Structural and functional analysis of Golgi in · Vinitha Joice Macwan Master of Science Department...

Structural and functional analysis of Golgi in

Legionella-infected macrophages

by

Vinitha Joice Macwan

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Cell and Systems Biology

University of Toronto

© Copyright by Vinitha Joice Macwan, 2018

ii

Structural and functional analysis of Golgi in Legionella-

infected macrophages

Vinitha Joice Macwan

Master of Science

Department of Cell and Systems Biology

University of Toronto

2018

Abstract

Legionella pneumophila is an accidental pathogen that replicates intracellularly within

the Legionella-containing vacuole (LCV). It hijacks important host regulators of early secretory

pathway including Rab1. To see the consequence of the trafficking disruption, we examined the

Golgi structure and function in Legionella-infected human U937 macrophages. Intriguingly, the

Golgi area in infected macrophages remained similar to non-infected macrophages. Furthermore,

TEM analysis also did not reveal any significant changes in the ultrastructure of the Golgi in

infected cells. Drug-induced Golgi disruption had differing effects on bacterial replication in

human macrophages. The glycosylation protein levels, visualized by fluorescent cis-Golgi lectin,

Helix promatia agglutinin (HPA), significantly decreased over time as infection progressed,

compared to control cells. This suggests that Legionella hijacks sugars which are possibly

important for bacteria growth. Collectively, our results indicate that the structure of Golgi is kept

intact whereas the function of Golgi is possibly impaired in Legionella-infected macrophages.

iii

Acknowledgements

First, I would like to thank my supervisor, Dr. Rene Harrison for her support and guidance

throughout my program as well as my co-supervisor, Dr. Mauricio Terebiznik for his valuable

help and advice. I am grateful for each of my colleagues: Durga for constant encouragement,

Reuben for strengthening my imaging skills, Urja for continual support and encouragement,

Roxy for her motivation and Mathieu for helpful tips and advice. I also thank Dr. Bebhinn

Treanor and Dr. Greg Vanlerberghe for being on my thesis advisory committee and for proving

helpful feedback on my research. I thank Bob for his help with TEM experiments as well as

Bruno for his help in the CNS. I would like to thank my husband Keith for his extreme support

and encouragement throughout my studies. I also thank all my family members for their love and

support.

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Table of Contents Acknowledgements .................................................................................................................................... iii

List of Figures ............................................................................................................................................. vi

List of Abbreviations ............................................................................................................................... viii

1. Introduction ......................................................................................................................................... 1

1.1 Legionella pneumophila .............................................................................................................. 1

1.2 Intracellular lifecycle of Legionella ........................................................................................... 1

1.2.1 Virulence system in Legionella ............................................................................................. 2

1.2.2 Intracellular growth stages of Legionella within macrophages ............................................ 2

1.2.3 Formation of the LCV ........................................................................................................... 3

1.3 Hijacking of host early secretory pathway by Legionella ........................................................ 4

1.3.1 Modulation of Rab1 at early stages of infection ................................................................... 4

1.3.2 Modulation of Rab1 at late stages of infection ..................................................................... 5

1.3.3 Recruitment of Arf1 to the LCV ........................................................................................... 6

1.3.4 Modification of lipids to resemble the Golgi ........................................................................ 6

1.3.5 Exploitation of SNAREs to mediate fusion of ER-derived vesicles to the LCV .................. 7

1.4 Golgi complex and its role in the secretory pathway ............................................................... 8

1.4.1 Structure ................................................................................................................................ 8

1.4.2 Function .............................................................................................................................. 11

1.5 Rationale and Objectives .......................................................................................................... 13

2. Materials and Methods ......................................................................................................................... 14

2.1 Reagents and Antibodies ................................................................................................................ 14

2.2 Cell culture ...................................................................................................................................... 15

2.3 Bacterial strains and cultivation .................................................................................................... 15

2.4 Bacterial infections .......................................................................................................................... 16

2.5 Intracellular growth assay.............................................................................................................. 16

2.6 Golgi disruption via pharmacological inhibitors ......................................................................... 16

2.7 Immunostaining and Spinning disk confocal microscopy ........................................................... 17

2.8 Transmission electron microscopy analysis .................................................................................. 18

2.9 Data analysis and Statistics ............................................................................................................ 18

3. Results .................................................................................................................................................... 19

3.1 Growth of Legionella with U937 macrophages ............................................................................ 19

3.2 Golgi structure remains intact in Legionella-infected U937 macrophages ................................ 21

v

3.3 Golgi disruption via pharmacological inhibitors differentially affect Legionella growth within

macrophages .......................................................................................................................................... 27

3.3.1 Golgi disruption induced by golgicide A decreases Legionella growth at late phases of

infection .............................................................................................................................................. 27

3.3.2 Golgi disruption induced by Brefeldin A does not impact Legionella growth within

macrophages ....................................................................................................................................... 30

3.3.3 Golgi disruption induced by nocodazole reduces Legionella growth at early and late phases of

infection .............................................................................................................................................. 32

3.4 The maturation process of LCVs in Legionella-infected macrophages is differentially affected

by different Golgi-disrupting drugs .................................................................................................... 35

3.4.1 The maturation of the LCV is delayed in NOC-treated macrophages infected with Legionella 36

3.4.2 Normal LCV maturation in GCA-treated macrophages infected with Legionella .................... 39

3.4.3 Infected macrophages treated with GCA and BFA had normal LCV maturation ..................... 41

3.4.4 LCVs are devoid of lysosomal components in Legionella-infected macrophages treated with

Golgi disrupting inhibitors .................................................................................................................. 43

3.5 Cis-Golgi lectin levels decrease over time in Legionella-infected macrophages ........................ 45

4. DISCUSSION ........................................................................................................................................ 48

4.1 Interaction of bacterial pathogens with the host secretory pathway .......................................... 49

4.2 How is Golgi integrity preserved in Legionella-infected macrophages? .................................... 50

4.3 Why is Golgi integrity preserved by Legionella? ......................................................................... 54

4.4 The effects of different Golgi-disrupting drugs on Legionella growth ....................................... 55

4.5 The effects of different Golgi-disrupting drugs on LCV maturation ......................................... 60

4.6 Why might Legionella co-opt sugars from the Golgi? ................................................................. 63

4.7 What causes the decrease in glycosylated protein levels of Golgi during Legionella infection?

................................................................................................................................................................ 64

4.8 Summary and future directions ..................................................................................................... 68

References .................................................................................................................................................. 71

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List of Figures

Figure 1: Formation of the LCV.. ................................................................................................... 4

Figure 2: Wild-type Legionella strain can multiply intracellularly within human U937

macrophages. ................................................................................................................................ 20

Figure 3: The cis-Golgi structure remains intact in Legionella-infected U937 macrophages. ..... 22

Figure 4: The trans-Golgi structure remains intact in Legionella-infected U937 macrophages.. 25

Figure 5: TEM of U937 macrophages infected with Legionella reveals no change in the Golgi

complex at an ultrastructural level. ............................................................................................... 26

Figure 6: Golgicide-induced Golgi disruption significantly decreases multiplication of Legionella

within U937 macrophages at late phase of infection. ................................................................... 29

Figure 7: Brefeldin A-induced Golgi disruption does not affect multiplication of Legionella

within U937 macrophages. ........................................................................................................... 31

Figure 8: Nocodazole-induced Golgi disruption significantly decreases multiplication of

Legionella within U937 macrophages at early and late time points of infection. ........................ 34

Figure 9: Increased FK2-positive LCVs in Legionella-infected macrophages treated with

nocodazole and golgicide A. ......................................................................................................... 38

Figure 10: Absence of KDEL-positive LCVs in Legionella-infected macrophages treated with

nocodazole and brefeldin A. ......................................................................................................... 40

Figure 11: Presence of calnexin-positive LCVs in Legionella-infected macrophages treated with

nocodazole or golgicide A or brefeldin A..................................................................................... 42

Figure 12: Absence of LAMP-1 in Legionella-infected macrophages treated with nocodazole or

golgicide A or brefeldin A. ........................................................................................................... 44

Figure 13: HPA lectin expression levels significantly decrease in Legionella-infected U937

macrophages. ................................................................................................................................ 47

Figure 14: Model explaining how Legionella could potentially preserve the Golgi complex

during infection. ............................................................................................................................ 53

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Figure 15: The effect of different Golgi disruption inhibitors on Legionella multiplication in

infected macrophages.................................................................................................................... 59

Figure 16: Model showing potential mechanisms to explain how Legionella impairs Golgi

function. ........................................................................................................................................ 67

viii

List of Abbreviations

ACES N-(2-acetamido)-2-amino-ethanesulfonic acid

ADP Adenosine diphosphate

AMP Adenosine monophosphate

ARF ADP ribosylation factor

AP-1 Adaptor protein 1

ATCC American type culture collection

BCV Brucella-containing vacuole

BFA Brefeldin A

BIG1/BIG2 BFA-inhibited guanine nucleotide exchange proteins 1 and 2

BCYE Buffered charcoal yeast extract

BYE Buffered yeast extract

C3 Complement component

CDC48 Cell division protein 48

CFU Colony forming unit

CPAF Chlamydial protease-like activity factor

CR3 Complement receptor 3

CREB3 Cyclic AMP-responsive element-binding protein 3

ED Entner-doudoroff

eEF1A Eukaryotic elongation factor 1A

ER Endoplasmic reticulum

ERAD ER-associated degradation

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ERGIC ER-Golgi intermediate compartment

FBS Fetal bovine serum

GalNAc N-acetyl-d-galactosamine

GAP GTPase-activating protein

GASE Golgi apparatus stress response element

GBF1 Golgi-specific BFA resistance guanine nucleotide exchange factor 1

GCA Golgicide A

GDP Guanosine diphosphate

GDI GDP dissociation inhibitor

GEF Guanine nucleotide exchange factor

GGA3 Golgi-localized-γ-ear-containing Arf binding protein 3

GTP Guanosine triphosphate

HPA Helix promatia agglutinin

HSP47 Heat shock protein 47

LAMP-1 Lysosome-associated membrane protein 1

LCV Legionella-containing vacuole

LGT Legionella glycosyltransferase

MIF Mature intracellular form

MOI Multiplicity of infection

NOC Nocodazole

PBS Phosphate-buffered saline

PFA Paraformaldehyde

PHB Poly-3-hydroxybutyrate

x

PI phosphatidylinositol

PIPs Phosphatidylinositol phosphates

PMA Phorbol 12-myristate 13-acetate

PPAR Peroxisome proliferator-activated receptor

RPMI Roswell park memorial institute

SCF Skp1/Cul1/F-box

SCV Salmonella-containing vacuole

SEM Standard error of mean

SNARE N-ethylmaleimide-sensitive factor activating protein receptors

SNX Sorting nexin

T4SS Type IVB secretion system

TCA Tricarboxylic acid

TEM Transmission electron microscopy

TFE3 Transcription factor E3

TGN Trans-Golgi network

UDP Uridine diphosphate

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1. Introduction

1.1 Legionella pneumophila

The intracellular pathogen Legionella pneumophila causes a severe, acute and often fatal

form of pneumonia known as Legionnaires’ disease (Suwwan De Felipe et al., 2008). It

replicates in highly diverse hosts that range from fresh water amoebae to alveolar macrophages

in humans (Zhu and Luo, 2016). Unlike most bacteria that get engulfed and destroyed by

macrophages, Legionella divides within its phagosome and subsequently kills the macrophage,

causing Legionellosis (Cazalet et al., 2004). The pathogen achieves this by the formation of a

replication-permissive membrane-bound compartment, the “Legionella-containing vacuole”

(LCV) (Rothmeier et al., 2013). This vacuole escapes the interaction with lysosomes. Legionella

replicates within this vacuole followed by disruption of vacuole membrane and subsequent

rupture of the host cell (Hubber and Roy, 2010).

1.2 Intracellular lifecycle of Legionella

The attachment of Legionella to the macrophage surface is mediated by the complement

receptor 3 (CR3) followed by phagocytosis (Clemens and Horwitz, 1992; Steinert et al., 1994).

The complement component (C3) covalently associates with the major outer membrane protein

(MOMP) of Legionella through the complement activation resulting from the alternative

pathway. C3 on the Legionella surface then serves as a ligand for complement receptors CR1 and

CR3, and mediates the uptake into the macrophage (Bellinger-Kawahara and Horwitz, 1987).

Following uptake, LCV is surrounded by small smooth vesicles that originate from the

endoplasmic reticulum (ER) (Kagan and Roy, 2002a; Swanson and Isberg, 1995; Tilney et al.,

2001). The membrane of the LCV then starts to resemble that of ER in terms of thickness and

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protein composition, and eventually becomes decorated with ribosomes (Tilney et al., 2001). In

addition, other host organelles including mitochondria are recruited to the LCV (Tilney et al.,

2001). Simultaneously, the LCV successfully evades the endocytic pathways as it avoids fusion

with endosomes and lysosomes (Horwitz and Maxfield, 1984). This vacuole provides Legionella

with required nutrients for replication and further protects it from being recognized by the

cellular immune system (Xu and Luo, 2013).

1.2.1 Virulence system in Legionella

The main virulence factor of Legionella is the Dot/Icm type IVB secretion system

(T4SS), which is known to manipulate host processes through numerous bacterial effectors that

are delivered to the host cytosol (Ensminger and Isberg, 2009). These effectors disrupt key

processes including vesicle trafficking and modify membrane lipids that are important in

membrane fission and fusion events. In addition to acting on the LCV, effectors are also known

to target endosomal vesicles throughout the cell to prevent interaction with the endosomal

system (So et al., 2015).

