Cytotoxic alkyl-quinolones mediate surface-induced virulence in Pseudomonas aeruginosa

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Open Access
Peer-reviewed

Research Article

Geoffrey D. Vrla, 

Mark Esposito, 

Chen Zhang, 

Yibin Kang, 

Mohammad R. Seyedsayamdost, 

Zemer Gitai

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Published: September 14, 2020

https://doi.org/10.1371/journal.ppat.1008867

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AbstractSurface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the downstream toxins that mediate surface-dependent P. aeruginosa virulence remain unclear, as do the signaling pathways that lead to their activation. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and act directly on host cells to cause cytotoxicity. Surface-induced AQ cytotoxicity is independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. We further show that packaging of AQs in outer-membrane vesicles (OMVs) increases their cytotoxicity to host cells but not their ability to stimulate downstream quorum sensing pathways in bacteria. OMVs lacking AQs are significantly less cytotoxic, suggesting these molecules play a role in OMV cytotoxicity, in addition to their previously characterized role in OMV biogenesis. AQ reporters also enabled us to dissect the signal transduction pathways downstream of the two known regulators of surface-dependent virulence, the quorum sensing receptor, LasR, and the putative mechanosensor, PilY1. Specifically, we show that PilY1 regulates surface-induced AQ production by repressing the AlgR-AlgZ two-component system. AlgR then induces RhlR, which can induce the AQ biosynthesis operon under specific conditions. These findings collectively suggest that the induction of AQs upon surface association is both necessary and sufficient to explain surface-induced P. aeruginosa virulence.
Author summary
Pseudomonas aeruginosa is one of the most intensely studied bacterial pathogens and is a leading cause of hospital-acquired infections in the United States. An intriguing aspect of P. aeruginosa is its ability increase its virulence following attachment to a solid surface, suggesting that these bacteria use mechano-transduction to regulate pathogenesis. However, the cytotoxins that mediate host-cell killing in response to surface attachment remain unknown. Here, we use a microscopy-based host-cell killing assay to show that the alkyl-quinolone (AQ) family of secreted small molecules is both necessary and sufficient to explain surface-induced virulence. We further show that these compounds are upregulated rapidly following bacterial surface attachment and that packaging of AQs into secreted outer membrane vesicles enhances AQ cytotoxicity. This work thus fills a major gap in our understanding of surface sensing in P. aeruginosa and provides new methods for investigating surface-dependent signaling pathways.

Citation: Vrla GD, Esposito M, Zhang C, Kang Y, Seyedsayamdost MR, Gitai Z (2020) Cytotoxic alkyl-quinolones mediate surface-induced virulence in Pseudomonas aeruginosa. PLoS Pathog 16(9):
e1008867.

https://doi.org/10.1371/journal.ppat.1008867Editor: Matthew C. Wolfgang, University of North Carolina at Chapel Hil, UNITED STATESReceived: April 29, 2020; Accepted: August 4, 2020; Published: September 14, 2020Copyright: © 2020 Vrla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the manuscript and its Supporting Information files.Funding: This work was funded by the NIH Pioneer Award DP1 AI124669-01 and the NIH NIGMS Award T32GM007388 to ZG, as well as the Princeton Catalysis Initiative to ZG and MRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this work.Competing interests: The authors have declared that no competing interests exist.

