key: cord-0272989-17tmxjcb
authors: Foulkes, Daniel M.; McLean, Keri; Hermann, Anne; Johnson, James; Winstanley, Craig; Berry, Neil; Fernig, David G.; Kaye, Stephen B.
title: A phospholipase assay screen identifies synergistic inhibitors of the P. aeruginosa toxin ExoU
date: 2022-02-21
journal: bioRxiv
DOI: 10.1101/2022.02.21.481271
sha: 680e23ec18404ec5aa498693f0e2a08192cc5b9c
doc_id: 272989
cord_uid: 17tmxjcb

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of disability and mortality worldwide and the World Health Organisation has listed it with the highest priority for the need of new therapies. P. aeruginosa strains that express ExoU are implicated in the worst clinical outcomes. ExoU is phospholipase that is secreted by P. aeruginosa directly into the cytoplasm of target host cells, where its catalytic activity, directed towards plasma membranes, causes rapid cell lysis. Inhibition of ExoU may be a novel strategy to combat acutely cytotoxic ExoU expressing P. aeruginosa infections. Using an in vitro phospholipase assay, we performed a high throughput screen to identify compounds that might be repurposed as therapeutic ExoU inhibitors. We discovered a panel of compounds that appeared to inhibit ExoU through distinct mechanisms. Compound C prevented ExoU membrane localisation in HEK293T cells and caused colocalization with lysosomes, whereas compound D prevented PIP2 dependent oligomerisation of ExoU in vitro suggestive of synergistic action. Indeed, the concentrations required by compounds C and D to inhibit in vitro ExoU catalytic activity, when used in combination, was in the nanomolar region. In corneal scratch and infection assays, these compounds reduced ExoU mediated cytotoxicity, as assessed by fluorescence microscopy and lactate dehydrogenase release assays.

The opportunistic pathogen P. aeruginosa is a major cause of nosocomial infection, associated with a wide range of debilitating and fatal diseases [1] [2] [3] . It is a leading cause of intensive care unit-acquired pneumonia (ICUAP) [4] , and is the second most frequent colonising bacteria in patients with COVID19 [5, 6] . It is also the primary causative agent of bacterial keratitis [7] . As a pathogen of current major concern, the World Health Organisation (WHO) has listed carbapenem-resistant P. aeruginosa (CRPA) with the highest priority for the development of new treatments [8] .

Pathogenic P. aeruginosa strains use the type III secretion system (T3SS) to inject exotoxins directly into the cytoplasm of compromised host epithelia [9] . The T3SS has been identified as a principal virulence determinant for poor clinical outcomes in pneumonia, sepsis, keratitis, and otitis externa [2-4, 10, 11] . Of the four effector proteins secreted by the T3SS, ExoS, ExoT, ExoU and ExoY, the most cytotoxic is ExoU. In P. aeruginosa infections where ExoU is expressed, disease severity is increased with poorer clinical outcomes [2, 4, 12, 13] .

ExoU is a potent phospholipase that is secreted directly into the target host cell cytoplasm via the P. aeruginosa T3SS [9] . Although the mechanisms of ExoU activation are not fully understood, it is established that ExoU binds to ubiquitin and phosphatidylinositol 4,5-bisphosphate (PIP2) to become activated [14, 15] . PIP2 induces ExoU oligomerisation, by which its ubiquitin-dependent catalytic activity is greatly enhanced [15] . In mammalian cells, binding of PIP2 causes ExoU localisation to the plasma membrane, via its C-terminal 4-helical bundle domain, where its catalytic activity induces cellular lysis [16, 17] . ExoU catalytic activity is directed towards phospholipids at the sn-2 position, and results in arachidonic acid release, which in turn stimulates NF-κB and MAPK signalling [18] [19] [20] . Increased expression of IL-8 and keratinocyte chemoattractant (KC) then causes acute localised inflammation, leading to infiltration of neutrophils and further tissue damage [18, 19] .

Inhibitors of ExoU might reduce acute cell lysis, tissue damage and inflammation associated with ExoU toxicity and provide a more substantial window for immune response or antibiotic action [21] . ExoU undergoes multiple activation steps including interaction with ubiquitin, DNAJC5 dependent host trafficking [22] and localisation to plasma membranes, which might be targetable with small molecules. In this study, we performed an in vitro phospholipase assay screen in order to identify novel ExoU inhibitors, interrogate their mechanisms of inhibition and test their efficacy in a corneal cell infection model.

