SENSING of HOST CELL CONTACT by the PSEUDOMONAS AERUGINOSA TYPE III SECRETION SYSTEM by ERIN ARMENTROUT Submitted in Partial

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SENSING OF HOST CELL CONTACT BY THE PSEUDOMONAS AERUGINOSA TYPE III SECRETION SYSTEM

ERIN ARMENTROUT

Submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy

Department of Molecular Biology and Microbiology

CASE WESTERN RESERVE UNIVERSITY

August 2017

Case Western Reserve University School of Graduate studies

We hereby approve the dissertation of

Erin Armentrout

Candidate for the degree of Doctor of Philosophy

Committee Chair

Piet de Boer

Committee Member

Arne Rietsch

Committee Member

Liem Nguyen

Committee Member

Pieter de Haseth

Date of Defense

June 30th, 2017

*We also certify that written approval has been obtained for any propriety material contain therein

1 Table of Contents

List of Tables 4

List of Figures 5

Abstract 7

Chapter 1: Pseudomonas aeruginosa, a human pathogen, and its 9 virulence factor, the Type III Secretion System Effectors 12 T3SS Structure 14 Powering the T3SS and Control of Secretion Rate 16 Regulation of T3SS Gene Transcription 17 Regulation of Secretion 18 Conclusion 22

Chapter 2: Sensing of Host Cell Contact by the Type III Secretion 24 System Introduction 25 Translocator Interactions 26 Models of initiation of effector secretion 28 Materials and Methods 32 Bacterial strains, cells, growth conditions 32 Plasmid and strain construction 32 Translocation assay 33 Crosslinking 34 Hemolysis 34 Red blood cell membrane isolation 35 Results and Discussion 36 Confirming interactions 36 Assigning functions 39 Conclusion 44

Chapter 3: Cellular Contribution to Host Cell Contact Signaling 53 Introduction 54 Targeting cellular components 54 Lipid rafts and endocytosis 55 Membrane curvature 58 Materials and Methods 59 Bacterial strains, cells, growth conditions 59 Plasmid and strain construction 60 siRNA knockdown 60 Translocation assay 61 Membrane damage repair assay 62 Microwell assay 63

2 Results and Discussion 64 Determining cellular targets of ExoS 64 Blocking endocytic pathways 68 Effector secretion trigger mechanism 72 Conclusion 74

Chapter 4: Conclusion and Future Works 90 Bacterial protein interactions 91 Cellular targets 93 Cellular processes: endocytosis and beyond 96 Membrane curvature 100

References 105

List of Tables

3-1: Cellular targets of ExoS and ExoT 76

List of Figures

1-1: The Type III Secretion System 23

2-1: Pseudomonas/Yersinia mismatch 46

2-2: Crosslinking PcrV and PopD & Translocation Defect 47

2-3: PopD dimer formation and associated translocation defect 48

2-4: PopB dimer formation and associated translocation defect 49

2-5: PopD-PcrV interaction effect on pore formation and insertion 50

2-6: PcrV and PopD Translocation Defect is not due to slow translocation 51

2-7: Proposed model 52

3-1: Feedback inhibition during infection with phagocytosed vs. non- 77 phagocytosed bacteria

3-2: Membrane damage repair 78

3-3: ExoS feedback inhibition in the presence of ExoT 79

3-4: ExoS feedback inhibition in the absence of ExoT 80

3-5: The effect of ExoT-GAP on ExoS feedback inhibition 81

3-6: The MLD affects feedback inhibition and the GAP domain is responsible 82 for feedback inhibition in the ExoS(2RD-N) mutant

3-7: The role of calcium during infection 83

3-8: Translocation is not affected by the absence of ASM 84

3-9: Absence of Caveolin-1 has varying effects on translocation 85

3-10: Absence of Caveolin-1 has varying effects on translocation 86

3-11: Knockdown of Flotillin-1 has no effect on translocation 87

3-12: Knockdown of Flotillin-2 has slight effect on translocation 88

5 3-12: Microwell assay 89

4-1: Host cell contribution to infection 103

4-2: Interplay between cellular processes and bacterial infection 105

Sensing of Host Cell Contact by the Pseudomonas aeruginosa Type III Secretion System

Abstract

ERIN ARMENTROUT

Pseudomonas aeruginosa uses a type III secretion system (T3SS), a syringe- like apparatus, to inject bacterial effector proteins into the host cell cytoplasm. Upon cell contact the T3SS assembles the translocon, which is essential for effector injection. The translocon creates a conduit for effectors to pass from the bacterium to the host cell and consists of three proteins: PopB and PopD, pore-forming translocators, and PcrV, the needle tip. Mismatch studies with Yersinia translocators identified interactions between PopD and PcrV, as well as PopB and PopD. The

PopD-PcrV interaction was confirmed by covalently crosslinking the interacting proteins during cell contact. We demonstrated that PopB and PopD also form homodimers. Tethering the PopD dimer or disrupting the PopD-PcrV interaction interferes with triggering of effector secretion. These data support the model that cell-contact is sensed by the translocation pore. The signal is then transmitted to the needle tip and from there propagated down the needle to initiate of effector secretion.

The host cell component that mediates sensing of the host cell contact has not yet been identified. Preliminary data from the lab suggested that the phagosome causes constant triggering of translocation, which may be related to membrane curvature. We aimed to identify this aspect using a three-pronged approach. First,

7 we attempted to recapitulate in vitro the membrane curvature that the bacterium experiences during phagocytosis. A curved membrane composed of only phosphatidylserine and phosphatidylcholine did not cause triggering of effector secretion, suggesting that there must be additional factors involved in sensing of host cell contact. Second, we demonstrated that blocking membrane damage repair- related endocytosis decreased the level of effectors translocated. However, this decrease was minor and implies that the membrane damage repair response is not solely responsible for triggering of effector secretion. Third, because ExoS can regulate its own translocation into host cells through its catalytic function, we aimed to identify cellular components that contribute to translocation by detecting targets of ExoS. We found that localization of ExoS, as well as both the GTPase activating protein- and the ADP-ribosyltransferase domains contribute to proper regulation of translocation.

Chapter 1

Pseudomonas aeruginosa, a human pathogen, and its virulence

factor, the Type III Secretion System

9 Pseudomonas aeruginosa is a Gram-negative, facultative anaerobic bacterium that is found in soil, in water, on surfaces, and as part of human skin biomes. It is an opportunistic human pathogen that is common in hospital-acquired infections. P. aeruginosa infects immunocompromised individuals, most commonly patients with cystic fibrosis, burn wounds, or ventilator-associated pneumonia. Cases of infection in healthy individuals are associated with damage at the site of infection; for example, contact lens-associated keratitis caused by P. aeruginosa occurs after the cornea becomes injured from contact lens wear (Wilcox, 2007). P. aeruginosa is a substantial problem in hospitals for two reasons 1) the bacterium likes to grow on surfaces, such as medical devices, and creates biofilms and 2) the bacterium has inherent antibiotic resistance. The high degree of antibiotic resistance in P. aeruginosa is due to two main factors. First, penetration of antibiotics is decreased by low permeability of its outer membrane, which is due to a lack of porins and inefficient transport by porins. Second, P. aeruginosa has a high adaptability to challenges by antibiotics, which is a result of efflux pumps, changes in gene expression, increases in production of beta-lactamase and other enzymes, and decreases in DNA damage repair that leads to an increase in mutation rate

(Breidenstein et al., 2011). Both its intrinsic antibiotic resistance and propensity to create biofilms makes P. aeruginosa difficult to treat and remove to prevent spread in hospitals. These qualities have driven researchers to search for alternatives to antibiotics for preventing infection by P. aeruginosa.

P. aeruginosa has several virulence factors. One of particular interest, the

Type III Secretion System (T3SS) and its accompanying effectors, is correlated with

10 poor clinical outcomes in ventilator-associated pneumonia and blood stream infections (Hauser et al., 2002; El-Solh et al., 2012). In one study, T3SS gene expression was lower for P. aeruginosa strains isolated from patients with chronic respiratory infections compared to strains isolated from patients with acute infections, suggesting that the T3SS is associated with acute infections (Roy-Burman et al., 2001). More recently, Jorth et al. (2015) found that T3SS gene expression varies between regions of the lung in individual patients in response to different selective pressures. Additionally, research suggests that the T3SS is important for establishing biofilms at the epithelial barrier (Tran et al., 2014). Together these data suggest that studying the T3SS can be useful for treatment and prevention for patients with acute or chronic infections.

The T3SS is related to the flagellar system and they likely share a common ancestor. This can be seen in the high degree of structural homology between the basal body of each structure (Saier, 2004). Both systems are designed to export bacterial proteins; however, the purpose of each system differs. Swimming motility in bacteria is controlled by the flagellum, which bacteria generally use in search of nutrients. On the other hand, T3SS is a molecular syringe that injects bacterial proteins called effectors into eukaryotic cells (Figure 1-1). Bacterial effectors manipulate eukaryotic cellular functions to increase survival. Genes encoding the

T3SS exists as a pathogenicity island on the chromosome or as a virulence plasmid and were likely passed among Gram-negative human pathogens by horizontal gene transfer (Nguyen et al., 2000). In P. aeruginosa, there are 36 genes organized in five operons that encode the T3SS and regulatory elements, while effectors and

11 corresponding chaperones are scattered throughout the chromosome (Hauser,

2009).

Effectors

Effectors are bacterial proteins that are translocated from the bacterium into the host cell cytoplasm. Once inside the host cell, effectors modify cellular proteins, thereby promoting the dissemination of the infection and evasion of the immune system. In P. aeruginosa, there are four T3SS-associated effectors: ExoS, ExoT, ExoY, and ExoU. ExoS and ExoU appear to be mutually exclusive. The reason strains do not produce both ExoS and ExoU is unknown (Hauser, 2009).

ExoS and ExoT have been extensively studied. Both ExoS and ExoT share

76% amino acid homology, and both contain a GTPase activating protein (GAP) domain and an ADP-ribosyl transferase (ADPR) domain (Hauser, 2009). Both contain a membrane localization domain (MLD), which modulates activity by localizing to the membrane (Hauser, 2009; Zhang and Barbieri, 2005). The GAP domain of both proteins target RhoA, Rac1, and Cdc42 (Pederson et al., 1999;

Goehring et al., 1999; Krall et al., 2002; Kazmierczak et al., 2002). On the other hand, the cellular targets of each protein’s ADPR domain differ. ExoS-ADPR targets a wide variety of molecules including Ras, vimentin, the ezrin/radoxin/moesin (ERM) family of proteins, and others, which have not yet been identified (Coburn and Gill,

1991; Coburn et al., 1989; Maresso et al., 2004). ExoT-ADPR has a smaller number of targets including CrkI and CrkII (Sun and Barbieri, 2003). The ADPR activity of the two proteins is modulated by binding of the host 14-3-3 protein through a cofactor

12 binding site (Coburn et al., 1991; Fu et al., 1993). In general, the modifications by both proteins affect the host cell cytoskeleton, cellular adhesion, and cellular tight junctions (Hauser, 2009). Dismantling of the host cell architecture prevents phagocytosis of the bacterium by immune cells. Disruption cell adhesion and cellular tight junctions of epithelial cells contributes to dissemination of the infection (Hauser, 2009).

ExoU has a patatin-like domain that acts like a phospholipase A2. ExoU has a variety of targets including phospholipids, lysophopholipids, and neutral lipids (Sato et al., 2003; Phillips et al., 2003). Much like ExoS or ExoT, ExoU requires binding to a eukaryotic cofactor, SOD1, in order to be active (Sato et al., 2003; Phillips et al.,

2003). ExoU causes rapid cell death triggered by loss of plasma membrane integrity

(Hauser et al., 1998). This helps the bacterium avoid phagocytosis and leads to a disruption in epithelial barriers, which contributes to dissemination of the infection

(Kurahasi et al., 1999).

ExoY is the least characterized effector. It was first identified as an adenylyl cyclase with homology to CyaA from Bordetella pertussis and EF from Bacillus anthracis (Yahr et al., 1998). Adenylyl cyclases hydrolyze ATP to cAMP and cAMP levels are important for signaling cascades in eukaryotic cells. Recently, ExoY has been identified as a promiscuous cyclase, hydrolyzing purine and pyrimidine cyclic nucleotide monophosphates (Morrow et al., 2017). ExoY contributes to increased endothelial permeability and cytoskeleton disruption, although its function seems to be dispensable for infection (Yahr et al., 1998; Cowell et al., 2005; Sun et al., 2012).

13 T3SS Structure

The T3SS is a complex structure that is highly regulated. The structure can be categorized into three portions: the basal body that spans the inner and outer membrane of the bacteria, the cytoplasmic sorting platform, and the extracellular portion, which is composed of the needle and translocon (Figure 1-1). The translocon attaches to the end of the needle and crosses the eukaryotic membrane to gain access to the target cell cytoplasm. The basal body is composed of several oligomeric rings. The outer membrane ring is constructed out of PscC subunits. PscD oligomerizes creating an inner membrane ring, which spans the periplasmic space, connecting it to the outer membrane ring (Dewoody et al., 2013).

The sorting platform is composed of an ATPase complex and an export apparatus and expands into the cytoplasm from the basal body (Figure 1-1). The platform is constructed before any protein can be recruited to and secreted from the apparatus (Deng et al., 2017). The ATPase complex is constructed out of PscN, PscL, and PscO. PscN is the ATPase, while the stator and stalk are PscL and PscO respectively (Deng et al., 2017). PscK bridges the basal body to the cytoplasmic-ring

(C-ring), which is made out of PscQ and creates the bottom of the sorting platform

(Figure 1-1)(Hu et al., 2015). The export apparatus is assembled partly within the inner membrane independent of the basal body and is made out of PcrD and

PscRSTU (Dietsche et al., 2016). The sorting platform is thought to prepare substrates for secretion and therefore plays a role in regulation of secretion substrates, which will be discussed later in this chapter. The first secretion substrates include PscJ and PscI. The inner membrane ring is completed with the

14 addition of PscJ. PscI composes the inner membrane rod through which all other secretion substrates are passed (Dewoody et al., 2013).

The needle extends from the basal body into the extracellular space. PscF subunits polymerize to create the needle shaft (Figure 1-1)(Hoiczyk and Blobel,

2001). The length of the needle is controlled by the ‘molecular ruler’ protein, PscP.

Exactly how this occurs is not known, but PscP could act as a physical ruler. This hypothesis was based on observations in Yersinina spp. that when YscP, the PscP homolog, was manipulated to include more or fewer repeats, the needle length was longer or shorter, respectively (Mota et al., 2005). The translocon sits at the end of the needle and creates a channel across the eukaryotic membrane into the host cell cytoplasm. The translocon comprises three proteins, PcrV, PopB, and PopD (Figure

1-1). The needle tip is composed of five PcrV subunits (Broz et al., 2007), while PopB and PopD work synergistically to create pores within the host membrane (Faudry et al., 2006). The pore is thought to form after PopB and PopD associate with the membrane, because PopB and PopD do not to form ring-like structures before membrane association (Schoehn et al., 2003). There is no consensus on stoichiometry of the pore, because there are few tools to study membrane- associated proteins. Using atomic force microscopy, pores have been visualized as 6-

8 subunit ring-like structures in membranes (Ide et al., 2001). Romano et al. (2016) suggested that pores are likely composed of eight PopB and eight PopD units. The needle tip is thought to dock to the pores, creating a continuous conduit for the bacterium to translocate its effectors into the host cell cytoplasm.

15 Powering the T3SS and Control of Secretion Rate

The width of both the needle and pore is 2-3 nm (Blocker et al., 2001;

Dacheux et al., 2001), which suggests proteins are transported through the apparatus unfolded. This was visualized in Salmonella enterica by attaching a tightly folded protein (glutathione S-transferase (GST)) to the substrate, which prevented the substrate from completing the secretion process (Radics et al., 2013).

How proteins are transported through the apparatus energetically is a point of controversy. The T3SS is unable to secrete effectors when its corresponding

ATPase was deleted along with selected negative regulators (Minamino and Namba,

2008; Paul et al., 2008). In contrast, deletion of flagellar ATPase homologs did not prevent secretion by the flagellar system, which suggests that the flagellar system is powered by the proton motive force (pmf). These data imply that ATP is necessary for secretion by the T3SS. Conversely, disruption of pmf also prevented secretion by the T3SS in Yersinia spp. and P. aeruginosa (Wilharm et al, 2004; Lee et al., 2014).