IntroductionThe opportunistic human pathogen P. aeruginosa infects a wide range of hosts such as mammals, plants, insects, and fungi [1], and is a major contributor to the morbidity of cystic fibrosis patients [2] and hospital-acquired infection [3]. P. aeruginosa uses a large set of secreted proteins and secondary metabolites to carry out the multiple requirements necessary for a successful infection, including host colonization, immune evasion, nutrient acquisition, and host cell killing (cytotoxicity) [4]. Given the multiple activities involved in pathogenesis, we recently developed a quantitative imaging-based host cell killing assay to specifically study the factors acutely required for killing host cells during short timescales [5]. This assay revealed that cytotoxicity is activated by attachment of P. aeruginosa to a solid surface [5]. This surface-induced cytotoxicity does not require the Type-IV Pilus (TFP), TFP-associated signaling (PilA-Chp-Vfr/cAMP), or Type III Secretion Systems (T3SS), but does require two regulatory proteins, LasR and PilY1 [5]. Since well-characterized cytotoxins such as T3SS and Vfr targets are not necessary for surface-induced host-cell killing in this assay, we sought to address the outstanding questions of which specific toxins mediate host cell killing in response to surface attachment.
Understanding the pathways that act downstream of LasR and PilY1 to trigger surface-induced virulence has been particularly challenging because these regulators are known to modulate many different targets [6,7,8]. P. aeruginosa possesses numerous candidate toxins that could mediate surface-induced virulence, including the type III secretion system (T3SS) and numerous other secreted proteins and secondary metabolites [4]. Many of these candidates were previously found not to be required for surface-induced virulence [5], which could reflect functional redundancy or the existence of a previously overlooked cytotoxin. Furthermore, while LasR is a direct transcriptional regulator, PilY1 is not, and the two best-characterized pathways that PilY1 regulates, those dependent on cAMP and c-di-GMP [9,10], are not necessary for surface-induced virulence.
Here we characterize the pathways that activate surface-induced virulence by first showing that a single family of cytotoxins, the alkyl-quinolones (AQs), are both necessary and sufficient to explain the surface-regulated killing of Dictyostelium discoideum by P. aeruginosa, and we extend these findings to mammalian host cells. AQs had been previously known to perform multiple functions that promote virulence [11], including activating quorum-sensing pathways [12,13,14], triggering the iron-starvation response [15,16], directly targeting host-cell functions [17,18,19], and stimulating the production of cytotoxic outer-membrane vesicles (OMVs) [20]. We show that surface-induced virulence results from direct AQ cytotoxicity, as opposed to other virulence-related functions. Furthermore, we extend the role AQs play in OMV-dependent virulence by demonstrating that AQs not only stimulate OMV production, but are themselves a major cytotoxic components of these vesicles, and that vesicle packaging significantly increases the potency of AQs. Supporting their importance in surface-induced virulence, we demonstrate that surface association triggers increased AQ accumulation. Using several reporters for AQ activity we further explain how the two previously known regulators of surface-induced virulence, LasR and PilY1, influence AQ production. Together our data indicate that surface-induced virulence results from induction of AQs, which act as toxins that directly kill host cells.