1.2.2 Intracellular growth stages of Legionella within macrophages

The two major stages of intracellular Legionella growth in macrophages include the

replicative form (RF) and the mature intracellular form (MIF), which switch through different

morphological intermediates within the development cycle of Legionella (Faulkner and Garduño,

2002; So et al., 2015). The RF is non-infectious (Joshi et al., 2001) and it is known to actively

undergo cell division. On the other hand, the MIF is highly infectious to cells in culture. These

bacteria can escape infected host cells and persist in the environment (Hoffmann et al., 2008).

During the initial time point of infection, Legionella starts adapting to the intracellular

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environment. After 6 hours, bacteria begin growing actively inside the macrophages (RFs) and

by 18 hours, they stop replicating and start to lyse the host cells (MIFs) (Faucher et al., 2011).

1.2.3 Formation of the LCV

After uptake, Legionella modulates different host cellular processes through several

translocated effectors, in order to form a specialized LCV which permits intracellular bacterial

growth within macrophages (Hubber et al., 2013). This vacuole is enriched in polyubiquitinated

proteins through the action of the effector AnkB, that exhibits molecular and functional mimicry

of eukaryotic F-box proteins. AnkB is delivered into the host cytosol via T4SS immediately upon

bacterial attachment to the host cell plasma membrane, where the polyubiquitinated proteins are

rapidly recruited (Price et al., 2009). This process ensures temporal regulation of effector

function within the macrophage and subsequently promotes optimal bacterial replication (Hubber

et al., 2013). As time progresses, this LCV resembles the ER by acquiring various different ER

components that can be marked by proteins with a KDEL motif initially within 30 min of

infection, followed by acquisition of resident ER protein calnexin by 4h post-infection (Robinson

and Roy, 2006).

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Figure 1: Formation of the LCV. The biogenesis of the LCV involves different stages. A)

During the initial stages of infection (0 – 3 h post-infection), a nascent LCV begins to form

which is enriched with ubiquitinated proteins. (1) Legionella translocates an effector named

AnkB which gets farnesylated (2), leading to its anchoring on LCV membrane, where it interacts

with the host E3 ubiquitin ligase component, SCF (3). This creates a platform for the docking of

polyubiquitinated host proteins to the LCV (4) which are broken down further by a proteasome

(5) to generate amino acids (6). B) The next stage involves LCV remodeling, which requires the

recruitment of Rab1 and Arf1 to the LCV (1) to mediate fusion of redirected ER-derived vesicles

with a KDEL tag to the LCV (2). C) During the final stages of infection, the LCV becomes

mature with ER-resident proteins like calnexin present on the membrane and containing a high

number of replicated Legionella.

1.3 Hijacking of host early secretory pathway by Legionella

1.3.1 Modulation of Rab1 at early stages of infection

The recruitment of ER-like vesicles to the LCV membrane has been observed during the

first 15-30 min of infection in U937 macrophages (Tilney et al., 2001). In order to redirect ER

membrane trafficking, Legionella hijacks and recruits Rab1, which is an important regulator of

vesicle trafficking in the secretory pathway between ER and Golgi, to the LCV (Kagan et al.,

5

2004). This is regulated by the effector SidM, which is translocated at initial stages of infection

and anchored to the LCV via a PI(4)P binding domain (Brombacher et al., 2009). Inactive Rab1

bound to guanosine diphosphate (GDP) associates with a GDP dissociation inhibitor (GDI),

which prevents nucleotide exchange and membrane association of the GTPase. The guanine

nucleotide exchange factor (GEF) domain of SidM induces the exchange of GDP for guanosine

triphosphate (GTP), causing the release of Rab1 from its GDI and recruitment to the LCV

membrane (Brombacher et al., 2009; Machner and Isberg, 2007, 2006). Furthermore, Rab1 is

also AMPylated by SidM which is necessary for its LCV localization (Hardiman and Roy, 2014).

This leaves Rab1 in a constitutively active GTP-bound state and blocks its interaction with

GTPase-activating proteins (GAPs) (Muller et al., 2010). Hence the duration of Rab1 activation

is regulated by AMPylation in order to modulate Rab1’s interactome to activate a particular

subset of Rab1-dependent signaling pathways, causing the tethering of ER-derived vesicles to

the LCV (So et al., 2015).

1.3.2 Modulation of Rab1 at late stages of infection

SidM and Rab1 levels on the LCV start to decrease 2 hours after infection in

macrophages (Ingmundson et al., 2007), suggesting that the AMPylation-dependent block in

access to Rab1 for GAPs is relieved. Legionella then utilizes SidD as an antagonistic effector to

efficiently deAMPylate Rab1 (Ingmundson et al., 2007; Muller et al., 2010). In addition, it

translocates its own Rab1 GAP, the effector LepB in order to have complete control of Rab1

function. This effector induces GTP hydrolysis and allows Rab1 to be susceptible to membrane

extraction by GDIs (Ingmundson et al., 2007). The release of these crucial effectors is regulated

temporally by Legionella, with SidM getting translocated at initial time of infection whereas

SidD and LepB being translocated at a later phase of infection to avoid an early inactivation of

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Rab1. Another effector that plays a regulatory role for Rab1 activity includes AnkX, which

carries out a post-translational phophocholination (Mukherjee et al., 2011). It does so when Rab1

is in its inactive GDP-bound form (Goody et al., 2012). As a result, the phosphocholinated GDP-

bound Rab1 is prevented from binding to multiple GEFs and GDIs, thereby inhibiting its

activation and membrane extraction.

1.3.3 Recruitment of Arf1 to the LCV

Legionella effectors spatially and temporally control host cell protein signaling by

modulating ER to Golgi trafficking pathway. In addition to Rab1, Legionella also targets another

important complex, Arf1, which is a small GTPase that is involved in regulating budding and

uncoating of vesicles in the ER-Golgi intermediate compartment and Golgi (Nagai et al., 2002).

A T4SS effector RalF has a role in activating Arf1 through its Sec7-homology domain, which is

present in several Arf1 GEFs to recruit it to the LCV during the initial phase of infection. The

function of this effector is under the control of an internal capping domain that localizes it to the

LCV and allows access to its Sec7-homology domain only upon membrane contact (Alix et al.,

2012). Once Arf1 is localized to the LCV, it promotes efficient fusion of ER-derived vesicles

(Robinson and Roy, 2006).

1.3.4 Modification of lipids to resemble the Golgi

Legionella manipulates membrane lipids, specifically phosphatidylinositol (PI) and its

phosphorylated derivatives, phosphatidylinositol phosphates (PIPs) in order to modulate host cell

proteins that are recruited to the LCV. Furthermore, these PIPs serve as anchors for multiple

T4SS effectors so that they can mediate fusion and fission events between the LCV and other

cellular membranes (So et al., 2015). For example, PI(4)P, normally found on Golgi membrane,

is enriched on the LCV membrane, which anchors effectors including SidM/DrrA, SidC, and

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SdcA to efficiently recruit ER-derived vesicles to the LCV (Ragaz et al., 2008; Weber et al.,

2006). On the LCV membrane, the PIP pattern is directly modified to PI(4)P by PI 3-

phosphatase effectors SidF and SidP. In addition, host cell PI 5-phosphatase is recruited to LCV,

where it binds to LpnE (Weber et al., 2009) to generate PI(4)P.

1.3.5 Exploitation of SNAREs to mediate fusion of ER-derived vesicles to the LCV

The fusion events between a vesicle and target membrane is controlled by SNAREs (N-

ethylmaleimide-sensitive factor activating protein receptors) (Jahn and Scheller, 2006). When a

vesicle comes close to its target membrane, v- and t-SNAREs interact to form a complex and

generate force along with supporting proteins in order to bring the v- and t membranes into close

contact and initiate fusion (So et al., 2015). Each v-SNARE is only able to interact with a small

subset of compatible t-SNAREs, challenging Legionella in its fusion process with ER SNAREs

such as Sec22b (Arasaki et al., 2012). To overcome this challenge, effector SidM not only

activates Rab1 to recruit ER-derived Sec22b-positive vesicles, but also directly interacts with

plasma membrane SNAREs, syntaxins (Arasaki et al., 2012). This allows these two SNARE

types to come into close proximity for a non-canonical SNARE pairing and subsequent fusion of

ER-derived vesicles with the LCV. In addition to manipulating host SNARE proteins, Legionella

also contains its own SNAREs that interact with eukaryotic SNAREs. For example, a T4SS

effector LseA mimics a host SNARE protein to directly interact with Golgi for efficient fusion

with ER SNARE (King et al., 2015).

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1.4 Golgi complex and its role in the secretory pathway

1.4.1 Structure

The Golgi apparatus is a membrane-bound organelle that is present in all eukaryotic cells. It

is composed of flattened, disk-shaped membrane-bound compartments called cisternae which are

organized in stacks (Lee et al., 2015). The cisternae contain different sets of resident proteins and

hence the stacks are divided into cis, medial, and trans sides. Several structural proteins play a

role in the proper organization of Golgi. For example, GM130 is a cis-Golgi structural protein

that is involved in promoting vesicle fusion to the Golgi membrane, along with p115, giantin and

GRASP65. On the other hand, TGN46 is a transmembrane protein that is usually present at the

trans-Golgi network (TGN) and shuttles between the TGN and plasma membrane (Banting and

Ponnambalam, 1997).

1.4.2.1 Golgi-associated Arfs

ADP ribosylation factors (Arfs) are small monomeric GDP/GTP-binding proteins that are

grouped into three classes: class I composed of Arf1, 2, and 3, and class II composed of Arf4 and

5 whereas class III comprises Arf6 (Kahn et al., 2006). They play a crucial role in the regulation

of vesicular transport by recruiting coat proteins that are required for vesicle formation (Bui et

al., 2009), anterograde ER-Golgi and retrograde Golgi-ER trafficking (D’Souza-Schorey and

Chavrier, 2006; Donaldson et al., 2005). For example, Arf1 localizes to all three Golgi

compartments: cis-, medial, and trans-Golgi, where its activation is spatially and temporally

regulated by specific guanine nucleotide exchange factors (GEFs). Specifically, the Arf1 GEF,

GBF1 (Golgi-specific BFA resistance guanine nucleotide exchange factor 1) localizes to the cis-

Golgi where it is involved in the assembly and maintenance of the Golgi stack. On the other

hand, BIG1/BIG2 (BFA-inhibited guanine nucleotide exchange proteins 1 and 2) associate with

9

the trans-Golgi in order to maintain the trans-Golgi network (Manolea et al., 2008; Sáenz et al.,

2009). Two commonly used pharmacological inhibitors to study Golgi function include brefeldin

A (BFA) and golgicide A (GCA), both of which target Arf1 GEFs that eventually leads to Golgi

fragmentation. BFA inhibits Arf1 by targeting both GBF1 and BIG1/BIG2 (Claude et al., 1999;

Kawamoto et al., 2002; Mansour et al., 1999; Yamaji et al., 2000) while GCA is highly specific

to only GBF1 (Sáenz et al., 2009).

1.4.2.2 Golgi Positioning: Microtubules

The structural integrity and position of Golgi complex is maintained by the microtubules.

The Golgi apparatus is situated at the minus ends of microtubules that are usually linked with the

microtubule-organizing center. Microtubules further constantly relocate Golgi elements inwards

towards the nucleus by dynein and dynactin motor complexes (Banting and Ponnambalam, 1997;

Martínez-Menárguez, 2013) in order to maintain a compact Golgi complex (Kreis, 1990).

Microtubules have a role in distributing cargo to the cell perimeter for secretion using the kinesin

motor (Hicks and Machamer, 2005). Nocodazole is a frequently used, reversible drug to

depolymerize microtubules (Dunlop et al., 2017)).

1.4.2.3 Importance of balanced anterograde and retrograde trafficking

Crucial for the steady-state structure of Golgi complex is the proper balance between

anterograde and retrograde transport through the Golgi (Hicks and Machamer, 2005). The

disruption of ER-to-Golgi traffic causes the Golgi to collapse into the ER because of the

unbalanced retrograde traffic (Hicks and Machamer, 2005). The inhibition of exit from either

cis-Golgi or trans-Golgi networks can lead to significant swelling as a result of anterograde

cargo build up in the cis-Golgi network or TGN (Hicks and Machamer, 2005; Kreis, 1990;

10

Moremen and Robbins, 1991). Hence, Golgi structure is a dynamic organelle maintained by the

appropriate balance of inward and outward membrane trafficking (Hicks and Machamer, 2005).

1.4.2.2 Golgi-associated stress response pathways

The Golgi complex is highly dynamic and rapidly disassembles and reassembles during

mitosis as well as under stress and physiological conditions (Wang and Seemann, 2011). Several

studies have identified multiple stress response pathways associated with Golgi disruption,

including the TFE3, HSP47, and the CREB3-ARF4 pathways. TFE3 is a transcription factor that

is typically located in the cytoplasm in a phosphorylated form. But when glycosylation is

perturbed within the Golgi via inhibition of glycosylation enzymes for example, TFE3 gets

dephosphorylated in the cytoplasm which leads to its translocation into the nucleus, where it

binds to a cis-actin enhance element known as the Golgi apparatus stress response element

(GASE). This eventually results in transcriptional activation of Golgi structural proteins,

glycosylation enzymes as well as a vesicular transport complex (Reiling et al., 2013). On the

other hand, HSP47 is an ER chaperone, whose expression is increased specifically when mucin-

type O-GalNAc glycosylation is inhibited (Zhang et al., 1993). The CREB3/ARF4 pathway is

activated when the function of ARF proteins is inhibited. Specifically, CREB3 translocates from

the ER to the nucleus to increase the transcription levels of ARF4, which ultimately leads to

Golgi stress-induced apoptosis and Golgi disruption (Hicks and Machamer, 2005). Due to their

close association with the ER, HSP47 and CREB3-ARF4 pathways might regulate the cis-Golgi

network, whereas the TFE3-GASE pathway might regulate the trans-Golgi network (Hicks and

Machamer, 2005).

