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bioRxiv
BIORXIV
bioRxiv
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Cold Spring Harbor Laboratory

39282278
10.1101/2024.09.03.611043
preprint
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Article
The Pseudomonas aeruginosa sphBC genes are important for growth in the presence of sphingosine by promoting sphingosine metabolism
DiGianivittorio Pauline 12
Hinkel Lauren A. 123
Mackinder Jacob R. 12
Schutz Kristin 1
http://orcid.org/0000-0003-3941-6233
Klein Eric A. 3
http://orcid.org/0000-0001-5659-749X
Wargo Matthew J. 1*
1 Department of Microbiology and Molecular Genetics, Larner College of Medicine, University of Vermont
2 Cellular, Molecular, and Biomedical Sciences Graduate Program, University of Vermont
3 Biology Department, Rutgers University-Camden
* Corresponding author: Matthew J. Wargo, 95 Carrigan Drive, 322 Stafford Hall, Burlington, VT 05405, mwargo@uvm.edu, F: 802-656-8749
03 9 2024
2024.09.03.611043https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format for noncommercial purposes only, and only so long as attribution is given to the creator.
nihpp-2024.09.03.611043.pdf
Sphingoid bases, including sphingosine, are important components of the antimicrobial barrier at epithelial surfaces where they can cause growth inhibition and killing of susceptible bacteria. Pseudomonas aeruginosa is a common opportunistic pathogen that is less susceptible to sphingosine than many Gram-negative bacteria. Here, we determined that deletion of the sphBCD operon reduced growth in the presence of sphingosine. Using deletion mutants, complementation, and growth assays in P. aeruginosa PAO1, we determined that the sphC and sphB genes, encoding a periplasmic oxidase and periplasmic cytochrome c, respectively, were important for growth on sphingosine, while sphD was dispensable under these conditions. Deletion of sphBCD in P. aeruginosa PA14, P. protegens Pf-5, and P. fluorescens Pf01 also showed reduced growth in the presence of sphingosine. The P. aeruginosa sphBC genes were also important for growth in the presence of two other sphingoid bases, phytosphingosine and sphinganine. In wild-type P. aeruginosa, sphingosine is metabolized to an unknown non-inhibitory product, as sphingosine concentrations drop in the culture. However, in the absence of sphBC, sphingosine accumulates, pointing to SphC and SphB as having a role in sphingosine metabolism. Finally, metabolism of sphingosine by wild-type P. aeruginosa protected susceptible cells from full growth inhibition by sphingosine, pointing to a role for sphingosine metabolism as a public good. This work shows that metabolism of sphingosine by P. aeruginosa presents a novel pathway by which bacteria can alter host-derived sphingolipids, but it remains an open question whether SphB and SphC act directly on sphingosine.

sphingosine
lipid
pathogenesis
NIH NIAID R01 AI103003 and Cystic Fibrosis Foundation WARGO24G0 (both to MJW) NIH NHLBI T32 HL076122 (supporting PD) and NIH NIAID T32 AI055402 (supporting LAH) NSF MCB-1553004 (to EAK)
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pmcIntroduction

In addition to their various cellular and signaling functions, some sphingolipids are key antimicrobial lipids with activity against both Gram-positive and Gram-negative bacteria(1–6). Antimicrobial sphingolipids are found at sites throughout the body including the lungs, the skin, and all mucosal surfaces(4, 7–13). Imbalances or deficiencies in barrier-associated sphingolipids, particularly sphingoid bases (examples in Fig 1A), increase chances of bacterial infection, illustrating the importance of these sphingolipids in defense against pathogens(14–16). The initial antibacterial action for sphingoid bases is predicted to be bacterial membrane disruption, due to their amphiphilic and detergent-like properties, followed by accumulation of sphingolipids in the cytosol, ultimately leading to cell death in both Gram-negative and Gram-positive bacteria(4, 6, 17).

In Gram-negative bacteria, sphingolipid exposure causes separation of the inner and outer membranes, similar to the type of damage caused by cationic peptides like cathelicidins(6). The concentrations of sphingoid bases needed to cause severe and cytotoxic membrane disruption in many bacteria is low, with Serratia marcescens and Pseudomonas aeruginosa as exceptions, requiring higher concentrations or specific media conditions. For example, the minimum bactericidal concentration for P. aeruginosa in most media is > 1 mM, more than 300-fold higher than for Staphylococcus aureus, which often co-infect in lungs and wounds(1), though P. aeruginosa killing can be seen with concentrations as low as 10 µM under distinct media and sphingoid base solubilization regimes(17) and sphingosine-dependent killing of P. aeruginosa can also be seen intracellularly(18). Although there are many factors that influence antimicrobial-bacterial interactions, the sphingolipid resistance profile of P. aeruginosa suggests that it possesses specific mechanisms for resistance to or detoxification of sphingoid bases.

P. aeruginosa is associated with a variety of infections, including hospital-acquired and ventilator-associated pneumonia and bacteremia(19–22), as well as chronic lung infection in individuals with cystic fibrosis (CF) and chronic obstructive pulmonary disorder (COPD)(22–28). Many of these infection niches contain abundant sphingosine, other sphingoid bases, and the sphingosine precursors sphingomyelin and ceramide(2, 29–33), though a decrease in sphingosine concentration due to ceramide accumulation has been shown in CF(34, 35). Therapeutic intervention to treat ceramide accumulation can rescue the susceptibility to P. aeruginosa infection in animal models(36). Within the context of pulmonary infections, P. aeruginosa’s ability to resist the antimicrobial effects of sphingosine is correlated with a survival advantage, due in part to the presence of sphingoid bases within the lung epithelium(23).

Exposure of P. aeruginosa to pulmonary surfactant leads to induction of a small set of sphingosine-responsive genes in an SphR-dependent manner, including a metabolic operon, sphBCD, encoding a predicted cytochrome c (sphB), predicted oxidoreductase enzyme (sphC), and predicted PLP-dependent aldolase enzyme (sphD)(23) (Fig 1B). We previously showed that loss of sphC led to a small but statistically significant reduction in P. aeruginosa survival in the presence of sphingosine(23). However, the conditions needed for sphingosine killing of P. aeruginosa are very specific. Thus, we sought to examine the effects of sphingosine conditions that may more closely mimic some infections sites. Here we demonstrate that, in addition to killing under specific conditions, sphingosine can strongly suppress growth of an P. aeruginosa sphBCD mutant, with follow-up experiments supporting the sphBC genes as important for P. aeruginosa growth in the presence of sphingosine via sphingosine detoxification. Sphingosine detoxification can function as a public good promoting growth of sphingosine-susceptible P. aeruginosa mutants.