One possible explanation is that the pmf is the main power source pushing substrates through the apparatus, while the ATPase is responsible for removing chaperones from substrates and unfolding the substrates before secretion (Akeda and Galan, 2005; Lee et al., 2014).

Another curious aspect of the T3SS is that translocators are released at a slow rate, sometimes labeled ‘leakage’, while effectors are released at a much faster rate. This increased rate can cause cytotoxicity within as little as 15 min (Dewoody et al., 2013). The rate of secretion is controlled by PcrG and PscO, which modulate the ability of the T3SS apparatus to utilize the pmf to power secretion (Lee et al.,

16 2014). This finding also supports the assertion that the pmf is the main power source for secretion.

Regulation of T3SS Gene Transcription

T3SS genes are regulated depending on environmental signals and conditions. Transcription of the T3SS genes occurs at low levels during the exponential growth phase in the presence of amino acids (Lee et al., 2001). During times of stress however, the bacteria suppress transcription of T3SS genes (Rietsch and Mekalanos, 2006). Yersinia spp. require calcium in order to grow exponentially at 37°C (Brubaker, 1983). This requirement was linked to the presence of the T3SS virulence plasmid/island (Gemski et al., 1980). After growth to log phase, the presence of calcium is no longer required and depletion of calcium causes secretion of T3SS substrates (Lee et al., 2001).

Additionally, transcription of T3SS genes increases upon stimulation of secretion either by calcium depletion or host cell contact (Hornef et al., 2000; Vallis et al., 1999). Transcription of T3SS genes is controlled by the master regulator operon exsACDE. Under non-inducing conditions, ExsC preferentially binds ExsE and ExsD is bound to ExsA (Rietsch et al., 2005; McCaw et al., 2002). Under inducing conditions, ExsE is exported by the T3SS, which allows ExsC to bind ExsD (Rietsch et al., 2005; Dasgupta et al., 2004). ExsA is freed to bind to promoter regions of T3SS genes, which increases transcription of T3SS genes (Frank and Iglewski, 1991).

17 Regulation of Secretion

There are three phases of secretion: early, middle, and late. Early secreted proteins include the inner rod proteins (PscI) and the needle shaft proteins (PscF).

Middle secreted proteins are translocon components: PcrV, PopB and PopD. Late secreted proteins are effectors: ExoS, ExoT, ExoY, and ExoU. The T3SS apparatus is regulated to ensure substrate fidelity before switching the secretion phases. Some elements of this regulation are known but others have yet to be discovered.

Many labs have tried to determine a secretion signal for substrates to elucidate the control of secretion hierarchy. It has been proven that approximately the first 15-20 residues are needed for secretion (Lloyd et al., 2001); however, these secretion signals do not appear to be conserved, i.e., the regions responsible for secretion share low sequence homology. Several groups have looked at both amino acid sequences and mRNA sequences, but results are inconclusive (Lloyd et al.,

2001). More specifically in Yersinia sp,, both the protein and mRNA sequence of the secretion signal in LcrV can be changed without consequence to secretion or cytotoxicity (Broms et al., 2007). In this same system, removing the sequence altogether does affect secretion and cytotoxicity (Broms et al., 2007). This observation is echoed by the observation that the secretion signals of a middle substrate (PopD) can be switched with that of a late substrate (ExoS) without changing secretion order (Tomalka et al., 2012). These data suggest that the

‘secretion sequence’ region is important for secretion of the substrate but does not play a role in secretion hierarchy. Alternatively, proper temporal secretion of PopD seems to be controlled by association with its chaperone PcrH (Tomalka et al.,

18 2012). On the other hand, this may not be the only explanation, because PcrV that is unbound to its chaperone is still able to secrete correctly, although at a lower efficiency (Lee et al., 2010). At this time, there is no overall consensus on how substrates and their chaperones contribute to secretion hierarchy.

The switch between early and middle secretion substrates is mediated by

PscU, which is part of the export apparatus. PscU associates with the basal body and undergoes auto-cleavage to facilitate substrate switching. An interaction with PscP, the molecular ruler, is thought to negatively affect this cleavage event for proper temporal regulation (Ho et al., 2017). However, the cleavage event is not necessarily the switch for change in secretion phases; instead, PscU is thought to undergo a conformational change to signal substrate switching and cleavage assists in causing this conformational change (Monjaras Feria et al., 2015).

The method of substrate switching between middle and late substrates is not well defined. PopN homologs were identified as a major component for this switch and were deemed the ‘gatekeeper’ protein (Deng et al., 2017). PopN forms a complex with Pcr1, Pcr2, and PscB (Yang et al., 2007) and export of PopN is needed for the switch from translocators to effectors (Sundin et al., 2004). Data from our lab suggests that the PopN complex blocks an acceptor site important for effector secretion on PcrD, a component of the export apparatus (Lee et al., 2014). The acceptor site becomes exposed when PopN is exported or if popN or pcr1 are deleted. Additionally, PcrG exerts control over secretion, which is not linked to its role as the chaperone to PcrV (Lee et al., 2010). PcrG negatively regulates secretion by stabilizing PcrD in an “off” conformation (Lee et al., 2014).

19 Effector secretion is polarized, meaning the bacterium only secretes effectors into the target cell and not into the extracellular space (Rosqvist et al., 1994). This implies that the bacterium relies on some signal that it has made contact with the host cell before switching secretion substrates. The translocon most likely contributes to sensing of host cell contact, because it mediates the interface between the T3SS and the target cell. The translocon is not well studied, but it is known that

PopB, PopD, and PcrV are all necessary for polarized translocation (Blocker et al.,

1999). Translocator insertion into the host membrane and pore formation is necessary but not sufficient for translocation (Verove et al., 2012; Russo et al.,

2016). The needle tip also needs to be stably docked to the pore in order to translocate effectors (Viboud and Bliska 2001; Russo et al., 2016). Additionally, data suggest that the purpose of the translocation is not to only provide a channel into the target cell but it is actively involved in sensing host cell contact. Specifically, the needle tip proteins have been implicated in this signaling of host cell contact, but the emergence of this signal is unknown (Veenendaal et al., 2007; Sato et al., 2011).

There is additional regulation of translocation after host cell contact. In P. aeruginosa, ExoS can inhibit translocation into a host cell by other P. aeruginosa cells, which is dependent on its catalytic activity (Cisz et al., 2008). This regulation has also been found for YopE, the ExoS homlog, in Yersinia spp. (Aili et al., 2006).

These data suggest that ExoS and YopE regulate translocation of effectors through manipulation of a cellular component. Contact with the host cell would be indicated by this cellular target and would trigger secretion of effectors. After ExoS or YopE modifies the target, translocation would be prevented. Identifying this component is

20 crucial to understanding how the bacterium senses host cell contact and it has not yet been identified.

Several groups have attempted to identify cellular components that are necessary for infection by T3SS+ bacteria using several different methods. When membranes are depleted of cholesterol, they are unable to support infection by

T3SS+ bacteria (Lafont et al., 2002; van der Goot et al., 2004; Hayward et al., 2005;

Kannan et al., 2008; Verove et al., 2012). However, this is most likely not due to a direct interaction with cholesterol by the bacterium, but rather an indirect effect.

Specifically, the cholesterol content of membranes controls the formation of lipid rafts. To further this point, T3SS pore proteins have been shown to associate with lipid rafts (Lafont et al., 2002; Verove et al., 2012). Isolated lipid rafts were found to be sufficient for triggering of effector secretion (van der Goot et al., 2004), but these lipid rafts were isolated from cells and, therefore, contain numerous unidentified elements that could be important for injection. For example, CD44 associates with lipid rafts and is important for infection (Lafont et al., 2002). In contrast, a study by

Bridge et al. (2010) suggests that neither cholesterol content nor lipid rafts are important for injection, but instead has to do with cellular adherence to tissue culture wells. They go on to explain that the leading edge of the cell during migration likely supports translocon formation and subsequent translocation by P. aeruginosa. Presently, there is no consensus on the cellular components that are necessary for infection.

21 Conclusion

There are still many questions left unanswered about the T3SS, but I chose to focus on how the bacterium senses host cell contact for polarized translocation of effectors. This is a crucial step during injection, and therefore makes an attractive candidate for development of drugs to treat P. aeruginosa and other T3SS+ bacterial infections. I approached this question two ways: 1) from the bacterial side, i.e., testing what interactions between bacterial proteins are needed for proper injection of effectors; 2) from the host side, i.e., what cellular components or processes are needed for injection of effectors.

Chapter 2

Sensing of Host Cell Contact by the Type III Secretion System

24 Introduction

Type III secretion systems (T3SS) have the ability to transfer effectors in a polarized manner, as characterized by a lack of effectors in the medium (Rosqvist et al., 1994). This observation implies that translocation is a one step process in which the effectors are transferred from the bacterial cytoplasm directly into the host cell.

In order for this to occur, the bacterium must have a mechanism to sense when contact with the host cell has been made. Despite the numerous studies on the subject, the mechanism remains poorly understood.

Mota et al. (2005) demonstrated that triggering was dependent on the ability of the apparatus to make contact with the host cell membrane. Yersinia spp. needle length and adhesin length seem to be coordinated. When T3SS needles were shortened through genetic manipulation, host cell contact by the apparatus was prevented while attachment was maintained by adhesins. Mota et al. (2005) did not find effectors in the medium, which indicates the T3SS apparatus is directly responsible for triggering effector secretion through cell contact and not an alternative method, such as attachment by the adhesins.

The eukaryotic membrane-spanning portion of the needle apparatus, the translocon, is of particular interest, because it facilitates host cell contact and delivery of effectors into the cytoplasm. The translocon consists of three translocator proteins: PopB, PopD, and PcrV. Deletion of any one translocator results in failure to translocate T3SS-associated effectors (Blocker et al., 1999), meaning proper assembly of the translocon is necessary for infection of host cells.

Purified PopB and PopD can individually create pores in artificial membranes

25 (Faudry et al., 2006). Conversely, if popB or popD is deleted, the bacteria cannot create pores in vivo (Goure et al., 2004), suggesting both proteins work synergistically to create pores for injection (Faudry et al., 2006). PcrV associates with the end of the needle shaft to act as a tip (Mueller et al., 2005). The needle tip is thought to facilitate insertion of both pore proteins into membranes by acting as an external chaperone or a scaffolding protein for proper formation of pores (Goure et al., 2004; Goure et al., 2005; Broz et al., 2007). The needle tip is thought to dock to the pore to create a continuous conduit into the host cell cytoplasm for translocation of effectors.

Translocator interactions

Translocators are classified by their functional homology, i.e., their ability to transport effectors across the eukaryotic cell membrane. Conversely, when looking at sequence homology between translocators from different T3SS families, the amino acid sequence similarity is low. Taking this a step further, previous data suggested there are genus- or species-specific interactions between translocators within T3SS families that are important for proper translocation. More specifically, certain Yersinia null mutants could not be complemented by corresponding P. aeruginosa translocators, e.g., ΔlcrV could not be complemented by pcrV (Broms et al., 2003a; Broz et al., 2007), but the entire P. aeruginosa translocon can complement a ΔlcrGV ΔlcrHyopBD Yersinia strain (Broms et al., 2003a). This suggests that the incompatibility was not from the inability of PcrV to be secreted and assemble a functional needle tip, but from an inability to interact with the

26 YopB/YopD pore. Additionally, Broz et al. (2007) found that certain sections of the needle tip were important for maintaining particular functions. LcrV-PcrV hybrid needle tips, containing the N-terminus from LcrV and the C-terminus of PcrV, were able to cause hemolysis of red blood cells with YopB/YopD pores, a test of functionality. Needle tips containing the PcrV N-terminus were unable to perform the same test, which suggests that PcrV N-terminus did not share enough homology with LcrV to maintain critical interactions between the needle tip protein and the pore proteins. However, these studies did not specify the key regions or residues for these interactions.

Previous work from our lab identified crucial interactions by utilizing incompatibility between Pseudomonas aeruginosa (PcrV, PopB, PopD) and corresponding translocators from the closely related Yersinia pseudotuberculosis

(LcrV, YopB, YopD). More specifically Pseudomonas-Yersinia hybrid proteins were used to detect interactions between the translocators and the specific regions of interactions important for injection. The ability of wild type PcrV or a hybrid needle tip, containing the N-terminus of LcrV and the C-terminus of PcrV (LcrV(N)-PcrV(C), denoted as LNPCV), to function with different combinations of translocator pore proteins, PopB or YopB with PopD or YopD, was tested (Figure 2-1A). The translocon’s ability to function was measured by a cytotoxicity assay, which monitors translocation of the effector, ExoS, into the host cell by observing cell rounding of lung epithelial (A549) cells in tissue culture after infection. ExoS causes depolymerization of actin, which collapses the host cell architecture and causes the cell to round up. The PopB/YopD pore was able to function with the LNPCV needle

27 tip but not the wild type needle tip, which implies that a critical interaction between the wild type needle tip and PopD was disrupted (Figure 2-1A). This is supported by supplemental material of Broz et al. (2007), which used Far Western blots to show that the N-terminus of LcrV interacted with YopD. Neither needle tip was able to work with the YopB/PopD pore, which is supported by Broms et al. (2003b), who found that wild type LcrV was unable to function with YopB and PopD pores within the Yersinia system. Because PopB and PopD work synergistically to create pores within the membrane (Goure et al., 2004; Faudry et al., 2006), this incompatibility suggests that lack of function of YopB/PopD pores is most likely caused by a disruption of the interaction between PopB and PopD.

The mismatch system was further utilized to narrow specific regions of interaction for PcrV-PopD and PopB-PopD. We created created Pseudomonas-

Yersinia hybrid proteins and testing whether or not they could intoxicate cells using the cytotoxicity assay described above. This assay determined that the last 27 amino acids of PopD were sufficient for its interaction with the N-terminus of PcrV (Figure

2-1B). The cytotoxicity data for the PopB-PopD interaction determined that PopB residues 274 through 297 and PopD residues 228 through 245 are critical for the interaction between the two proteins (Figure 2-1B).

Models of initiation of effector secretion

It is unknown whether the translocon has a passive (e.g., allows small host cell specific molecules to enter the bacterial cytoplasm) or active role (e.g., physically acts as the sensor) in initiation of effector secretion upon cell contact. An

28 early model for effector triggering is the ‘plug’ model, which states that a protein sitting within the channel would block the passage of effectors into the host cell. The blockage is then displaced when the needle tip reaches the host membrane. YopN, the secretion of which signals substrate switching from translocators to effectors, has been proposed as the protein in question for Yersinia (Ferracci et al., 2005).

YopN was suggested to attach at the base of the needle apparatus and partially insert into the secretion channel either physically blocking entry of other proteins or the acceptor site for effectors (Ferracci et al., 2005). Another version of this model suggests that the needle tip switches from a ‘closed’ to an ‘open’ conformation when inserted into the eukaryotic membrane. This model was originally supported by Blocker et al. (2008), but was later invalidated by the same group (Cheung et al., 2015), which found tip complexes in only one conformation.

Both versions of the plug model are invalid, because they cannot account for secretion of translocators before effector secretion (Cisz et al., 2008), which relies on the assumption that the channel is too small for proteins to travel through. A modification of the ‘plug’ model suggests that the bacterium can sense varying levels of a small molecule specific to host cells present in the cytoplasm through the small opening of the needle tip. The pore opening is estimated to be between 2.0-3.0 nm

(Blocker et al., 2001; Dacheux et al., 2001), which is large enough for ions and small molecules to pass through. Consistent with this idea, Dacheux et al. (2001) demonstrated that PopB/PopD pores were able to release a small molecule from macrophages that created a chemotactic gradient, which caused P. aeruginosa strains to swarm the perforated macrophage. Lower calcium levels in the host cell

29 cytoplasm have been proposed as the trigger in Yersinia (Lee et al., 2001), but this hypothesis was disproven for P. aeruginosa (Cisz et al., 2008). At this point, no concrete evidence has been provided to support the hypothesis of a cell specific molecule as the sensor of host cell contact.

An alternative model for the injectisome model of T3SS is that pore proteins and effectors form an intermediate complex, which associates with the bacterial membrane and are transferred to host cells during close contact (Akopyan et al.,

2011). This would be similar to how AB toxins are delivered into host cells and implies that there is no specific signal to initiate effector secretion. This model has only been demonstrated for YopH in Yersinia sp. and EspC in Enteropathogenic

Escherichia coli (EPEC) (Akopyan et al., 2011; Tejeda-Dominguez et al., 2017). This model seems unlikely because effectors have not been found in the medium and cell contact by the apparatus is needed (Rosqvist et al., 1994). This model has been discounted by co-infection experiments, where two strains are mixed and incubated with target cells. One strain releases translocators but does not produce effectors, while the second strain does not secrete translocators, but does secrete effectors into the medium. If this AB toxin-like model where true, the strains alone would be unable to translocate effectors into host cells, but co-infection with both strains should complement each other. However, these experiments have been unsuccessful (Rosqvist et al., 1994; Sory et al., 1995; this study), demonstrating that this model is incongruous and unconvincing.