The pOPINF E. coli expression vector was purchased from Addgene. The pEGFP-C3 vector, in which ExoU was cloned for expression of GFP-tagged ExoU in human cells, was a gift from Dr. Michael Batie (University of Liverpool, UK). Arachidonoyl thio-PC was purchased from Cayman Chemical (Michigan, USA). PIP2 was purchased from Avanti Polar Lipids (Alabama, USA). Bovine ubiquitin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DNTB) were purchased from Sigma-Aldrich. ExoU screening compounds were provided by LifeArc (UK) and Professor Patrick Eyers (University of Liverpool, UK).

The α-GFP and α-tubulin antibodies were purchased from Sigma Aldrich. Live/Dead assay reagents were purchased from Invitrogen. LDH assay reagents and propidium iodide were purchased from Thermofisher. The P. aeruginosa strain PA103 was a gift from Professor Dara Frank (Medical College of Wisconsin).

Full-length ExoU, expressed in pOPINF, was purified from C43(DE3) E. coli as previously described [23] . Briefly, transformed C43(DE3) bacteria were grown in Terrific broth (Melford) supplemented with ampicillin (100 μg/ml) and grown to an 

Recombinant ExoU sn-2 directed phospholipase activity was detected using an adapted Caymen chemical cPLA2 assay kit in a 384-well plate format, as previously described [23] . A master mix contained 1mM ATPC, 1 µM PIP2, 25 µM mono ubiquitin, 1.25 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and 100 nM ExoU. The master mix was dispensed into 384-well plates at a volume of 10 µL per well. An Echo acoustic liquid handling robot was then used to dispense 0.2 µL of screening compound to each well. The final concentration of compound for each condition 10 µM. ExoU activity was detected as percentage to that of DMSO controls, 4 hours after the start of the assay.

Compounds or DMSO (2% v/v) were added to 1 µg of ExoU with 2. Cells were transfected with either pcDNA5/FRT/TO plasmid encoding Flag-tagged WT ExoU, or pEGFP-C3 to express GFP-tagged S142A ExoU, using polyethylenimine (PEI) at a ratio of 3:1 PEI:cDNA.

Transfected HeLa cells were collected 8 h after transfection and tetracycline (TET) induced ExoU expression, by combining the culture medium (to obtain suspended cells) and adherent cells released by trypsinisation. The resulting HeLa cell and medium suspension was mixed in a 1:1 ratio with trypan blue reagent (Thermo Scientific) and a Countess II Automated Cell Counter (Thermo Scientific) was employed to detect the percentage of lysed cells .

were stained with propidium iodide (PI) and diluted with PBS so that samples contained less than 500 cells/µl for 10 000 events per run. After gating to select whole cells, employing a BD Accuri C6 flow cytometer, the total cell numbers were evaluated with forward scatter and PI-stained cells were detected by using an appropriate laser for fluorescence. Results were given as relative florescence units for PI uptake.

Western blotting was used to detect total GFP-S142A ExoU in transfected HEK293T cells. whole-cell lysates were generated using a modified RIPA buffer (50 mM Tris-

glycerol, a complete protease inhibitor cocktail tablet (Roche). After resuspension in lysis buffer, HEK293T cells were briefly sonicated and centrifuged at 16 000g prior to quantification of protein concentration using Bradford reagent. Samples were boiled in sample buffer (50 mM Tris-Cl pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and 10 mM DTT) and 40 μg of total protein, for each sample, resolved by SDS-PAGE prior to transfer onto nitrocellulose membranes (Bio-Rad).

Membranes were blocked in Tris-buffered saline (pH 7.4) with 0.1% (v/v) Tween 20 (TBS-T) in 5% (w/v) milk followed by incubation with primary (α-GFP) and secondary (α-mouse-HRP) antibodies. Proteins were detected using enhanced chemiluminesence reagent (Bio-Rad).

HEK 293T cells were seeded on 4 compartment glass-bottom imaging dishes (Ibidi) and transfected with pEGFP-C3 to express GFP-tagged S142A ExoU and RFP-Lamp1 (ThermoFisher), when 70% confluent. 2 hours after transfection, cells were incubated with DMSO (0.1% v/v) or compound C. Intracellular localisation of treated or untreated ExoU was monitored live and longitudinally for 24h by confocal microscopy (Zeiss LSM780) at 37 °C and 5% CO2. Colocalisation, as well as colocalisation coefficients for ExoU and lysosomes, were analysed with ImageJ.

HCE-T cells were seeded in 6-well plates and grown until a fully confluent monolayer had formed. A 10 µl pipette tip was used to make a scratch across the diameter of the wells. PA103 that had previously been expanded in LB culture to an OD600 of 0.8 were added to the scratched HCT cells at an MOI of 10. Compounds or DMSO (0.1 % v/v) were also added and infection was allowed to take place for 6 hours, after which Live/Dead fluorescence microscopy and LDH assay analyses were performed. 