Results

The importance of sphBCD genes for P. aeruginosa growth in the presence of sphingosine and sphingosine analogs

We previously reported the importance of sphR and sphA for resistance to sphingosine-dependent killing of P. aeruginosa PAO1 with a minor impact of sphC mutation(23). Killing of P. aeruginosa by sphingosine requires specific media conditions (high divalent cation concentration) and/or micellular sphingosine(17, 23). We observed that even in the absence of these very particular conditions and thus the absence of killing, sphingosine could strongly inhibit growth of P. aeruginosa ∆sphBCD deletion mutants and that inhibition was stronger when growth was conducted in glass rather than in plastic at a given concentration of sphingosine (Fig 2A). The same protective role of sphBCD can be observed during growth in the presence of sphinganine (Fig 2B) and phytosphingosine (Fig 2C).

Deletion of the sphBCD operon increased susceptibility to sphingosine and close analogs when measured at 18 hours post inoculation (Fig 2), and we wanted to examine the kinetics of this growth inhibition by measuring growth over time. We measured growth with 100% set as WT OD600 in the absence of sphingosine at 18 hours. At 200 µM sphingosine, the sphBCD deletion shows initial growth that starts to plateau after about 10 hours, while WT has a delay in growth before resumption of a nearly normal growth rate. The complementation strain has no substantial delay, growing at a rapid rate after lag phase (Fig 3A). The sphBCD deletion is also defective for growth in the presence of sphinganine (Fig 3B) and phytosphingosine (Fig 3C). Neither of these sphingosine analogs shows the strong delay in WT growth and, while ∆sphBCD growth in phytosphingosine shows the same plateau as for sphingosine (compare Fig 3C with 3A), the ∆sphBCD strain can grow slowly in the presence of sphinganine with a substantial delay. Growth of all strains in the absence of sphingosine is presented in Supplemental Figure S1.

The critical role for sphC for growth in the presence of sphingosine

Deletion of sphBCD can be complemented by plasmids carrying sphBCD or a plasmid carrying sphBC, but not other single genes from the locus (Fig 4A), supporting sphB and sphC as required components for resistance to sphingosine. Deletion of sphC alone phenocopies ∆sphBCD and sphC complements this phenotype in ∆sphC (Fig 4B). Similar to sphingosine, deletion of sphC results in growth inhibition by the sphingosine analogs sphinganine and phytosphingosine, and these phenotypes can be complemented (Fig 4C & D). These data support a role for sphBC in resistance to growth inhibition by antimicrobial sphingoid bases.

sphBC are important for metabolism of sphingosine to a non-toxic metabolite

While there were many potential mechanisms by which sphBC could provide sphingosine resistance, one potential mechanism was metabolism of sphingoid bases to a compound that was not growth inhibitory. The sphC gene encodes a TAT-secreted periplasmic oxidoreductase(37) and sphB encodes a sec-secreted periplasmic cytochrome c5-like protein, predicted to be a lipoprotein. These predicted functions suggested a role for oxidation of some compound in the periplasm, potentially sphingosine or a compound required for subsequent sphingosine metabolism. Sphingosine is depleted from supernatants and cell culture extracts of WT cells (Fig 5 and also seen in(38)), while sphingosine and close analogs accumulate in cell culture extracts of ∆sphBCD, as measured by bioassay (Fig 5A–C). The bioassay measures are supported by liquid-chromatography mass spectrometry (Fig 5D) and thin-layer chromatography (TLC) (Fig 5E). These data support a role for sphBC in sphingosine metabolism to a non-toxic product, as functional sphBC (WT) leads to no substantial growth inhibition and absence of the added sphingosine.

Phylogenetic distribution of the sphBCD genes and their roles in other species

The sphBCD genes are present in all sequenced P. aeruginosa and are also present in most non-aeruginosa Pseudomonads using the Pseudomonas genome browser(39). As sphB and sphC encode proteins in large families, true orthology is difficult to assess, particularly in the absence of any direct understanding of substrate interaction in the case of SphC. Co-occurrence searches with String(40) yield quite a large number of hits in the Firmicutes, Actinobacteria, and Alpha-, Beta-, and Gamma-Proteobacteria, but nothing outside of those groups. Manual searching through the resultant genes suggests some could be orthologs, including a putative SphC of Caulobacter crescentus, described below, while others are likely unrelated to sphingosine. Therefore, we first focused on assessing function of the sphBCD genes in other Pseudomonads, including P. fluorescens WCS365 which does not have an sphD ortholog in its sphBC operon. Deletion of the sphBCD genes from P. aeruginosa PA14 and P. protogens Pf-5 showed a growth defect in the presence of 200 µM sphingosine regardless of culture vessel material (Fig 6A&B). The sphBCD deletion mutant of P. fluorescens Pf01 was lower than WT in each vessel material, but the difference was only significant in glass (Fig 6A). Deletion of sphBC in P. fluorescens WCS365 did not show a phenotype. These data support a role for sphBC in resistance to sphingosine beyond P. aeruginosa, but presence of these genes does not necessarily predict importance for growth in the presence of sphingosine. We deleted the sphC gene from C. crescentus but the measured effect was significant only within a very small sphingosine concentration range (Supplemental Figure S2A). We also tested heterologous expression of C. crescentus sphBC to attempt complementation of P. aeruginosa ∆sphBCD. For C. crescentus putative sphBC, the native sec- and TAT-signal sequences encoded in sphB and sphC, respectively, were swapped for the sec- and TAT-signal sequences from P. aeruginosa sphB and sphC. While data show a trend towards partial rescue, the impact of C. crescentus sphBC in P. aeruginosa is not statistically significant (Supplemental Figure S2B).