The model for Shigella spp. proposed by Murillo et al. (2016) states that the signal indicating host cell contact relies on conformational changes of apparatus

30 components. Shigella spp. start out with a pentameric IpaD, the PcrV homolog, needle tip. IpaB, the PopB homolog, is then exported and displaces an IpaD monomer to create a new needle tip composed of four IpaD molecules and one IpaB molecule (Veenendaal et al., 2007; Cheung et al., 2015). IpaD facilitates pore formation by IpaB and IpaC, the PopD homolog (Picking et al., 2005). IpaB partially inserts into the host cell membrane causing a conformational change, which is transferred to the IpaD molecules (Veenendaal et al., 2007; Murillo et al., 2016). The signal is transmitted from the IpaD molecules to the needle shaft proteins (Murillo et al., 2016). The needle shaft proteins then propagate the signal down to the basal body (Kenjale et al., 2005; Torruellas et al., 2005; Deane et al., 2006). For the Ysc family, which includes P. aeruginosa and Yersinia spp., it is possible that a similar mechanism occurs because the needle tip and needle shaft proteins have been found to regulate secretion (Sato et al., 2011; Torruellas et al., 2005). For PcrV, this regulatory function is not connected with its cytoplasmic regulation (Sato et al.,

2011). However, YopB has not been found to associate with the needle tip (Mueller et al., 2005), implying the initial step differs from the Shigella spp. model.

Because the translocon is necessary for infection, it is central to establish how it functions mechanistically. I wanted to test how these aforementioned interactions (Figure 2-1) contributed to infection by looking at what role they played during the translocon function. By comparing these data with the previously listed models for effector secretion triggering, I should be able to elucidate the mechanism of translocation. The majority of the data presented in this chapter has been published in PLoS Pathogens (Armentrout and Rietsch, 2016).

31 Materials and Methods

Bacterial strains, cells, and growth conditions

E. coli strains were grown in LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter), supplemented with 15 μg/ml gentamicin when necessary. P. aeruginosa strains were grown on “high salt” LB (10 g/L tryptone, 5 g/L yeast extract, 200 mM NaCl, 5 mM MgCl2, 0.5 mM CaCl2), supplemented with 30 μg/ml of gentamicin when necessary. Assembly of the T3SS is controlled by the osmolarity of the medium. Use of “high salt” LB allows for consistent production of the T3SS under laboratory conditions (Rietsch and Mekalanos, 2006). Production of translocator proteins was induced through the addition of IPTG to the growth medium.

A549 cells (American Type Culture Collection, Cat. #CCL-185) were grown in

RPMI1640 (ThermoFisher) medium supplemented with 10% fetal bovine serum

(FBS) (RP10) at 37°C in a 5% CO2 atmosphere. Cells were maintained in the presence of penicillin and streptomycin. Before experiments, cells to be infected were washed once with Dulbecco’s phosphate buffered saline (DPBS,

ThermoFisher), and the medium was exchanged with RP10 lacking antibiotics.

Plasmid and strain construction

Mutations were introduced into the chromosome of strains through allelic exchange as described previously (Rietsch et al., 2005). Plasmids were constructed using standard molecular biological techniques. The indicated translocator (or portion of a translocator) was amplified by polymerase chain reaction using the appropriate primers. Y. pseudotuberculosis YPIII DNA was used as template for the

32 amplification of yopD, yopB, and lcrV. Hybrid translocators were generated using splicing by overlap extension (SOE) PCR. PCR products were cut with the appropriate restriction enzymes and ligated into plasmids pEXG2 (allelic exchange vector) or pPSV37 (plasmid that can replicate in P. aeruginosa with lacUV5 promoter and lacIq).

Translocation assay

P. aeruginosa strains were grown to mid-logarithmic phase in “high salt” LB, pelleted, and resuspended in PBS with 5mM Mg2+ and 0.5 mM Ca2+ (PBS-MC). A549 epithelial cells were infected with P. aeruginosa strains at a multiplicity of infection

(MOI) of 25 for 2 hours. The cells were then washed three times with PBS-MC and rinsed with 1ml of 250 μg/mL proteinase K in PBS-MC. The protease solution was then removed, and the cells were incubated at room temperature for 15 minutes to digest extracellular protein. The protease-treated cells were resuspended in 1ml of

PBS-MC with 2mM phenylmethylsulfonyl fluoride (PMSF) and pelleted (3 minutes,

5000 rpm). The cells were resuspended in 300 μL of PBS-MC with 0.5% Triton X-

100 and incubated on ice for 15 minutes. 150 μL of the cell suspension was removed and mixed with 50 μl of 4X SDS sample buffer (SDS sample). The remaining cells were pelleted, and 150 μL of supernatant weas removed and combined with 50 μL of 4X SDS sample buffer (Triton solubilized fraction). The samples were separated by SDS-PAGE and analyzed by Western blot. Membranes were probed with antibodies directed against ExoS, RpoA (Neoclone) and either human glucose-6- phoshate isomerase (G6PI, Santa Cruz Biotechnology Inc.), actin (Developmental

33 Studies Hybridoma bank), or tubulin (Santa Cruz Biotechnology Inc.). Antibodies were detected using a horseradish peroxidase labeled secondary antibody, and

WesternBright Quantum reagent and imaged using a GE ImageQuant LAS4000 imaging system. Protein levels were quantitated using ImageJ (NIH), and ExoS levels were normalized to G6PI (or actin, or tubulin)-levels.

Crosslinking

P. aeruginosa strains producing cysteine mutants of PopD, PopB, or PcrV (as indicated) were grown to mid-logarithmic phase in “high salt” LB with 200 μM IPTG, pelleted and resuspended in PBS-MC. A549 epithelial cells were infected for 2 hours in the presence of 200 μM IPTG and either 25 μM copper phenathroline or 1 mM tris(2-carboxyethyl)phosphine (TCEP). After infection cells were washed with 25 mM iodoacetamide for 3 min to alkylate free cysteine residues. Cells were then washed once with high salt PBS (PBS-MC + 1 M KCl) before the cells were scraped up in 1 ml of PBS-MC with 2 mM phenylmethylsulfonyl fluoride (PMSF), and pelleted

(3 minutes, 5000 rpm). The cells were resuspended in 45 μL of PBS-MC with 0.5%

Triton X-100 and incubated on ice for 15 minutes. The cells were pelleted, and 45 μL of supernatant was removed and combined with 15 μL of 4x SDS sample buffer without Dithiothreitol (DTT). Samples were analyzed by Western blot. Membranes were probed for the presence of PcrV, PopB, or PopD using affinity-purified antibodies.

34 Hemolysis

The protocol we used was based on the assay published by Blocker et al.

(1999). Sheep red blood cells (Quadfive) were washed three times with PBS-MC, and resuspended in RPMI without phenol red or FBS to a concentration of 5 x108 cells/mL. Bacterial cultures were diluted 1:250 from overnight cultures into high salt LB and allowed to grow to mid-log phase. Bacterial cultures were spun down and resuspended in PBS MC, the OD600 was measured and bacteria were resuspended to a concentration of 2.5 x 109 cells/mL. Bacteria and red blood cells were mixed 1:1 (MOI of 5) in a v-bottomed 96-well plate. Samples were centrifuged at 2000 g for 10 minutes and then incubated for 1 hour at 37°C. The samples were then resuspended and centrifuged at 2000 g for 10 minutes before collecting 100 μL of supernatant. Supernatants were placed in a clean flat-bottomed 96-well plate and then hemoglobin release was calculated by measuring absorbance at 415 nm.

Baseline lysis was determined by red blood cells mixed and incubated with PBS MC.

Percent lysis was determined by comparing samples to 100% lysis using 0.1% SDS.

Red blood cell membrane isolation

Red blood cells and bacterial samples were prepared as noted for the hemolysis assay. Samples were prepared by mixing 1.25 mL of a 8x108 cells/mL red blood cell suspension with 250 μL of a 2x1010 cells/mL bacterial suspension in the presence of 1X cOmplete protease inhibitor (Sigma-Aldrich). They were then centrifuged at 2000 g for 10 minutes and incubated at 37°C for 1 hour. Samples were resuspended and centrifuged at 2000 g for 10 min before lysing with distilled

35 water on ice for 10 minutes. Intact cells were centrifuged at 5000 rpm for 10 minutes. The supernatant containing the lysed membranes was removed. A 30 μl aliquot was removed and mixed with 10 μL of 4X SDS sample buffer to serve as input control. A sucrose gradient was formed by layering 0.5 mL 50%, 1 mL 41%, and 1 mL 20% sucrose in 20 mM HEPES pH7.6 1 mM EDTA. Red blood cell membranes were isolated by separating the supernatant on a sucrose gradient

(Beckman Coulter Optima Max-XP ultracentrifuge; MLS 50 rotor; 34,407rpm

(95,000 rcf for 2 hours), and collecting the membranes at the interface between the

41% and 20% sucrose phases. Samples were split into three parts. One part was diluted with ~10X volume of 10 mM HEPES pH 7.6 1 mM EDTA buffer, one with 1 M

KCl, and the last part with 1 M Na2CO3. The membranes were pelleted by ultracentrifugation (TLA 100.3 rotor, 40,000 rpm for 1h), resuspended in 50 μL 1X

SDS sample buffer, and boiled at 95°C for 10 min before. Samples were desalted using Zebra spin desalting columns (Thermo) and mixed with 4X SDS sample buffer

(80 μl final volume). Samples were analyzed by Western blot (input samples were diluted 1:2 and 10 μl were loaded on the gels, 20 μl of the membrane preps were loaded). Membranes were probed with antibodies directed against PopB (MW: 40.1 kDa) and PopD (MW: 31.3 kDa).

Results and Discussion

Confirming interactions

Previous data from the lab identified possible interactions between translocator proteins important for infection (Figure 2-1B). I wanted to confirm

36 these interactions by covalently binding them during infection. I was able to do this by introducing a cysteine residue in each of the two target proteins, which can form a disulfide bond when an oxidant, Cu2+, is added. A disulfide bond would only be able to develop if the cysteine residues are within close proximity, demonstrating that the proteins must be interacting. The P. aeruginosa translocators are naturally devoid of cysteine residues making this a useful tool to study protein interactions.

All cysteine-substituted proteins used were secreted at wild type levels and did not affect translocation (Figures 2-2A, 2-3A, & 2-4A).

Cysteine residues replaced a selected residue in each protein’s region of interaction. When copper ion was added, a band that migrated at a height expected for a PcrV(Q87C)-PopD(A292C) complex was present only in samples that contained both cysteine-substituted proteins (Figure 2-2A). The PcrV(Q87C)-

PopD(A292C) band was found in samples collected from infected cells but not in the supernatant, which suggests that this interaction only occurs in the context of the host cell (Figure 2-2A). Additionally, PopD(R243C) was also able to crosslink to

PcrV(Q87C). The R243 residue lies outside the identified region of interaction

(Figure 2-1B), which could suggest that the interaction is flexible and that exact residues used for the crosslinking might not necessarily be associating for the interaction. This discrepancy could also be due to the methods used for mapping the interactions. If the residues in Yersinia were similar enough to that found in

Pseudomonas, the substitution would not be found to affect cytotoxicity, meaning the region of interaction could be much larger or there could be multiple interactions but only one was identified.

37 The same experiment was conducted for the PopB-PopD interaction.

Residues PopB(A280) and PopD(R243) were chosen because they fell within the regions of interaction for PopB-PopD (Figure 2-1B). Unexpectedly, the crosslinking rendered conflicting results. High-molecular weight bands that could correspond to crosslinked PopB-PopD complexes were found in samples that only contained one cysteine substitution but not the other (PopB (A280C) or PopD (R243C)). These data establish that PopB and PopD are crosslinking to another protein with a cysteine residue but not to each other. There are three explanations to this confusing result: PopB and PopD could be crosslinking to (i) a host cell protein, (ii) a serum protein, or (iii) a bacterial protein containing a cysteine residue.

I was able to demonstrate that the high-molecular weight band corresponds to a homodimer of PopB or PopD for each substitution (Figures 2-4A & 2-3A, respectively). This was achieved by using a combination of cysteine-substituted and/or size-tagged PopB or PopD. The size-tagged proteins were created by adding four VSV-G peptides inserted at permissible sites. High-molecular weight bands were found only when proteins containing cysteine substitutions were exposed to

Cu2+, an oxidant (lanes 4, 6, 7, and 8)(Figures 2-3A & 2-4A). Size-tagged proteins samples that had cysteine-substitution contained high-molecular weight bands that migrated more slowly than the high-molecular weight bands seen in the non-size tagged protein samples (lane 6 vs. 7)(Figures 2-3A & 2-4A). When both size-tagged and non-size-tagged cysteine-substituted proteins were expressed, three bands were found (lane 8). The slowest migrating high-molecular weight band traveled at the same rate as the size-tagged cysteine-substituted protein sample band (lane 8

38 vs. 6). The fastest migrating molecular band traveled at the same rate as the non- size-tagged cysteine-substituted protein sample band (lane 8 vs. 7)(Figures 2-3A &

2-4A). The third band had an intermediate molecular weight and was about twice as dense. Statistically, a heterodimer containing a size-tagged and non-size-tagged protein is twice as likely to form, indicating the middle band is most likely a heterodimer. This pattern would only occur if PopB and PopD each formed homodimers in the cysteine crosslinking experiments. These three bands are made by disulfide bonds as proven by the addition of dithiothreitol (DTT) to the sample; this reductant is used to remove disulfide bonds in proteins (lane 9)(Figures 2-3A &

2-4A).

Assigning functions

After confirming that these interactions occur during infection, the next step was to discover how these interactions contribute to certain activities associated with the translocon’s function. The known functions that can be tested include insertion of pore proteins and subsequent formation of the pore, docking of the needle tip to the pore, and triggering of effector secretion. An insertion defect can be determined functionally by pore formation or directly by changes in the amount of

PopB or PopD found in the host cell membrane. Docking and triggering can be determined by monitoring translocation of effectors. To discern a difference between a docking or triggering defect, pcr1 can be deleted and effector levels can be measured. Under normal conditions, Pcr1 suppresses effector secretion before the needle contacts the host membrane. Deleting pcr1 causes the bacterium to

39 secrete constitutively, allowing it to overcome a triggering defect but not a docking defect. If the bacterium has a docking defect deleting pcr1 causes the bacterium to secrete into the medium instead of the host cell.

Pore formation was ascertained by a hemolysis assay, which assesses proper insertion of PopB and PopD into membranes by detecting hemoglobin release from red blood cells. These methods have been well established in the field as an appropriate way to measure pore formation (Blocker et al., 1999; Dacheux et al.,

2001; Broms et al., 2003a; Goure et al., 2004; Picking et al., 2005; Goure et al., 2005;

Broz et al., 2007). Recently one group called the legitimacy of the assay into question, because the test resulted in a false negative (Ekestubbe et al., 2016). They found that hemolysis was minimal and low levels of pore proteins were detected, which implied there was no pore formation. However, translocation did occur and pore proteins were found in HeLa cell membranes. This discrepancy indicates that loss of function results need to be more carefully scrutinized, but results demonstrating functional pores can still be considered valid. A PopD-YopD hybrid

(PYD268) with the last 27 amino acids of PopD replaced by corresponding YopD residues was used to test the effect of disrupting the previously identified PopD-

PcrV interaction (Figure 2-1). In the strain producing PYD268, the level of hemolysis was similar to wild type levels versus the levels in the negative control, ΔpcrV

(ΔV)(Figure 2-5A). Deleting pcrV acts as a negative control because PcrV is important for proper pore formation (Goure et al., 2004; Goure et al., 2005; Broz et al., 2007). Additionally, strains producing a PcrV-LcrV hybrid needle tip (LNPCV) or a strain that produces both LNPCV and PYD268 had hemolysis levels similar to wild

40 type compared to the negative control. The LNPCV needle tip restores the interaction between PopD and PcrV. These data suggest that the identified PopD-

PcrV interaction was not important for insertion of translocators into membranes or pore formation.

