In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores

ABSTRACT The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cells. We demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection. IMPORTANCE Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.


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Reviewer #1 (Comments for the Author): In this study, the cytotoxic effect of pyoverdine and pyochelin was investigated using a human bronchial epithelial cells (16HBE) model by comparing spent media of P. aeruginosa wild type and isogenic pyoverdine (pvd) or pyochelin (pch) defective mutants.P. aeruginosa pvd mutant supernatant was less cytotoxic than wild type or pch mutant supernatants.In addition, the pyoverdine synthesis inhibitor 5-fluorocytosine reduced the toxicity of high-pyoverdine producers and highly virulent P. aeruginosa clinical isolates.However, purified pyoverdine was not cytotoxic.Since previous studies showed that pyoverdine itself can modulate the expression of other virulence factors, authors postulated that pyoverdine could positively affect the production of other toxins/cytotoxic factors.However, toxA or prpL genes inactivation did not affect P. aeruginosa toxicity.Removal of lipidic fraction from P. aeruginosa wild type spent medium by chloroform extraction abrogated cytotoxicity, suggesting involvement of rhamnolipids.In accordance with this hypothesis, purified rhamnolipids were cytotoxic in the 16HBE model.C4-HSL-dependent QS is a major positive controller of rhamnolipids biosynthesis.However, the pvd defective mutant produced the same C4-HSL levels as the wild type.Authors conclude that pyoverdine modulates the rhamnolipids production independently from QS.Other experiments (less convincing, see major comments) were carried out to rule out the possibility that mexEF efflux could play a role in the pyoverdine-dependent regulation of rhamnolipids.In the final part of the study, authors investigated the effect of different iron chelators on 16HBE inflammatory response.Results showed that pyochelin and other small MW iron chelators (es.deferoxamine) were cytotoxic, Cytotoxicity was abrogated by gallium.However, pyoverdine was not cytotoxic.Interestingly, the combination of pyoverdine and deferoxamine showed increased cytotoxicity compared to deferoxamine alone.
Overall, experiments are well conducted, and results deserves to be published.My major issues are below: 1) The work is written in good english but in a convoluted manner.Some logical steps are not made explicit.Each experiment should be described by first explaining why it is done and then what the conclusions are.
2) The order in which the experiments are described is not linear and contributes to the reader's confusion.The authors should reorder the description of the results following the order described in the summary above.
3) The majority of the experiments concerning the mexEF system with transposon mutants are unnecessary and confounding.The only result Authors should describe, in this case, is that mexT and mexEF genes are not mutated in PAO1 ∆pvdF.The result should be shown in supplementary.4) The secondary metabolite pyocyanin is very cytotoxic and present, together with siderophores and other virulence factors, in membrane vesicles produced by P. aeruginosa.Vesicles and their content content could be retained by filtering.Authors should consider the possibility that, in addition to rhamnolipids, also pyocyanin production could be reduced in the pvd mutant.Pyocyanin should be measured in P. aeruginosa filtered supernatants.5) Levels of rhlA transcription should be measured in the wild type and in the pvd mutant.6) Swarming motility is related to virulence and biofilm production, and rhamnolipids play a major role in this phenotype.Authors should compare swarming in wild type and pvd mutant.
Reviewer #2 (Comments for the Author): Minor comments: 1.A graphical plot of the in vitro model would help to understand much easier the "infection" set up. 2. From line 112 in text , as pvdF-pchBA mutant did not provide further protection compare to pvdF alone, Fig S1 C and E figure should show all statistical comparison including the (NS, if that is the case) between pvdF and pvdF-pchBA double mutant.3. Line 170 which "mutants" are you describing?Is the PAO1cat vs PAO1pvdf?Please refer it as the control vs the pyoverdineproducing counterparts as in line 157.4. Line 173 -174 I am not sure I understand why the authors imply that pyoverdine regulates rhamnolipid production though an alternative pathway when the results were analyzed from simple mutants of rhlI.The results should be addressed from a double mutant pvdf -rhlI perspective.5. Line 189 -think you are referring to Fig S3E and not Fig 3E. 6. WGS raw reads should be deposited into a repositorium such as SRA from NCBI. 7. A description on how the quantification of lipids was performed, should be included in the material and methods sections.8. Considering the interesting results, the way by which the authors support their conclusions, and because the role of different molecules is being assessed, I suggest that a graphical diagram showing the most important results achieved in this manuscript is included.
