Submitted Manuscript: Confidential
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Host monitoring of quorum sensing during Pseudomonas aeruginosa
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infection
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Authors: Pedro Moura-Alves
1,2*
, Andreas Puyskens
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, Anne Stinn
1,3,4,5
, Marion Klemm
1
, Ute
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Guhlich-Bornhof
1
, Anca Dorhoi
1,6,7
, Jens Furkert
8
, Annika Kreuchwig
8
, Jonas Protze
8
, Laura
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Lozza
1,9
, Gang Pei
1
, Philippe Saikali
1
, Carolina Perdomo
1
, Hans J. Mollenkopf
10
, Robert
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Hurwitz
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, Frank Kirschhoefer
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, Gerald Brenner-Weiss
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, January Weiner 3
rd1
, Hartmut
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Oschkinat
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, Michael Kolbe
3,4,5
, Gerd Krause
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, Stefan H.E. Kaufmann
1,13*
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Affiliations:
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1
Max Planck Institute for Infection Biology, Department of Immunology, Charitéplatz 1, 10117
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Berlin, Germany.
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2
Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University
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of Oxford, Oxford, UK.
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3
Max Planck Institute for Infection Biology, Structural Systems Biology, Charitéplatz 1, 10117
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Berlin, Germany.
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4
Department of Structural Infection Biology, Centre for Structural Systems Biology (CSSB),
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Helmholtz-Centre for Infection Research (HZI), Notkestraße 85, 22607 Hamburg, Germany.
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5
Faculty of Mathematics, Informatics and Natural Sciences, University of Hamburg,
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Rothenbaumchaussee 19, 20148 Hamburg, Germany.
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6
Institute of Immunology, Friedrich-Loeffler Institut, Greifswald - Insel Riems, Germany.
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7
Faculty of Mathematics and Natural Sciences, University of Greifswald, Greifswald,
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Germany.
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8
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10,
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13125 Berlin, Germany.
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9
Epiontis GmbH - Precision for Medicine, Barbara-McClintock- Str. 6, 12489 Berlin,
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Germany.
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10
Microarray Core Facility, Max Planck Institute for Infection Biology, Department of
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Immunology, Charitéplatz 1, 10117 Berlin, Germany.
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Submitted Manuscript: Confidential
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11
Protein Purification Core Facility, Max Planck Institute for Infection Biology, Charitéplatz
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1, 10117 Berlin, Germany.
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12
Institute of Functional Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany.
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13
Hagler Institute for Advanced Study at Texas A&M University, College Station, TX 7843.
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*Correspondence to: pedro.mouraalves@ludwig.ox.ac.uk ; kaufmann@mpiib-berlin.mpg.de
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Submitted Manuscript: Confidential
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Abstract: Pseudomonas aeruginosa (P.aeruginosa) rapidly adapts to altered conditions by
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quorum sensing (QS), a communication system used to collectively modify its behaviour, via
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production, release and detection of signalling molecules. QS molecules can also be sensed by
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hosts, however respective receptors and signalling pathways are poorly understood. We
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describe a unique pattern of regulation in the host by the Aryl hydrocarbon Receptor (AhR),
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critically dependent on qualitative and quantitative sensing of P.aeruginosa quorum. QS
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molecules bind to the AhR and distinctly modulate its activity. This is mirrored upon infection
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with P.aeruginosa collected from diverse growth stages and with QS-mutants. We propose that
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by spying on bacterial quorum, the AhR is a major sensor of infection dynamics, capable of
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orchestrating host defence according to the status quo of infection.
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One Sentence Summary: By sensing bacterial communication the Aryl hydrocarbon Receptor
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modulates host defence according to the level of threat.
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Main Text:
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Pseudomonas aeruginosa (P.aeruginosa) is a resourceful and ubiquitous gram-
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negative bacterium that causes infectious diseases in a broad spectrum of organisms, including
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plants, animals and humans(1). Its prevalence in burn victims, Cystic Fibrosis (CF) patients
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and immunocompromised individuals, such as AIDS patients, is commonly associated with a
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poor, often fatal outcome(2). P.aeruginosa is also a major cause of nosocomial infections, such
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as bacterial pneumonia, urinary tract infection and surgical-wound contamination(1). Because
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of its profound antibiotic resistance, therapy of P.aeruginosa is extremely difficult(1).
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Moreover, this pathogen possesses a wide range of mechanisms to adapt to different and
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sometimes harsh environments, further aggravating its eradication, even by antibiotic
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treatment(1). One such important and unifying mechanism is the capacity of P.aeruginosa to
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perform quorum sensing (QS)(1, 3, 4). QS is a cell-to-cell signalling mechanism employed by
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different bacteria to coordinate their activities in response to changes in community density,
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via chemical communication using different diffusible molecules, so called autoinducers (AI),
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and their receptors (Fig.1A)(3, 4). In P.aeruginosa, QS regulates production of a vast set of
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virulence factors, such as extracellular proteases and phenazines, and is crucial for colonization
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and infection, regulating diverse mechanisms such as biofilm formation and antimicrobial
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resistance(1, 3-5). Differences in P.aeruginosa virulence and transition from acute to chronic
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infection have been linked to changes in autoinducer levels, as well as in the expression of QS
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Submitted Manuscript: Confidential
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regulated genes(1, 3, 6-8). Consequently, QS constitutes an obvious target in the current search
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for novel treatment options for P.aeruginosa infections(3, 4, 9). Noteworthy, changes in
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expression of AI, and QS regulated genes, may not only impact on bacterial community
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dynamics, but also on the host response during infection. It has been previously reported that
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different QS regulated molecules, such as homoserine lactones (HSL), quinolones and
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phenazines, can interact with host cells, influencing a broad range of responses, including
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immunomodulation(9). Thus far, the host receptors and signalling pathways, as well as the
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mechanisms involved in monitoring infection dynamics are incompletely understood.