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1.4.2 Function

1.4.2.1 Trafficking, sorting and secretion

The Golgi complex is a central membrane organelle that primarily functions as the post-

translational modification factory as well as trafficking hub for proteins and lipids within the cell

(Huang and Wang, 2017). The cis-Golgi compartment is responsible for receiving transport

vesicles containing newly synthesized proteins and lipids from the ER and ER-Golgi

intermediate (ERGIC) compartments (Reynders et al., 2011). These proteins then mature as they

travel through the different cisternae. They are then sorted and targeted into various transport

carriers at the TGN. There is also cargo transport in the retrograde direction within Golgi stacks

as well as towards the ER, which makes sure that escaped ER resident proteins can locate back to

their correct sites of action (Lippincott-Schwartz et al., 2000; Shorter and Warren, 2002).

Furthermore, this also promotes continuous quality control and removal of damaged proteins via

the ER-associated degradation (ERAD) pathway (Hebert et al., 2010).

1.4.2.2 Models for protein trafficking in the Golgi

There are two different models that exist to describe how proteins traffic through the

different compartments of Golgi: the cisternal maturation (or progression) model and the

anterograde vesicular transport (Emr, 2009; Glick and Nakano, 2009; Pelham and Rothman,

2000). The former model portrays cisternae as transient stacks which move out in a ‘conveyer

belt’ fashion. The fusion of COPII vesicles or other ER-derived vesicles (Bannykh and Balch,

1997; Mironov et al., 2003) leads to the generation of a new cis-cisterna, which eventually

matures into a TGN cisterna. Next, this compartment is dissociated into secretory vesicles and

other types of carriers. The resident Golgi enzymes are believed to be recycled from older to

12

younger cisternae via COPI vesicles (Glick and Malhotra, 1998; Rabouille and Klumperman,

2005). On other hand, the vesicular transport model portrays cisternae as being stable

compartments (Dunphy and Rothman, 1985; Farquhar, 1985; Farquhar and Palade, 1981;

Rothman, 1981), each consisting of specific sets of resident Golgi enzymes that process

secretory cargoes (Kleene and Berger, 1993; Nilsson et al., 2009; Rabouille et al., 1995). COPII

vesicles deliver newly synthesized proteins to the cis-Golgi, which then move to the next

compartment via COPI-vesicles. This model has been updated to newer versions that suggest

bidirectional roles for COPI vesicles, where anterograde COPI vesicles carry secretory cargoes

forward whereas retrograde COPI vesicles recycle trafficking components (Orci et al., 2000;

Pelham and Rothman, 2000).

1.4.2.3 Post-translational modifications

As the cargo proteins are packaged and sorted throughout the Golgi stack, they undergo further

post-translational modifications including glycosylation, sulfation and phosphorylation

(Reynders et al., 2011). The process of glycosylation generates highly distinct and diverse types

of glycosylated structures which are categorized into glycoproteins, glycolipids and

proteoglycans (Hart, 1992; Spiro, 2002; Varki, 1998). Glycosylation involves the linking of

monosaccharides together, transferring sugars from one substrate to another and trimming sugars

from the glycan structure (Walsh, 2006). Glycosylation is regulated by numerous players and

factors including glycosyltransferases (glycosylation enzymes), nucleotide sugar synthases, and

transporters, the Golgi lumenal environment as well as the structure and organization of Golgi

membranes (Stanley, 2011).

13

1.5 Rationale and Objectives

The successful intracellular replication of Legionella relies heavily on recruiting proteins and

lipids trafficking between the ER and Golgi to the LCV membrane. The disturbance in vesicle

trafficking and improper function of Rab GTPases is likely to have a major impact on the

structural integrity of Golgi complex. Since the cargo trafficking from ER to Golgi is recruited to

the LCV, few studies have attempted to analyze the localization and presence of Golgi-

associated proteins on the LCV membrane. However, these proteins including p115 and GM130

were not found to be present on the LCV at any time point after the infection (Arasaki et al.,

2012; Hay et al., 1997). While the LCV does not recruit Golgi directly, this does not necessarily

mean that the Golgi is unaffected during infection. Therefore, my project involved a detailed

study of the structure and function of Golgi complex during Legionella infection in human

macrophages. My hypothesis was that the Golgi complex will disperse/spread apart and

eventually fragment due to perturbed anterograde trafficking between the ER and Golgi caused

by Legionella. To test this hypothesis, I have first performed a thorough analysis of Golgi

structure via immunofluorescence and electron microscopy during different intracellular growth

stages of Legionella within U937 macrophages. Then, I examined the effect of

pharmacologically induced Golgi disruption on Legionella multiplication within the

macrophages by immunofluorescence microscopy. Next, I was interested in investigating the

correlation between Golgi disruption, Legionella growth and LCV maturation. Lastly, I

determined whether Legionella would impair Golgi function by analyzing the glycosylated

protein levels within Golgi in infected cells, compared to control human macrophages.

14

2. Materials and Methods

2.1 Reagents and Antibodies

Roswell Park Memorial Institute (RPMI) media was purchased from American Type

Culture Collection (Manassas, VA). Fetal Bovine Serum (FBS) was purchased from Wisent Inc.

(Quebec City, QC). Phorbol 12-Myristate 13-Acetate (PMA), nocodazole (NOC) and golgicide

A (GCA) were purchased from Sigma Aldrich Canada Co. (Oakville, ON). N-(2-acetamido)-2-

amino-ethanesulfonic acid (ACES) and α-ketoglutarate were purchased from BioShop Canada

Inc. (Burlington, ON). GIBCO® phosphate-buffered saline (PBS) and brefeldin A (BFA) were

purchased from ThermoFisher Scientific Inc. (Waltham, MA). Paraformaldehyde (PFA) was

purchased from Electron Microscopy Sciences (Hatfield, PA). Mouse monoclonal anti-GM130

(610823) was purchased from BD Biosciences (San Jose, CA). Mouse monoclonal anti-

Legionella pneumophila (MAB10223) was purchased from EMD Millipore Canada Ltd.

(Etobicoke, ON). Rabbit polyclonal anti-Legionella pneumophila was obtained from Mauricio

Terebiznik lab (Toronto, ON). Mouse monoclonal anti-FK2 (04-263) and mouse monoclonal

anti-calnexin (MAB3126) were purchased from EMD Millipore Corporation (Temecula, CA).

Rabbit polyclonal anti-TGN46 (ab50595) was purchased from Abcam Inc. (Toronto, ON).

Saponin was purchased from Bio Basic Canada Inc. (Markham, ON). AffiniPure donkey

polyclonal anti-rabbit, donkey anti-mouse CyTM2 or CyTM3 or CyTM5 secondary antibodies were

purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Mouse

monoclonal anti-KDEL (10C3) was purchased from Enzo Life Sciences Inc. (Farmingdale, NY)

and rat monoclonal anti-LAMP-1 (1D4B) was purchased from Santa Cruz Biotechnology Inc.

(Dallas, TX). Lectin Helix promatia agglutinin (HPA), an Alexa FluorTM 488 conjugate

15

(L11271) was purchased from Life Technologies (Carlsbad, CA). Activated charcoal was

purchased from Sigma Aldrich Co. (St. Louis, MO).

2.2 Cell culture

The U937 human monocyte cell line was obtained from American Type Culture

Collection (ATCC). U937 cells were thawed from liquid nitrogen storage and grown in RPMI

medium, containing 10% heat-inactivated FBS. The suspension cells were passaged every 3-4

days in T-75 tissue culture flasks. They were incubated in 5% CO2, at 37C until 80 – 90%

confluency was reached. Once confluent, 100 ng/ml of PMA was used to differentiate them into

macrophages (Passmore et al., 2001) for 24 hours at 37C with 5% CO2. After differentiation,

U937 macrophages were counted using a hemocytometer and 400,000 / 800,000 / 1,000,000

cells were plated on 18 mm / 35 mm / 25 mm glass coverslips, respectively, for incubation at

37ᵒC with 5% CO2 for 24 hours.

2.3 Bacterial strains and cultivation

All strains of Legionella pneumophila were obtained from Mauricio Terebiznik’s lab

(University of Toronto, ON). Bacteria from frozen glycerol stocks were streaked onto BCYE

agar plates, containing ACES. To obtain the short rod form of Legionella, bacteria were scraped

from BCYE plates after growth for 3 days and cultured in buffered yeast extract (BYE) broth for

24 h at 37C with shaking at 100 rpm. To further enrich growth of short rods, this culture was

sub-cultured in fresh BYE broth supplemented with additional amount (50%) of α-ketoglutarate

reagent at an OD600 of 0.05 and grown at 37C with shaking at 100 rpm to an OD600 of 2.0 – 3.0.

16

2.4 Bacterial infections

For infections, Legionella strain Lp01 at an OD600 of 2.0 – 3.0 was added onto

differentiated U937 macrophages with a Multiplicity of Infection (MOI) of 50. Plates were spun

at 4C for 5 min at 300 g and incubated further at 4C for 5 more min to promote bacterial

attachment. They were next incubated at 37ᵒC for 30 min followed by three 1X PBS washes and

addition of fresh RPMI media. Infections were allowed to proceed at 37C with 5% CO2 and at

indicated times, cells were fixed using 4% PFA for microscopy analysis.

2.5 Intracellular growth assay

U937 macrophages adhered to 12-well plates were infected with Lp01 Legionella strain

at MOI of 50 as previously described. To obtain colony forming units (CFUs), monolayers were

incubated with distilled water for 15 min at indicated time points of infection at 37C with 5%

CO2 for collection of supernatant. For cell lysis, monolayers were next incubated with RPMI

media containing 0.2% saponin at 37C with 5% CO2 for 15 min followed by scraping cells and

collection of bacteria, which were then diluted and plated on BCYE plates.

2.6 Golgi disruption via pharmacological inhibitors

U937 macrophages were infected with Lp01 strain as described above. At indicated time

points of infection, infected cells were washed three times with 1X PBS followed by incubation

in media containing either 1 µM nocodazole, 1 µM golgicide A or 10 µM (3 µg / ml) brefeldin A

for 3 h, followed by three washes with 1X PBS. Cells were then fixed using 4% PFA for

microscopy analysis.

17

2.7 Immunostaining and Spinning disk confocal microscopy

Non-infected and Legionella-infected macrophages were fixed with 4% PFA at indicated

time points post infection for 20 min, followed by permeabilization using 0.1% Triton X-100 in

PBS supplemented with 100 mM glycine for 20 min. Next, cells were blocked using PBS

consisting of 5% milk for 1 h. All primary antibodies were diluted in PBS with 2% milk and

incubated for 1 h. To label Legionella, 1:500 mouse monoclonal antibody or 1:3000 rabbit

polyclonal antibody was used. To label the Golgi, 1:200 GM130 antibody or 1:200 TGN46

antibody was used. To label the LCV, 1:100 KDEL antibody or 1:100 calnexin antibody or 1:50

FK2 antibody was used. To label lysosomal marker LAMP-1, 1:200 LAMP-1 antibody was used.

All secondary staining was carried out with CyTM2-, CyTM3- or CyTM5- conjugated antibodies in

PBS with 1% FBS for 1 h. To label cis-Golgi lectin, 1:500 fluorescent Helix promatia agglutin

(HPA) was added to cells and incubated for 1 h after blocking in PBS with 5% FBS. To label the

nucleus, cells were washed 2 times with double distilled water and incubated with 1:10,000

DAPI for 10 min. Next, cells were mounted using Dako Fluorescent Mounting Medium (Dako

Cytomation, CA) for microscopy analysis. Z-stack images at 0.2 µm per slice were acquired at

40X (1.4 N.A) using a WaveFX-X1 Spinning Disk Confocal Microscope (Quorum

Technologies) and all imaging parameters including exposure, laser intensity and gain were kept

constant across samples in each trial. Images were captured with MetaMorph (Molecular

Devices) and were next imported into Volocity Image Analysis (PerkinElmer) for Golgi

analyses. To quantify cis- or trans-Golgi area, threshold for GM130 or TGN46 intensity was

defined such that only fluorescent signal was included. The area covered by these fluorescent

components and total fluorescence levels of proteins were calculated using Volocity software for

50 infected cells in four independent experiments. To quantify number of Legionella per infected

18

macrophage, infected cells were stained for Legionella as described above and counted visually

using 63X oil-immersion objective using an inverted Zeiss epifluorescent microscope in 50

different cells from three independent experiments.

2.8 Transmission electron microscopy analysis

Legionella-infected macrophages on coverslips were fixed in 2% glutaraldehyde in 0.1 M

Sorenson’s phosphate buffer at pH 7.2 for 2 h. Next, cells were post-fixed in 1% osmium

tetroxide and 1.25% potassium ferrocyanide in sodium cacodylate buffer at room temperature for

45 min and stained for 30 min with 1% uranyl acetate in water. Then, dehydration and

embedding of samples into Epson resin was carried out followed by sectioning. These sections

were collected on Formvar-coated copper grids and stained with uranyl acetate and lead citrate.

Sections were then imaged using the H-7500 transmission electron microscope (Hitachi). The

number of individual Golgi stacks per cell and the number of cisternae per Golgi stack were then

quantified.

2.9 Data analysis and Statistics

Statistical analysis was carried out using Prism from GraphPad software Inc. (La Jolla,

CA). A one-way Anova or two-way ANOVA was used followed by Tukey’s test for multiple

comparisons. Data shown represent mean ± S.E.M. from three independent experiments unless

stated otherwise. All figures were constructed using Adobe Illustrator CS6 (San Jose, CA).