In this manuscript, Donghoon Kang and col., have explored the role of relevant virulence factors from P. aeruginosa by using an in vitro model of bronchial epithelial cell line monolayer.The project focuses on the host -induced responses and mechanisms involved upon interaction of the host cells with condition media from different mutants of the bacteria.The manuscript includes thorough supporting information for each of the result sections which are supported by several molecular assays such as qRT PCR, pyoverdine production, growth curves, host cell death, as well as confocal microscopy.They also explore the differences between some of the mutants by whole genome sequencing.Data is well integrated and clearly discussed.

Major comments:
My main concern relies on the degree to which the current results can be extrapolated to an in vivo scenario, such as the chronic infections that P. aeruginosa produce within the lung of patients with cystic fibrosis.A significant number of studies (including this one) have explored the interaction of P. aeruginosa and airway epithelial cells using two-dimensional models of cell lines on non-permeable surfaces that do not allow polarization (review Crabbé et al. 2014).Such models, fail to mimic epithelial cell polarity and mucus production, which has a significant advantage for human cells to protect against bacterial invasion.Furthermore, the results and conclusions achieved here could be less drastic or even disappear in the context of a pseudostratified epithelium.The authors should deep-in this matter on the Discussion section.6. WGS raw reads should be deposited into a repositorium such as SRA from NCBI. 7. A description on how the quantification of lipids was performed, should be included in the material and methods sections.8. Considering the interesting results, the way by which the authors support their conclusions, and because the role of different molecules is being assessed, I suggest that a graphical diagram showing the most important results achieved in this manuscript is included.

Reviewer #1 (Comments for the Author):
In this study, the cytotoxic effect of pyoverdine and pyochelin was investigated using a human bronchial epithelial cells (16HBE) model by comparing spent media of P. aeruginosa wild type and isogenic pyoverdine (pvd) or pyochelin (pch) defective mutants.P. aeruginosa pvd mutant supernatant was less cytotoxic than wild type or pch mutant supernatants.In addition, the pyoverdine synthesis inhibitor 5-fluorocytosine reduced the toxicity of high-pyoverdine producers and highly virulent P. aeruginosa clinical isolates.However, purified pyoverdine was not cytotoxic.
Since previous studies showed that pyoverdine itself can modulate the expression of other virulence factors, authors postulated that pyoverdine could positively affect the production of other toxins/cytotoxic factors.However, toxA or prpL genes inactivation did not affect P. aeruginosa toxicity.Removal of lipidic fraction from P. aeruginosa wild type spent medium by chloroform extraction abrogated cytotoxicity, suggesting involvement of rhamnolipids.In accordance with this hypothesis, purified rhamnolipids were cytotoxic in the 16HBE model.C4-HSL-dependent QS is a major positive controller of rhamnolipids biosynthesis.However, the pvd defective mutant produced the same C4-HSL levels as the wild type.Authors conclude that pyoverdine modulates the rhamnolipids production independently from QS.Other experiments (less convincing, see major comments) were carried out to rule out the possibility that mexEF efflux could play a role in the pyoverdine-dependent regulation of rhamnolipids.
In the final part of the study, authors investigated the effect of different iron chelators on 16HBE inflammatory response.Results showed that pyochelin and other small MW iron chelators (es.deferoxamine) were cytotoxic, Cytotoxicity was abrogated by gallium.However, pyoverdine was not cytotoxic.Interestingly, the combination of pyoverdine and deferoxamine showed increased cytotoxicity compared to deferoxamine alone.
Overall, experiments are well conducted, and results deserves to be published.My major issues are below: 1) The work is written in good english but in a convoluted manner.Some logical steps are not made explicit.Each experiment should be described by first explaining why it is done and then what the conclusions are.
We appreciate the reviewer's feedback.We made relevant improvements to the manuscript.For instance: • We clearly state the hypothesis before discussing the effects of 5-fluorocytosine:"Since genetic disruption of pyoverdine biosynthesis decreased toxicity of the conditioned medium, we hypothesized the same result could be accomplished using a chemical inhibitor.To that end, we tested whether the pyoverdine biosynthetic inhibitor and FDA-approved antimycotic drug 5-fluorocytosine (5-FC) inhibited pyoverdine-dependent virulence."• We clearly explain why we are conducting the pyocyanin supplementation experiment: "We also examined whether conditioned medium cytotoxicity could be attributed to pyocyanin content since pyocyanin is known to cause acute oxidative damage to host cells" • We clearly state the hypothesis before discussing the phenotypes of the rhamnolipid biosynthetic mutants in EMEM: "Based on previous studies, we posited that the relevant secreted lipid factors were rhamnolipids.We recently demonstrated that P. aeruginosa secretes rhamnolipids that rapidly induce membrane rupture and permeabilization in a wide range of host cells, including murine macrophages, human bronchial epithelial cells, and erythrocytes.