Data directly measuring PopB and PopD protein levels from isolated red blood cell membranes after infection correlated well with the hemolysis data.

Strains producing PYD268 inserted PopB and PYD268 into membranes similarly to wild type PopB and PopD levels versus the negative control, ΔV (Figure 2-5B). The negative control demonstrates that PcrV is needed for insertion of PopD, which is consistent with previous data (Goure et al., 2004; Picking et al., 2005). This result also indicates that PcrV is needed for PopB insertion, which is consistent with data from Picking et al. (2005) and Broz et al. (2007). PopB and PopD or PYD268 were also present after high salt (1 M KCl) or high pH (1 M Na2CO3) washes, demonstrating that the proteins were tightly associated with the membranes and not peripherally attached. A high salt wash can remove proteins that are associating with membranes through ionic interactions, while a high pH wash removes proteins associated with membranes through hydrophobic interactions. At first glance, these data seem to disagree with the idea that PcrV is necessary to insert PopD, but they merely demonstrate that the identified interaction is not important for insertion, while another interaction would be responsible for insertion. Additionally, I tested the ability of PopD and PYD268 to insert into membranes in the absence of PopB

(Figure 2-5C). Both PopD and PYD268 can insert into membranes with or without

PopB present. This experiment was to control for the possibility that PYD268 did

41 affect insertion, but the defect was overcome by assistance of PopB. These data further support the conclusion that the PopD-PcrV interaction doesn’t affect insertion or pore formation.

Next I tested whether the PopD-PcrV interaction had an effect on docking or triggering of effector secretion. I did this by testing the ability of strains expressing

PYD268 to properly translocate. Bacteria expressing PYD268 had a significant decrease in translocation compared to wild type, on average 12% (Figure 2-2B).

This defect can be overcome by restoring the interaction between PopD and PcrV by pairing it with the LNPCV hybrid needle tip, resulting in 102 + 29% translocation compared to wild type. This defect is likely due to a block in the signaling that initiates effector secretion, which is demonstrated by the recovery of secretion when pcr1 is deleted in a strain expressing PYD268 (Figure 2-2B). This decrease and recovery is indicative of a disruption of effector secretion triggering by an interruption of the PcrV-PopD interaction. An alternative explanation is that the hybrid needle tip has a higher rate of translocation seen in the LNPCV-only lane,

168% compared to wild type (Figure 2-2B), which compensates for the low translocation levels seen in the PYD268-expressing strains. However, the PYD268 translocation defect cannot be overcome when the secretion rate is increased

(Figure 2-6A). Secretion rate can be increased by introducing a mutation into pscO,

(E88K), which allows the T3SS to better utilize the proton motive force (pmf) for protein transport and therefore secretes at a faster rate (Lee et al., 2014). This suggests that the low levels of translocation seen in strains expressing PYD268 is not due to a slow secretion rate, which would have been compensated by

42 PscO(E88K), but rather that the defect is a result of an inability to initiate effector secretion.

An alternate explanation for how deletion of pcr1 compensates for the

PYD268 translocation defect relies on the AB toxin model. According to the AB toxin model discussed earlier, the deletion of pcr1 could mask the PYD268 defect by secreting effectors into the medium, which are then taken up by the pores and translocated. To determine if this was the case, cells were co-infected with a Δpcr1 strain without translocators along with the PYD268 strain. According to the AB toxin model, the PYD268 pores would take up the effectors secreted by the Δpcr1 strain and translocation would be restored. However, co-infection with the Δpcr1 strain did not compensate for the defect seen in PYD268 (Figure 2-6B), which is further evidence against the alternative AB toxin infection model proposed by

Akopyan et al. (2011).

When investigating the PopB and PopD homodimer interactions, I took a slightly different approach. I had already demonstrated the ability to crosslink and tether the proteins together during infection (Figures 2-3A & 2-4A). I then determined whether such crosslinking would affect translocation. Tethering PopD proteins together during infection causes a substantial defect in translocation: reduction to 21% compared to wild type PopD (Figure 2-3B). PopD(R243C) is able to translocate properly in the presence of a membrane-impermeable reducing agent, tris(2-carboxyethyl)phosphine (TCEP), which prevents crosslinking of

PopD(R243C) molecules. This observation demonstrates that the defect in translocation is not due to a possible interaction that is inhibited by the substituted

43 residue. This translocation defect can be overcome by the deletion of pcr1, which demonstrates that it is largely associated with triggering of effector secretion

(Figure 2-3B). Deletion of pcr1 does not affect the formation of the disulfide bond between PopD(R243C) molecules (lane 10)(Figure 2-3A). This same translocation defect can be found for tethering of PopB homodimers; however, the defect is much less pronounced (Figure 2-4B). The crosslinked proteins were still able to translocate 75 + 11% of ExoS compared to wild type. Taken together, these data suggest that the pore proteins need to move or have a certain amount of flexibility in order to function.

Conclusion

The PcrV-PopD, PopB-PopB, and PopD-PopD interactions have been confirmed by crosslinking the proteins together during infection (Figures 2-2A, 2-

3A, & 2-4A). When these interactions were individually disrupted or locked in place, translocation of ExoS decreased (Figures 2-2B, 2-3B, & 2-4B). Restoration of translocation was achieved either by restoring the interaction (pairing two hybrid proteins) or removing a negative regulator (deleting pcr1). The PopD-PopD, PcrV-

PopD, and to a lesser extent PopB-PopB interactions are involved with triggering of effector secretion (Figure 2-2B, 2-3B & 2-4B). If the pore molecules are unable to move (Figure 2-3B & 2-4B) or the signal is incapable of transferring from the pore to the needle tip (Figure 2-2B), then triggering of effector secretion does not occur. I propose a model of effector secretion, where an unknown factor causes a conformational change with the pore proteins (PopD-PopD), transfers to the needle

44 tip (PcrV-PopD), and then the needle components, which initiates effector secretion

(Figure 2-7). This model is echoed in the model proposed by the Blocker group

(Murillo et al., 2016).

Chapter 3

Cellular Contribution to Host Cell Contact Signaling

53 Introduction

An important question in the field is how T3SS+ bacteria know they have made contact with target cells. One way to approach this question is to see what components of the host cell are necessary for injection. P. aeruginosa have been found to trigger on a variety of target cells ranging from amoebae (Abd et al., 2008;

Pukatzki et al., 2006), to mouse immune cells and fibroblasts, human immune and epithelial cells (our studies). The diversity of targets suggests that the cellular component important for injection is ubiquitous. Numerous studies have tried to answer this question by broad screening studies or more focused studies that target a specific component. In this chapter, I detail my attempt to identify the cellular component using both broad and targeted strategies.

Targeting cellular components

ExoS is the best-studied effector of P. aeruginosa. ExoS contains two catalytic domains: a GTPase-activating protein (GAP) domain and an ADP-ribosylation

(ADPR) domain. The GAP domain mimics that of a eukaryotic GAP, which regulates

G-proteins by hydrolyzing GTP to GDP, effectively turning off the activity of the G- protein. The ADPR domain adds one or more ADP moieties to eukaryotic proteins, which can regulate the protein’s activity. ExoT shares a high sequence homology with the GAP and ADPR domains of ExoS. The targets of ExoS-GAP and ExoT-GAP domains are similar, while the targets of ExoS-ADPR and ExoT-ADPR domains differ

(Table 3-1). Additionally, the GAP domain of YopE, the Yersinia spp. homolog of

ExoS, shares a high sequence homology with ExoS and they have similar eukaryotic

54 targets (Table 3-1). The targets of the GAP and ADPR domains influence the host cell architecture; this targeting of cellular architecture is thought to promote survival by counteracting phagocytosis by immune cells (Hauser, 2009).

During infection of epithelial cells, P. aeruginosa bacteria appear to translocate low levels of ExoS; however, if both catalytic domains of ExoS are inactive (R146K/E379D/E381D, GAP-/ADPR-, denoted here as GA-), P. aeruginosa bacteria translocate high levels of ExoS (Figure 3-1 left panel; Cisz et al., 2008). The same observation has been made for YopE (Aili et al., 2008). These results indicate that active ExoS and YopE regulate their own translocation. For Yersinia spp., this feedback inhibition is thought to limit the host immune response, which increases survival (Isaksson et al., 2009). The simplest explanation for the mechanism of feedback inhibition is that ExoS and YopE target the cellular component that is responsible for triggering of effector secretion. The target of ExoS that modulates its secretion is unknown. Identification of this target is crucial because it can give further insight into how the cell contributes to sensing of host cell contact.

Lipid rafts and endocytosis

At this time, the consensus theory is that cholesterol is the cellular component necessary for injection. This conclusion is based on several studies that found that cholesterol depletion prevented infection (Lafont et al., 2002; van der

Goot et al., 2004; Hayward et al., 2005; Kannan et al., 2008; Verove et al., 2012).

Cholesterol and cholesterol esters have been identified as important for P. aeruginosa adherence to host cells (Rostand and Esko, 1993). Additionally, PopB,

55 IpaB, and SipB bind cholesterol in vitro (Faudry et al., 2006; Hayward et al., 2005), but PopB and PopD can insert into membranes devoid of cholesterol (Faudry et al.,

2006). These observations do not directly point to cholesterol as being a ‘receptor’ for the bacteria, but rather, they demonstrate an indirect role for cholesterol during infection. To further this point, P. aeruginosa cannot translocate effectors into undifferentiated HL-60 cells, a Human promyelocytic leukemia cell line, even though the cells have cholesterol in their membranes (Rucks and Olson, 2005; Ip and

Cooper, 1980). This observation demonstrates that cholesterol is not directly responsible for infection by P. aeruginosa.

Cholesterol is a major player in membrane fluidity and composition, which can affect many aspects of cellular function, including the formation of microdomains or lipid rafts. Several studies have suggested that lipid rafts play a part in infection (Lafont et al., 2002; van der Goot et al., 2004; Hayward et al., 2005;

Kannan et al., 2008). In addition to cholesterol, lipid rafts are also defined by the presence of sphingolipids, which are suggested to play a role in phagocytosis

(Tafesse et al., 2015). Acid sphingomyelinase (ASM) was found to be released upon injection by P. aeruginosa and this ASM release created ceramide clustering, which was hypothesized to be important for internalization of P. aeruginosa (Grassme et al., 2003). Furthermore, inhalation of different ASM inhibitors prevented P. aeruginosa infection in the lungs of cystic fibrosis mice, supporting the importance of ceramide formation for infection by P. aeruginosa (Becker et al., 2009). These studies have demonstrated a role of ceramide during P. aeruginosa infection, but they have not specified how it would contribute mechanistically to injection.

56 The release of ASM and conversion of surface sphingolipids to ceramide is associated with the membrane damage repair (MDR) pathway (Figure 3-2) (Tam et al., 2010; Corrotte et al., 2013). The MDR pathway is a process by which the cell can repair mechanical damage to the membrane or excise pathogenic pores from the membrane, both of which can lead to a loss of membrane integrity. The MDR pathway involves sensing of increased cytoplasmic calcium levels by the cell, which triggers exocytosis of liposomes carrying ASM. Outside the cell, ASM converts sphinoglipids into ceramide. Clustering of ceramide initiates invagination, which leads to Caveolin-1-dependent endocytosis that removes the damaged membrane, restoring membrane integrity (Tam et al., 2010; Corrotte et al., 2013). It could be possible that P. aeruginosa manipulates this process in order to infect the host cell.

Adenovirus has been found to subvert the MDR to gain entry into the host cell

(Luisoni et al., 2015).

Additionally, Sheahan and Isberg (2015) screened for host cell components important for injection and saw an enrichment of trafficking and endocytotic pathways. This is in agreement with data mentioned above that demonstrates the importance of phagocytosis during injection, which is related to endocytosis. It is possible that other or additional endocytic pathways are also involved during injection.

Membrane curvature

Our lab examined effector secretion feedback during injection of epithelial cells or phagocytic cells. P. aeruginosa strains that infected phagocytic cells did not

57 exhibit the same feedback inhibition by ExoS seen during injection of epithelial cells

(Figure 3-1). When cytochalasin D, which depolymerizes actin, was added to prevent phagocytosis by macrophages, feedback inhibition by ExoS was restored

(Figure 3-1 right panel). These data suggest that phagocytosed bacteria receive a constant trigger, whereas bacteria that remain on the surface have a transient trigger. This conclusion implies that phagocytosis somehow contributes to triggering of effector secretion, which is underlined by the fact that ExoS targets proteins that contribute to phagocytosis.

Phagocytosis and endocytosis both have been implicated in contributing to triggering of effector secretion. These processes transport extracellular entities into the cell, meaning they both manipulate membrane curvature. It is possible that the conformational change of the pore proteins, stated in Chapter 2, is caused by a change in membrane curvature and this is what causes initiation of effector secretion. This hypothesis is supported by Viboud and Bliska (2001), who speculated that ruffling of the membrane around bacterial attachment sites caused

Yersinia spp. to secrete YopE, which inhibited actin polymerization in order to prevent disruption of the conduit formed by the translocon.

Previous studies have been unable to determine the cellular components or processes that contribute to infection. I took a three-pronged approach to clarifying how the host cell contributes to triggering of effector secretion. The first strategy was to determine how ExoS modulates its own translocation. The second strategy was to determine if the MDR or other defined endocytotic pathways were important for triggering of effector secretion. The third strategy was to see if I could

58 recapitulate membrane curvature seen by the bacteria during phagocytosis to determine if this was sufficient to trigger effector secretion.

Materials and Methods

Bacterial strains, cells, and growth conditions

A549 cells (American Type Culture Collection, Cat. #CCL-185) were grown in

RPMI1640. Murine embryonic fibroblasts (MEF) (cav-1+ and cav-1-; ATCC, Cat.

#CRL-2752 & #CRL-2753, respectively) were grown in Dulbecco’s Modified Eagle’s

Medium (DMEM, Invitrogen). BJ (human normal fibroblast from foreskin; ATCC, Cat.

#CRL-2522) and NPA (Human fibroblast Niemann Pick Type A positive; Coriell

Institute, Cat. #GM00112) were grown in Eagle’s Minimum Essential Medium

(EMEM, ATCC Cat. #30-2003). All media were supplemented with 10% fetal bovine serum. All cells were kept at 37°C in a 5% CO2 atmosphere. Cells were maintained in the presence of penicillin and streptomycin. Before experiments, cells to be infected were washed once with Dulbecco’s phosphate buffered saline (DPBS, Cat. #14190), and the medium was exchanged with corresponding media lacking antibiotics.

59 Plasmid and strain construction

Mutations were introduced into the chromosome of strains through allelic exchange as described previously (Rietsch et al., 2005). Plasmids were constructed using standard molecular biological techniques. PCR products were cut with the appropriate restriction enzymes and ligating into plasmids pEXG2 (allelic exchange vector) or pPSV37 (plasmid that can replicate in P. aeruginosa with lacUV5 promoter and lacIq).

siRNA knockdown

The protocol used was modified from the manual for Lipofecamide RNAiMAX

(Invitrogen). A549 cells were plated at 1.5 x105 cells per well in a 6-well plate in

RPMI media without serum. Cells were allowed to attach for 18-24 hours. For each well, Lipofectamine (5 μL, Invitrogen, Cat. #13778) was added to 250 uL opi-MEM

(Thermo Fisher, Cat. #31985062), while siRNA (3 μL, 30 pmol) was added to 250 uL opi-MEM. The two solutions were mixed together and incubated for 10-20 minutes at room temperature. 500 μL of the Lipofectamine and siRNA mixture was added to each well and incubated for 18-24 hours. siRNA targeted Caveolin-1 (Sigma-Aldrich,

Cat. #SASI_Hs01_00199504), Flotillin-1 (Sigma-Aldrich, Cat.

#SASI_Hs01_00229858), Flotillin-2 (Sigma-Aldrich, Cat. #SASI_Hs01_00222343), or

Control sequence (Sigma-Aldrich, Mission Universal Control #1, Cat. #SIC001).

Knockdown was confirmed by Western blot with corresponding antibodies,

Caveolin-1 (Santa Cruz Biotechnology Inc.), Flotillin-1 (Sigma-Aldrich), and Flotillin-

60 2 (Sigma-Aldrich). The protein of interest was compared to an internal control of tubulin (Santa Cruz Biotechnology Inc.).