19

3. Results

3.1 Growth of Legionella with U937 macrophages

Legionella infects various different mammalian host cells including macrophage-like

tissue culture cells such as U937 and THP-1 cells (Newton et al., 2010). To test proper growth of

Legionella within U937 human macrophages, in our hands, an intracellular growth assay was

performed by infecting differentiated U937 macrophages with wild-type Lp01 Legionella strain

at MOI of 10 and 100, and obtaining CFUs/ mL at 2, 4, 6, 8 and 10 h post-infection. As

expected, the growth rate of Legionella over time was lower in macrophages infected at a MOI

of 10 compared to growth rate at MOI of 100 (Figure 2A). Furthermore, there was a gradual

increase in the number of viable bacteria in macrophages over time with both MOIs, where

bacterial growth was 10-fold greater with macrophages infected at a MOI of 100 compared to

MOI of 10, as expected. Next, Legionella-infected U937 macrophages were fixed at 1, 3, 6, and

10 h post-infection and stained for external and total Legionella to visualize growth and

multiplication over time. The number of Legionella per infected macrophage increased from 1–2

at 1 h to about 15–20 at 10 h post infection, suggesting bacterial growth over time within the

macrophages (Figure 2B and C). The percentage of infected macrophages is shown in Figure 2D

and was about 15% at all time points of infection investigated.

20

Figure 2: Wild-type Legionella strain can multiply intracellularly within human U937

macrophages. (A) U937 cells were infected with wild type Legionella strain at a MOI of 10 or

100. After internalization and removal of external bacteria, cells were incubated for periods of

time indicated, and the number of viable bacteria (after replication in host cells) was determined

in duplicates, by disrupting host cells using 0.2% saponin followed by plating supernatants on

BCYE agar plates. (B) Infected U937 macrophages were fixed at indicated time points for

immunofluorescence analysis to visualize multiplication of Legionella within macrophages.

External Legionella is shown in green, total Legionella in white, nucleus in blue followed by

merged images. Yellow arrowheads point to total Legionella within the macrophage at each time

point. Scale bars = 10 µm. (C) Growth of Legionella within macrophages represented by

number of Legionella per infected cell at each time point. Legionella number was analyzed using

an inverted Zeiss epifluorescent microscope at 63x oil-immersion objective. (D) The percentage

of infected U937 macrophages at indicated time points post infection. Data represent mean ±

S.E.M. from three independent experiments (N = 3, n = 50).

21

3.2 Golgi structure remains intact in Legionella-infected U937 macrophages

Legionella hijacks important ER to Golgi trafficking regulators such as Rab1 and Arf1 in

order to establish a specialized vacuole for efficient intracellular growth (Kagan et al., 2004;

Nagai et al., 2002). This in turn will disturb the balance between anterograde and retrograde

trafficking, hypothetically leading to Golgi fragmentation. In order to test this hypothesis, we

performed detailed analysis of Golgi structure in Legionella-infected macrophages using

spinning disk confocal microscopy. Specifically, U937 macrophages were infected with wild-

type Lp01 Legionella strain at MOI of 50 and fixed at different times for subsequent

immunostaining of external and total Legionella, the cis-Golgi marker GM130 and the nuclei

(Figure 3). The overall structure of the cis-Golgi remained unchanged throughout the infection

and looked similar to the Golgi structure in uninfected cells (Figure 3A). In order to validate this

visually unchanged cis-Golgi compartment between non-infected and infected macrophages, the

Volocity program was used to perform the following quantification. First, a threshold for GM130

signal intensity was defined such that only fluorescent Golgi components and not the

background, were included in the quantification. Next, cis-Golgi in each infected and non-

infected cell was manually traced with a selection tool. Then the measurements function in

Volocity was used to calculate the mean area of this specific traced region in selected cells for

both non-infected and infected macrophages. This mean output number generated by this

function in Volocity would then reflect the distribution of cis-Golgi components, as visualized by

GM130, in a particular cell. In addition to quantifying the area covered by these Golgi

components, the sum fluorescence intensity of these components was also calculated for the

comparison between macrophages with and without Legionella. Analysis of the data using the

Prism program showed no significant difference in cis-Golgi area between non-infected and

22

infected cells at any time point after infection (Figure 3B). Furthermore, there was no significant

difference in the total amount of GM130 protein levels, determined by measuring the

fluorescence intensity, between non-infected and infected macrophages (Figure 3C).

Figure 3: The cis-Golgi structure remains intact in Legionella-infected U937 macrophages.

(A) U937 macrophages infected with Legionella were fixed at indicated time points followed by

immunostaining to visualize external Legionella (green), total Legionella (red), cis-Golgi

marker, GM130 (white) and the nucleus (yellow). Scale bars = 5 µm. Yellow arrowheads point

to intact cis-Golgi structures. Quantifications of cis-Golgi area (B) and fluorescence of GM130

(C) in non-infected (Lp -) and infected (Lp +) cells at each time point. ‘Lp’ refers to Legionella

pneumophila. ‘-’ and ‘+’ refer to non-infected and infected macrophages, respectively. Data

represent mean ± S.E.M. from four independent experiments (N = 4, n = 50, One-way ANOVA

test).

23

This same procedure was followed to examine any differences in area and fluorescence

levels of trans-Golgi compartment between non-infected and infected macrophages. Infected

macrophages were fixed and immunostained for TGN46, a marker of the trans Golgi network

(TGN) (McCrossan et al., 2001). According to the microscopic images shown in Figure 4A,

there were no visual differences in the overall trans-Golgi compartment between macrophages

with and without Legionella. Furthermore, comparison of data generated by Volocity, in the

same manner as described above for cis-Golgi, suggested no significant difference in trans-Golgi

area between non-infected and infected macrophages (Figure 4A and B). In addition, the total

amount of TGN46 protein levels, as measured by total TGN46 fluorescence intensity, in infected

macrophages also remained the same as levels in non-infected macrophages (Figure 4C).

Although our fluorescent imaging results suggested that the area covered by Golgi and

the levels of different resident proteins did not differ between non-infected and infected

macrophages, these data was based on fluorescent signals emitted by immunostained Golgi

proteins which lacked high resolution details about the Golgi complex such as the number of

Golgi stacks and the number of cisternae. In order to confirm and examine these Golgi

components at an ultrastructural level, we used transmission electron microscopy (TEM). U937

macrophages were infected with Legionella and fixed at different times after infection for

subsequent TEM processing and sectioning. The structural integrity of the Golgi complex was

unchanged in macrophages infected with Legionella and was similar to the Golgi in uninfected

macrophages. For example, infected macrophages contained normally organized stacks that were

located near the nucleus, and consisted of similar number of cisternae, just like in non-infected

macrophages (Figure 5A). For detailed analysis, the number of Golgi stacks per cell was

analyzed in 50 macrophages infected with or without Legionella. Within the TEM sections of

24

uninfected or infected macrophages at different times of infection, the number of Golgi stacks

present in one macrophage was counted in a total of 50 macrophages. The number of Golgi

stacks per macrophage without and with Legionella ranged from 6-8 and 4-9, respectively

(Figure 5B). Data analysis revealed no significant difference in this Golgi stack number between

non-infected and infected macrophages. Next, these Golgi stacks were further analyzed by

counting the number of cisternae present per stack in 50 macrophages with or without

Legionella. As shown in the enlarged insets of Figure 5A, the average number of cisternae per

Golgi stack in both non-infected and infected macrophages was 5. Comparison of data suggested

that there was no significant difference in the number of cisternae per Golgi stack between

macrophages with and without Legionella (Figure 5C).

Together, these results suggested that the structure of Golgi complex is unaffected in

Legionella-infected macrophages despite the hijacking of important ER-Golgi regulators such as

Rab1.

25

Figure 4: The trans-Golgi structure remains intact in Legionella-infected U937

macrophages. (A) U937 macrophages infected with Legionella were fixed at indicated time

points followed by immunostaining to visualize external Legionella (green), total Legionella

(red), trans-Golgi marker, TGN46 (white) and the nucleus (yellow). Yellow arrowheads point to

intact trans-Golgi structure. Scale bar = 5 µm. Quantifications of trans-Golgi area (B) and

fluorescence of TGN46 (C) in non-infected (Lp -) and infected (Lp +) cells at each time point.

‘Lp’ refers to Legionella pneumophila. ‘-’ and ‘+’ refer to non-infected and infected

macrophages, respectively. Data represent mean ± S.E.M. from four independent experiments (N

= 4, n = 50, One-way ANOVA test).

26

Figure 5: TEM of U937 macrophages infected with Legionella reveals no change in the

Golgi complex at an ultrastructural level. (A) Golgi stacks in non-infected and infected U937

macrophages at different time points post-infection. Panels on right represent zoomed images of

dashed region in original image. Yellow arrowheads in zoomed images point to individual

cisternae within the Golgi stack. Scale bar = 500 nm. (B) Number of Golgi stacks per cell in non-

infected (Lp -) and infected (Lp +) macrophages at different time points of infection. (C) Number

of cisternae per Golgi stack in non-infected (Lp -) and infected cells (Lp +) macrophages at

different time points of infection. ‘Lp’ refers to Legionella pneumophila. ‘-’ and ‘+’ refer to non-

infected and infected macrophages, respectively. Data represent mean ± S.E.M. from two

independent experiments (N = 2, n = 25, One-way ANOVA test).

27

3.3 Golgi disruption via pharmacological inhibitors differentially affect

Legionella growth within macrophages

The structure of the Golgi in Legionella-infected macrophages was determined to be

intact and unchanged based on our microscopy analysis, which contradicted our hypothesis that

Golgi would be fragmented as infection progressed over time. This suggested that Legionella

possibly requires a proper Golgi structure for its survival, and therefore maintains the structural

integrity of this complex despite hijacking of important vesicle trafficking regulators and inputs.

In order to test this possibility, we perturbed the structure of Golgi complex using three different

pharmacological inhibitors including golgicide A (GCA), brefeldin A (BFA) and nocodazole

(NOC) and examined the subsequent effect on Legionella growth within U937 macrophages.

3.3.1 Golgi disruption induced by golgicide A decreases Legionella growth at late phases

of infection

The first drug we used was GCA, which is a highly specific and reversible inhibitor of

the cis-Golgi ArfGEF, GBF1 (Sáenz et al., 2009). GCA-treatment causes rapid dissociation of

COPI vesicle coat from Golgi membranes, ultimately resulting in disassembly of the Golgi

complex. In order to examine the effect of GCA on Legionella growth, we infected U937

macrophages with Legionella followed by GCA treatment for 3 h at early and late time points of

infection, (6 h and 9 h), since this is when Legionella begins to grow and rapidly increase in

number within the LCV, respectively (Faucher et al., 2011). Next, we immunostained for

external and total Legionella, cis-Golgi and the nucleus for microscopy analysis. Uninfected

macrophages treated with GCA had Golgi components that were dispersed throughout the

cytoplasm compared to a more compact Golgi seen in cells without GCA (Figure 6A). This

dispersed Golgi phenotype was confirmed by quantifying the area covered by cis-Golgi in GCA-

28

treated cells, which was significantly larger compared to area covered by cis-Golgi in untreated

cells (Figure 6B). At the earlier time point of infection, GCA-induced Golgi disruption did not

perturb the number of Legionella present in macrophages (Figure 6C) in contrast to the later time

point of infection, where Legionella number was reduced in macrophages treated with GCA

(Figure 6D, arrow). To verify whether this difference in Legionella growth between cells treated

with and without GCA at 6 h was significant, we visually quantified the number of Legionella

per infected macrophage in 50 macrophages. At 6 h post-infection, macrophages consisted of 4-6

Legionella and 3-5 Legionella in cells without and with GCA, respectively. On the other hand,

macrophages without and with GCA at 9 h post infection consisted of 9-16 and 6-10 Legionella,

respectively. This difference in Legionella number per infected macrophage between untreated

and GCA-treated macrophages at 9 h was significant (Figure 6E). Therefore, Golgi disruption

induced by GCA significantly reduced Legionella growth at late phases of infection.

29

Figure 6: Golgicide-induced Golgi disruption significantly decreases multiplication of

Legionella within U937 macrophages at late phase of infection. (A) U937 macrophages

treated with or without 1 µM golgicide A (GCA) for 3 h followed by fixation and

immunostaining to visualize the cis-Golgi marker GM130 (red) and nucleus (yellow). (B)

Quantification of cis-Golgi area in cells without (- GCA) and with golgicide A (+ GCA) to show

Golgi dispersion phenotype induced by GCA. Legionella-infected U937 macrophages treated

with or without 1 µM GCA for 3 h followed by fixing and staining at (C) 6 h and (D) 9 h post-

infection to visualize external Legionella (green), total Legionella (white), the cis-Golgi marker

GM130 (red) and nucleus (yellow). Yellow arrowheads point to dispersed Golgi whereas arrows

point to decreased Legionella growth in GCA treated macrophages. Scale bars = 5 µm. (E) Data

analysis representing number of Legionella per infected macrophage in U937 cells treated with

or without GCA at 6 h and 9 h post infection. Data represent mean ± S.E.M. from three

independent experiments (N = 3, n = 50, ***, P = 0.0001, two-way ANOVA test).