Detoxification of sphingosine is a public good

The sphBC genes have a role in metabolism of sphingosine to a product that is not growth inhibitory, which suggests that cells capable of sphingosine metabolism could potentially protect cells that cannot otherwise metabolize sphingosine from its antimicrobial effects. Wild-type P. aeruginosa partially protects ∆sphBCD from sphingosine growth inhibition as measured by both fluorescent signal (Fig 7A) and CFU (Fig 7B) and the same effect was seen when the fluorescent markers were swapped between the strains (Supplemental Fig S3). P. aeruginosa could likewise protect the sphingosine-susceptible Staphylococcus aureus (Fig 8A). While protection of S. aureus by ∆sphBCD trended lower than wild type (Fig 8B), this did not reach significance given the assay variability.

Discussion

Sphingoid bases, including sphingosine, are important antimicrobial compounds on epithelial surfaces of mammals(12, 41) and are also produced by plants and released into the rhizosphere(42). Here, we report the identification of the P. aeruginosa sphBCD operon as necessary for metabolism, and thus detoxification, of sphingosine and other sphingoid bases, showing that functional sphBCD is needed for wild-type levels of growth in the presence of sphingoid bases. These conclusions are supported by growth studies, complementation, and measurements of sphingosine metabolism. Wild-type P. aeruginosa can also protect susceptible bacteria from sphingosine pointing to a potential role in mixed microbial communities.

The work presented here focuses on conditions wherein sphingosine inhibits growth but is not bactericidal for either wild-type or ∆sphBCD. These conditions are quite different than those required for P. aeruginosa killing by sphingosine by us and others, which typically use very high divalent cation concentrations and is dependent on the phase of the lipid(17, 23). In our current data, sphingosine is dried onto the vessel surface allowing vehicle evaporation prior to adding media and P. aeruginosa and results in growth inhibition rather than killing, though others have also noted bacterial growth inhibition rather than killing for sphingoid bases(42). Therefore, while the concentration of sphingosine in the entire well is listed in our experiments, the concentration of free sphingosine in the liquid phase at any given point in time is unknown. Our model may not mimic the antimicrobial activity of sphingosine in liquid covered epithelium, like in the lung(12), and might be a closer mimic to the antimicrobial activity of sphingosine on the skin with a temporary covering of sweat(3). In a similar manner, our model is likely closer to the behavior of plant-derived sphingoid bases in non-saturated soils. The importance and properties of the vessel material underlines the difference of our model, where there are noticeable differences in concentration-dependent inhibition and growth phenotype depending on whether the culture vessel was glass or plastic. Because of the very different conditions in our model, the phenotypes shown here are not directly comparable to the killing phenotypes we previously reported(23) or to the P. aeruginosa killing presented by others(17). In our previous work, an sphC mutant had a small but measurable defect in the sphingosine killing assay while ∆sphR and ∆sphA mutants were very susceptible to sphingosine killing. However, in the growth inhibition assay, the sphA mutant has no phenotype (Supplemental Figure S4A). Additionally and interestingly, while sphBCD can be induced by sphingosine in an sphR-dependent manner(23), sphR is not important for growth in the presence of sphingosine (Supplemental Figure S4B) suggesting either that basal transcription is sufficient for growth or that there is another regulator inducing sphBCD, perhaps related to envelope stress response. We think that these differences in phenotypes for sphingosine-related mutants in the two sphingosine response models, killing vs growth inhibition, are likely biologically important and may reflect management of sphingosine under different environmental conditions. We also note that the carbon source in minimal media impacts the effect of sphingosine on PAO1 growth with less impact of sphingosine when grown using a carbon source which enables faster growth (Supplemental Figure S5), though complementation with sphBCD still improves growth even when there is little overall inhibition (i.e. in MOPS + Succinate). This effect of carbon source could be due to either faster growth rate or more rapid accumulation of cell mass that could dilute the effectiveness of sphingosine – these are conjecture and would need to be formally tested.

Our genetic analysis implicates SphC and SphB as the critical proteins for sphingosine resistance encoded in the sphBCD operon, as deletion of sphC phenocopies ∆sphBCD (Fig 4B–D) and only vectors containing both sphC and sphB can complement ∆sphBCD (Fig 4A). SphC is predicted to be an FMN-linked oxidoreductase that is known to be TAT secreted and localized to the periplasm(37). SphB is a predicted lipoprotein cytochrome c with a Sec signal sequence. Based on the data presented here and the presence of the sphBCD operon in the sphingosine:SphR regulon(23), we predict that SphC can oxidize sphingosine to a metabolite that is non-toxic and the electron needed for this oxidation is replenished by SphB. Some evidence supporting that SphC and SphB might be partners is that while plasmid-borne sphC can complement ∆sphC, it is not as strong a complementation as plasmid-borne sphBC complementation of ∆sphBCD (Fig 4). This could be explained by a stoichiometry mismatch between SphC and SphB. As to why the plasmid carrying sphBC does not complement as well as the plasmid carrying sphBCD, we are not sure, though since we have not measured transcript and protein levels generated from these constructs, it could simply be a difference in functional expression. It is interesting to note that the two organisms that we tested that carry only sphBC in an operon P. fluorescens WCS365 and C. crescentus, compared to those with sphBCD, show little to no effect of the sphBC deletion on growth in the presence of sphingosine (Fig 6 and Supplemental Figure S2).

Multiple attempts to identify the direct metabolite of sphingosine were unsuccessful, perhaps because one potential initial product would be a very reactive aldehyde aldol. While our data here underscore the necessity of sphBC for sphingosine metabolism and normal levels of P. aeruginosa growth in the presence of sphingosine, we currently have no evidence that sphBC are sufficient for sphingosine metabolism. This leaves open the possibility that SphB and SphC act indirectly to detoxify sphingosine. Complementation of P. aeruginosa ∆sphBCD with secretion adapted sphBC homologs from Caulobacter crescentus showed no significant effect (Supplemental Figure S2). There are many reasons this heterologous complementation might have failed yet be non-informative, including poor protein folding in the heterologous host, secretion failure despite the attempt at secretion adaptation of each sequence to the heterologous host, rapid degradation of one or both proteins, or, in the case of SphB, failure to interact with the unknown inner membrane electron donor in the heterologous host. Additionally, while C. crescentus putative SphB and SphC are homologous to P. aeruginosa SphB (44% identity, 55% positive) and SphC (42% identity, 58% positive), it is unknown whether they are orthologous.