Translocation assay

P. aeruginosa strains were grown to mid-logarithmic phase in “High salt” LB, pelleted and resuspended in PBS-MC. In ASM inhibitor experiments, cells were preincubated with 30 μM of desipramine (DPA, Sigma-Aldrich, Cat #D3900), fluoxetine (FLX, Sigma-Aldrich, Cat. #F132), or vehicle control for 30 minutes before bacteria were added without changing the media. A549 epithelial cells were infected with P. aeruginosa strains at an MOI of 25 for 2 hours. The cells were then washed three times with PBS-MC, and rinsed with 1 ml of 250 μg/mL proteinase K in PBS-MC. The protease solution was then removed, and the cells were incubated at room temperature for 15 minutes to digest extracellular protein. The protease- treated cells were resuspended in 1ml of PBS-MC with 2 mM phenylmethylsulfonyl fluoride (PMSF), and pelleted (3 minutes, 5000 rpm). The cells were resuspended in

300 μL of PBS-MC with 0.5% Triton X-100 and incubated on ice for 15 minutes. 150

μL of the cell suspension were removed and mixed with 50 μl of 4X SDS sample buffer (SDS sample). The remaining cells were pelleted, and 150 μL of supernatant were removed and combined with 50 μL of 4X SDS sample buffer (Triton solubilized fraction). The samples were separated by SDS-PAGE and analyzed by Western blot.

Membranes were probed with antibodies directed against ExoS, RpoA (Neoclone) and either human glucose-6-phoshate isomerase (G6PI, Santa Cruz Biotechnology

Inc.), actin (Developmental Studies Hybridoma bank), or tubulin (Santa Cruz

61 Biotechnology Inc.). Antibodies were detected using a horseradish peroxidase labeled secondary antibody, and WesternBright Quantum reagent and imaged using a GE ImageQuant LAS4000 imaging system. Protein levels were quantitated using

ImageJ, and ExoS levels were normalized to G6PI (or actin, or tubulin)-levels.

Membrane damage repair assay

Streptolysin O (SLO; from Stepococcus pyogenes; Sigma Aldrich, Cat. #S5265) was activated by adding 10mM DTT and incubating for 15 minutes. Chilled medium

(0.5 mL) with or without 0.5 U/uL of activated SLO was added to cells. The plate was incubated for 5-15 minutes at 4°C. Warm medium (0.5 mL) was added to cells. One plate was incubated for 3 minutes in a 37°C cabinet (4 min condition). The other plate had media removed and replaced with 0.5 mL media and 50ug/mL propidium odide (PI, Sigma Aldrich, Cat. #P4170) for 1 minute. The same procedure was conducted for the other plate. Media was removed, cells washed once, and cells were fixed with 3.7% formaldehyde for 15 minutes in the dark. Cells were mounted with Prolong Gold anti-fade (Thermo Fisher #P36935) and incubated overnight at room temp.

Microwell assay

Microwells were manufactured and treated by the Gurkan lab (Dept. of

Mechanical & Aerospace Engineering, Case Western Reserve University). The wells are made out of polydimethylsiloxane (PDMS) and were plasma treated to create a hydrophobic surface to attract the lipids (Andreasson-Ochsner and Reimhult, 2013).

62 The lipid mixture comprised phosphatidylcholine (PC, Avanti Product #840054C) and phosphatidylserine (PS, Avanti Product # 840034C) at a ratio of 8 mol: 2 mol

(Faudry et al., 2006). Marina Blue 1,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine (Thermo Fisher Product #M12652), a fluorescent lipid marker, was added to the lipid mixture at 1% of the final concentration by weight

(Faudry et al., 2006). The lipids were mixed and then chloroform was evaporated under air flow for 20-30 minutes (Andreasson-Ochsner et al., 2011). The lipid was suspended in 500 μL of buffer (50 mM, Tris pH 7.5, 5 mM MgCl2) (Deleon-Rangel et al., 2013). The lipid was passed through an extruder with a pore size of 400 nm, 20 times to create uniform vesicles (Avanti). The lipid mixture was diluted to a final concentration of 5 mg/mL (Andreasson-Ochsner and Reimhult, 2013). The lipid bilayer was created by incubating the lipid mixture with the microwells for 30 minutes. The solution was pipetted every 5 minutes to keep the solution mixed. No lipid control wells were incubated with buffer. The wells were washed twice with buffer.

Bacteria were grown up in high salt LB media until mid-log. Bacteria were diluted to 5 x108 cfu/mL. After lipid overlay, bacteria were incubated with the microwells for 15 minutes to allow bacteria to enter microwells. The bacteria mixture was removed and replaced with fresh media and incubated for 75 minutes.

The wells were washed once with high salt LB and once with PBS. The cells were then fixed with 2% paraformaldehyde (PFA) for 20 minutes. The microwells were then mounted upside down on with Prolong Gold anti-fade. Wild-type bacteria were compared to a positive control (Δpcr1) and a negative control (ΔpopB). No lipid

63 control is an additional sham control for some aspects of the experimental set up that are not the lipid or cell curvature. In order to allow ample time for detection, I included a condition with the addition of EGTA during the 75 min incubation.

Samples were imaged the same day on an inverted confocal Leica DM6000 confocal microscope.

Results and Discussion

Determining cellular targets of ExoS

The first strategy was to figure out which cellular target of ExoS was influencing feedback inhibition. This was accomplished by first narrowing down which catalytic domain was largely responsible for feedback inhibition. I did this by comparing the amount of translocated ExoS for strains expressing wild-type ExoS

(S+), or ExoS with either or both mutations in its catalytic domains (G-, A-, or GA-)

(Figure 3-3A). Initially, it appeared as if the ADPR domain is mainly responsible for the feedback inhibition, because when only the ADPR domain is active the phenotype more closely resembles wild-type ExoS (G- vs. S+). This conclusion was supported by the observation that when the ADPR domain is inactive, the bacteria have deregulated translocation (A- vs GA-). However, these data were collected with a strain that produced ExoT, and because ExoT has similar activity to ExoS (Table 3-

1), it could obscure results from the translocation experiments.

When the experiments were conducted in the absence of ExoT (Figure 3-4A), the GAP domain of ExoS appeared to contribute principally to the feedback inhibition. This can be seen when comparing the strain deficient in GAP activity to

64 the catalytically inactive ExoS (G- vs. GA-) or comparing the strain only containing active GAP to wild-type (A- vs. S+). This reversal could be explained by the similarity in targets of the GAP domains of ExoS and ExoT (Table 3-1). It is possible that in the presence of ExoT, the GAP domain of ExoS is functionally redundant, but the ADPR contributes to feedback inhibition in a non-redundant manner. This gives the appearance that ADPR is the main source of feedback inhibition. These data demonstrate that both domains of ExoS can contribute to feedback inhibition in a non-redundant manner. This implies that the domains target similar processes through different pathways. Additionally, this conclusion is not exclusive to epithelial cells or human cells, because the same pattern is observed in mouse embryonic fibroblasts (MEF cells)(Figure 3-3B & Figure 3-4B), which implies that this regulatory control could be applicable to most or all cell types.

To confirm the redundancy of the ExoS-GAP and ExoT-GAP domains, I looked at the effect the ExoT-GAP domain has on ExoS translocation. The level of translocation of ExoS(GA-) in the absence of ExoT is similar to the level of translocation of ExoS(GA-) with ExoT(G-) (Figure 3-5). This observation supports the idea that the effect of ExoT is largely a result of its GAP domain, which is consistent with the hypothesis stated above. These data suggest that ExoT also contributes to regulation of translocation. This was consistent with previous data that demonstrated that preintoxication of ExoT was able to moderately prevent triggering by P. aeruginosa, although ExoS was able to completely prevent triggering

(Cisz et al., 2008). These data suggest ExoT has the capacity to regulate translocation, although, ExoS is the key regulator.

65 The conclusion that the GAP domain is responsible for the modulation of translocation into host cells is consistent with previous research, which implicated the GAP domain as the main driver of the feedback inhibition (Cisz et al., 2008). In agreement, high levels of effectors are translocated when YopE, ExoS, and ExoT GAP mutants are expressed compared to corresponding wild-type proteins in the

Yersinia spp. system (Aili et al. 2006; Aili et al. 2008).

Not only are the catalytic domains important for ExoS function, but localization of ExoS is also important for its function (Zhang and Barbieri, 2005).

This work identified a leucine-rich domain of ExoS, which was termed the membrane localization domain (MLD), because when deleted, ExoS no longer localized to the membrane. They found that if four leucine residues within the MLD were mutated to asparagine residues (4L-N), the localization of ExoS was similar to when MLD was deleted. This mislocalization did not occur if two arginine residues and a glutamic acid residue within the MLD were mutated to asparagine residues

(2RD-N). Bacteria expressing ExoS(ΔMLD) or ExoS(4L-N) were unable to modify

Ras, a target of ExoS, nor were they able to cause cell rounding in the same time frame as wild type ExoS (Zhang and Barbieri, 2005). Cell rounding is not the best measurement of feedback inhibition, because cell rounding is found in samples where the bacteria impaired feedback inhibition (unpublished observation).

To test whether deleting the MLD would affect feedback inhibition of ExoS translocation, we generated a strain of P. aeruginosa that produced a version of ExoS lacking its MLD, ExoS(ΔMLD). ExoS(∆MLD) did not feedback inhibit its own translocation, much like catalytically inactive ExoS (GA-)(Figure 3-6A). This is in

66 agreement with Isaksson et al. (2009). The authors concluded that the MLD of YopE contributed to the feedback inhibition of YopE because they found high levels of effector expression, which is associated with deregulated effector secretion.

However, the authors did not see increased translocation by the YopE(ΔMLD) mutant. They concluded that YopE regulation of effectors was separate from the catalytic function. This does not match our data in which ExoS(ΔMLD) had high levels of translocation similar to deregulated effector secretion caused by catalytically inactive ExoS(GA-). Although I agree that the MLD is important for feedback inhibition of ExoS, I think this phenotype is linked with its catalytic function and cannot be uncoupled.

Additionally, the ExoS(4L-N) mutant exhibited a translocation phenotype similar to ExoS(GA-) and ExoS(ΔMLD). This observation reiterates that the leucine residues are necessary for localization of ExoS (Zhang and Barbieri, 2005) and that localization is important for feedback inhibition. The ExoS(2RD-N) mutant had a moderate translocation phenotype, ~30% translocation compared to ExoS(GA-) similar to the level of translocation for ExoS(A-) (Figure 3-6A & Figure 3-4A).

Previously, ExoS(2RD-N) was found to have altered localization compared to wild type ExoS and that this difference in localization affected the ability of the ExoS-GAP domain to target Rac1 (Zhang et al., 2007). This suggests that the defect in localization of ExoS(2RD-N) could affect the ability of ExoS-GAP domain to control effector secretion through modulation of Rac1.

Because previous data demonstrated that the altered localization of

ExoS(2RD-N) affects the ability of ExoS to modify eukaryotic targets, I wanted to test

67 whether it affected the activities of the GAP and/or the ADPR domain. Strains expressing ExoS with the 2RD-N mutation along with mutations in either or both catalytic domains were created (Figure 3-6B). The level of ExoS(2RD-N/G-) translocation was similar to ExoS(G-) compared to ExoS(GA-); this suggests that the

ExoS(2RD-N) mutation had no effect on the ADPR domain’s activity. On the other hand, the level of ExoS(2RD-N/A-) translocation had an intermediate phenotype, meaning that the 2RD-N mutation does affect the ability of the GAP domain to modify targets that lead to feedback inhibition. This corresponds with observations made by Zhang et al. (2007); more specifically, targeting Rac1 by ExoS affects control of effector secretion into host cells.

Blocking endocytic pathways

The second approach focused on examining if the membrane damage repair

(MDR) pathway (Figure 3-2) or another endocytic pathway influenced triggering of effector secretion. MDR is a process by which a cell can repair damage to its membrane. It involves several steps, starting with detection of damage by an influx of calcium ions from the extracellular space. Second, the cell exocytoses acid- sphingomyelinase (ASM), which converts sphingomyelin to ceramide. Third, clustering of ceramide on the surface initiates invagination. Fourth, the damage is then endocytosed in a Caveolin-1-dependent manner (Figure 3-2)(Tam et al., 2010;

Corrotte et al., 2013). The connection between MDR and P. aeruginosa is based on previous research that suggested that treatment with ASM inhibitors helped prevent

P. aeruginosa infection in cystic fibrosis mouse model lungs (Becker et al., 2009). I

68 hypothesized that the pore formed by T3SS causes an influx of calcium, which in turn initiates the MDR process, which triggers effector secretion.

To test this hypothesis, I first monitored intracellular calcium levels during infection using a calcium sensitive fluorescent probe. I found that the cells infected with bacteria had higher levels of intracellular calcium compared to a media-only control (Figure 3-7A). However, this increase from baseline was not dependent on pore formation by the bacterium as hypothesized; i.e. an increase in intracellular levels from the baseline were seen in cells infected with ΔpcrV strains, which do not form pores within the host cell membrane. Also, calcium levels appear to be higher in strains that do form pores in host cells (ΔexsE); however, the difference in calcium levels is small and likely insignificant. These data suggest that there are calcium fluxes that occur during infection but are not caused by T3SS pore formation. To test whether intracellular calcium fluxes contributed to injection, I selectively chelated intracellular but not extracellular calcium and infected cells with P. aeruginosa strains. If the calcium fluxes were important for triggering of effector secretion, then chelating intracellular calcium would block effector translocation. Chelating intracellular calcium did not significantly affect translocation (Figure 3-7B); however, the variability between experiments was high, which makes drawing conclusions difficult. It is possible that the calcium fluxes are important for injection but in a localized manner, which neither method is sensitive enough to detect. In order to determine this, calcium levels and injection would have to be imaged for each individual cell. Alternatively, it is possible that infection by P. aeruginosa causes changes in calcium concentrations, but those fluxes

69 are dispensable for injection. However, the importance of calcium during infection of Salmonella sp., a T3SS+ bacteria, is well documented (Pace et al., 1993), arguing against the latter explanation.

The second approach I used to test if the MDR was involved in triggering was to see what role ASM played during infection. I did this by measuring the difference between translocation into Niemann-Pick Type A (NPA) cells that do not express

ASM because of a genetic defect and a wild-type control cell line (BJ) (Figure 3-8A).

If the MDR pathway is involved in triggering of effector secretion, then translocation into the NPA cells should be defective compared to the wild-type control. There was no significant difference in translocation when comparing the two cell types, suggesting that the MDR is not involved. To confirm this conclusion, I transiently blocked ASM using two different ASM inhibitors, desipramine (DPA) or fluoxetine

(FLX) (Figure 3-8B). FLX had a moderate inhibitory effect on translocation, while

DPA had no significant effect. These data are inconsistent, but they could differ because of the two drugs target ASM by different mechanisms. These data suggest that ASM is not the main driver in triggering of effector secretion, which is unexpected because previous data found that infection by P. aeruginosa was prevented with treatment of ASM inhibitor (Becker et al., 2009). This discrepancy could be because our data focused on translocation of effectors and Becker et al.

(2009) looked at the bacterial load in lungs of a mouse model. It is possible that ASM has an effect in vivo that does not relate to translocation of effectors into host cells.

The last approach I implemented was to establish if Caveolin-1-dependent endocytosis is involved in injection of effectors. I compared translocation between a

70 cell line with a stable deletion of cav-1 (Cav-1 (-)) and a wild-type control cell line

(Cav-1 (+)) (Figure 3-9A). There was no significant difference between the two cell lines, suggesting that Caveolin-1 has no effect on injection by P. aeruginosa. On the other hand, because Cav-1(-) is a stable knockout cell line, it is possible that the cells developed compensating methods for the loss of cav-1. To account for this, I transiently knocked down cav-1 using specific siRNA against the gene. An average knockdown of 29% of wild-type levels was achieved using this method (Figure 3-

9B). To determine whether this level of knockdown is sufficient to have a physiological effect, I assayed membrane damage repair after pore-formation by streptolysin O (SLO)(Tam et al., 2010). Knockdown of cav-1 had a modest effect on translocation (Figure 3-9B) and prevented MDR, as shown by propidium iodide (PI) staining after SLO exposure (Figure 3-10). This decrease in translocation after cav-1 knockdown supports the idea that the MDR contributes or possibly alternative

Caveolin-1-dependent endocytic process contributes to triggering of effector secretion; however, this effect is moderate and implies that there are other elements involved that have yet to be defined.