30

3.3.2 Golgi disruption induced by Brefeldin A does not impact Legionella growth within

macrophages

In order to validate whether Legionella growth would be compromised with other Golgi-

disrupting drugs, we next used brefeldin A (BFA), which inhibits the GTP-binding protein Arf1

and blocks anterograde trafficking from the ER to Golgi apparatus (Ivessa et al., 1995). As

previously described for GCA, BFA was added to Legionella-infected macrophages at early (3 h)

and late phase (6 h) of infection followed by immunostaining of Legionella and cis-Golgi, and

subsequent analysis of Legionella growth by microscopy. As expected, the cis-Golgi structure

was completely dispersed throughout the cytoplasm in BFA-treated U937 macrophages

compared to a more compact structure in untreated macrophages (Figure 7A). This dispersed

Golgi in BFA-treated macrophages covered a significantly larger area than Golgi components in

untreated macrophages (Figure 7B). At both early (Figure 7C) and late phases of infection

(Figure 7D), the growth of Legionella in BFA-treated macrophages was similar to Legionella

growth in untreated macrophages. Quantifications were carried out as previously described and

Legionella number per infected macrophage ranged from 6-9 and 5-7 in cells without and with

BFA at 6 h post infection (Figure 7E). At later stages of infection, there was an increase in the

number of Legionella per infected macrophage, ranging from 13-19, as expected in untreated

cells. Similarly, BFA-treated cells also had an increase in Legionella growth number, ranging

from 15-20. The difference in Legionella growth between untreated and BFA-treated

macrophages at both time points of infection was not significant. These results suggested that

BFA-induced Golgi disruption did not affect Legionella growth at any time point post-infection.

31

Figure 7: Brefeldin A-induced Golgi disruption does not affect multiplication of Legionella

within U937 macrophages. (A) U937 macrophages treated with or without 3 mg/ml brefeldin A

(BFA) for 3h followed by immunostaining to visualize cis-Golgi marker GM130 (red) and

nucleus (yellow). (B) Quantification of cis-Golgi area in cells without (- BFA) and with brefeldin

A (+ BFA) to represent Golgi disrupted phenotype induced by BFA. Legionella-infected U937

macrophages treated with or without 3 mg/ml BFA for 3h followed by fixing and staining at 6 h

(C) and 9 h (D) post-infection to visualize external Legionella (green), total Legionella (white),

cis-Golgi marker GM130 (red) and nucleus (yellow). Yellow arrowheads point to dispersed

Golgi in BFA treated macrophages. Scale bar = 5 µm. (E) Quantifications representing number

of Legionella per infected macrophage in U937 cells treated with or without BFA at 6 h and 9 h

post-infection. Data represent mean ± S.E.M. from three independent experiments (N = 3, n =

50, two-way ANOVA test).

32

3.3.3 Golgi disruption induced by nocodazole reduces Legionella growth at early and late

phases of infection

Due to the contradicting effects of GCA and BFA on Legionella multiplication within

macrophages, it was not completely clear whether an intact Golgi complex was inevitably

beneficial for Legionella growth. We therefore turned to another inhibitor that causes Golgi

dispersal, nocodazole (NOC), to analyze its effect on Legionella multiplication. This drug

primarily interferes with microtubule polymerization, thereby disrupting the trafficking of Golgi

elements from ER, in turn causing formation of Golgi ministacks that remain distributed

throughout the cell (Dunlop et al., 2017). This phenotype was clearly observed in macrophages

treated with NOC, where Golgi ministacks were distributed throughout the cytoplasm (Figure

8A) in contrast to a more compact Golgi in untreated macrophages (Figure 8A). The dispersal of

Golgi components in macrophages with and without NOC was quantified followed by statistical

analysis, which suggested a significant increase in the area covered by Golgi components in

NOC-treated macrophages compared to area covered by Golgi components in untreated

macrophages (Figure 8B). To examine Legionella multiplication following NOC-induced Golgi

disruption, macrophages were first infected with Legionella followed by incubation with NOC

for 3 hours at early (6 h) and late phases (9 h) of infection. Next, infected cells treated with or

without NOC were fixed for subsequent immunostaining of Legionella and cis-Golgi

compartment. As expected, Legionella-infected macrophages treated with NOC at both time

points of infection consisted of Golgi ministacks that were dispersed throughout the cell (Figure

8C and D). Interestingly, there was a lower number of Legionella in macrophages treated with

NOC at both early and late time phases of infection (Figure 8C and D). The number of

Legionella per infected macrophage was counted in a total of 50 macrophages as described

33

previously and data analysis confirmed that Legionella number per macrophage was significantly

reduced in NOC-treated macrophages at both phases of infection. At 6 h post-infection, NOC-

treated macrophages consisted of 2-5 Legionella, which was significantly lower compared to

untreated macrophages which generally contained 5-9 Legionella. Similarly, at 9 h post-

infection, the number of Legionella per macrophage in NOC-treated macrophages ranged from

9-14, which was also significantly lower than Legionella number in untreated macrophages,

where it ranged from 14-19 (Figure 8E).

34

Figure 8: Nocodazole-induced Golgi disruption significantly decreases multiplication of

Legionella within U937 macrophages at early and late time points of infection. (A) U937

macrophages treated with or without 1 µM nocodazole (NOC) for 3 h followed by

immunostaining to visualize cis-Golgi marker GM130 (red) and nucleus (yellow). (B)

Quantification of cis-Golgi area in cells without (- NOC) and with nocodazole (+ NOC) to

represent Golgi disruption induced by NOC. Legionella-infected U937 macrophages treated with

or without 1 µM NOC for 3 h followed by fixing and staining at 6 h (C) and 9 h (D) post-

infection to visualize external Legionella (green), total Legionella (white), cis-Golgi marker

GM130 (red) and nucleus (yellow). Yellow arrowheads point to dispersed Golgi whereas arrows

point to decreased Legionella growth in NOC treated macrophages. Scale bar = 5 µm. (E)

Quantifications representing number of Legionella per infected macrophage in U937 cells treated

with or without NOC at 6 h and 9 h post-infection. Scale bar = 5 µm. Data represent mean ±

S.E.M. from three independent experiments, (N = 3, n = 50, ***, P = 0.0001, ****, P < 0.0001,

two-way ANOVA test).

35

Collectively, these results suggested that Golgi disruption induced by GCA and NOC had

a negative effect on Legionella growth whereas BFA-induced Golgi disruption did not

compromise Legionella growth within U937 macrophages (Table 1).

3.4 The maturation process of LCVs in Legionella-infected macrophages is

differentially affected by different Golgi-disrupting drugs

Once Legionella is internalized by phagocytosis into a macrophage, the proper formation

and maturation of LCV is crucial for the pathogen to replicate and survive intracellularly. This

process of the LCV establishment occurs in different stages that can in turn be identified by

specific markers. For example, during early stages of infection, the LCV is decorated by

ubiquitinated proteins that regulate important processes such as intracellular replication (Dorer et

al., 2006). In addition, attachment of early secretory vesicles to the LCV takes place, which is

marked by proteins containing the ER retention signal, KDEL (Robinson and Roy, 2006) As

infection progresses, the LCV matures into an ER-derived vacuole that is highly enriched in the

ER resident integral membrane protein, calnexin (Kagan and Roy, 2002b). Our previous results

suggested that different inhibitors that induce Golgi disruption either reduced Legionella

multiplication at different infection stages or did not impact bacterial growth at all. In order to

relate this difference in observations to the LCV maturation process, we stained for several LCV

markers to examine the characteristics of this vacuole in infected macrophages treated with

GCA, BFA, or NOC.

36

3.4.1 The maturation of the LCV is delayed in NOC-treated macrophages infected with

Legionella

We first examined the accumulation of ubiquitinated proteins on the LCV in Legionella-

infected macrophages that were treated with either GCA or BFA or NOC at 6 h post-infection,

by staining with FK2, an antibody that binds both mono- and poly-ubiquitinated proteins (Ivanov

and Roy, 2009), in addition to immunostaining of total and external Legionella. As expected,

Legionella was seen to replicate extensively in untreated and BFA-treated macrophages.

Although there was growth of Legionella in macrophages treated with NOC and GCA, it was

comparatively lower than Legionella growth in untreated macrophages (Figure 9A). Moreover,

we wanted to exclusively choose an infected macrophage with moderately replicated Legionella

so that we could examine the presence or absence of FK2 on the LCV. Immunofluorescence

microscopy analysis revealed the presence of ubiquitinated proteins surrounding the LCV in only

macrophages treated with NOC (Figure 9A), but not in untreated or GCA- or BFA-treated

macrophages. In order to validate this observation, the number of LCVs that were positive for

FK2 staining was counted in 50 infected macrophages for each condition. Data analysis

confirmed that nearly all of the vacuoles containing Legionella were FK2-positive in NOC-

treated macrophages whereas only about 10% of LCVs were FK2-positive in untreated

macrophages. This difference in the percentage of FK2-positive LCVs between untreated and

NOC-treated macrophages seems significant although it needs to be confirmed with more

replicates. On the other hand, GCA- and BFA-treated macrophages consisted of about 35% and

6% of LCVs that stained positive for FK2, which did not seem to be significantly different

compared to the percentage of FK2-positive LCVs in untreated macrophages (Figure 9B). These

results were consistent with our previous data since we saw reduced Legionella growth in NOC-

37

treated macrophages at the late phase of infection, which correlates with higher percentage of

FK2-positive LCVs. Together, this experiment suggested that the maturation process of LCV is

delayed in NOC-treated macrophages However, it needs to be repeated for conclusive results.

38

Figure 9: Increased FK2-positive LCVs in Legionella-infected macrophages treated with

Nocodazole and Golgicide A. (A) Legionella-infected macrophages treated without (Con) or

with 1 µM NOC,1 µM GCA, or 3 mg/ml BFA at 6 h post-infection for 3 hours followed by

immunostaining to visualize external Legionella (red), total Legionella (green), LCV marker

FK2 (white) and nucleus (blue). Scale bar = 10 µm. (B) Quantifications representing percentage

of LCVs that were FK2-positive for each treatment. Data represent mean ± S.D. from one

independent experiment (N = 1, n = 25). Arrowhead represents FK2 positive LCV.

39

3.4.2 Normal LCV maturation in GCA-treated macrophages infected with Legionella

The ER-retention signal KDEL is usually present on the LCV during early stages of

Legionella infection and persists until late phases of infection (Kagan and Roy, 2002b). In order

to examine whether LCVs acquire this ER marker in Legionella-infected macrophages treated

with GCA, BFA or NOC, we followed the same protocol as previously described for the

immunostaining of KDEL and Legionella, after inducing Golgi disruption using the three

inhibitors at 6 h post-infection. In agreement with previous studies (Kagan and Roy, 2002b),

LCVs in untreated macrophages stained positive for KDEL (Figure 10A). Interestingly, the

LCVs in GCA-treated macrophages were KDEL-positive despite the reduction in Legionella

growth that we previously observed for this particular condition (Figure 10A). On the other hand,

LCVs in NOC- and BFA-treated macrophages did not stain positively for KDEL. To validate

these microscopy observations, we quantified the percentage of KDEL-positive LCVs for all

conditions and data analysis revealed that in untreated and GCA-treated macrophages, 42% and

about 20% of LCVs stained positive for KDEL, respectively. In contrast, none of the vacuoles in

NOC- and BFA-treated macrophages showed robust staining for KDEL (Figure 10B).

40

Figure 10: Absence of KDEL-positive LCVs in Legionella-infected macrophages treated

with Nocodazole and Brefeldin A. (A) Legionella-infected macrophages treated without (Con)

or with 1 µM NOC, 1 µM GCA, or 3 mg / ml BFA at 6 h post-infection for 3 hours followed by

immunostaining to visualize external Legionella (red), total Legionella (green), ER marker

KDEL (white) and nucleus (blue). Yellow arrowheads represent KDEL positive LCVs. Scale bar

= 10 µm. (B) Quantifications representing percentage of LCVs that were KDEL-positive for

each treatment. Data represent mean ± S.D. from one independent experiment (N =1, n = 25).

41

3.4.3 Infected macrophages treated with GCA and BFA had normal LCV maturation

We next analyzed whether the ER marker, calnexin would be present on LCVs in

infected macrophages treated without or with GCA, BFA and NOC in a similar manner as

described above. Briefly, Legionella infected macrophages were treated with Golgi disrupting

inhibitors for 3 hours at 6 h post-infection followed by fixing and immunostaining for calnexin

and Legionella. Consistent with our previous results, Legionella replicated extensively in

untreated and BFA-treated macrophages compared to moderate Legionella growth in NOC- and

GCA-treated macrophages (Figure 11A). As expected, LCVs in untreated and BFA-treated

macrophages were highly enriched in calnexin (Figure 11A). Furthermore, this observation was

consistent with our previous data where we showed normal Legionella growth in macrophages

treated with BFA at 9 h post-infection (Figure 7D). In contrast, LCVs in NOC-treated

macrophages were not positive for calnexin, which further correlated with our previous data

NOC-inhibited Legionella growth at later phase of infection (Figure 8D). Strikingly however,

although GCA-treated macrophages also had reduced Legionella growth at late phases of

infection, they still contained LCVs that were enriched in calnexin (Figure 11A). The percentage

of LCVs that were calnexin-positive in untreated and BFA-treated macrophages was about 33%

and 48%, respectively. Although the percentage of calnexin-positive LCVs present in NOC- and

GCA-treated macrophages was lower (about 11% and 30%, respectively) than the percentage of

calnexin-positive LCVs in untreated macrophages (about 33%), this difference did not seem

significant (Figure 11B). However, this experiment needs to be repeated for conclusive results.