When we examined other strains and other Pseudomonas species, we noted that while sphBCD deletion led to poorer growth in the presence of sphingosine for P. aeruginosa PA14, P. fluorescens Pf01, and P. protegens Pf-5, deletion of sphBC in P. fluorescens WCS365 had no phenotype (Fig 6). Additionally, the magnitude of the phenotype differed between species and, like for P. aeruginosa, was dependent on the culture vessel material. Combining these findings with the observation that P. aeruginosa ∆sphBCD can still grow in the presence of sphingosine, albeit not to the same extent as wild type, we conclude that there are other proteins or cellular processes that can function independently of sphBCD. In P. fluorescens WCS365, there is no sphD homolog at the locus and there is very minimal decrease in growth of either wild-type or ∆sphBC. This strain must have an alternate mechanism to resist growth inhibition by sphingosine.

Regardless of whether SphC and SphB directly act on sphingosine, sphBC dependent sphingosine metabolism depletes sphingosine from the media (Fig 5). Such sphingosine depletion led us to hypothesize that metabolism of sphingosine by wild-type cells would protect sphingosine-susceptible bacteria in co-culture which we did indeed observe in a co-culture of wild-type and ∆sphBCD cells (Fig 7). Similarly, S. aureus is completely killed by 50 µM sphingosine under the conditions of our assay, but a small percentage can be protected by P. aeruginosa. While fewer S. aureus are protected by ∆sphBCD, variation in the means makes the contribution of sphBCD to this protection not statistically significant. One of the caveats of this P. aeruginosa-S. aureus co-culture is that, for these lab isolates, P. aeruginosa eventually kills the S. aureus(43–45). Future work will look at co-isolates of these species from the same patient samples, where apparently peaceful co-existence is common(46). Since many bacteria and some fungi are susceptible to sphingoid bases(3, 42), sphingoid base detoxification in areas of very high sphingosine concentration (skin, rhizoplane) might contribute to community structure and composition.

Our identification and characterization of the sphingoid base-dependent phenotype of sphBCD and sphC mutants is an important step in our understanding of bacterial manipulation of sphingolipids. However, there remain a number of important and unaddressed issues identified during our work, including the biochemical mechanism behind SphC and SphB function, the identity and role of sphBC-independent sphingosine management systems in P. aeruginosa and other Pseudomonads, and the contributions of sphingosine detoxification to spatial architecture in sessile communities.

Materials and Methods

Strains and growth conditions

Pseudomonas aeruginosa PA14, PAO1, and related mutant strains were maintained at 37°C on Pseudomonas Isolation Agar (PIA) plates with 20 μg/ml gentamicin added when appropriate. Pseudomonas fluorescens, Pseudomonas protegens, and strains of those species were maintained at 30 °C on lysogeny broth-Lennox formulation (LB) plates. Prior to assay set up, strains were grown shaking either at 37 °C or 30 °C overnight in a 1X MOPS medium (47), modified as previously described (48), and supplemented with 25 mM pyruvate and 5 mM glucose, adding in 20 μg/ml gentamicin when appropriate. For competition assays, Pseudomonas aeruginosa PAO1 and Staphylococcus aureus strains were maintained at 37 °C on LB plates. Prior to co-culture experiments, P. aeruginosa and S. aureus were grown shaking at 37 °C in 1X MOPS medium with 20 mM pyruvate and 5 mM glucose. All strains are listed in Table 1.

General Allelic Exchange, Chromosomal Alterations, and Electroshock Transformations

All allelic exchanges were completed using the pMQ30 non-replicative and counter-selectable vector(49). Briefly, once constructs were cloned into the pMQ30 backbone, they were transformed into chemically competent S17 λpir E. coli by heat shock. For conjugation, donor and recipient cells were mixed, collected by centrifugation, and resuspended in a small volume of LB and spotted onto LB plates to dry after which they were incubated overnight at 30°C. Single crossover merodiploids were selected by plating on PIA with 50μg/ml gentamicin at 37°C, which also kills the donor E. coli. Two independent single crossovers for each allele were inoculated into LB and incubated at 37°C for 3–4 hours with shaking before plating on LB and LB with no NaCl and including 5% sucrose and incubated at 30°C overnight. Sucrose-resistant colonies were then patched to LB with 5% sucrose and no NaCl plates (incubated at 30°C) and LB with 50μg/ml gentamicin plates (incubated at 37°C) to identify and discard remaining merodiploids. Verification of strains from double crossovers was completed using PCR as described.

Allelic exchange vectors for deletion of sphBCD or sphC in PAO1 and PA14 were created by splice overlap extension as we have described previously for other sphingosine related genes(23). Briefly, PCR fragments were amplified for both upstream and downstream of sphBCD or sphC and ligated into pMQ30 cut with either KpnI/HindIII or BamHI/HindIII. For sphBCD PCR fragment amplification, the upstream region was amplified with primers 2080 and 2083, while the downstream region was amplified using 2081 and 2083. For sphC PCR fragment amplification, the upstream region was amplified with primers 1022 and 1024, while the downstream region was amplified using 1023 and 1025. After verification by digest screening, plasmids were sequenced by Plasmidsaurus. Sequence verified plasmids were transformed into chemically competent S17 λpir E. coli and allelic exchange was completed as described above, resulting in strains LAH 83.2 (PAO1 ∆sphBCD), PD49 (PAO1 ∆sphBCD), and PD47 (PAO1 ∆sphC).

Allelic exchange vectors for sphBCD deletion in P. fluorescens Pf-01 and P. protegens Pf-5 and sphBC deletion in P. fluorescens WCS365 were created using splice overlap extension (SOE) as described above. After amplification and splice overlap, fragments were ligated into pMQ30 cut with KpnI/HindIII (for P. fluorescens WCS365 and P. protegens Pf-5) or XbaI /KpnI (for P. fluorescens PF-01). The P. fluorescens WSC365 sphBC upstream region was amplified with primers 2736 and 2737, while the downstream region was amplified using primers 2738 and 2739. The P. fluorescens Pf-01 sphBCD upstream region was amplified with primers 2740 and 2741, while the downstream region was amplified using 2742 and 2743. The P. protegens Pf-5 sphBCD upstream region was amplified using primers 2732 and 2733, while the downstream region was amplified using 2734 and 2735. After verification by digest screening, plasmids were sequenced by Plasmidsaurus. Sequence verified plasmids were transformed into chemically competent S17 λpir E. coli and allelic exchange was completed as described above, resulting in strains LAH 323 (WSC365 ∆ sphBC), LAH 362 (Pf-01 ∆sphBCD), and LAH 349 (Pf-5 ∆sphBCD).