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Recently, we have demonstrated that the Aryl Hydrocarbon Receptor (AhR), a highly
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conserved ligand dependent transcription factor, directly recognizes P.aeruginosa phenazines,
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and thereby plays an important role in infection control(10). AhR binds to phenazines, mediates
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their degradation and regulates the expression of several host genes including detoxifying
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enzymes, chemokines and cytokines. Accordingly, resistance of AhR deficient (AhR
-/-
) mice to
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P.aeruginosa is diminished(10). Taking into consideration the vast set of ligands that the AhR
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is able to detect and the numerous biological roles it can exert, we hypothesized that AhR
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monitors the course of bacterial infection and disease by sensing different bacterial QS
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molecules expressed at various stages of infection (Fig.1A), and thereby orchestrates the most
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appropriate immune response against different stages of infection.
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Results
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AhR senses bacterial QS molecules in vitro
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Using luciferase AhR reporter cells(10), we infected THP-1 macrophages (THP-1 AhR
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reporter) and A549 alveolar type II pneumocytes (A549 AhR reporter) with P.aeruginosa
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laboratory wild type (WT) UCBPP-PA14 (PA14 WT) and GFP labelled (PA14 WT-GFP)
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strains collected from distinct stages of bacterial growth (early log: OD600<0.3; mid log:
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0.5<OD600<0.8; late log: OD600>1). AhR was more profoundly activated by bacteria from
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later growth phases (Fig.1B and Fig.S1A), while multiplicity of infection (MOI, Fig.S1B) and
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percentage of infected cells remained comparable over the different growth stages
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(Fig.S1C,D).Similar results were obtained with filtered bacterial supernatants from PA14-WT
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strains (Fig.1C and Fig.S1E), pointing to different AhR signalling by distinct P.aeruginosa
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molecules. A comparable phenotype was observed using supernatants from PAO1, a different
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commonly used P.aeruginosa laboratory strain (Fig.S1F). Amongst the obvious candidates are
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Submitted Manuscript: Confidential
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the P.aeruginosa phenazines, previously identified as AhR ligands (10). Consistently,
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increasing concentrations of the P.aeruginosa phenazine pyocyanin (Pyo) were detected in
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PA14 supernatants along bacterial growth (Fig.S1G, H), correlating with the observed AhR
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activation (Fig.1B,C and Fig.S1A,E).
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Phenazines are part of the QS regulated molecules expressed by P.aeruginosa, with
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Pyo providing a terminal signal of QS(3, 4, 11, 12). P.aeruginosa QS is regulated by four
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tightly controlled pathways, namely Las, Rhl, Pqs and Iqs (Fig.1A)(3, 4, 12). These pathways
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are tightly interconnected and their cognate autoinducer molecules are capable of activating a
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distinct downstream transcriptional pathway (Fig.1A). In brief, N-3-oxo-dodecanoyl-
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homoserine lactone (3-o-C12-L-HSL) and N-butanoyl-homoserine lactone (C4-L-HSL) are
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produced in a sequential manner via Las and Rhl systems, and activate the receptors LasR and
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RhlR, respectively(3, 4, 12). A third pathway, Pqs, leads to the synthesis of the Pseudomonas
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quinolone signalling molecule (2-heptyl-3-hydroxy-4-quinolone, PQS), and its precursor 4-
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hydroxy-2-heptylquinoline (HHQ), which signal via the PqsR(3, 4, 12). Recently, the Iqs
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pathway has been discovered, however the mechanism of 2-(2-hydroxyphenyl)-thiazole-4-
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carbaldehyde (IQS) production and its receptor are less understood(1, 3). Using high-
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performance liquid chromatography (HPLC), we confirmed a sequential autoinducer
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abundance in the supernatants of PA14 (Fig.1D). Considering the unique expression profiles
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of the QS molecules 3-o-C12-L-HSL, C4-L-HSL, HHQ and PQS, we determined their ability
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to modulate canonical AhR signalling. Stimulation of THP-1 and A549 AhR reporter cells with
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the different P.aeruginosa QS molecules resulted in differential modulation of AhR signalling
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(Fig.1E). The 3-o-C12-L-HSL and HHQ potently inhibited AhR activation by the known
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Pseudomonas AhR ligand 1-hydroxyphenazine (1-HP)(10), in a dose dependent manner
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(Fig.1F,G). Several QS molecules have been reported to induce apoptosis in host cells,
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depending on the concentration, cell type and exposure time(13, 14). No major differences in
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cell viability were detected for the majority of the conditions tested here, as measured by lactate
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dehydrogenase (LDH) release (Fig.S2A). An exception occurred after 24h stimulation of THP-
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1 cells with high concentrations of 3-o-C12-L-HSL (Fig.S2A). These results are in agreement
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with previous studies showing that epithelial cells, such as A549, are more resistant to 3-o-
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C12-L-HSL induced apoptosis than macrophages(13, 14). All experiments with THP-1 cells in
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the presence of 3-o-C12-L-HSL were performed at earlier time points, when no differences in
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cell viability were detected. Yet, we decided to further exclude a possible relationship between
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