42

Figure 11: Presence of calnexin-positive LCVs in Legionella-infected macrophages treated

with nocodazole or golgicide A or brefeldin A. (A) Legionella-infected macrophages treated

without (Con) or with 1 µM NOC, 1 µM GCA, or 3 mg / ml BFA at 6 h post-infection for 3

hours followed by immunostaining to visualize external Legionella (red), total Legionella

(green), ER marker calnexin (white) and nucleus (blue). Yellow arrowheads represent calnexin

positive LCVs. Scale bar = 10 µm. (B) Quantifications representing percentage of LCVs that

were calnexin-positive for each treatment. Data represent mean ± S.D. from one independent

experiment (N = 1, n = 25).

43

3.4.4 LCVs are devoid of lysosomal components in Legionella-infected macrophages

treated with Golgi disrupting inhibitors

An important factor that allows intracellular replication of Legionella within

macrophages is the ability to avoid fusion with the lysosomal compartment (Horwitz, 1983). In

order to examine whether LCVs acquire lysosomal components due to disrupted Golgi at late

phases of infection, we followed the same protocol described previously by treating Legionella-

infected macrophages with GCA, BFA or NOC for 3 hours at 6 h post-infection and subsequent

immunostaining of external and total Legionella as well as the lysosomal marker LAMP-1 (Cella

et al., 1996). As expected, LCVs in Legionella-infected macrophages with an intact Golgi

complex did not acquire LAMP-1 protein, which suggested proper multiplication of bacteria

within the macrophage. Similarly, LCVs in infected macrophages treated with either GCA, BFA

or NOC also did not stain positive for LAMP-1 (Figure 12A), which indicated that a disrupted

Golgi structure at late phases of infection did not affect Legionella’s ability to escape the

canonical lysosomal pathway. These observations were confirmed by data analysis which

indicated that none of the LCVs in infected macrophages that were either treated with or without

Golgi disrupting inhibitors, stained positive for LAMP-1 (Figure 12B).

Collectively, examination of LCVs with various markers suggested that reduced

Legionella growth in NOC-treated macrophages was consistent with delayed maturation of

LCVs that were composed of a higher percentage of ubiquitinated proteins and lower percentage

of ER marker proteins, in contrast to untreated macrophages infected with Legionella. Similarly,

GCA-treated infected macrophages had LCVs with a slightly higher percentage of ubiquitinated

proteins and lower percentage of ER proteins compared to control macrophages. On the other

hand, LCVs in BFA-treated macrophages were enriched with ER marker proteins and had a very

44

low percentage of ubiquitinated proteins, resembling LCVs in untreated macrophages (Table 1).

This observation was consistent with our previous results where we showed normal growth of

Legionella in BFA-treated macrophages.

Figure 12: Absence of LAMP-1 in Legionella-infected macrophages treated with

nocodazole or golgicide A or brefeldin A. (A) Legionella-infected macrophages treated without

(Con) or with 1 µM NOC, 1 µM GCA, or 3 mg / ml BFA at 6 h post-infection for 3 hours

followed by immunostaining to visualize external Legionella (red), total Legionella (green),

LAMP-1 (white) and nucleus (blue). Scale bar = 10 µm. (B) Quantifications representing

percentage of LCVs that were LAMP-1-positive for each treatment. Data represent mean ± S.D.

from one independent experiment (N = 1, n = 25).

45

Table 1: Summary of Legionella growth and LCV maturation due to Golgi-disrupting

inhibitors

3.5 Cis-Golgi lectin levels decrease over time in Legionella-infected

macrophages

Our second main objective of this study was to analyze the function of Golgi in

Legionella-infected macrophages. The primary function of the Golgi complex is to modify and

secrete proteins and lipids that arrive from the ER. Glycosylation is one of the post-translational

modifications carried out within Golgi which can be detected experimentally using the sugar

binding proteins, lectins (Yamamoto, 2014). For example, Helix promatia agglutinin (HPA) is a

cis-Golgi lectin that is highly specific for N-Acetyl-Galactosamine (GalNAc) carbohydrate

moiety (Mitchell and Schumacher, 1999) on glycosylated proteins. In order to examine the

glycosylation activity of Golgi in Legionella-infected macrophages, we fixed infected

macrophages at 1, 3, 6 and 10 h time points after infection for subsequent immunostaining of

external and total Legionella as well as fluorescently-labelled HPA lectin. Our microscopy

images visually showed a gradual increase in the number of Legionella per infected macrophage

as infection progressed, which indicated proper intracellular growth of Legionella (Figure 13A).

At 1 h after infection, the intensity of fluorescent HPA was similar to the HPA fluorescence

Recruitment of LCV markers (%)

Legionella growth (%, relative

to control)

FK2 KDEL Calnexin LAMP-1

Control 100 10 42 33 0

Nocodazole 66 (early phase) & 76 (late phase) 100 0 11 0

Golgicide 85 (early phase) & 75 (late phase) 35 20 30 0

Brefeldin 100 6 0 48 0

46

intensity in uninfected macrophages (Figure 13A, arrowheads). Intriguingly, HPA fluorescence

levels were so low at 3, 6, and 10 h after infection that they were visually undetectable (Figure

13A). We validated these observations by carrying out data analysis which confirmed that the

intensity of HPA fluorescence significantly decreased at 3, 6, and 10 h after infection, compared

to the fluorescence levels in uninfected macrophages (Figure 13B). This experiment suggested

that Legionella severely impairs the glycosylation activity within the Golgi during mid to late

stages of infection within macrophages.

47

Figure 13: HPA lectin expression levels significantly decrease in Legionella-infected U937

macrophages. (A) U937 macrophages infected with Legionella were fixed at indicated time

points followed by immunostaining to visualize external Legionella (green), total Legionella

(red), cis-Golgi lectin, Helix promatia agglutinin (HPA) (white) and the nucleus (yellow). Scale

bar = 5 µm. Yellow arrowheads indicate presence of HPA lectin fluorescence signal. (B)

Quantifications of HPA lectin fluorescence levels in non-infected (Lp -) and infected (Lp +) cells

at each time point. ‘Lp’ refers to Legionella pneumophila. ‘-’ and ‘+’ refer to non-infected and

infected macrophages, respectively. Data represent mean ± S.E.M. from three independent

experiments (n = 50, ****, P < 0.0001, One-way ANOVA test).

48

4. DISCUSSION

The main objective of this study was to examine the fate of the Golgi structure and

function in Legionella-infected human macrophages. The hallmarks for efficient survival of

Legionella within the host are its ability to escape the lysosomal pathway primarily (Derré and

Isberg, 2004) and the subsequent manipulation of ER to Golgi vesicle transport to form the LCV.

Legionella achieves this by injecting effectors that transiently modulate the activity of important

regulators of these pathways such as Rab1 and Arf1 (Kagan and Roy, 2002b; Neunuebel et al.,

2011; Tilney et al., 2001). The resulting disturbance in the balance of anterograde and retrograde

transport should hypothetically cause Golgi to collapse into the ER (Hicks and Machamer,

2005). Although previous studies have shown that Golgi associated proteins such as GM130 are

not recruited to the LCV (Arasaki et al., 2012; Hay et al., 1997), whether the structure or

function of Golgi is affected in Legionella-infected macrophages remains unknown. Considering

Legionella’s manipulation of the early secretory pathway, I hypothesized that the Golgi

apparatus would eventually be altered and fragmented in Legionella-infected macrophages.

Our immunofluorescence and electron microscopy studies suggested that the structure of

the Golgi was unaltered and Golgi stack number as well as number of cisternae per Golgi stack

was also unchanged, respectively. We have determined that disrupting Golgi structure via

nocodazole or golgicide A, but not via brefeldin A, caused a reduction in Legionella growth at

late phases of infection. Furthermore, the maturation of LCV was delayed in nocodazole-treated

macrophages but not in golgicide-treated macrophages infected with Legionella. Hence, the

Golgi integrity is not affected by Legionella, but its disruption leads to defects in LCV

biogenesis. Most interestingly, we have demonstrated that Legionella decreases the glycosylated

49

protein levels within Golgi, suggesting the function of Golgi, but not its structure, is perturbed in

Legionella-infected macrophages.

4.1 Interaction of bacterial pathogens with the host secretory pathway

Several pathogens intercept the secretory pathway within the host cell in order to

establish a specialized replication-permissive vacuole including Brucella, Chlamydia and

Salmonella. Specifically, Brucella mainly interacts with the retrograde recycling pathway

whereas Chlamydia localizes between the TGN and the plasma membrane. Salmonella is known

to associate with lysosomes and localize to the peri-Golgi region (Hilbi and Haas, 2012). Like

Legionella, Brucella replicates in an ER-derived compartment known as the Brucella-containing

vacuole (BCV) via the recruitment of Rab2 by an effector protein, RicA (de Barsy et al., 2011).

BCVs closely interact with the ER exit sites and eventually associate with the ER without the

need for Arf1 and COPI-dependent retrograde Golgi-ER transport, unlike Legionella. Instead,

BCV biogenesis depends on the small GTPase Sar1 and formation of COPII-dependent transport

vesicles (Celli et al., 2005). This highlights the importance of Rab1 and Arf1 for intracellular

pathogens and suggests that Golgi might be involved during Legionella infection, as these host

regulators are closely associated with Golgi membranes.

Chlamydia utilizes T3SS to translocate proteins that promote development and

pathogenesis within the host cells. For example, the protein called IncD associates with the

ceramide transfer protein, CERT, and hence interferes with the non-vesicular ER-TGN lipid

transport machinery in order to obtain the sphingomyelin precursor ceramide (Derre et al., 2011;

Elwell et al., 2011). Interestingly, this pathogen causes Golgi fragmentation via the cleavage of

the integral Golgi matrix protein golgin-84 by chlamydial protease-like activity factor (CPAF)

(Christian et al., 2011), in order to promote its intracellular growth and maturation (Heuer et al.,

50

2009). Like Legionella, Chlamydia inclusions are also decorated with Rab1, which is important

for ER to Golgi vesicle trafficking. Since Chlamydia directly causes Golgi fragmentation to

acquire lipids, it will be interesting to find out if any of the Legionella effectors also directly

target the Golgi.

Salmonella establishes its SCV (Salmonella-containing vacuole) in the vicinity of the

Golgi apparatus, specifically the exocytic post-Golgi vesicles (Kuhle et al., 2006). During late

stages of infection, Salmonella-induced filaments (SIFs) emerge from the SCVs that are

localized at the peri-Golgi region, and interact with endocytic and secretory pathway (Schroeder

et al., 2011). For example, secretory carrier membrane protein 3, that usually cycles between the

TGN and recycling compartments colocalizes with SIFs (Mota et al., 2009). Although Golgi

components were not visualized near Legionella, the presence of an intact Golgi structure seems

crucial for possibly acquiring nutrients such as sugars.

4.2 How is Golgi integrity preserved in Legionella-infected macrophages?

The presence of an intact Golgi structure in Legionella-infected macrophages despite the

disturbance in the early secretory trafficking induced by the bacteria, was in contrast to our

hypothesis that Golgi would fragment due to resulting unbalanced anterograde and retrograde

trafficking. Through immunofluorescence microscopy, we have measured the area taken up by

cis-and trans-Golgi components, visualized by GM130 and TGN46, respectively in non-infected

and Legionella-infected U937 macrophages. Data analysis suggested that there was no

significant difference in area covered by either GM130 or TGN46 between non-infected and

infected macrophages, indicating the existence of an intact Golgi structure in infected

macrophages. This quantification method has been used in several recent studies done to

examine structural and morphological changes in Golgi caused either by mitosis or

51

pharmacological inhibitors (Farber-Katz et al., 2014; Fourriere et al., 2016; Wortzel et al., 2017).

Hence, it seems to be a valid and accurate method to analyze possible structural changes in Golgi

in Legionella-infected macrophages. In addition, ultrastructural analysis of Golgi via TEM also

revealed no change in either the Golgi stack number or the number of cisternae per stack,

between non-infected and Legionella-infected macrophages. It was very intriguing that

Legionella can keep this highly dynamic organelle together within the infected macrophages.

Knowing that this pathogen can not only efficiently manipulate important host regulators such as

Rab1 temporally, but also can regulate protein function and signalling via exploitation of the

ubiquitination process, phosphorylation or ribosylation (So et al., 2015), it is possible that

Legionella effectors could maintain the structure of Golgi during infection despite the deviation

of early secretory trafficking. The presence of an intact Golgi structure even after disturbed ER-

Golgi trafficking in infected macrophages suggests that there probably exists enough Rab1 and

Arf1 protein levels in order to preserve the early secretory pathway and ultimately the structure

of Golgi. On the other hand, it is possible that there exist certain Legionella effectors that target

Golgi structural proteins in order to prevent the Golgi from fragmenting during infection (Figure

14).

Furthermore, the intact structure of Golgi in Legionella-infected macrophages suggests

that the overall balance between anterograde from the ER to Golgi to post-Golgi secretion and

retrograde trafficking back to the ER is maintained. Rab1 and Arf1, which regulate anterograde

trafficking from ER to Golgi and retrograde trafficking from Golgi to ER, respectively, are

recruited to the LCV membrane and hence the trafficking in both directions should

hypothetically be slowed down or suppressed. On the other hand, Legionella can inhibit

retrograde trafficking from the endosomal compartments to the TGN (Finsel et al., 2013), in

52

order to prevent its fusion with the endosome and subsequently with the lysosomes, that would

otherwise lead to the degradation of LCV within the lysosomal compartments. To avoid this

pathway, Legionella utilizes the effector RidL, which specifically binds to Vps29, a subunit of

the heterotrimeric retrograde cargo recognition (retromer) complex that is crucial for cargo

sorting and function of the retrograde pathway. This prevents the association of Vps29 with the

other key proteins such as cargo recognition receptors (Vps10 and CIMPR) and sorting nexins

(SNXs), in turn blocking the formation of a cargo complex (Finsel et al., 2013). Based on this

information, endosomal compartments are not transported to the TGN in Legionella-infected

macrophages. Hence, the only possible way that the Golgi complex can stay intact is if secretion

from the TGN is also blocked.