The sphBCD, sphBC, sphCD, and sphC complementation constructs, pPD8, pPD54, pPD55, and pPD23, were generated by amplifying the appropriate region from genomic DNA using primer pairs 2726 & 2727, 2882 & 2883, 2726 & 2727, and 2511 & 2512, all cut with EcoRI and HindIII and independently ligated into similarly cut pMQ80. Plasmids with correct insert determined by PCR were sequenced (Plasmidsaurus) and correct plasmids electrotransformed into target strains (Table 1). The empty vector control for all complementations was the empty pMQ80 vector.

The sGFP2 construct, pJM18, and mScarlet-1 construct, pKSmScar6, were built using HiFi (NEB) assembly using synthetic gene fragments (gBlocks) and ligated into pUCP22 digested with BamHI and EcoRI. pJM18 and pKSScar6 assemblies were verified by digest screening using HindIII and SacI and digest-correct clones were sequenced (Plasmidsaurus) before electrotransformation into target strains (Table 1).

Chemicals and notes on sphingolipid stability, solubility, and handling

All media, media components, and standard chemicals were purchased from ThermoFisher or Sigma. The sphingoid bases sphingosine, phytosphingosine, and sphinganine were purchased from Cayman Chemicals and dissolved in 95% ethanol as aliquots of 50 mM stocks and stored at −20 °C. Storing as aliquots is important, as multiple freeze-thaw cycles lead to loss of each of the sphingoid bases’ antimicrobial capacity and ability to stimulate gene induction via SphR(23). Sphingoid bases were delivered to the vulture vessel in ethanol and then the solvent evaporated to dryness, using air drying for multi-well plastic plates and a gentle stream of nitrogen gas for glass tubes.

Determining IC50 for sphingosine, sphinganine, and phytosphingosine in glass and plastic

P. aeruginosa strains were grown overnight at 37 °C shaking in MOPS media 25 mM sodium pyruvate, 5 mM glucose, and 20 μg/ml gentamicin. Cells from overnight cultures were collected via centrifugation, washed with MOPS media, and resuspended in MOPS with 25 mM sodium pyruvate and 20 μg/ml gentamicin. Starting at an OD600 of 0.05, Pa strains were grown for 18 hours at 37°C with horizontal shaking in either plastic 48-well plates or 13×100mm glass tubes in the presence or absence of various concentrations of each sphingoid base. For the incubation periods, plates were covered with a sterile, breathable, adhesive microporous sealing film (USA Scientific) to allow for equal gas exchange for each well, while glass tubes were covered loosely with aluminum foil. After 18-hour incubations, OD600 was measured using a Synergy H1 (Biotek) plate reader. IC50 values calculated in GraphPad Prism using the log(inhibitor) vs response – Variable slope (four parameter) curve fitting analysis.

Kinetic growth assays

To measure growth kinetics, sphingoid bases were used at 200 μM. Prior to inoculation, P. aeruginosa strains were grown overnight at 37 °C, shaking in MOPS media with 25 mM sodium pyruvate, 5 mM glucose, and 20 μg/ml gentamicin. Cells were collected via centrifugation, washed in MOPS media, and resuspended in MOPS with 25 mM pyruvate and 20 μg/ml gentamicin, at a starting OD600 of 0.05 in 48-well plates sealed with breathable adhesive films. Absorbance for the film was removed by determining the difference between the absorbance post film application to the read pre-application and subtracting that difference for each well and applying that to all reads for that well. Growth was measured via OD600 taken every 30 minutes with a Synergy 2 H1 Biotek hybrid plate reader set at 37°C with orbital shaking before each read.

Growth assays for other Pseudomonads, Caulobacter, and heterologous complementation

To investigate the importance of sphBCD in other Pseudomonas strains and species, overnight cultures in MOPS media with 25 mM sodium pyruvate and 5mM glucose were grown at 37 °C for P. aeruginosa strains and 30 °C for P. protegens and P. fluorescens strains. Cells were collected via centrifugation, washed in MOPS media, and resuspended in MOPS media with 25 mM pyruvate (or 20 mM pyruvate, 10 mM glucose, or 10 mM succinate when assessing catabolite repression, shown in supplemental figures). Pseudomonas strains and species were grown in sterile 13×100mm boroscilicate glass tubes or plastic 48-well plates for 18 hours at 37 °C (for P. aeruginosa strains) or 30 °C (for P. protegens and P. fluorescens strains), with orbital shaking, in the presence or absence of sphingoid bases (200 µM final concentration) at a starting OD600 of 0.05. After 18-hour incubations, growth was measured by OD600 using a Synergy H1 Biotek plate reader.

Caulobacter crescentus WT NA1000 and related ∆sphC were maintained at 30 °C on PYE (peptone-yeast extract) plates containing 2 g/L Bacto Peptone, 1 g/L Yeast Extract, 1 mM MgSO4, and 0.5 mM CaCl2. Prior to assay set up, strains were grown shaking at 30 °C overnight in M2 minimal salts medium (6.1 mM Na2HPO4 , 3.9 mM KH2PO4, 9.3 mM NH4Cl, 0.5 mM MgSO4, 10 uM FeSO4 (EDTA chelate), and 0.5 mM CaCl2) with 0.2% glucose as the sole carbon source. To investigate the importance of sphBC in other gram-negative bacterium, such as C. crescentus, overnight cultures in M2 minimal media were grown at 30 °C. Cells were collected via centrifugation and resuspended again in M2 minimal media at an OD600 of 0.05 and grown in sterile 13 X100 mm borosilicate glass tubes at 30 °C, with orbital shaking, in the presence or absence of sphingosine, at varying concentrations. After 18-hour incubations, growth was measure by OD600 using a Synergy H1 Biotek plate reader.