I also tested to see if other endocytic processes might be involved during infection. Pore proteins were found to associate with Flotillin positive lipid rafts

(Verove et al., 2012), and Flotillin-1 and -2 define an endocytic pathway that is independent of Clathrin or Caveolin (Glebov et al., 2005). Furthermore, Flotillin-1 was found to colocalize with Chlamydia pneumonia, a T3SS+ bacterium, inclusions and it plays a role in intracellular growth (Korhonen et al., 2012). To test if Flotillin- dependent endocytosis plays a role in triggering of effector secretion, I transiently

71 knocked down flotillin-1 or flotillin-2 with specific siRNA and measured levels of translocation into the cell. I was able to reduce Flotillin-1 or -2 levels to 32% or 17% of wild type levels, respectively (Figure 3-11B & Figure 3-12B). Translocation was not significantly affected by knockdown of flotillin-1 (Figure 3-11A). On the other hand, translocation did decrease after knockdown of flotillin-2, suggesting Flotillin-2 does influence triggering of effector secretion (Figure 3-12A). These data suggest that Flotillin-dependent endocytosis is not mainly responsible for triggering of effector secretion, but it may play a small role.

The MDR and Flotillin-dependent endocytic pathways individually have a slight effect on triggering of effector secretion. The minor influence means that these pathways are not solely responsible, but it is possible that these pathways could be sufficient to trigger effector secretion. Furthermore, the bacterium could manipulate several different pathways in order to initiate effector secretion, which means that when testing the effect of one pathway on a population, only a slight decrease is seen.

Effector secretion trigger mechanism

The third approach I took was to recapitulate a minimal system in which the bacterium can trigger. In order to determine if a curved membrane is sufficient to indicate host cell contact, I tested the ability of lipid-coated curved surfaces to trigger effector secretion in P. aeruginosa. The wells were coated with a PC:PS lipid mix with marina blue lipid dye for visualization (Figure 3-13A). This lipid mixture was shown previously to support PopB and PopD insertion (Faudry et al., 2006).

72 The strains constitutively expressed mCherry, while GFP expression acted as an indicator of effector secretion triggering. Past studies have demonstrated that ExoS is upregulated at the transcriptional level upon triggering of effector secretion

(Frank, 1997). I used GFP as a transcriptional reporter for effector secretion by replacing exoS with gfp while maintaining the exoS promoter regions (Cisz et al.,

2008). Simply, if effector secretion is triggered, then the cell will express GFP.

Initial experiments revealed low levels of GFP expression in wild type strains, leading to optimization of the system. Optimization included longer incubation time to increase the GFP signal and utilizing a superfolder GFP that was optimized specifically for P. aeruginosa. Additionally, the promoter region of exsA was mutated to improve promoter binding and therefore enhance transcription of T3SS genes.

This enhancement would increase transcription upon activation and increase the amount of T3SS apparati, which would increase signal by providing more instances that an apparatus would interface with the lipid membrane. Conversely, these modifications did not increase the GFP signal seen in wild type (WT) conditions

(Figure 3-13B). The wild type bacteria more closely resembled the negative controls

(ΔpopB or WT no lipid) than any of the positive controls (Δpcr1, ΔpopB +EGTA, or

WT +EGTA). The EGTA control demonstrates the capacity to which the strains can be induced. The low level of triggering after optimization of the system suggests that the curved membrane is not sufficient for inducing effector secretion. Previous data have suggested that additional elements are needed for infection, such as cholesterol or vimentin (Lafont et al., 2002; Russo et al., 2016). The addition of cholesterol or vimentin to the lipid mixture did not increase the level of triggering

73 (data not shown). These data suggest that membrane curvature and a minimal lipid bilayer that supports PopB and PopD insertion are not sufficient for triggering of effector secretion. In fact, several groups have suggested that additional membrane proteins are needed for pore formation or translocon stability (Sheahan and Isberg,

2015; Lafont et al., 2002).

Conclusion

At this time, lipid rafts, endocytosis, and phagocytosis have all been implicated in contributing to infection by P. aeruginosa. Exactly how these entities contribute to translocation has not been examined. One way to elucidate these details is to discover how ExoS contributes to regulation of effector secretion. My studies showed both GAP and ADPR domains of ExoS have the capacity to influence feedback inhibition; however, the GAP domain of ExoS is the major contributor to feedback inhibition when looking solely at ExoS translocation. Proper localization of

ExoS affects the ability of the GAP domain to regulate feedback inhibition of effector secretion. This suggests that the main eukaryotic target that contributes to triggering of secretion is Rac1. Additionally, I hypothesized that certain endocytic processes such as the membrane repair pathway or Flotillin-dependent endocytosis contributed to triggering of effector secretion. There was a modest decrease in translocation when the MDR pathway or Flotillin-dependent endocytosis were blocked, which suggest that these processes have role in triggering of effector secretion, but they are not the main cause of triggering. Also, membrane curvature alone was not sufficient to trigger effector secretion. It is possible that in

74 combination with certain lipids or receptors, membrane curvature could play an important role.

75 Table 3-1: Cellular targets of ExoS and ExoT

Protein Domain Eukaryotic targets ExoS GAP RhoA, Rac, Cdc42 ADPR Ras, vimentin, ezrin, radixin, moesin, Rab family proteins ExoT GAP RhoA, Rac, Cdc42 ADPR CrkI, CrkII

Chapter 4

Conclusion and Future works

90 My thesis work revolved around answering how Pseudomonas aeruginosa detects host cell contact to engage the Type III Secretion System (T3SS). This question is helpful for the development of new treatments for infections by P. aeruginosa and other T3SS+ pathogens. I attempted to answer this question by exploring how the bacterial-host cell interface functions and what eukaryotic components are necessary for injection by the T3SS.

Bacterial protein interactions

To understand the bacterial involvement in sensing of host cell contact, I studied the interactions between the bacterial proteins that form the translocon, which creates a conduit from the T3SS into the host cell cytoplasm. I determined that an interaction between PopD and PcrV as well as the ability of the pore proteins to move freely was important for triggering of effector secretion. These data argue that the translocon actively participates in sensing host cell contact, rather than simply serving as a conduit for effector injection. More specifically, the pore undergoes a conformational change within the host cell membrane, which indicates cell contact. Next, the signal is transferred to the needle tip through the PopD-PcrV interaction, and then transferred to the needle shaft and basal body to activate substrate switching.

Our proposed model is echoed in the model proposed by Murillo et al.

(2016), which based their model on constitutively active mutants. They concluded that IpaB, a PopB homolog, undergoes a conformational change upon insertion into the eukaryotic membrane. This change transfers a signal to IpaD, a PcrV homolog,

91 and IpaD transmits the signal to MxiH, a PscF homolog. Altogether their data suggests a signal transferring from the cellular side to the bacterial side. This model is likely to occur during P. aeruginosa infection, because constitutively secreting mutants of PcrV and PscF have been discovered (Sato et al., 2011; Torruellas et al.,

2005). These mutations force the protein into a conformation that resembles the

‘on’ state. Because our model postulates that the pore is involved in signaling of host cell contact, we should be able to obtain PopD mutants that are resistant to feedback inhibition by ExoS, i.e., stuck in the ‘on’ conformation. However, designing a screen to find these mutants would be difficult, because the interaction between the pore and apparatus is most likely transient, which would cause the signal to be weak.

What is needed is a reporter that turns on and amplifies the signal.

Additionally, isolating translocons during injection would give more insight into the specific state of the translocon during injection. This could be achieved by introducing a His-tag into PcrV at permissible sites. The protein can then be isolated after infection by running lysates over a nickel column. Pore proteins would be expected to purify along with PcrV. However, if the interaction is weak, few pore proteins would purify along with PcrV. This could be addressed by crosslinking the pore proteins to PcrV before purification of the complex. This purification could give insight into conformation of the translocon during infection, stoichiometry of the pore, and orientation of the pore proteins. Additionally, several groups have suggested that eukaryotic proteins interact with the translocon, which could increase stability of translocon, which would increase translocation (Sheahan and

Isberg, 2015; Skoudy et al., 2000; Russo et al., 2016). In order to identify

92 interactions with eukaryotic transmembrane proteins that are important for translocation, we could crosslink translocators to these host proteins, followed by purification and identification. A promiscuous crosslinker such as N-γ- maleimidobutyryl-oxysuccinimide ester (GMBS), succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), or p-benzoyl-l-phenylalanine

(BPA) could be used to hit a wide variety of targets. Isolating the protein and then sending the sample for analysis by mass spectrometry would be used to identify these host proteins. After the component is identified additional experiments could be performed to determine how it contributes to translocation.

Cellular targets

When studying the cellular contribution to injection by the T3SS, I took a three-pronged approach. The first prong was aimed at determining the cellular component that influenced the feedback inhibition of translocation by ExoS. In the presence of ExoT, the ADPR domain of ExoS was the major influence on feedback inhibition. However, in the absence of ExoT, the GAP was the main contributor to feedback inhibition by ExoS. This difference is most likely because the GAP domains of ExoS and ExoT share similar targets within the host cell. Data from Yersinia spp. found that the GAP domain of YopE, the ExoS homolog, was important for feedback inhibition in the Yersinia system (Aili et al., 2008). For P. aeruginosa, both catalytic domains of ExoS are capable of causing feedback inhibition, which suggests that the two domains influence feedback inhibition in a non-redundant manner. For example, many targets of both the GAP and ADPR domains target proteins that

93 influence the host cell cytoskeleton, which is thought to counteract phagocytosis

(Hauser, 2009). This observation is consistent with our hypothesis that the trigger that initiates effector secretion is related to phagocytosis.

The targets of the GAP domain are well defined, which is in contrast to the

ADPR domain that modifies a variety of cellular components, not all of which have been identified. In order to determine the target modified by the ADPR domain that is responsible for feedback inhibition, we can tag, isolate, and identify novel targets.

Cells are injected with ExoS by P. aeruginosa strains. The cells are then permeabilized and incubated with biotinylated NAD. This allows ExoS to add an ADP moiety with a biotin tag to its targets. This tag can then be used to track and isolate the protein; the protein can then be sent for analysis by mass spectrometry. Strains expressing ExoS(G-) or ExoS(GA-) in the absence of ExoT would be compared to narrow down the possible targets. After identifying novel targets, we could conduct further studies to determine how the identified targets contribute to injection by P. aeruginosa.

Additionally, I confirmed that the localization of ExoS to the membrane was important for its regulatory effect. When the membrane localization domain was deleted, ExoS(ΔMLD) was unable to feedback inhibit its translocation. Additionally, when two arginine residues and an aspartic acid residue within the MLD were changed to asparagines (ExoS(2RD-N)), the ability of the GAP domain to control feedback inhibition was reduced. This observation is consistent with previous data, which suggested that ExoS(2RD-N) had an altered localization compared to wild type ExoS and this mislocalization impaired the GAP domain’s ability to target

94 eukaryotic components (Zhang et al., 2007). In contrast, ExoS(2RD-N) did not affect the activity of the ADPR domain.

Combined data from our lab and the Barbieri group suggest that Rac1 is the target of ExoS responsible for feedback inhibition (Zhang et al., 2007). In order to confirm this hypothesis, we can test whether a dominant active form of Rac1 prevents feedback inhibition by ExoS. Zhang et al. (2007) previously used dominant active proteins to determine that Rac1 was responsible for phenotypic differences in cell rounding. However, the authors did not look at the effect on effector translocation or feedback inhibition. Although, cell rounding is associated with functional translocation of wild type ExoS, cell rounding and feedback inhibition are not correlated. However, if Rac1 is not the target, we could also test the ability of dominant active RhoA or Cdc42 to block feedback inhibition.

Rac1 is an attractive candidate, because data has suggested that Rac1- mediated macropinocytosis could be important for infection by Salmonella spp. and

Shigella spp. (Francis et al., 1993; Sansonetti et al., 2001). This is partially based on the observation that membrane ruffling similar to the initial steps of macropinocytosis is seen around sites of bacterial contact (Lim and Gleeson, 2011).

Macropinocytosis is an actin-mediated endocytic process, which the cell uses to non-selectively uptake extracellular solutes and nutrients (Lim and Gleeson, 2011).

Actin clustering, like that seen in the early steps of macropinocytosis, could contribute to formation of a functional translocon (Bridge et al., 2010; Viboud and

Black, 2001). Additionally, antigen-presenting cells use this process to sample the environment for antigens (Lim and Gleeson, 2011). Macropinocytosis is regulated

95 through a variety of molecules including Ras, Rac1, and Cdc42 (Lim and Gleeson,

2011). This observation is notable because all of the listed molecules are targeted by

ExoS (Hauser, 2009), suggesting a possible answer to how ExoS could modulate effector translocation (Figure 4-1). This process is evolutionarily conserved and has been studied in Dictyoselium disoideum as well as mammalian immune cells (Lim and Gleeson, 2011), which could explain how P. aeruginosa can trigger on a variety of target cells.

Initially, our lab disregarded this process, because it relies on actin polymerization and our lab demonstrated that P. aeruginosa strains are able to translocate into cells with a depolymerized actin cytoskeleton. However, initial steps of macropincytosis involve sorting nexins, which are recruited to the plasma membrane and deform the membrane to create a macropinocytic pit (Lim and

Gleeson, 2011). The macropinocytic pit formed by sorting nexins could be sufficient to trigger effector secretion. In support of this hypothesis, sorting nexin 1 has been imaged at bacterial entry sites during early stages of Salmonella sp. infection (Bujny et al., 2008). We could test this hypothesis by depleting sorting nexins utilizing specific siRNA and then testing how translocation is affected.

Cellular processes: endocytosis and beyond

The second prong was focused on determining if specific endocytic processes played a part during injection by P. aeruginosa. This was based on previous data that suggested that lipid rafts were important for infection and more specifically, that ceramide clustering occurred during infection by P. aeruginosa (van der Goot et al.,

96 2004; Kannan et al., 2008; Grassme et al., 2003). These observations led us to test whether membrane damage repair was involved during infection. MDR is a process by which the cell can excise damaged membrane in order to restore membrane integrity. This process includes an influx of calcium from the extracellular space, exocytosis of acid sphingomyelinase (ASM), ceramide production, ceramide clustering, and Caveolin-1-dependent endocytosis (Tam et al., 2010; Corrotte et al.,

2013). Additionally, other data suggested that the alternative Flotillin-dependent endocytosis could contribute to infection (Korhonen et al., 2012; Glebov et al.,

2005). I used a combination of mutant cell lines, ASM inhibitors, and siRNA targeting of specific genes to determine if the MDR or Flotillin-dependent endocytosis were required for injection by the T3SS. Together the data demonstrated a slight effect of the MDR pathway and Flotillin-dependent endocytosis during translocation of effectors. This suggests that these processes are involved but are not the major contributor to triggering of effector secretion. It is possible that P. aeruginosa has the ability to utilize several endocytic pathways to inject eukaryotic cells (Figure 4-

1). This would make results difficult to interpret, because when one pathway is blocked the cells are able to compensate with an alternative pathway. The broad range of intercellular targets for the effectors supports this hypothesis.

Furthermore, several of these eukaryotic targets are involved in phagocytic, endocytic, or trafficking pathways in the cell, suggesting that any one of these processes could control effector secretion by the T3SS (Figure 4-1).

In an effort to determine the cause of effector secretion, several studies have screened for cellular components important for infection (Figure 4-2). A study using

97 CRISPR/Cas9 screen for Vibrio parahaemolyticus T3SSs found that surface sulfation and fucosylation was important for killing by the T3SS1 and T3SS2, respectively

(Blondel et al., 2016). In this study, these modifications were important for bacterial attachment and insertion of translocators, respectively. This study used a short infection period along with the output of cell death, resulting in selection criteria biased towards early steps of infection. Additionally, Russo et al. (2016) found that surface vimentin was central to stabilizing the translocon for injection of effectors.

These studies underline the importance of eukaryotic cell surface composition for infection in cells.

Alternatively, a study by Sheahan and Isberg (2015) used an shRNA screen, which selected for cellular components that affected the activity of YopE within the host cell. This study differs from the ones mentioned before, because the authors selected for factors that affect translocation of YopE, an ExoS homolog, or downstream steps that affect YopE function. This study found that CCR5 was important for pore formation and, therefore, translocation. Although, the more interesting finding of this screen was that there was enrichment in factors involved in vesicle trafficking. This could affect translocation in a couple of different ways.

For example, the screen identified the vesicular trafficking protein, CopB1.

Previously, this protein was found to block the activity of SopE, an ExoS homolog, during Salmonella sp. infection (Misselwitz et al., 2011). Misselwitz et al. (2011) found that depletion of this protein resulted in poor cholesterol and sphingolipid trafficking to the outer membrane. Furthermore, Skoudy et al. (2000) found that

CD44 was recruited to sites of Shigella sp. entry and bound IpaB, a PopB homolog.