There are several Rab GTPases that regulate trafficking from the TGN to the plasma

membrane including Rab 8, 10 and 14 (Hutagalung and Novick, 2011). Previous studies have

shown the presence of Rab8 and Rab 14 around the LCV (Urwyler et al., 2009), indicating that

Legionella intercepts this specific transport route as well. Rab8 is mainly involved in the

regulation of late secretory transport in mammalian cells, specifically the export of cargo from

the TGN to the plasma membrane through recycling endosomes (Henry and Sheff, 2008). In

Legionella-infected macrophages, Rab8 might be important for modulation of PtdIns(4)P levels

on LCVs (Ragaz et al., 2008). Similarly, Rab14 has also been shown to be important for lipid

manipulation on the LCV although it is not important for Legionella intracellular replication

(Hoffmann et al., 2014). In contrast, Rab14 is crucial for intracellular growth of Salmonella and

Chlamydia (Capmany and Damiani, 2010). Rab10, which is important for Golgi-endosome

trafficking, has a role in promoting efficient Legionella replication (Hoffmann et al., 2014). Due

to the manipulation of these Rab proteins that regulate trafficking from the TGN by Legionella,

53

this transport route might also be blocked or interrupted in infected macrophages. Hence,

Legionella efficiently takes control of multiple Rabs involved in both anterograde and retrograde

trafficking so that the overall balance between these vesicular trafficking pathways is maintained

(Figure 14), which might in turn prevent the fragmentation of Golgi complex.

Figure 14: Model explaining how Legionella could potentially preserve the Golgi complex

during infection. In an uninfected macrophage, anterograde trafficking from endoplasmic

reticulum (ER) to Golgi to plasma membrane (PM) is balanced by retrograde trafficking in the

reverse direction of cargo towards the ER. In an infected macrophage, an intact Golgi despite

redirection of Rab1 and Arf1 to the LCV can be explained by three hypothetical mechanisms. (i)

There could still exist low levels of Rab1 and Arf1 that could maintain the balance in trafficking

between both directions. (ii) Early and late secretory trafficking in both directions could be

impaired or suppressed significantly. (iii) Potential Legionella effectors (black hexagon with a

question mark) could affect Golgi structural proteins (purple diamonds) that in turn prevent

Golgi fragmentation.

54

4.3 Why is Golgi integrity preserved by Legionella?

The proper organization and juxta-nuclear positioning of Golgi complex is not very

crucial for its function. For example, the formation of ministacks caused by the microtubule

depolymerization agents like nocodazole, are capable of carrying out glycosylation as well as

transporting constitutive secretory cargo (Cole et al., 1996b; Rogalski et al., 1984). Hence,

Legionella would still possibly benefit from a functional Golgi even if this organelle was

dissociated due to disrupted secretory trafficking in infected macrophages. Legionella perhaps

keeps Golgi from disintegrating in order to prevent possible activation of stress response

pathways that would eventually trigger intracellular defences and lead to apoptosis. Several

Golgi stress response pathways have been identified by previous studies including the TFE3

pathway and the HSP47 pathway, which are both activated when glycosylation in the Golgi is

attenuated or inhibited (Miyata et al., 2013; Oku et al., 2011; Taniguchi et al., 2015).

Additionally, the CREB3-ARF4 pathway of Golgi stress response has recently been identified by

Reiling et al. (2013), which is activated when the function of ARF proteins is inhibited.

Specifically, proteolytic activation of CREB3 transcription factor releases it from the ER so that

it can translocate into the nucleus in order to upregulate ARF4 transcription, which in turn leads

to Golgi stress-induced apoptosis and disruption of the Golgi (Reiling et al., 2013). Hence it is

possible that Legionella can prevent the activation of this pathway by possibly blocking the

proteolytic activity of CREB3 through its effectors or inhibiting the transcription of ARF4,

ultimately to prevent cell death.

In summary, the structural organization of the Golgi complex is unchanged in Legionella

infected macrophages despite the disturbance in the early (ER to Golgi) secretory trafficking

pathway. The importance of the existence of an intact Golgi in infected macrophages is possibly

55

to prevent the intracellular defense mechanisms resulting in the activation of stress response

pathways that would otherwise be turned on by Golgi fragmentation, leading to cell death, which

in turn would be detrimental for intracellular growth of Legionella.

4.4 The effects of different Golgi-disrupting drugs on Legionella growth

Our thorough examination of Golgi structure in Legionella-infected macrophages

suggested that it is intact despite hijacking of important regulators of the early secretory

pathway. We therefore wanted to test if an intact Golgi is vital for bacterial growth, by disrupting

the Golgi complex and investigating whether this impacts the multiplication of Legionella within

the macrophages. The first inhibitor we used to disintegrate Golgi was golgicide A (GCA),

which specifically targets GBF1, an Arf1 GEF (Sáenz et al., 2009). We determined that GCA

significantly reduced Legionella growth at only late (9 h) but not early (6 h) phases of infection.

On Golgi membranes, Arfs recruit many coat and cargo proteins including COPI, that mediate

vesicular biogenesis as well as anterograde and retrograde trafficking (D’Souza-Schorey and

Chavrier, 2006; Donaldson et al., 2005). Specifically, Arf1 localizes to all Golgi compartments

(Jackson and Casanova, 2000) and its activation is spatially and temporally regulated by certain

GEFs. For example, GBF1 localizes to cis-Golgi where it plays a role in the assembly and

maintenance of the Golgi complex (Manolea et al., 2008; Sáenz et al., 2009). GCA is highly

specific for inhibiting GBF1, which causes rapid cytoplasmic redistribution of COPI, leading to

dispersion of cis- and medial-Golgi (Sáenz et al., 2009). Importantly, GCA only causes the

dispersal of COPI but not the TGN-associated coat protein complexes including AP-1 (clathrin

adaptor protein 1) and GGA3 (Golgi-localized-γ-ear-containing Arf binding proteins) (Zhao et

al., 2006). The second inhibitor we used was brefeldin A (BFA), which disrupts Golgi by

inhibiting anterograde/retrograde transport between the ER and the Golgi and causes tubulation

56

of recycling endosomes that mix with the TGN (Klausner et al., 1992; Lippincott-Schwartz et al.,

1989). In contrast to GCA, BFA targets multiple Golgi-associated GEFs including cis-Golgi

GBF1 as well as the GEFs localized at the TGN, namely BIG1 and BIG2. Our results showed

that Legionella multiplication was not affected during early stages of infection in cells treated

with either BFA or GCA. Since these inhibitors were added at 3 h post-infection, it is possible

that Legionella has already acquired the necessary host proteins such as Rab1 and Arf1 by this

time in order to establish the LCV for intracellular replication (Figure 15B). These regulators are

recruited to the LCV within 30 min after infection, and the resident ER proteins such as calnexin

are associated with the LCV within 1-2 h (Kagan and Roy, 2002a). Although Legionella

replication initiates around 3 h post-infection (Kagan et al., 2004), the acquisition of important

host regulators takes place in early stages of infection, which is probably why there was no effect

on intracellular multiplication of Legionella within macrophages treated with either BFA or

GCA at 3 h after the infection. A study done by Kagan et al. (2004) showed that Legionella

intracellular growth was inhibited when macrophages were pretreated with BFA for 45 min

before infection as well as when BFA was added at the time of infection. On the other hand,

when BFA was added to macrophages after 30 min post-infection, Legionella growth was not

affected (Kagan et al., 2004). From our work, it was surprising to notice that the replication was

significantly reduced during late phases of infection in macrophages treated with GCA, but not

BFA. The range of Legionella number within infected macrophages treated with and without

GCA was 6-10 and 9-16, respectively. The main difference between the mechanism of action of

BFA and GCA is that Golgi-associated Arfs are dissociated from all compartments in the

presence of BFA whereas only cis- and medial Arfs are dissociated from the membranes as a

result of GCA treatment. Since the Legionella effector RalF mimics GBF1 to recruit Arf1 to the

57

LCV, it is possible that GCA could inhibit RalF in addition to inhibiting GBF1, dissociating Arf1

from the LCV membrane and causing a reduction of Legionella growth during late stages of

infection. Consistent with this explanation, Arf1 was shown to be essential for fusion of ER-

derived vesicles with the LCV and was not crucial for the interaction of early secretory vesicles

exiting the ER and the LCV (Robinson and Roy, 2006). Hence, during the late stages of

infection, GCA could target the RalF effector, thereby interfering with activation of Arf1 on the

LCV. This could prevent proper fusion of ER-derived vesicles with the LCV, preventing access

to necessary proteins and lipids required for intracellular multiplication of Legionella (Figure

15C). However, this explanation does not correlate with unaffected Legionella number in

macrophages treated with BFA during late stages of infection. Since BFA targets the GEFs,

BIG1 and BIG2 in addition to GBF1, it is possible that higher BFA concentrations might result

in inhibition of RalF and the subsequent reduction in Legionella growth.

The third inhibitor used to induce Golgi disruption was nocodazole (NOC) which

primarily depolymerizes microtubules, leading to a disturbance in vesicle trafficking that

ultimately results in the formation of Golgi ministacks that remain dispersed throughout the cell

(Dunlop et al., 2017). There was a significant reduction in Legionella number in macrophages

treated with NOC, during both early and late stages of infection (Figure 15A). This suggests the

importance of intact microtubules for proper intracellular Legionella replication. A study done

by Rothmeier et al (2013) showed that a Legionella effector, LegG1 is involved in activating the

small GTPase Ran in order to promote microtubule polymerization and LCV motility. This

enabled the LCV to efficiently localize to its interacting compartments such as the ER.

Accordingly, Legionella growth rate was reduced in NOC-treated RAW264.7 macrophages,

indicating that LegG1-mediated microtubule polymerization may be required for the intracellular

58

replication of Legionella. Our results suggest microtubules might play a significant role during

early and late stages of Legionella infection within macrophages. During early stages,

microtubules might aid in localizing the LCV towards the ER whereas at later stages of infection,

they might promote processes that require vesicle trafficking away from the LCV for interaction

with the host vesicles. NOC-treated macrophages form dispersed Golgi ministacks remain

functional (Dukhovny et al., 2008), which is not the case of the fully dispersed Golgi found in

BFA- or GCA-treated macrophages (Cole et al., 1996a). In NOC-treated cells, cargo proteins and

the recycling Golgi proteins are sorted and concentrated in ER exit sites and move through the

ministacks to reach the plasma membrane in the absence of microtubules (Dukhovny et al.,

2008). On the contrary, BFA causes an inhibition of secretion and results in the resorption of

Golgi complex into the ER (Langhans et al., 2007). Based on this information, although secretion

might probably be occurring upon nocodazole treatment in Legionella-infected macrophages, it

can be hindered due to the lack of important components such as Arf1. Another possibility is that

the dispersed mini-stacks consists of potentially a larger surface area compared to an intact Golgi

complex which could potentially compete with the LCV as it would provide more docking sites

for the incoming ER-derived vesicles than the LCV. This would cause a delay in LCV formation

and hence might result in decreased Legionella multiplication in NOC-treated macrophages.

Although these explanations emphasize the role of microtubules more than Golgi in Legionella

multiplication, they do highlight the importance of these cytoskeletal components during late

stages of infection which has not been investigated previously.

Together, these results suggest that an intact Golgi structure and microtubules might be

necessary for proper intracellular Legionella replication within U937 macrophages during late

stages of infection. Although Legionella multiplication is not completely inhibited due to Golgi

59

disruption, the reduction in Legionella number is significant and hence suggests an important

role of Golgi structural components for efficient intracellular growth of Legionella.

Figure 15: The effect of different Golgi disruption inhibitors on Legionella multiplication in

infected macrophages. (A) The significantly reduced Legionella growth in nocodazole-treated

macrophages could be a result of the absence of functional microtubules necessary for ER-

derived vesicle trafficking towards the LCV. (B) Due to the inhibition in ER to Golgi trafficking

caused by brefeldin A, most of the ER-derived vesicles might be recruited to the LCV,

unaffecting Legionella growth. (C) During late stages of infection, in addition to inhibiting Arf1

GEF, GBF1, golgicide A might also be inhibiting the function of Legionella effector RalF, that

usually recruits Arf1 regulator to the LCV, eventually interfering with optimal Legionella

multiplication.