To investigate the importance of homologous sphBC from C. crescentus in rescuing P. aeruginosa ∆sphBCD growth inhibition in the presence of sphingosine, overnight P. aeruginosa cultures in MOPS media with 25 mM sodium pyruvate, 5 mM glucose, and 20 μg/ml gentamicin were grown shaking overnight at 37 °C. sphBCD complementation was assessed with native P. aeruginosa genes (PD128; PAO1 ∆sphBCD with PasphBCD on pUCP22) or C. crescentus homologues (PD117; PAO1 ∆sphBCD with CcsphBC on pUCP22). Cells were collected via centrifugation, washed in MOPS media, and resuspended in MOPS media with 25 mM pyruvate with 20 μg/ml gentamicin. P. aeruginosa strains were grown in sterile 13×100mm borosilicate glass tubes for 18 hours at 37 °C, with orbital shaking, in the presence or absence of sphingosine, at a starting OD600 of 0.05. After 18-hour incubations, growth was measured by OD600 using a Synergy H1 Biotek plate reader.

sphA-lacZ reporter assay

To determine the amount of sphingoid base remaining in culture when sphBCD is deleted, sphA transcriptional induction was measured using our previously described sphA-lacZ reporter assay and construct(23). P. aeruginosa was electrotransformed with the sphA promoter construct (pAL4)(23), and resultant colonies were grown overnight at 37 °C, shaking, in MOPS media with 25 mM sodium pyruvate, 5 mM glucose, and 20 μg/ml gentamicin prior to induction. Cells were collected by centrifugation, washed in MOPS media, and resuspended in MOPS media with 25 mM sodium pyruvate and 20 μg/ml gentamicin with or without lipid extracts from strains to be tested. Lipid extracts were collected for each strain after 18 hours incubation in the presence or absence of sphingoid bases (200 μM final concentration). β-galactosidase assays were then completed as previously described(50, 51) using Miller’s method(52).

Thin layer chromatography

To visualize the amount of sphingosine remaining in culture in the presence or absence of sphBCD, we used thin layer chromatography. Pa strains were grown overnight at 37 °C, shaking in MOPS media with 25 mM sodium pyruvate, 5 mM glucose, and 20 μg/ml gentamicin. Cells were collected by centrifugation, washed in MOPS media, and resuspended in MOPS media with 25 mM pyruvate and 20 μg/ml gentamicin at a starting OD600 of 0.05. Strains were grown for 18 hours at 37 °C, with orbital shaking, in sterile foil-covered borosolicate 13×100mm glass tubes with or without 200 μM sphingosine. After incubation period, lipids were extracted from whole cell culture using the Bligh and Dyer method (53). Briefly, chloroform:methanol (1:2; v:v) was added, samples were vortexed, and one volume of water was added. After briefly vortexing, samples were centrifuged for 10 minutes at 14,000 x g. After centrifugation, the lower organic fraction was collected and dried using N2 gas before final resuspension in 20 μL of ethanol. TLC silica gel 60 F254 plates (Sigma Aldrich) were pre-run with acetone, dried, and lipid extracts spotted onto the plate. After samples dried, plates were run in a closed glass chamber with chloroform:methanol:water (65:25:4; v:v:v) as the mobile phase. After the mobile phase approached top of the plate, the plate was removed, dried, and was sprayed with Ninhydrin Solution (Acros Organics) to detect sphingosine by its primary amine group.

LC/ESI-MS/MS

To directly quantify the levels of sphingosine remaining in culture in the presence and absence of sphBCD, LC/ESI-MS/MS was completed by Lipotype, Inc (Germany). Strains were grown as per TLC and, after incubation, samples were lysed at 4 °C for 10 minutes via bead beating with vortex cell disruptor using 0.5 mm glass beads. Samples were stored at −80 °C until shipment to Lipotype, Inc. Before LC/ESI-MS/MS, samples were spiked with deuterated internal standards (including 0.25 ng sphingosine-d7). Methanol/isopropanol was added for protein precipitation and the cleared solutions were analyzed using an Agilent 1290 HPLC system with binary pump, multisampler, and column thermostat with a Kinetex EVO C-18, 2.1 × 100 mm, 2.6 μm column using a gradient solvent system of ammonium carbonate (2 mM) and methanol. The flow rate was set at 0.4 mL/min and the injection volume was 1 uL. The HPLC was coupled with an Agilent 6495 Triplequad mass spectrophotometer (Agilent Technologies, Santa Clara, USA) with electrospray ionization source. Analysis was performed with Multiple Reaction Monitoring in positive mode, with at least two mass transitions for each compound. All sphingolipids were calibrated using individual standards. The Agilent Mass Hunter Quant software was used for quantification.

P. aeruginosa Competition Assays

P. aeruginosa strains were grown overnight at 37 °C, shaking in MOPS media with 20 mM sodium pyruvate, 5 mM glucose, and 20 μg/ml gentamicin prior to competition assay set up. Cells were collected via centrifugation, washed three times in MOPS media, and resuspended in MOPS media with 20 mM pyruvate and 20 μg/ml gentamicin and normalized to an OD600 of 0.5. Sterile borosilicate 13×100 mm glass tubes had vehicle or sphingosine, for a final concentration of 200 µM, dried as described above. To these tubes, 900 mL of MOPS media with 20 mM sodium pyruvate and 20 μg/ml gentamicin was added, followed by 50 µl each of 0.5 OD600 GFP and mScarlet expressing P. aeruginosa for total starting OD600 of 0.05. All cultures were grown at 37 °C, shaking for 18 hours. At 0 and 18-hour timepoints, OD600 and GFP (485/528 nm) and mScarlet (550/610 nm) fluorescent signals were measured using a Synergy H1 plate reader (BioTek). Background fluorescence for GFP and mScarlet was corrected by subtracting the signal from WT monoculture carrying the opposite fluorescent protein (mScarlet or GFP, respectively). Corrected fluorescence values were expressed as a percentage of monoculture of WT carrying GFP or mScarlet (set to 100%). Additionally, 20 µL aliquots of each culture were serially diluted in R2B and spot plated onto MOPS media agar plates with 20 mM sodium pyruvate and 5 mM glucose for colony forming unit (CFU) counts at each timepoint. Total colony forming units were counted, GFP-expressing colonies were detected by UV transillumination and appropriate excitation filter and imaged using a ChemiDoc XRS+ Gel Imaging System (BIO RAD). mScarlet expressing colonies were calculated by subtracting GFP-expressing colonies from the total CFU/mL.

