Sphingosylphosphorylcholine (SPC) is a substrate for the Pseudomonas aeruginosa phospholipase C/sphingomyelinase, PlcH

Sphingosylphosphorylcholine (SPC) is a substrate for the Pseudomonas aeruginosa phospholipase C/sphingomyelinase, PlcH | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Sphingosylphosphorylcholine (SPC) is a substrate for the Pseudomonas aeruginosa phospholipase C/sphingomyelinase, PlcH View ORCID Profile Pauline DiGiannivittorio , Kristin Schutz , View ORCID Profile Lauren A. Hinkel , View ORCID Profile Matthew J. Wargo doi: https://doi.org/10.1101/2025.03.27.645745 Pauline DiGiannivittorio 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 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pauline DiGiannivittorio Kristin Schutz 1 Department of Microbiology and Molecular Genetics, Larner College of Medicine, University of Vermont Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lauren A. Hinkel 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 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lauren A. Hinkel Matthew J. Wargo 1 Department of Microbiology and Molecular Genetics, Larner College of Medicine, University of Vermont Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthew J. Wargo For correspondence: mwargo{at}uvm.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Sphingolipids are critical to eukaryotic cell membrane structure and function and play important roles in a variety of host processes that impact infection. Thus, it is not surprising that many pathogens can perturb host sphingolipid homeostasis, often to promote pathogenesis. Pseudomonas aeruginosa is a common opportunistic pathogen that, among many virulence factors, secretes the dual-functioning hemolytic phospholipase C/sphingomyelinase, PlcH. PlcH contributes to P. aeruginosa pathogenesis in several ways and plcH mutants are defective in nearly every infection model, wherein PlcH has been shown to hydrolyze both phosphatidylcholine and sphingomyelin, resulting in inflammation and rupture of host cell membranes. Here, we demonstrate that PlcH can also hydrolyze sphingosylphosphocholine (SPC, also known as lysosphingomyelin), an important host signaling sphingolipid responsible for regulating cellular and tissue responses such as inflammation and endothelial barrier function. PlcH hydrolyzes sphingomyelin to generate phosphocholine and ceramide, and analogously, here we demonstrate that PlcH hydrolyzes SPC to sphingosine and putatively, phosphocholine. We provide evidence that SPC induction of PlcH is primarily regulated by the sphingosine-responsive SphR regulator and that resultant sphingosine liberated from SPC induces transcription from the other genes in the SphR regulon. This work introduces another way that P. aeruginosa can alter the host sphingolipidome, potentially a different mechanism to promote pathogenesis. The capacity for the hemolytic Clostridium perfringens alpha toxin to also cleave SPC suggests that SPC may be a common substrate for phosphocholine-specific phospholipases C. Importance PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of P. aeruginosa . Here we show that both P. aeruginosa PlcH and C. perfringens alpha toxin can hydrolyze the signaling phospholipid sphingosylphosphorylcholine (SPC), also called lysosphingomyelin. Thus, SPC should be considered a potential target for such phospholipases during infection whose resulting hydrolysis can induce sphingosine-sensitive genes. Introduction The secreted enzyme PlcH is an important virulence factor for P. aeruginosa pathogenesis and infection 1 - 4 and has both phosphocholine-specific phospholipase C (PLC) and sphingomyelinase activity 5 . PlcH hydrolyzes phosphatidylcholine into phosphocholine and diacylglycerol and hydrolyzes sphingomyelin into phosphocholine and ceramide 1 , while lipids with other headgroups are very poor PlcH substrates 6 , 7 . Secreted PlcH, primarily in complex with the chaperone PlcR2 7 - 10 , causes cellular and tissue damage, promotes inflammation, and disrupts lung surfactant function, enhancing the pathogenesis and in vivo survival of P. aeruginosa 11 - 19 . Transcription of plcH is controlled by three independent activators, whose inducing signals are present in the host: the glycine betaine and dimethylglycine-responsive transcriptional activator GbdR 20 - 22 , the sphingosine-binding transcriptional activator SphR 23 , and the phosphate starvation-responsive transcriptional activator PhoB 24 . Choline induces plcH transcription and resultant enzyme secretion only after choline metabolism to glycine betaine 21 , 25 . Phosphatidylcholine and sphingomyelin are considered the primary PlcH substrates in vivo 6 , 7 , however the ability of PlcH to remove phosphocholine from other molecules, like the colorimetric substrate nitrophenylphosphorylcholine (NPPC) 7 , 26 , suggest that other phosphocholine-containing molecules in the host might be physiologically relevant PlcH substrates. Sphingosylphosphocholine (SPC) ( Fig 1 ), also called lysosphingomyelin, is a bioactive sphingolipid with functional similarity to sphingosine-1-phosphate (S1P) 27 , 28 . SPC can be generated through the removal of the N-acyl tail from sphingomyelin via sphingomyelin deacylase and can also be synthesized de novo in specific cell types, such as platelets 29 - 31 , and is associated with both high-density (HDL) and low-density (LDL) lipoproteins 32 . As a signaling molecule, SPC can bind and activate S1P receptors 1-5 33 and has also been implicated as a second messenger regulating intracellular Ca 