ExoU with an N-terminal histidine tag was purified to homogeneity from E. coli by In vitro ExoU forms oligomers in the presence of PIP2, which in turn greatly enhances ExoU catalytic activity. Using native-PAGE and immunoblotting we found that compound D, but not compound C, E or F, was able to prevent ExoU oligomerisation in vitro ( Figure 2) . SpcU, the cognate inhibitory chaperone of ExoU, also prevented ExoU oligomerisation, serving as a positive control (Figure 2 ). We hypothesised that the mechanism of inhibition of ExoU by compound D was distinct to that of compound C, E and F, and therefore might be used in combination to further increase ExoU inhibitory activities. Alone, compounds C and D inhibited

ExoU phospholipase activity, with IC50 values of 1.7 ± 0.5 µM and 16.3 ± 3.4 µM, respectively ( Figure 3A) . However, the inhibitory activity for these compounds, when used in combination, is markedly improved to an IC50 of 310 ± 16 nM ( Figure 3B ). 

Human cells transfected to express WT ExoU undergo membrane blebbing and rapid cell lysis [24] . Using a previously established protocol [23] , we sought to determine whether or not compounds C, D, E or F could protect transfected HeLa cells from ExoU mediated cell lysis (Figure 4 ). Brightfield microscopy was employed to observe morphology and quantity of adherent HeLa cells after FLAG-tagged ExoU expression, with and without compounds present in HeLa cells ( Figure 4A ). The percentage LDH release ( Figure 4B ) and propidium iodide (PI) uptake ( Figure 4C 

In a previous study, we determined that established ExoU inhibitors, Pseudolipasin A, compound A and compound B, caused a reduction in the total amount of ExoU observed in transfected human cells. We sought to determine how the compounds identified in the present screen influenced ExoU stability and localisation in transfected HEK293T cells. As wild-type ExoU causes rapid lysis when expressed in human cells,

HEK293T cells were transfected with pEGFP-C3 to express a GFP-tagged catalytically inactive (S142A) ExoU mutant ( Figure 5 ). Compound C caused a dosedependent reduction in the total quantity of GFP-S142A ExoU, detectable by western blotting, in the lysates of transfected HEK293T cells. Employing fluorescence microscopy, we observed that without compound present (DMSO 0.1% v/v), GFP-ExoU localised almost exclusively to plasma membrane ( Figure 5B ). However, in the presence of compound C, GFP-S142A ExoU was observed to be punctate throughout the cytoplasm ( Figure 5B ). Our previous study indicated that ExoU was not degraded by proteasomes, so we hypothesised that certain compounds might cause lysosomal degradation of ExoU. To this end, we co-transfected HEK293T cells to express GFP-S142A ExoU and RFP-tagged lysosomal associated protein 1 (RFP-LAMP1) ( Figure   5C ). We found that GFP-S142A ExoU colocalised RFP-LAMP1 ( Figure 5C ), which might indicate that, in the presence of compound C, ExoU is targeted for lysosomal destruction. 

Employing a scratch and infection assay [23] , we sought to determine whether or not any of the compounds had the ability to reduce the extent of HCE-T cell lysis caused by endogenous ExoU secreted from the PA103 strain of P. aeruginosa ( Figure 6 ). Six 

In the present study, we employed a high throughput phospholipase assay screen to identify small molecules that have the ability to inhibit recombinant ExoU in vitro.

Inhibitors of ExoU activity may provide a means by which to interrogate the mechanisms of ExoU activation as well as avenues for potential therapies, in P.

aeruginosa infections were ExoU is a determinant of poor clinical outcome. Of 6,500 compounds screened, we discovered ~25 compounds that could inhibit ExoU at micromolar concentrations ( Figure 1 ). From this panel we selected compounds C, D, E and F for further analysis.

Aside from modulating catalytic activity by binding to the active site, compounds may possess alternative modes of ExoU inhibition [21] , for instance by preventing oligomerisation, ubiquitin or phospholipid binding. ExoU oligomerisation in the presence of phospholipids is an important step in its full activation in the host cell. In this study, we showed that of the panel of compounds selected, compound D was able to prevent recombinant ExoU oligomerisation ( Figure 2 ). This distinct mechanism of ExoU inhibition suggested that in combination with other compounds, including compound C synergistic inhibition of ExoU inhibition could be achieved. This was indeed the case as together compounds C and D had a 310 nM IC50 ( Figure 3 ). As compound D prevented ExoU oligomerisation, it could be hypothesised that that this molecule disrupts interaction with PIP2, perhaps by binding to the ExoU C-terminus.