To test this hypothesis, we used a rhamnolipid biosynthetic mutant, MPAO1rhlA, to measure the lipid content of conditioned media."• We clearly state the purpose of the swarming motility experiment: "In addition to killing host cells, rhamnolipids are known to regulate P. aeruginosa swarming motility, which promotes pathogen proliferation and biofilm formation within the host.To test whether pyoverdine production affects swarming motility, we measured the area of lawn growth on EMEM semisolid agar (0.5%) for wild-type PAO1, the pyoverdine biosynthetic mutant ΔpvdF, and the rhamnolipid biosynthetic mutant rhlA." 2) The order in which the experiments are described is not linear and contributes to the reader's confusion.The authors should reorder the description of the results following the order described in the summary above.
The figures and manuscript text have been rearranged accordingly.As the reviewer suggested, we first discuss the role of pyoverdine in conditioned medium toxicity in Fig. 1 then discuss how supplementing the P. aeruginosa growth medium (EMEM) with 5-fluorocytosine mitigates that toxicity in Fig. 2. We then investigate the identity of the toxic factor in the conditioned medium in Fig. 3 and validate these findings using rhamnolipid biosynthetic mutants in Fig. 4. We demonstrate that purified rhamnolipids kill 16HBE cells and that rhamnolipids contribute to swarming motility in EMEM in Fig. 5. Finally, we study the consequences of long-term exposure (~24-60 h instead of 30 min as in Fig. 1-5) to siderophores (pyoverdine, pyochelin) -their relative toxicities (Fig. 6) and their effects on the activation of proinflammatory pathways (Fig. 7).
3) The majority of the experiments concerning the mexEF system with transposon mutants are unnecessary and confounding.The only result Authors should describe, in this case, is that mexT and mexEF genes are not mutated in PAO1 ∆pvdF.The result should be shown in supplementary.
We appreciate the reviewer for pointing this out.Based on these concerns, we removed all results regarding the mexEF system.
4) The secondary metabolite pyocyanin is very cytotoxic and present, together with siderophores and other virulence factors, in membrane vesicles produced by P. aeruginosa.Vesicles and their content content could be retained by filtering.Authors should consider the possibility that, in addition to rhamnolipids, also pyocyanin production could be reduced in the pvd mutant.Pyocyanin should be measured in P. aeruginosa filtered supernatants.
We appreciate the suggestion.We measured pyocyanin content in the conditioned media (see new materials and methods subsection "Pyocyanin Measurement") and found that WT PAO1 produced significantly greater levels of pyocyanin than the pyoverdine mutant.However, this concentration (~6 µM) was not sufficient to kill 16HBE cells within the time we observe substantial cell death by the conditioned medium (~15 min).In fact, even concentrations 10-folds greater (60 µM) was not sufficient to kill cells.These results reinforce our findings that pyoverdine-dependent production of rhamnolipids drives conditioned medium toxicity.We included these results in Fig. S4 (lines 215-230).
5) Levels of rhlA transcription should be measured in the wild type and in the pvd mutant.
We measured rhlA, rhlB, rhlR, and rhlI mRNA levels in WT PAO1 and PAO1ΔpvdF grown in EMEM.For all genes, the pyoverdine mutant did not exhibit substantial (fold change ≥ |1.5|) decrease in transcription.We concluded in the manuscript that this may be due to pyoverdine regulating rhamnolipid production via other biosynthetic or regulatory factors or pyoverdine regulating rhamnolipid egress rather than biosynthesis (lines 364-371).The relevant figure has been incorporated as Fig. S6E.
6) Swarming motility is related to virulence and biofilm production, and rhamnolipids play a major role in this phenotype.Authors should compare swarming in wild type and pvd mutant.
graphical plot of the in vitro model would help to understand much easier the "infection" set up. 2. From line 112 in text , as pvdF-pchBA mutant did not provide further protection compare to pvdF alone, Fig S1 C and E figure should show all statistical comparison including the (NS, if that is the case) between pvdF and pvdF-pchBA double mutant.3. Line 170 which "mutants" are you describing?Is the PAO1cat vs PAO1pvdf?Please refer it as the control vs the pyoverdine-producing counterparts as in line 157.4. Line 173 -174 I am not sure I understand why the authors imply that pyoverdine regulates rhamnolipid production though an alternative pathway when the results were analyzed from simple mutants of rhlI.The results should be addressed from a double mutant pvdf -rhlI perspective.5. Line 189 -think you are referring to Fig S3E and not Fig 3E.