Results
Alkyl-quinolones are necessary and sufficient for surface-induced virulence of P. aeruginosa towards D. discoideum
Surface attachment strongly stimulates the ability of P. aeruginosa PA14 to kill D. discoideum amoebae [5]. Time-lapse imaging of D. discoideum exposed to planktonic P. aeruginosa demonstrates that D. discoideum completely clears the bacterial population through phagocytosis (S1 Movie). However, similar treatment of D. discoideum with P. aeruginosa that had been previously attached to a glass surface results in reduced D. discoideum motility followed by cell lysis (S2 Movie). This behavior is not unique to glass surfaces, as a variety of surfaces with different stiffnesses similarly induce P. aeruginosa virulence towards D. discoideum (S1 Fig and [5]). These results suggested that surface association leads to the induction of a factor (or factors) that makes P. aeruginosa more virulent.
To identify candidates for the toxin responsible for surface-induced virulence we used our surface-induced virulence assay to screen a number of mutants in secreted effectors or global regulatory proteins known to promote pathogenesis (S2 Fig). Specifically, we grew each mutant to the same density, allowed it to associate with a glass surface for 1 hour, added D. discoideum host cells, and monitored host cell death by fluorescence microscopy using the live-cell-impermeant dye, calcein-AM. Loss of many candidate P. aeruginosa cytotoxins, including phenazines, rhamnolipids, and hydrogen cyanide, or global regulators of virulence, including Vfr, GacA, and PvdS, did not significantly reduce surface-induced killing of D. discoideum (S2 Fig). In contrast, PqsA was absolutely required for surface-induced virulence (Fig 1A and S2 Fig). PqsA is an enzyme required for the biosynthesis of AQs such as PQS, HHQ, and HQNO [21], suggesting that AQs play a key role in surface-induced virulence. To determine if other factors might promote virulence at lower multiplicities of infection (MOI), we measured the killing of D. discoideum by wild type and pqsA mutant bacteria at a range of MOIs. Under no conditions did we detect significant killing by the pqsA mutant, while wild type bacteria effectively killed D. discoideum after 1 hour at MOIs as low as 50 to 1 (S3 Fig).
Fig 1. Alkyl-quinolone production is necessary and sufficient for surface-induced killing of D. discoideum by P. aeruginosa.(A) Representative images of D. discoideum infected with surface-attached wild type and ΔpqsA P. aeruginosa after 1 h co-culture (scale bars=30 μm). Fluorescent calcein-AM staining indicates cell death. (B) Schematic of the PQS pathway depicting the functions of relevant genes. Solid and dotted arrows represent biosynthetic reactions and gene regulation, respectively. Genes above arrows indicate genetic requirements for pathways. (C) Quantification of D. discoideum killing by PQS pathway mutants. Expression of pqsABCDE is driven by the endogenous pqsA promoter or a strong constitutive promoter inserted upstream of the pqsA gene (Pconst−pqsABCDE). Values are mean ± SEM from 3 biological replicates, and approximately 200–300 cells were analyzed for each measurement. (D) Cytotoxicity of purified HHQ and PQS to D. discoideum in axenic cultures grown for 3 days at 22°C. Values are mean ± SD of five biological replicates (n=5). E) MTT cell viability assay of TIB-67 mouse monocytes after 48 h of treatment with various concentrations of the alkyl-quinolones HHQ, PQS, or HQNO in a 96-well format. Percent survival is relative to an untreated control. Values are mean ± SEM of three biological replicates (n=3). (F) Quantification of survival of TIB-67 monocytes after 18 h co-cultured with surface-attached P. aeruginosa. Values are mean ± SEM of 4–5 biological replicates (n=4–5). Approximately 2500–3000 cells were analyzed for each measurement. For statistical analysis, mutants were compared to wild type unless shown otherwise. Statistical analyses are Student’s t-test (two-tailed, *=p2,500 cells analyzed per sample). Cell stains (Hoescht and FM 4–64) and GFP+P. aeruginosa were not used for experiments quantified in (G) to prevent interference with biosensor signal. Statistical tests are Student’s two-tailed t-test. Biosensor signal is the mean PpqsA-YFP / PrpoD-mKate fluorescence per cell subtracted by this value for the DMSO (0 μM HHQ) control condition.

https://doi.org/10.1371/journal.ppat.1008867.g002Having validated our AQ biosensor, we used it to compare AQ levels between planktonic and surface-attached P. aeruginosa populations. Because the AQ biosensor responds to both HHQ and PQS, we focused on quantifying AQs from ΔpqsH, which makes HHQ but not PQS. We note that this strain is less virulent than wild type but retains 40% of its virulence and its virulence is still specifically induced by surface-association (Fig 1C). We doped the AQ biosensor (1:50) into surface-attached and planktonic populations of ΔpqsH at the time of D. discoideum addition. The AQ biosensor is itself avirulent such that doping it at low levels (1:50) enabled us to quantify AQ production without disrupting the assay. Comparing the biosensor signal, we observed a significant increase in biosensor signal in surface-attached populations as compared to planktonic ΔpqsH populations (Fig 2D and 2E). Conversion of biosensor signal to HHQ concentration using the standard curve (Fig 2C) indicated that the HHQ concentration in surface-attached P. aeruginosa population during D. discoideum infection is 2.4 ± 0.2 μM (Fig 2E), while HHQ was undetectable in the planktonic condition (statistically indistinguishable from the 0 HHQ control) (Fig 2E). This reflects a fold increase by a factor of at least 20 and indicates that surface attachment stimulates AQ accumulation.
Given our results that association with a glass surface stimulated AQ accumulation, we wanted to determine if a biological surface such as a host-cell monolayer can also stimulate AQ accumulation. First, we confirmed the levels of bacterial attachment to a monolayer of A549 human lung epithelial cells after co-culture for 1 hour with P. aeruginosa constitutively expressing GFP (S9 Fig). We then used our fluorescent AQ biosensor to compare AQ accumulation in P. aeruginosa cells that were attached to a monolayer of A549 human lung epithelial cells for 1 hour to that of planktonic cells immediately after exposure to an A549 monolayer (Fig 2F). We observed significantly higher AQ biosensor signal when doped into populations of A549-attached bacteria (Fig 2G), suggesting that association with human cells can also stimulate AQ accumulation.