However, the exact binding sites of these compounds on ExoU is not known, therefore, crystal structures and mutagenesis studies will be pivotal in elucidating the modes and mechanisms of compound interaction.

Once inside the host cytoplasm, ExoU binds to ubiquitin and uses its C-terminal 4helical bundle to localise with and insert into plasma membranes [17] . Consistent with this mechanism, using fluorescence microscopy we observed that GFP-tagged S142A

ExoU was localised almost entirely to the plasma membrane. However, in the presence of compound C, much less GFP-ExoU could be detected, and the little ExoU present was found to be colocalised with lysosomes. This result suggests that the binding of compound C to ExoU might cause ExoU to traffic to lysosomes where it is degraded. The human chaperone protein DNAJC5 has recently been elucidated to be indispensable for ExoU cytotoxicity through its trafficking activity, rather than direct chaperoning [22] . Compound C might interfere with ExoU trafficking, or induce an ExoU conformation that promotes its trafficking to lysosomes.

Compounds C, D, E and F, as well as inhibiting ExoU phospholipase activity in vitro could also mitigate ExoU induced cell lysis in transfected HeLa cells, which suggests potential for therapeutic use. To further explore this, we sought to determine whether or not these compounds had the ability to protect human corneal cells from ExoU secreted by P. aeruginosa. Compounds C and D reduced the extent and severity of HCE-T cell lysis after PA103 infection and were more efficacious when used in combination ( Figure 6 ). A combinational therapeutic approach that target distinct mechanisms of ExoU activation might be an attractive strategy. Having nanomolar potency, when compound C and D are used in combination, lends credence to the prospect that they might be efficacious in in vivo studies.

ExoU is characterised as the major virulence factor responsible for acute epithelial injury in numerous diseases. Therefore, inhibition of ExoU with small molecules could be an important novel strategy in combating acutely cytotoxic ExoU expressing P.

aeruginosa infections. Targeting of ExoU may also have the advantage of a decreased risk for selecting resistance, as inhibition of ExoU mediated virulence may synergise with and allow the host immune system to adequately respond [21] . Pharmacological targeting of ExoU may be compatible with antibiotic usage, whereby inhibitors of ExoU serve as an adjuvant therapy. In this way, ExoU inhibitors could mitigate the acute cytotoxic effects whilst conventional antibiotics eliminate the P. aeruginosa. ExoU inhibitors, discovered from in vitro screening, demonstrate distinct mechanisms of inhibition. These inhibitors should be further investigated, using biochemical and cellular based assays, to advance our understanding of the currently poorly characterised mechanisms of ExoU activity as well as efficacy testing in in vivo model disease systems.

Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia

Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections

Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections

Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa

Co-infections in people with COVID-19: a systematic review and metaanalysis

Persistent Bacterial Coinfection of a COVID-19 Patient Caused by a Genetically Adapted Pseudomonas aeruginosa Chronic Colonizer. Front Cell Infect Microbiol

Bacterial keratitis: perspective on epidemiology, clinico-pathogenesis, diagnosis and treatment

Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis

Structure and function of the Type III secretion system of Pseudomonas aeruginosa

Clinical outcomes of type III Pseudomonas aeruginosa bacteremia

Difference of Type 3 secretion system (T3SS) effector gene genotypes (exoU and exoS) and its implication to antibiotics resistances in isolates of Pseudomonas aeruginosa from chronic otitis media

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury

Intensive care unit-acquired pneumonia due to Pseudomonas aeruginosa with and without multidrug resistance

Identification of a ubiquitin-binding interface using Rosetta and DEER

Phosphatidylinositol 4,5-Bisphosphate-Dependent Oligomerization of the Pseudomonas aeruginosa Cytotoxin ExoU

A novel phosphatidylinositol 4,5-bisphosphate binding domain mediates plasma membrane localization of ExoU and other patatin-like phospholipases

Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A2 (PLA2) Enzyme, ExoU

ExoU activates NF-kappaB and increases IL-8/KC secretion during Pseudomonas aeruginosa infection

Central role of PAFR signalling in ExoU-induced NF-kappaB activation

The type III pseudomonal exotoxin U activates the c-Jun NH2-terminal kinase pathway and increases human epithelial interleukin-8 production

Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections. Microorganisms

The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis

A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U

Intoxication of host cells by the T3SS phospholipase ExoU: PI(4,5)P2-associated, cytoskeletal collapse and late phase membrane blebbing