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4.5 The effects of different Golgi-disrupting drugs on LCV maturation

In order to test whether differences in Legionella reduction after Golgi disruption by

different inhibitors would correlate with any changes in LCV maturation process, we analyzed

the presence or absence of different LCV markers in Legionella-infected macrophages at 9 h

post-infection via immunofluorescence. Our results indicated that LCVs in NOC-treated

macrophages consisted of a higher percentage of ubiquitinated proteins and lower percentage of

ER marker proteins, in contrast to untreated macrophages infected with Legionella. It is essential

for Legionella to interact with the host ubiquitin-proteasome system in order to achieve optimal

intracellular replication (Qiu and Luo, 2017). Effectors translocated through the T4SS hijack the

host ubiquitination pathway to efficiently enrich the LCV with ubiquitinated proteins (Qiu and

Luo, 2017). A study done by Dorer et al (2006) determined that the ubiquitinated proteins

accumulate on the LCV membrane shortly after its formation and persist until the replication

stage of Legionella growth. For example, Legionella effector AnkB consists of an F-box domain

that mediates its direct interaction with the host SCF1 E3 ubiquitin ligase complex acquired by

the LCV. This leads to AnkB-mediated enrichment of the LCV with lysine48-linked

polyubiquitinated proteins (Price et al., 2009), which are then subjected to host-mediated

proteasomal degradation into free amino acids. This becomes a rich source of nutrients for the

intracellular replication of Legionella (Price et al., 2011). The enrichment of K48-linked

polyubiquitinated proteins on the LCVs in NOC-treated macrophages during late stages of

Legionella infection, detected by the FK2 antibody, suggests that the degradation of these

ubiquitinated proteins is probably inhibited, thereby hindering the generation of amino acids

required for proper intracellular Legionella replication. Alternatively, it could be a result of

increased ubiquitination present on the LCV. The degradation of the ubiquitinated proteins in

61

Legionella infected macrophages might be catalyzed by the host factor Cdc48/p97, an ATPase

which has a role in recognizing and extracting ubiquitinated proteins from membranes for their

subsequent delivery to the proteasome (Jarosch et al., 2002). The depletion of Cdc48/p97

impaired intracellular replication in permissive host cells, which correlated with an accumulation

of ubiquitinated proteins on the LCV (Dorer et al., 2006). This suggests an important role of

Cdc48/p97 in regulating turnover of ubiquitinated substrates on the LCV membrane during the

replicative phase of Legionella intracellular growth (Dorer et al., 2006). According to our results,

it is possible that microtubules might potentially play a role in the recruitment of this ATPase

towards the LCV for subsequent degradation of ubiquitinated proteins generated by the AnkB

Legionella effector, for proper intracellular replication of Legionella. A link between Cdc48 and

microtubules has been shown in a different context where this ATPase along with its interacting

proteins regulates spindle disassembly by changing microtubule dynamics and bundling during

mitosis (Cao et al., 2003). Absence of the ER retention signal, KDEL in addition to low levels of

the ER marker, calnexin on the LCVs of NOC-treated macrophages further suggests that LCV

maturation is delayed as a result of microtubule depolymerization. The absence of these

important cytoskeletal components that are crucial for vesicle trafficking poses a challenge for

Legionella to acquire the necessary proteins and lipids needed from the ER for optimal

intracellular growth within the macrophages. Since the drugs that induced Golgi disruption were

added to infected macrophages at 6 h post-infection, Legionella’s ability to avoid the lysosomal

pathway was not affected as this typically occurs during very early stages of infection in order to

survive intracellularly. Hence, the absence of LAMP-1 on LCVs in NOC-, GCA- and BFA-

treated macrophages was an expected result.

62

The presence of low and high amounts of ubiquitinated proteins and ER marker proteins,

respectively, on LCVs in GCA-treated macrophages suggests that Golgi disruption does not

affect the maturation process of the LCV. However, although the number of KDEL-positive

LCVs in GCA-treated macrophages was about 50% lower than the number of KDEL-positive

LCVs in untreated macrophages, the difference did not seem significant. On the other hand, the

percentage of LCVs that were calnexin-positive was similar in untreated and GCA-treated

macrophages, suggesting that Legionella can still efficiently acquire ER-derived proteins upon

Golgi disruption. However, these observations are only based on a single trial and hence more

replicates of this experiment are necessary for further validation and confirmation. If this

observation of unchanged LCV maturation in GCA-treated macrophages is true, it does not

correlate with our previous result where we showed decreased Legionella number due to GCA-

induced Golgi disruption at late stages of infection. But it is possible that different pathways are

involved in LCV biogenesis and Legionella replication, which could explain normal LCV

maturation but decreased Legionella growth in GCA-treated macrophages. In macrophages

treated with BFA, LCV maturation was not perturbed as the vacuoles contained low amounts of

ubiquitinated proteins and high amounts of calnexin, resembling the pattern on vacuoles in

untreated macrophages. This observation supports our previous results that showed normal

Legionella replication at late stages of infection in BFA-treated macrophages.

Collectively, our results suggest that the existence of an intact Golgi structure might not

be crucial for proper LCV biogenesis in Legionella-infected macrophages. However,

microtubules might play a primary role in the formation of the LCV.

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4.6 Why might Legionella co-opt sugars from the Golgi?

Glycosylation is vital for many biological processes such as cell attachment to the

extracellular matrix, cell recognition as well as regulation of protein activity in the cell. The

majority of glycoproteins are either secreted from the cell or localized to the cell surface with

their glycans serving as the molecular frontier of the cell (Stanley, 2011). There are several

factors that regulate oligosaccharide biosynthesis and lead to altered cell surface glycan display.

For example, endogenous regulation coupled with external signaling events can result in

differential expression of cell surface oligosaccharides. In addition, external inputs that include

exogenously delivered metabolites can also change cell surface glycan composition. Importantly,

pathogens also can utilize components of the host sugar metabolic pathway in order to survive

and replicate within the host cell, including streptococci, Salmonella enterica and Brucella

abortus.

Salmonella and Brucella hijack glucose or glucose phosphate from the host cells in order

to feed their energy-generating pathways such as glycolysis and the TCA cycle (Passalacqua et

al., 2016). Through bacterial metabolic deletion mutant studies, it has been previously shown

that Salmonella Typhimurium is highly dependent upon glucose during infection in mice

(Bowden et al., 2009; Tchawa Yimga et al., 2006). Specifically, the lack of phosphofructokinase,

an important enzyme in the glycolysis pathway, significantly reduced the replication of this

bacteria with macrophages (Bowden et al., 2009). Furthermore, mutations in glucose uptake

systems and the glucose catabolism component, glucokinase, impaired bacterial growth during

infection. The subsequent studies have determined that within the TCA cycle, the conversion of

fumarate to malate as well as the conversion of malate to oxaloacetate and pyruvate were the

most important steps for full virulence of S. Typhimurium (Mercado-Lubo et al., 2009). This

64

pathogen might also hijack succinate, ornithine or arginine from host cells for malate generation.

On the other hand, Brucella abortus caused an increased expression of a gene for PPAR

(peroxisome proliferator-activated receptors)-gamma regulator in host cells that eventually leads

to elevated intracellular glucose concentrations important for the colonization of this bacteria

(Xavier et al., 2013). Hence, it is possible that Legionella also acquires certain sugar molecules

from the macrophages in order to feed its metabolic pathways to generate energy necessary for

its intracellular replication and survival.

4.7 What causes the decrease in glycosylated protein levels of Golgi during

Legionella infection?

In order to determine whether Legionella affects Golgi function, we stained U937

macrophages infected with Legionella for the cis-Golgi lectin, HPA. We used fluorescent

imaging to quantify the levels of fluorescent HPA in cells at different time points after the

infection. Our results and data analysis suggested the level of glycosylated proteins, represented

by the total HPA fluorescence, was significantly reduced in infected macrophages at 3, 6, and 10

h post-infection, compared to HPA fluorescence levels in non-infected macrophages. These

observations are novel and suggest an important role of sugars for Legionella growth within

macrophages. Interestingly, a recent study has determined that during the late post-exponential

growth phase of infection (10-12 h post-infection), glucose serves as a carbon substrate for the

biosynthesis of poly-3-hydroxybutyrate (PHB), an important carbon source for transmissive

Legionella (Gillmaier et al., 2016). Furthermore, exogenous glucose is used in the bacterial

Entner-Doudoroff (ED) pathway in order to generate acetyl-CoA for PHB biosynthesis (Eylert et

al., 2010). Additionally, glucose uptake was highly increased during late phases of Legionella

growth (Harada et al., 2010). Since the level of glycosylated proteins in cis-Golgi significantly

65

decreased during mid to late stages of infection, it is possible that the sugars on these proteins are

an important source for Legionella to feed into this metabolic pathway to generate PHB. On the

other hand, Legionella also contains some glycosyltransferases (Lgts) that specifically use UDP-

glucose as a donor substrate to modulate the eukaryotic Elongation Factor 1A (eEF1A) that

ultimately inhibits host protein synthesis (Belyi et al., 2006). Lgt1 and Lgt2 specifically are

produced in the post-exponential phase of Legionella growth, which might function as lethal

toxins in order to aid the bacteria’s ability to kill and escape from the host cell (Belyi et al.,

2011). So, it is possible that these glycosyltransferases could also potentially acquire the

necessary UDP-glucose substrates from Golgi during late stages of infection, although the

inferred decrease in sugar levels might not be significantly high enough to impair glycosylation.

Since the HPA lectin specifically detects sugar moieties on glycosylated proteins in cis-

Golgi, its decreased fluorescence levels in Legionella infected macrophages could possibly

suggest that cis-Golgi glycosylation enzymes responsible for adding the specific sugar molecules

on target proteins, are either down-regulated or absent. This can be a consequence of the

redirected contents in the ER vesicles to the LCV by Legionella, which then could abolish either

the recycling of Golgi glycosylation enzymes from the ER to the Golgi compartment (Stanley,

2011) or the transport of newly synthesized glycosylation enzyme components towards the Golgi

(Hassinen and Kellokumpu, 2014). This could possibly explain a potential absence of

glycosylation enzymes and their subsequent catalytic activity, which in turn could correlate with

decreased HPA fluorescence observed in infected macrophages. Alternatively, other important

factors necessary for normal glycosylation within Golgi are the availability of nucleotide sugars

as well as functional nucleotide sugar transporters. Hence, other explanations for decreased

glycosylation activity in the cis-Golgi in Legionella infected macrophages include possible

66

inhibition or modulation of the function of these sugar transporters, and direct recruitment of

nucleotide sugars to the LCV through putative effector proteins (Figure 16). Experimental

approaches and techniques to test these possibilities are described in detail in the following

section.

67

Figure 16: Model showing potential mechanisms to explain how Legionella impairs Golgi

function. In an uninfected macrophage (left), the newly synthesized Golgi glycosyltransferases

in the endoplasmic reticulum (ER) arrive at the Golgi membrane on which they are inserted.

They catalyze glycosylation reactions by adding nucleotide sugars transported by the nucleotide

sugar transporter, to the target protein to generate a glycosylated protein. In a Legionella-infected

macrophage (right), the reduced levels of glycosylated substrates could result from three

potential scenarios: (a) Glycosyltransferases synthesized in the ER could be re-directed to the

LCV or (b) Legionella could translocate potential effectors that can directly target and hijack the

nucleotide sugars in the cytosol or (c) a specific putative Legionella effector can inhibit the

function of sugar nucleotide transporters.

68

4.8 Summary and future directions

The main objective of this study was to conduct a detailed analysis of the Golgi structure

and function in Legionella-infected macrophages. We have showed that although Legionella

does not perturb the Golgi structure during infection, the existence of an intact Golgi structure

seems necessary for Legionella intracellular replication during late stages of infection. On the

other hand, the process of LCV biogenesis in infected macrophages was dependent primarily on

microtubules. Most interestingly, we have discovered that Legionella significantly impairs the

function of Golgi during mid to late stages of infection, which is a novel finding.

In order to identify the putative effectors involved in decreasing glycosylated proteins in

Golgi, macrophages can be infected with different Legionella strains containing deletions of

specific sets of genes. There are more than 300 Legionella effectors with the vast majority of

them lacking a specific phenotype when deleted individually. Hence, O’Connor et al (2011) has

constructed multiple single cluster deletion mutants as well as combinations of cluster deletion

mutants in an attempt to identify which group of effectors are important for intracellular growth

of Legionella in various different host organisms (O’Connor et al., 2011). These deletion mutants

can be obtained for infection of macrophages to identify which gene cluster(s) will cause

changes in glycosylated protein levels. Once this correlation between deletion mutants and

glycosylation levels is made, a set of putative effectors can then be further identified.

To understand how these putative effectors cause changes in glycosylation, a genomic

and bioinformatic analysis could be performed which will help reveal any presence of specific

protein domains that relate to functions of specific eukaryotic proteins such as the nucleotide

sugar transporters or glycosyltransferases. For example, if the putative effectors are potential

glycosyltransferases, then a ‘DXD’ motif is most likely to be present since it is conserved in

69

many prokaryotic and eukaryotic glucosyltransferases (Belyi et al., 2006). On the other hand, if

the putative effectors are potential nucleotide sugar transporters, then the structural features such

as the protein length (300-350 amino acids) / molecular weight, high number of hydrophobic

residues, presence of 6-10 transmembrane α-helical domains, are more likely to be present

(Baldwin et al., 2001; Descoteaux et al., 1995; Norambuena et al., 2002). Although these are

some general structural features for sugar transporters, there also exists certain domains within

transporters that are highly specific for a particular type of sugar. For example, if the identified

Legionella effector is a transporter of UDP-galactose, then it would have the highly conserved

‘GL’ sequence in one of the transmembrane domains (Handford et al., 2006).

Other experimental approaches to confirm this glycosylation defect of Golgi in

Legionella-infected macrophages include analyzing the trans-Golgi lectin expression levels

which will clarify whether there is an inhibition in post-glycosylation modifications in this

compartment. Since Golgi function involves secretion of modified proteins to the plasma

membrane, lysosomal compartments as well as secretory vesicles, the presence of post-

translational modifications in certain proteins localized to each of these compartments can be

tested via immunoprecipitation and Western blotting in infected macrophages. Flow cytometry

can also be used to probe the surface glycocalyx in a quantitative manner. Since the sugars

recognized by HPA lectin begin to disappear from the cis-Golgi during mid to late stages of

infection, the localization of this lectin should be closely examined at different infection stages

with approximately 1 h intervals between time points.

In summary, we have presented a novel finding that links Legionella infection to a defect

in Golgi function in human macrophages. This sheds light on the importance of Golgi during

Legionella infections that has not been examined in detail to date. Hence, further studies in this

70

area will help us gain better insight into pathways involved in altered Golgi function during

Legionella infections. In addition, it will enhance our understanding of mechanisms utilized by

Legionella effectors to manipulate important host cell regulators.

71

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