Packaging of AQs in secreted outer membrane vesicles increases AQ cytotoxicity
One difference between bacterially produced and purified AQs is that when bacteria make AQs they are often packaged into OMVs [20]. AQs stimulate the production of secreted outer-membrane vesicles (OMV) and are abundant in OMVs themselves [20]. Furthermore, OMV’s can induce rapid cell death in mammalian cell lines [27,28,29], but the specific cytotoxin responsible for this killing is unclear. To determine if OMVs are cytotoxic to D. discoideum, and if AQs are responsible for this cytotoxicity, we treated axenic D. discoideum cultures with equal amounts of OMVs purified from wild type or ΔpqsA P. aerguinosa and monitored D. discoideum death (Fig 3A). OMVs containing AQs resulted in nearly complete D. discoideum death after 1 hour, while no significant cell death was observed in the D. discoideum treated with OMVs from the pqsA mutant (Fig 3A). Thus, AQs are a major contributor to the cytotoxicity of OMVs towards D. discoideum.
Fig 3. AQs are responsible for OMV cytotoxicity and OMV packaging enhances AQ potency.(A) Quantification of D. discoideum death following treatment of axenic cultures with outer membrane vesicles (OMVs) isolated from wild type (WT) and ΔpqsA mutant P. aeruginosa or a vehicle control (t=1 h). (B) Images of D. discoideum treated with fluorescently labelled OMVs (oprM::mNeonGreen) from wild type and pqsA backgrounds in the absence of live-dead calcein-AM dye after 1 h (scales bars=10 μm). (C) Quantification of D. discoideum death following treatment of axenic cultures with lysed or unlysed outer membrane vesicles (OMVs) isolated from wild type (WT) P. aeruginosa (t=1 h). (D) Representative images of TIB-67 mouse monocyte at various timepoints following treatment with OMVs isolated from wild type (WT) and pqsA mutant P. aeruginosa or a vehicle control. Propidium iodide (red) staining of nuclei indicates cell death (scale bars=100 μm). (E) Quantification of monocyte death in (D). Percent death is the increase in PI-stained nuclei divided by the total PI-negative cells at 1 h. Values are mean +/- SEM. Approximately 2500–3000 cells were analyzed for each measurement. Data shown in (B) and (C) are mean and SD of two independent experiments. Approximately 300–500 cells were analyzed for each condition. Vehicle control is solution collected by filtering OMV samples in PBS (pH=7) through 100k MWCO filters. Statistical analysis in B and E is Student’s two-tailed t-test (***=p 500 cells) of surface-attached P. aeruginosa expressing a (PpqsA−mCherry) promoter fusion normalized by the expression of a constitutive Ptac-mCherry reporter. Values are mean ± SEM of three biological replicates (n=3). (E) LC/MS-based quantification of HHQ and PQS in extracts of wild type, ΔpilY1, and ΔlasR liquid cultures grown to OD=1.5. Values are mean ± SEM of three biological replicates (n=3), and concentrations were calculated using a standard curve constructed from purified AQ standards (n.d.=not detected). (F) Quantification D. discoideum killing by mutants in the AlgR-PilY1 pathway after 1 h co-culture. (G) qRT-PCR analysis comparing relative transcript abundance in surface-attached to planktonic P. aeruginosa after 1 h growth on a surface, with diagram showing relative amplicon positions (red lines) in the pqsA gene and upstream regions. Values are mean ΔΔCt values ± SEM (n=3 biological replicates) relative to the gapA control transcripts. (H) Mean fluorescence intensity per cell (>500 cells) of surface-attached P. aeruginosa expressing a (Plrs1−mCherry) promoter fusion. Values are mean ± SEM of three biological replicates (n=3). Values in (B) and (F) are mean ± SEM of three biological replicates (n=3). Statistical analysis was performed against wild type (Student’s t-test, two-tailed, *=p
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