P. aeruginosa – S. aureus Competition Assays

P. aeruginosa was grown overnight in MOPS media with 20 mM sodium pyruvate and 5 mM glucose, shaking at 37 °C. Cells were collected by centrifugation, washed three times with MOPS media, and added to 1 mL MOPS media with 20 mM pyruvate, 5 mM glucose, and 20 µM sphingosine at a final OD600 of 0.05 to allow time for sphBCD induction. During this incubation, overnight 37 °C LB cultures of S. aureus were collected via centrifugation, washed three times with R2B, and adjusted to an OD600 of 0.5 in R2B. P. aeruginosa diluted into R2B +/− 100 µM sphingosine for one hour, shaking at 37 °C. After one hour, S. aureus was added to an OD600 of 0.05 to the P. aeruginosa-containing media or R2B +/− 100 µM sphingosine, and grown for five hours shaking at 37 °C. At 0 and 5 hours of co-culture, 20 µL aliquots serially diluted in R2B, and spot plated onto both PIA and tryptic soy agar (TSA) +7.5% NaCl to select for growth of P. aeruginosa and S. aureus, respectively, and colony forming unit (CFU) counted.

Supplementary Material

Supplement 1

Funding:

NIH NIAID R01 AI103003 and Cystic Fibrosis Foundation WARGO24G0 (both to MJW) NIH NHLBI T32 HL076122 (supporting PD) and NIH NIAID T32 AI055402 (supporting LAH) NSF MCB-1553004 (to EAK)

Figure 1: Sphingoid bases and arrangement of the sphBCD operon.

(A) Structures of the sphingoid bases used in this study noting the head-group differences. All of the sphingoid bases used in this study are C18 versions, though there is tail length variation in naturally occurring versions from different body sites or organisms. (B) Organization of the sphBCD operon in P. aeruginosa showing the relative gene sizes, predicted functions, and the gene numbers in the PAO1 and PA14 genome. The SphR bs denotes the binding site for the sphingosine-responsive transcriptional activator SphR and the hairpin at the right edge is the predicted rho-independent terminator.

Figure 2: Concentration dependent inhibition for sphingoid bases is dependent on the base and the culture vessel material.

All panels show relative growth measured by OD600 as compared to the WT with empty vector (pEV) in the absence of sphingosine at the 18-hour timepoint. The data shown here are for (A) sphingosine, (B) sphinganine, and (C) phytosphingosine in either glass (open symbols) or plastic (closed symbols). The IC50 curve and estimated IC50s to the right of the plots generated using variable-slope curve fitting in GraphPad Prism. If calculated IC50 was above the solubility of sphingosine, it was listed as NA. Data points denote means summarizing three independent experiments and error bars mark standard deviation. Abbreviations: pEV, empty vector pMQ80.

Figure 3: Kinetic growth assessment of wild-type, mutant, and complemented strains in the presence of sphingoid bases.

All panels are 18-hour timecourses measuring relative growth of each strain at each timepoint as measured by OD600 compared to the WT with empty vector (pEV) in the absence of sphingosine at the 18h timepoint. The data shown here are for (A) sphingosine, (B) sphinganine, and (C) phytosphingosine. Growth curves in MOPS pyruvate in the absence of sphingoid bases are presented in Supplemental Figure S1. Data points denote means summarizing three independent experiments and error bars mark standard deviation with only the bars above the mean shown for figure clarity. Abbreviations: pEV, empty vector pMQ80; pBCD, vector containing sphBCD.

Fig 4. The sphBC genes are critical for wild-type levels of growth in the presence of sphingoid bases.

(A) Complementation analysis of ∆sphBCD shows significant complementation only with plasmids expressing sphB and sphC, while sphD appears dispensable for growth at 18 hour normalized to WT empty vector growth in MOPS pyruvate set as 100%. (B-D) Deletion of sphC phenocopies deletion of sphBCD and can be complemented by sphC on a plasmid. This phenotype is shared between the sphingoid bases (B) sphingosine, (C) sphinganine, and (D) phytosphingosine. 18-hour growth was normalized to WT empty vector growth in MOPS pyruvate set as 100%. For all panels, all data points are shown and are colored by experiment with white circles for all replicates from experiment #1, gray from experiment #2, and black from experiment #3. Only the means for each experiment are used in the statistical analyses for these panels (n = 3 per condition). For (A), significance noted as (**, p<0.01; ***, p<0.001; ****, p<0.0001) calculated from ANOVA with Dunnett’s post-test with ∆sphBCD pEV as the comparator. For (B-D), significance noted as (**, p<0.01; ***, p<0.001; ****, p<0.0001) for comparisons of each complemented strain to its empty vector control, while significant difference to WT with empty vector noted as (a, p<0.0001; b, p<0.01). Analysis of B-D conducted with ANOVA and Tukey’s post-test comparing all groups. Abbreviations: pEV, empty vector pMQ80; pBCD, vector containing sphBCD; pC, vector containing sphC.

Figure 5: Metabolism of sphingoid bases is dependent on the presence of sphBCD.

(A-C) Determination of sphingoid bases remaining in the culture after 18 hours of incubation as measured using the sphA-lacZ reporter assay, (D) LC-MS, and (E) TLC. Statistical significance noted as ****P < 0.0001 using a two-way ANOVA with Tukey’s post-test comparing all groups. For panels A-D, all data points are shown and are colored by experiment with white circles for all replicates from experiment #1, gray from experiment #2, and black from experiment #3. Only the means for each experiment are used in the statistical analyses for these panels (n = 3 per condition, except the LC-MS, for which only two replicates were run and are therefore not statistically analyzed). The spot that runs below sphingosine in the ∆sphBCD mutant TLC lane (in E) is an unknown amine-containing lipid and did not run similarly to any of our sphingolipid standards. Abbreviations: ns, not significant; P, pyruvate (control); S, sphingosine; ∆BCD, ∆sphBCD; pEV, empty vector pMQ80; pBCD, vector containing sphBCD.

Fig 6. The role of sphBC in other Pseudomonads.