98 The same group later found that the CD44-IpaB interaction occurred in lipid rafts and that the presence of sphingolipids was important for infection (Lafont et al.,

2002). The purpose of CD44-IpaB binding for infection was not specified, although, the authors speculated that it could contribute to signaling events contributing to cytoskeleton rearrangements important for infection. These data suggest that the formation of lipid rafts and the proteins associated with the rafts are crucial for translocation (Figure 4-1). Additionally, CopB1 depletion also affected the ability of

Rac1 and Cdc42 to localize to the membrane after contact with bacteria (Misselwitz et al., 2011). This finding suggests that vesicular trafficking is important for localization of cellular targets, which could affect translocation by the T3SS. In order to determine if certain identified factors contribute to effector translocation or feedback inhibition in the P. aeruginosa system, we could test how depleting the protein using a specific siRNA affects pore formation, docking, triggering, and feedback inhibition.

Additionally, many studies implicated lipid rafts as important for infection, but how has not been specified. We can test the effect of sphingolipids and lipid rafts during injection by P. aeruginosa utilizing inhibitors of enzymes involved in sphingolipid synthesis, myriocin or fumonisin B1. These inhibitors were previously shown to block phagocytosis of Candida albicans, which is dependent on the presence of sphingolipids in the phagocytic cup (Tafesse et al., 2015). Furthermore, van der Goot et al. (2004) found that purified lipid rafts were able to trigger secretion of Shigella flexneri. Previous studies focused on depletion of cholesterol and the effect on infection (Lafont et al., 2002; van der Goot et al., 2004; Hayward et

99 al., 2005; Kannan et al., 2008; Verove et al., 2012). In contrast, we could deplete sphingolipids and measure translocation. Alternatively, we could perform similar tests with a cell line that has deficiency in sphingolipid biosynthesis, SPB-1 cells.

These experiments would give us a better idea about how lipid rafts affect infection, whether they affect insertion of translocators into the eukaryotic membrane, pore formation, docking, triggering, or regulation of translocation.

Membrane curvature

The third prong was to recapitulate the conditions that lead to triggering of effector secretion. Data from our lab suggests that phagocytosed bacteria experience a constant trigger compared to bacteria that remain on the surface of the eukaryotic cell. This suggests that the trigger for effector secretion is not simply a protein located on the surface acting as a receptor, but rather an unidentified aspect of phagocytosis. Because P. aeruginosa triggers on a variety of targets, the cellular component that causes triggering must be widespread. These observations led our lab to hypothesize that the trigger may be membrane curvature sensed by the pore proteins to initiate effector secretion. Conversely, the system I used to test this hypothesis did not reveal any positive results, suggesting that membrane curvature alone is not the trigger. It is possible that membrane curvature does cause a conformational change in the pore, which leads to initiation of secretion, but the reason the bacteria did not trigger within our system is due to an additional unidentified factor.

100 A simple lipid mixture was utilized for this experiment, because it was previously shown to support insertion of PopB and PopD as well as pore formation

(Faudry et al., 2006). On the other hand, PopB and PopD insertion is not the only prerequisite for translocation, i.e. additional factors are needed to promote stable translocon formation and injection (Lafont et al., 2002; Hayward et al., 2005; Verove et al., 2012; Russo et al., 2016; Blondel et al., 2016). To test this hypothesis, a more complex lipid mix could be used for future experiments, such as the addition of cholesterol and sphingolipids, which have been stated as important for infection

(Lafont et al., 2002; van der Goot et al., 2004; Hayward et al., 2005; Kannan et al.,

2008; Verove et al., 2012). The addition of cholesterol to the system did not cause the bacteria to trigger, suggesting that cholesterol alone is not sufficient for triggering. It is more likely that cholesterol as well as sphingolipids contribute to the formation of the phagocytic cup (Kannan et al., 2008; Tafesse et al., 2015).

Sphingolipids encapsulate several lipids, and therefore, the kind of sphingolipid could be chosen through binding assays with PopB and PopD (Faudry et al., 2006).

Previous data also stated the need for vimentin for proper stability of the translocon, but during my studies the addition of vimentin to the lipid mixture did not affect triggering of effector secretion. This observation does not necessarily discount the previous study but simply implies that vimentin is not sufficient for triggering or not important for P. aeruginosa injection. Several studies propose that additional eukaryotic membrane proteins are needed for proper translocation

(Skoudy et al., 2000; Sheahan and Isberg, 2015). Therefore, in order to trigger in this system additional purified proteins might need to be included. Conversely, because

101 P. aeruginosa can trigger on a variety of targets, additional proteins that are specific to mammals would be unlikely to be needed.

102

103

104 References

Abd, H., B. Wretlind, A. Saeed, E. Idsund, K. Hultenby and G. Sandstrom (2008). "Pseudomonas aeruginosa utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii." J Eukaryot Microbiol 55(3): 235-243.

Aili, M., E. L. Isaksson, S. E. Carlsson, H. Wolf-Watz, R. Rosqvist and M. S. Francis (2008). "Regulation of Yersinia Yop-effector delivery by translocated YopE." Int J Med Microbiol 298(3-4): 183-192.

Aili, M., E. L. Isaksson, B. Hallberg, H. Wolf-Watz and R. Rosqvist (2006). "Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis." Cell Microbiol 8(6): 1020-1033.

Akeda, Y. and J. E. Galan (2005). "Chaperone release and unfolding of substrates in type III secretion." Nature 437(7060): 911-915.

Akopyan, K., T. Edgren, H. Wang-Edgren, R. Rosqvist, A. Fahlgren, H. Wolf-Watz and M. Fallman (2011). "Translocation of surface-localized effectors in type III secretion." Proc Natl Acad Sci U S A 108(4): 1639-1644.

Andreasson-Ochsner, M. and E. Reimhult (2013). "Mobile and three-dimensional presentation of adhesion proteins within microwells." Methods Mol Biol 1046: 123- 132.

Andreasson-Ochsner, M., G. Romano, M. Hakanson, M. L. Smith, D. E. Leckband, M. Textor and E. Reimhult (2011). "Single cell 3-D platform to study ligand mobility in cell-cell contact." Lab Chip 11(17): 2876-2883.

Armentrout, E. I. and A. Rietsch (2016). "The Type III Secretion Translocation Pore Senses Host Cell Contact." PLoS Pathog 12(3): e1005530.

Becker, K. A., J. Riethmuller, A. Luth, G. Doring, B. Kleuser and E. Gulbins (2010). "Acid sphingomyelinase inhibitors normalize pulmonary ceramide and inflammation in cystic fibrosis." Am J Respir Cell Mol Biol 42(6): 716-724.

Blocker, A., P. Gounon, E. Larquet, K. Niebuhr, V. Cabiaux, C. Parsot and P. Sansonetti (1999). "The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes." J Cell Biol 147(3): 683-693.

Blocker, A., N. Jouihri, E. Larquet, P. Gounon, F. Ebel, C. Parsot, P. Sansonetti and A. Allaoui (2001). "Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton." Mol Microbiol 39(3): 652-663.

105 Blocker, A. J., J. E. Deane, A. K. Veenendaal, P. Roversi, J. L. Hodgkinson, S. Johnson and S. M. Lea (2008). "What's the point of the type III secretion system needle?" Proc Natl Acad Sci U S A 105(18): 6507-6513.

Blondel, C. J., J. S. Park, T. P. Hubbard, A. R. Pacheco, C. J. Kuehl, M. J. Walsh, B. M. Davis, B. E. Gewurz, J. G. Doench and M. K. Waldor (2016). "CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated Cytotoxicity." Cell Host Microbe 20(2): 226-237.

Breidenstein, E. B., C. de la Fuente-Nunez and R. E. Hancock (2011). "Pseudomonas aeruginosa: all roads lead to resistance." Trends Microbiol 19(8): 419-426.

Bridge, D. R., M. J. Novotny, E. R. Moore and J. C. Olson (2010). "Role of host cell polarity and leading edge properties in Pseudomonas type III secretion." Microbiology 156(Pt 2): 356-373.

Broms, J. E., A. L. Forslund, A. Forsberg and M. S. Francis (2003a). "PcrH of Pseudomonas aeruginosa is essential for secretion and assembly of the type III translocon." J Infect Dis 188(12): 1909-1921.

Broms, J. E., A. L. Forslund, A. Forsberg and M. S. Francis (2003b). "Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis." Microbiology 149(Pt 9): 2615-2626.

Broms, J. E., M. S. Francis and A. Forsberg (2007). "Diminished LcrV secretion attenuates Yersinia pseudotuberculosis virulence." J Bacteriol 189(23): 8417-8429.

Broz, P., C. A. Mueller, S. A. Muller, A. Philippsen, I. Sorg, A. Engel and G. R. Cornelis (2007). "Function and molecular architecture of the Yersinia injectisome tip complex." Mol Microbiol 65(5): 1311-1320.

Brubaker, R. R. (1983). "The Vwa+ virulence factor of yersiniae: the molecular basis of the attendant nutritional requirement for Ca++." Rev Infect Dis 5 Suppl 4: S748- 758.

Bujny, M. V., P. A. Ewels, S. Humphrey, N. Attar, M. A. Jepson and P. J. Cullen (2008). "Sorting nexin-1 defines an early phase of Salmonella-containing vacuole- remodeling during Salmonella infection." J Cell Sci 121(Pt 12): 2027-2036.

Cheung, M., D. K. Shen, F. Makino, T. Kato, A. D. Roehrich, I. Martinez-Argudo, M. L. Walker, I. Murillo, X. Liu, M. Pain, J. Brown, G. Frazer, J. Mantell, P. Mina, T. Todd, R. B. Sessions, K. Namba and A. J. Blocker (2015). "Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex." Mol Microbiol 95(1): 31-50.

106 Cisz, M., P. C. Lee and A. Rietsch (2008). "ExoS controls the cell contact-mediated switch to effector secretion in Pseudomonas aeruginosa." J Bacteriol 190(8): 2726- 2738.

Coburn, J., S. T. Dillon, B. H. Iglewski and D. M. Gill (1989). "Exoenzyme S of Pseudomonas aeruginosa ADP-ribosylates the intermediate filament protein vimentin." Infect Immun 57(3): 996-998.

Coburn, J. and D. M. Gill (1991). "ADP-ribosylation of p21ras and related proteins by Pseudomonas aeruginosa exoenzyme S." Infect Immun 59(11): 4259-4262.

Coburn, J., A. V. Kane, L. Feig and D. M. Gill (1991). "Pseudomonas aeruginosa exoenzyme S requires a eukaryotic protein for ADP-ribosyltransferase activity." J Biol Chem 266(10): 6438-6446.

Corrotte, M., P. E. Almeida, C. Tam, T. Castro-Gomes, M. C. Fernandes, B. A. Millis, M. Cortez, H. Miller, W. Song, T. K. Maugel and N. W. Andrews (2013). "Caveolae internalization repairs wounded cells and muscle fibers." Elife 2: e00926.

Cowell, B. A., D. J. Evans and S. M. Fleiszig (2005). "Actin cytoskeleton disruption by ExoY and its effects on Pseudomonas aeruginosa invasion." FEMS Microbiol Lett 250(1): 71-76.

Dacheux, D., J. Goure, J. Chabert, Y. Usson and I. Attree (2001). "Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa- infected macrophages." Mol Microbiol 40(1): 76-85.

Dasgupta, N., G. L. Lykken, M. C. Wolfgang and T. L. Yahr (2004). "A novel anti-anti- activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system." Mol Microbiol 53(1): 297-308.

Deane, J. E., P. Roversi, F. S. Cordes, S. Johnson, R. Kenjale, S. Daniell, F. Booy, W. D. Picking, W. L. Picking, A. J. Blocker and S. M. Lea (2006). "Molecular model of a type III secretion system needle: Implications for host-cell sensing." Proc Natl Acad Sci U S A 103(33): 12529-12533.

DeLeon-Rangel, J., R. R. Ishmukhametov, W. Jiang, R. H. Fillingame and S. B. Vik (2013). "Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking." FEBS Lett 587(7): 892-897.

Deng, W., N. C. Marshall, J. L. Rowland, J. M. McCoy, L. J. Worrall, A. S. Santos, N. C. J. Strynadka and B. B. Finlay (2017). "Corrigendum: Assembly, structure, function and regulation of type III secretion systems." Nat Rev Microbiol 15(6): 379.

Dewoody, R. S., P. M. Merritt and M. M. Marketon (2013). "Regulation of the Yersinia type III secretion system: traffic control." Front Cell Infect Microbiol 3: 4.

107

Dietsche, T., M. Tesfazgi Mebrhatu, M. J. Brunner, P. Abrusci, J. Yan, M. Franz- Wachtel, C. Scharfe, S. Zilkenat, I. Grin, J. E. Galan, O. Kohlbacher, S. Lea, B. Macek, T. C. Marlovits, C. V. Robinson and S. Wagner (2016). "Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus." PLoS Pathog 12(12): e1006071.

Ekestubbe, S., J. E. Broms, T. Edgren, M. Fallman, M. S. Francis and A. Forsberg (2016). "The Amino-Terminal Part of the Needle-Tip Translocator LcrV of Yersinia pseudotuberculosis Is Required for Early Targeting of YopH and In vivo Virulence." Front Cell Infect Microbiol 6: 175.

El-Solh, A. A., A. Hattemer, A. R. Hauser, A. Alhajhusain and H. Vora (2012). "Clinical outcomes of type III Pseudomonas aeruginosa bacteremia." Crit Care Med 40(4): 1157-1163.

Faudry, E., G. Vernier, E. Neumann, V. Forge and I. Attree (2006). "Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa." Biochemistry 45(26): 8117-8123.

Ferracci, F., F. D. Schubot, D. S. Waugh and G. V. Plano (2005). "Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion." Mol Microbiol 57(4): 970-987.

Francis, C. L., T. A. Ryan, B. D. Jones, S. J. Smith and S. Falkow (1993). "Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria." Nature 364(6438): 639-642.

Frank, D. W. (1997). "The exoenzyme S regulon of Pseudomonas aeruginosa." Mol Microbiol 26(4): 621-629.

Frank, D. W. and B. H. Iglewski (1991). "Cloning and sequence analysis of a trans- regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa." J Bacteriol 173(20): 6460-6468.

Fu, H., J. Coburn and R. J. Collier (1993). "The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family." Proc Natl Acad Sci U S A 90(6): 2320-2324.

Gemski, P., J. R. Lazere and T. Casey (1980). "Plasmid associated with pathogenicity and calcium dependency of Yersinia enterocolitica." Infect Immun 27(2): 682-685.

Glebov, O. O., N. A. Bright and B. J. Nichols (2006). "Flotillin-1 defines a clathrin- independent endocytic pathway in mammalian cells." Nat Cell Biol 8(1): 46-54.

108 Goehring, U. M., G. Schmidt, K. J. Pederson, K. Aktories and J. T. Barbieri (1999). "The N-terminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein for Rho GTPases." J Biol Chem 274(51): 36369-36372.

Goure, J., P. Broz, O. Attree, G. R. Cornelis and I. Attree (2005). "Protective anti-V antibodies inhibit Pseudomonas and Yersinia translocon assembly within host membranes." J Infect Dis 192(2): 218-225.

Goure, J., A. Pastor, E. Faudry, J. Chabert, A. Dessen and I. Attree (2004). "The V antigen of Pseudomonas aeruginosa is required for assembly of the functional PopB/PopD translocation pore in host cell membranes." Infect Immun 72(8): 4741- 4750.

Grassme, H., V. Jendrossek, A. Riehle, G. von Kurthy, J. Berger, H. Schwarz, M. Weller, R. Kolesnick and E. Gulbins (2003). "Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts." Nat Med 9(3): 322-330.

Hauser, A. R. (2009). "The type III secretion system of Pseudomonas aeruginosa: infection by injection." Nat Rev Microbiol 7(9): 654-665.

Hauser, A. R., E. Cobb, M. Bodi, D. Mariscal, J. Valles, J. N. Engel and J. Rello (2002). "Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa." Crit Care Med 30(3): 521-528.

Hauser, A. R., P. J. Kang and J. N. Engel (1998). "PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence." Mol Microbiol 27(4): 807-818.

Hayward, R. D., R. J. Cain, E. J. McGhie, N. Phillips, M. J. Garner and V. Koronakis (2005). "Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells." Mol Microbiol 56(3): 590-603.

Ho, O., P. Rogne, T. Edgren, H. Wolf-Watz, F. H. Login and M. Wolf-Watz (2017). "Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System." J Biol Chem 292(8): 3299-3311.

