………………………..41

In vitro Pull Down………………………………………………………….41

Identification of AnkB Substrates by Two-Step Immunoprecipitation..48

AnkB Substrate Identification by Formaldehyde Cross-Linking………53

II. Discussion………………………………………………………………….56

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