The growth WT and mutant for each strain in 200 µM sphingosine shown normalized to that strain’s growth in MOPS pyruvate media. As seen for P. aeruginosa PAO1 (Fig 2), the culture vessel material impacts the potency of sphingosine for some strains. Significance noted as (**, p<0.01; ***, p<0.001; ****, p<0.0001) calculated from ANOVA with Sidak’s post-test comparing WT to mutant within each strain. For both panels, all data points are shown and are colored by experiment with white circles for all replicates from experiment #1, gray from experiment #2, and black from experiment #3. Only the means for each experiment are used in the statistical analyses for these panels (n = 3 per condition) Abbreviations: ∆BCD, ∆sphBCD; ∆BC, ∆sphBC; Pa, P. aeruginosa; Pf, P. fluorescens; Pp, P. protogens.

Fig 7. Wild type sphingosine detoxification can protect co-cultured ∆sphBCD from growth inhibition by sphingosine.

(A) Fluorescence signal for GFP and mScarlet normalized to monoculture of WT carrying GFP or mScarlet, respectively. GFP signal shown with light green bars and mScarlet signal shown with dark red bars. The strain carrying each fluorescent protein is labeled below graph. (B) CFU counts of GFP-expressing colonies in the presence (S) or absence (P) of sphingosine. The strain carrying each fluorescent protein is labeled below graph. Significance noted as (***, p<0.001; ****, p<0.0001) calculated from ANOVA with Tukey’s post-test comparing within and between co-culture groups. For both panels, all data points are shown and are colored by experiment with white circles for all replicates from experiment #1, light gray from experiment #2, dark gray from experiment #3, and black from experiment #4. Only the means for each experiment are used in the statistical analyses for these panels (n = 4 per condition) Abbreviations: ∆BCD, ∆sphBCD; P, pyruvate (control); S, sphingosine; N.D., not detectable.

Fig 8. P. aeruginosa can protect S. aureus from complete killing by sphingosine.

(A) Titration of WT P. aeruginosa into the sphingosine-containing media for 1h prior to S. aureus inoculation protected a small proportion of S. aureus from the lethal effects of 5 hours in the presence of 50 µM sphingosine in a P. aeruginosa inoculum-dependent manner. (B) The proportion of the initial S. aureus population protected by P. aeruginosa WT trended higher than the proportion protected by ∆sphBCD. All data points are shown and are colored by experiment with white circles for all replicates from experiment #1, gray from experiment #2, and black from experiment #3. Only the means for each experiment were used for a t-test to statistically analyze these data (n = 3 per condition) and thus the technical replicates with no CFU are averaged to a non-zero number for each experiment (even for experiment #1, where only one replicate had countable colonies).

Table 1: Strains and plasmids used in this study

Lab Strain ID	Genotype	Plasmid	Source	
MJ79	Pseudomonas aeruginosa PAO1 wild-type	-	(PMID: 10984043) Stover et al.	
LAH 83.2	∆sphBCD in PAO1	-	this study	
PD47	∆sphC in PAO1	-	this study	
PD132	Pseudomonas aeruginosa PAO1 wild-type	pPD1	this study	
PD136	∆sphBCD in PAO1	pPD1	this study	
PD139	∆sphBCD in PAO1	pPD8	this study	
PD121	∆sphC in PAO1	pPD1	this study	
PD134	∆sphC in PAO1	pPD23	this study	
LAH304	∆sphBCD in PAO1	pPD54	this study	
LAH301	∆sphBCD in PAO1	pPD55	this study	
PD12	∆sphBCD in PAO1	pPD23	this study	
AL51	Pseudomonas aeruginosa PAO1 wild-type	pAL5	(PMID: 24465209) LaBauce et al.	
MJ984	Pseudomonas aeruginosa PA14 wild-type (new stock of MJ101)	-	(PMID: 7604262) Rahme et al.	
PD49	∆sphBCD in PA14	-	this study	
LAH 311	Pseudomonas fluorescens WCS365 wild-type	-	this study	
LAH 323	∆sphBC in WCS365	-	this study	
LAH 313	Pseudomonas fluorescens Pf-01 wild-type	-	this study	
LAH 362	∆sphBCD in Pf-01	-	this study	
LAH 314	Pseudomonas protegens Pf-5 wild-type	-	this study	
LAH 349	∆sphBCD in Pf-5	-	this study	
JR124	Pseudomonas aeruginosa PAO1 wild-type	pJM18	this study	
JR125	Pseudomonas aeruginosa PAO1 wild-type	pKSmScar6	this study	
JR129	∆sphBCD in PAO1	рJM18	this study	
JR131	∆sphBCD in PAO1	pKSmScar6	this study	
JR268	∆sphBCD in PAO1	-	this study	
MJ661	Staphylcoccus aereus wild-type	-	ATCC	
PD207	Caulobacter crescentus WT NA1000	-	this study	
PD209	∆sphC in Caulobacter crescentus	-	this study	
PD113	Pseudomonas aeruginosa PAO1 wild-type	pPD34	this study	
PD108	∆sphBCD in PAO1	pPD34	this study	
PD128	∆sphBCD in PAO1	pPD49	this study	
PD117	∆sphBCD in PAO1	pPD35	this study	
Lab Plasmid ID	Description	Source	
pPD34	pUCP22, Pa replicative vector	(PMID: 1899844) Schweizer et al.	
	pMQ30, allelic exchange	(PMID: 1899844) Schweizer et al.	
pPD1	pMQ80, Pa replicative vector	(PMID: 16820502) Shanks et al.	
pPD8	sphBCD in pMQ80, Pa replicative vector	this study	
pPD23	sphC in pMQ80, Pa replicative vector	this study	
pPD54	sphBC in pMQ80, Pa replicative vector	this study	
pPD55	sphCD in pMQ80, Pa replicative vector	this study	
pAL5	sphA-lacZYA reporter in pMQ80, Pa replicative vector	(PMID: 24465209) LaBauve et al.	
pJM18	sGFP2 in pUCP22	this study	
pKSmScar6	mScarlet-1 in pUCP22	this study	
pPD49	Pa sphBC in pUCP22, Pa replicative vector	this study	
pPD35	Cc sphBC in pUCP22, Pa replicative vector	this study
==== Refs
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