Hoiczyk, E. and G. Blobel (2001). "Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells." Proc Natl Acad Sci U S A 98(8): 4669-4674.

Hornef, M. W., A. Roggenkamp, A. M. Geiger, M. Hogardt, C. A. Jacobi and J. Heesemann (2000). "Triggering the ExoS regulon of Pseudomonas aeruginosa: A GFP-reporter analysis of exoenzyme (Exo) S, ExoT and ExoU synthesis." Microb Pathog 29(6): 329-343.

109

Hu, B., D. R. Morado, W. Margolin, J. R. Rohde, O. Arizmendi, W. L. Picking, W. D. Picking and J. Liu (2015). "Visualization of the type III secretion sorting platform of Shigella flexneri." Proc Natl Acad Sci U S A 112(4): 1047-1052.

Ide, T., S. Laarmann, L. Greune, H. Schillers, H. Oberleithner and M. A. Schmidt (2001). "Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli." Cell Microbiol 3(10): 669-679.

Ip, S. H. and R. A. Cooper (1980). "Decreased membrane fluidity during differentiation of human promyelocytic leukemia cells in culture." Blood 56(2): 227- 232.

Isaksson, E. L., M. Aili, A. Fahlgren, S. E. Carlsson, R. Rosqvist and H. Wolf-Watz (2009). "The membrane localization domain is required for intracellular localization and autoregulation of YopE in Yersinia pseudotuberculosis." Infect Immun 77(11): 4740-4749.

Jorth, P., B. J. Staudinger, X. Wu, K. B. Hisert, H. Hayden, J. Garudathri, C. L. Harding, M. C. Radey, A. Rezayat, G. Bautista, W. R. Berrington, A. F. Goddard, C. Zheng, A. Angermeyer, M. J. Brittnacher, J. Kitzman, J. Shendure, C. L. Fligner, J. Mittler, M. L. Aitken, C. Manoil, J. E. Bruce, T. L. Yahr and P. K. Singh (2015). "Regional Isolation Drives Bacterial Diversification within Cystic Fibrosis Lungs." Cell Host Microbe 18(3): 307-319.

Kannan, S., A. Audet, H. Huang, L. J. Chen and M. Wu (2008). "Cholesterol-rich membrane rafts and Lyn are involved in phagocytosis during Pseudomonas aeruginosa infection." J Immunol 180(4): 2396-2408.

Kazmierczak, B. I. and J. N. Engel (2002). "Pseudomonas aeruginosa ExoT acts in vivo as a GTPase-activating protein for RhoA, Rac1, and Cdc42." Infect Immun 70(4): 2198-2205.

Kenjale, R., J. Wilson, S. F. Zenk, S. Saurya, W. L. Picking, W. D. Picking and A. Blocker (2005). "The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus." J Biol Chem 280(52): 42929-42937.

Korhonen, J. T., M. Puolakkainen, R. Haivala, T. Penttila, A. Haveri, E. Markkula and R. Lahesmaa (2012). "Flotillin-1 (Reggie-2) contributes to Chlamydia pneumoniae growth and is associated with bacterial inclusion." Infect Immun 80(3): 1072-1078.

Krall, R., J. Sun, K. J. Pederson and J. T. Barbieri (2002). "In vivo rho GTPase- activating protein activity of Pseudomonas aeruginosa cytotoxin ExoS." Infect Immun 70(1): 360-367.

110 Kurahashi, K., O. Kajikawa, T. Sawa, M. Ohara, M. A. Gropper, D. W. Frank, T. R. Martin and J. P. Wiener-Kronish (1999). "Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia." J Clin Invest 104(6): 743-750.

Lafont, F., G. Tran Van Nhieu, K. Hanada, P. Sansonetti and F. G. van der Goot (2002). "Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft- mediated CD44-IpaB interaction." Embo j 21(17): 4449-4457.

Lee, P. C., C. M. Stopford, A. G. Svenson and A. Rietsch (2010). "Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV." Mol Microbiol 75(4): 924-941.

Lee, P. C., S. E. Zmina, C. M. Stopford, J. Toska and A. Rietsch (2014). "Control of type III secretion activity and substrate specificity by the cytoplasmic regulator PcrG." Proc Natl Acad Sci U S A 111(19): E2027-2036.

Lee, V. T., S. K. Mazmanian and O. Schneewind (2001). "A program of Yersinia enterocolitica type III secretion reactions is activated by specific signals." J Bacteriol 183(17): 4970-4978.

Lim, J. P. and P. A. Gleeson (2011). "Macropinocytosis: an endocytic pathway for internalising large gulps." Immunol Cell Biol 89(8): 836-843.

Lloyd, S. A., A. Forsberg, H. Wolf-Watz and M. S. Francis (2001). "Targeting exported substrates to the Yersinia TTSS: different functions for different signals?" Trends Microbiol 9(8): 367-371.

Luisoni, S., M. Suomalainen, K. Boucke, L. B. Tanner, M. R. Wenk, X. L. Guan, M. Grzybek, U. Coskun and U. F. Greber (2015). "Co-option of Membrane Wounding Enables Virus Penetration into Cells." Cell Host Microbe 18(1): 75-85.

Maresso, A. W., M. R. Baldwin and J. T. Barbieri (2004). "Ezrin/radixin/moesin proteins are high affinity targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS." J Biol Chem 279(37): 38402-38408.

McCaw, M. L., G. L. Lykken, P. K. Singh and T. L. Yahr (2002). "ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon." Mol Microbiol 46(4): 1123-1133.

Minamino, T. and K. Namba (2008). "Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export." Nature 451(7177): 485-488.

Misselwitz, B., S. Dilling, P. Vonaesch, R. Sacher, B. Snijder, M. Schlumberger, S. Rout, M. Stark, C. von Mering, L. Pelkmans and W. D. Hardt (2011). "RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42." Mol Syst Biol 7: 474.

111

Monjaras Feria, J. V., M. D. Lefebre, Y. D. Stierhof, J. E. Galan and S. Wagner (2015). "Role of autocleavage in the function of a type III secretion specificity switch protein in Salmonella enterica serovar Typhimurium." MBio 6(5): e01459-01415.

Morrow, K. A., D. W. Frank, R. Balczon and T. Stevens (2017). "The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant." Handb Exp Pharmacol 238: 67-85.

Mota, L. J., L. Journet, I. Sorg, C. Agrain and G. R. Cornelis (2005). "Bacterial injectisomes: needle length does matter." Science 307(5713): 1278.

Mueller, C. A., P. Broz, S. A. Muller, P. Ringler, F. Erne-Brand, I. Sorg, M. Kuhn, A. Engel and G. R. Cornelis (2005). "The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles." Science 310(5748): 674-676.

Murillo, I., I. Martinez-Argudo and A. J. Blocker (2016). "Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip." Sci Rep 6: 27649.

Nguyen, L., I. T. Paulsen, J. Tchieu, C. J. Hueck and M. H. Saier, Jr. (2000). "Phylogenetic analyses of the constituents of Type III protein secretion systems." J Mol Microbiol Biotechnol 2(2): 125-144.

Pace, J., M. J. Hayman and J. E. Galan (1993). "Signal transduction and invasion of epithelial cells by S. typhimurium." Cell 72(4): 505-514.

Paul, K., M. Erhardt, T. Hirano, D. F. Blair and K. T. Hughes (2008). "Energy source of flagellar type III secretion." Nature 451(7177): 489-492.

Pederson, K. J., A. J. Vallis, K. Aktories, D. W. Frank and J. T. Barbieri (1999). "The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins." Mol Microbiol 32(2): 393-401.

Phillips, R. M., D. A. Six, E. A. Dennis and P. Ghosh (2003). "In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors." J Biol Chem 278(42): 41326- 41332.

Picking, W. L., H. Nishioka, P. D. Hearn, M. A. Baxter, A. T. Harrington, A. Blocker and W. D. Picking (2005). "IpaD of Shigella flexneri is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes." Infect Immun 73(3): 1432-1440.

Pukatzki, S., A. T. Ma, D. Sturtevant, B. Krastins, D. Sarracino, W. C. Nelson, J. F. Heidelberg and J. J. Mekalanos (2006). "Identification of a conserved bacterial

112 protein secretion system in Vibrio cholerae using the Dictyostelium host model system." Proc Natl Acad Sci U S A 103(5): 1528-1533.

Radics, J., L. Konigsmaier and T. C. Marlovits (2014). "Structure of a pathogenic type 3 secretion system in action." Nat Struct Mol Biol 21(1): 82-87.

Rietsch, A. and J. J. Mekalanos (2006). "Metabolic regulation of type III secretion gene expression in Pseudomonas aeruginosa." Mol Microbiol 59(3): 807-820.

Rietsch, A., I. Vallet-Gely, S. L. Dove and J. J. Mekalanos (2005). "ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa." Proc Natl Acad Sci U S A 102(22): 8006-8011.

Rietsch, A., M. C. Wolfgang and J. J. Mekalanos (2004). "Effect of metabolic imbalance on expression of type III secretion genes in Pseudomonas aeruginosa." Infect Immun 72(3): 1383-1390.

Romano, F. B., Y. Tang, K. C. Rossi, K. R. Monopoli, J. L. Ross and A. P. Heuck (2016). "Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex." J Biol Chem 291(12): 6304-6315.

Rosqvist, R., K. E. Magnusson and H. Wolf-Watz (1994). "Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells." Embo j 13(4): 964-972.

Rostand, K. S. and J. D. Esko (1993). "Cholesterol and cholesterol esters: host receptors for Pseudomonas aeruginosa adherence." J Biol Chem 268(32): 24053- 24059.

Roy-Burman, A., R. H. Savel, S. Racine, B. L. Swanson, N. S. Revadigar, J. Fujimoto, T. Sawa, D. W. Frank and J. P. Wiener-Kronish (2001). "Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections." J Infect Dis 183(12): 1767-1774.

Rucks, E. A. and J. C. Olson (2005). "Characterization of an ExoS Type III translocation-resistant cell line." Infect Immun 73(1): 638-643.

Russo, B. C., L. M. Stamm, M. Raaben, C. M. Kim, E. Kahoud, L. R. Robinson, S. Bose, A. L. Queiroz, B. B. Herrera, L. A. Baxt, N. Mor-Vaknin, Y. Fu, G. Molina, D. M. Markovitz, S. P. Whelan and M. B. Goldberg (2016). "Intermediate filaments enable pathogen docking to trigger type 3 effector translocation." Nat Microbiol 1: 16025.

Saier, M. H., Jr. (2004). "Evolution of bacterial type III protein secretion systems." Trends Microbiol 12(3): 113-115.

113 Sansonetti, P. J. (2001). "Microbes and microbial toxins: paradigms for microbial- mucosal interactions III. Shigellosis: from symptoms to molecular pathogenesis." Am J Physiol Gastrointest Liver Physiol 280(3): G319-323.

Sato, H. and D. W. Frank (2011). "Multi-Functional Characteristics of the Pseudomonas aeruginosa Type III Needle-Tip Protein, PcrV; Comparison to Orthologs in other Gram-negative Bacteria." Front Microbiol 2: 142.

Sato, H., D. W. Frank, C. J. Hillard, J. B. Feix, R. R. Pankhaniya, K. Moriyama, V. Finck- Barbancon, A. Buchaklian, M. Lei, R. M. Long, J. Wiener-Kronish and T. Sawa (2003). "The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU." Embo j 22(12): 2959-2969.

Schoehn, G., A. M. Di Guilmi, D. Lemaire, I. Attree, W. Weissenhorn and A. Dessen (2003). "Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas." Embo j 22(19): 4957-4967.

Sheahan, K. L. and R. R. Isberg (2015). "Identification of mammalian proteins that collaborate with type III secretion system function: involvement of a chemokine receptor in supporting translocon activity." MBio 6(1): e02023-02014.

Skoudy, A., J. Mounier, A. Aruffo, H. Ohayon, P. Gounon, P. Sansonetti and G. Tran Van Nhieu (2000). "CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells." Cell Microbiol 2(1): 19-33.

Sory, M. P., A. Boland, I. Lambermont and G. R. Cornelis (1995). "Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach." Proc Natl Acad Sci U S A 92(26): 11998-12002.

Sun, J. and J. T. Barbieri (2003). "Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins." J Biol Chem 278(35): 32794-32800.

Sun, Y., M. Karmakar, P. R. Taylor, A. Rietsch and E. Pearlman (2012). "ExoS and ExoT ADP ribosyltransferase activities mediate Pseudomonas aeruginosa keratitis by promoting neutrophil apoptosis and bacterial survival." J Immunol 188(4): 1884- 1895.

Sundin, C., J. Thelaus, J. E. Broms and A. Forsberg (2004). "Polarisation of type III translocation by Pseudomonas aeruginosa requires PcrG, PcrV and PopN." Microb Pathog 37(6): 313-322.

Tafesse, F. G., A. Rashidfarrokhi, F. I. Schmidt, E. Freinkman, S. Dougan, M. Dougan, A. Esteban, T. Maruyama, K. Strijbis and H. L. Ploegh (2015). "Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans." PLoS Pathog 11(10): e1005188.

114

Tam, C., V. Idone, C. Devlin, M. C. Fernandes, A. Flannery, X. He, E. Schuchman, I. Tabas and N. W. Andrews (2010). "Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair." J Cell Biol 189(6): 1027- 1038.

Tejeda-Dominguez, F., J. Huerta-Cantillo, L. Chavez-Duenas and F. Navarro-Garcia (2017). "A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins." MBio 8(2).

Tomalka, A. G., C. M. Stopford, P. C. Lee and A. Rietsch (2012). "A translocator- specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function." Mol Microbiol 86(6): 1464- 1481.

Torruellas, J., M. W. Jackson, J. W. Pennock and G. V. Plano (2005). "The Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion." Mol Microbiol 57(6): 1719-1733.

Tran, C. S., S. M. Rangel, H. Almblad, A. Kierbel, M. Givskov, T. Tolker-Nielsen, A. R. Hauser and J. N. Engel (2014). "The Pseudomonas aeruginosa type III translocon is required for biofilm formation at the epithelial barrier." PLoS Pathog 10(11): e1004479.

Vallis, A. J., T. L. Yahr, J. T. Barbieri and D. W. Frank (1999). "Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions." Infect Immun 67(2): 914-920. van der Goot, F. G., G. Tran van Nhieu, A. Allaoui, P. Sansonetti and F. Lafont (2004). "Rafts can trigger contact-mediated secretion of bacterial effectors via a lipid-based mechanism." J Biol Chem 279(46): 47792-47798.

Veenendaal, A. K., C. Sundin and A. J. Blocker (2009). "Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton." J Bacteriol 191(2): 563-570.

Verove, J., C. Bernarde, Y. S. Bohn, F. Boulay, M. J. Rabiet, I. Attree and F. Cretin (2012). "Injection of Pseudomonas aeruginosa Exo toxins into host cells can be modulated by host factors at the level of translocon assembly and/or activity." PLoS One 7(1): e30488.

Viboud, G. I. and J. B. Bliska (2001). "A bacterial type III secretion system inhibits actin polymerization to prevent pore formation in host cell membranes." Embo j 20(19): 5373-5382.

115 Wilharm, G., V. Lehmann, K. Krauss, B. Lehnert, S. Richter, K. Ruckdeschel, J. Heesemann and K. Trulzsch (2004). "Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB." Infect Immun 72(7): 4004-4009.

Willcox, M. D. (2012). "Management and treatment of contact lens-related Pseudomonas keratitis." Clin Ophthalmol 6: 919-924.

Yahr, T. L., A. J. Vallis, M. K. Hancock, J. T. Barbieri and D. W. Frank (1998). "ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system." Proc Natl Acad Sci U S A 95(23): 13899-13904.

Yang, H., Z. Shan, J. Kim, W. Wu, W. Lian, L. Zeng, L. Xing and S. Jin (2007). "Regulatory role of PopN and its interacting partners in type III secretion of Pseudomonas aeruginosa." J Bacteriol 189(7): 2599-2609.

Zhang, Y. and J. T. Barbieri (2005). "A Leucine-Rich Motif Targets Pseudomonas aeruginosa ExoS within Mammalian Cells." Infection and Immunity 73(12): 7938- 7945.

Zhang, Y., Q. Deng, J. A. Porath, C. L. Williams, K. J. Pederson-Gulrud and J. T. Barbieri (2007). "Plasma membrane localization affects the RhoGAP specificity of Pseudomonas ExoS." Cell Microbiol 9(9): 2192-2201.

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