2+ levels 34 - 36 . SPC is involved in the maintenance of cell proliferation 37 , differentiation 38 , and regulation of apoptosis 39 , and has impacts on regulation of the immune response 40 and endothelial barrier function 36 , 41 , 42 . Download figure Open in new tab Figure 1: (A) Structures of sphingomyelin, SPC, NPPC, and sphingosine and (B) Schematic of the plcH locus with SphR and GbdR binding sites. Chemical structures generated with ChemDraw and gene organization diagram generated with BioRender. Abbreviations: bs, binding site; sph, sphingosine; tss, transcription start site; term, transcriptional terminator. Here, we show that SPC is a PlcH substrate and that SPC induces plcH transcription and thus, PlcH activity primarily via SphR detection of the sphingosine produced by SPC hydrolysis. Additionally, the purified PLC alpha toxin from Clostridium perfringens also hydrolyzes SPC, pointing to SPC as a phosphocholine-PLC substrate more generally. These findings add an important host signaling molecule to the substrate repertoire of PlcH and other bacterial PLCs. Results SPC induces PlcH expression and transcription of the other SphR-regulon members Following up on our identification of sphingosine as an inducer of plcH transcription via SphR 23 , we first investigated whether SPC could induce PlcH activity, as measured by p -nitrophenyl phosphorylcholine (NPPC) hydrolysis. After a 4-hour incubation of PA14 WT with SPC, there was a significant increase in PlcH activity compared to the pyruvate negative control ( Figure 2A ). Sphingosine and choline conditions were included as positive controls, as we have previously shown that sphingosine induces plcH in an SphR -dependent manner, whereas choline induces plcH in a GbdR-dependent manner 20 - 22 . Download figure Open in new tab Figure 2: SPC induces PlcH activity and transcription of the SphR regulon (A) SPC induces extracellular PlcH activity as measured by nitrophenylphosphorylcholine (NPPC) hydrolysis as normalized to the level induced by SPC. (B) β-galactosidase activity normalized to signal from the SPC induction condition using the plasmid-borne sphA promoter lacZ reporter (left), the chromosomal lacZ integration into the cerN locus generating a synthetic lacZcerN operon (middle), and the chromosomal lacZYA integrated into the sphBCD locus replacing the native genes and generating a reporter of the sphB promoter (right). Statistical significance for both A & B noted as * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001) using 1-way ANOVA and Dunnett’s post-test with pyruvate as the comparator. For all panels, all collected data points are shown and are colored by experiment with white circles for all replicates from experiment #1, grey 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: Pyr, pyruvate (control); SPC, sphingosylphosphorylcholine; Sph, sphingosine; Sphn, sphinganine; Phyto, phytosphingosine; Cer, ceramide. Since sphingosine induces expression of P. aeruginosa genes involved in ceramide and sphingosine metabolism in addition to plcH 23 , 43 , we next investigated whether SPC induced these same sphingosine-responsive genes. After a 4-hour incubation with SPC, the reporter constructs for the sphA, cerN , and sphB promoters were each induced, relative to the negative control pyruvate-only condition ( Figure 2B ). Sphingosine and its analogues sphinganine and phytosphingosine were included as positive induction controls 44 , while ceramide was included as a control for a compound that must be metabolized to sphingosine (by the ceramidase, CerN) to allow induction of the sphingosine-responsive genes 43 , 45 . Given the similarity between SPC induction and sphingosine induction, we predicted that SPC is likely hydrolyzed to sphingosine to enable induction. SPC hydrolysis is plcH dependent and generates sphingosine PlcH can hydrolyze sphingomyelin to ceramide and phosphocholine. Considering the structural similarity of SPC to sphingomyelin ( Figure 1A ), we predicted that PlcH would also hydrolyze SPC, generating sphingosine and phosphocholine as products. To test this hypothesis, we first used the sphA promoter lacZ reporter construct (from Figure 2B , left panel) in both P. aeruginosa WT and Δ plcHR . Upon exposure to SPC, the Δ plcHR mutant showed substantially reduced reporter induction compared to WT ( Figure 3A ). Sphingosine induction was not different between these strains. These data supported a role for PlcH in SPC hydrolysis. Download figure Open in new tab Figure 3: plcH is necessary for SPC hydrolysis to sphingosine. (A) WT carrying the P sphA -lacZ reporter responds to SPC, while Δ plcHR carrying the same reporter does not. (B) Using thin layer chromatography of lipid extracts from strains supplied with SPC, when SPC is supplied to the Δ plcHR strain, sphingosine is not generated (see migration of the standards on either side of the TLC) . TLC was done on silica plates with a chloroform:methanol:water (65:25:4) mobile phase and after drying, were stained with ninhydrin to detect the primary amine on the sphingoid base. Data shown as means of three independent experiments. Statistical significance noted as * or # (p < 0.05) and ** (p < 0.01) using 2-way ANOVA with Sidak’s post-test showing comparisons within reporter strain compared to the pyruvate condition (asterisks) or comparing WT + SPC to Δ plcHR + SPC (#). For (A), all collected data points are shown and are colored by experiment with white circles for all replicates from experiment #1, grey 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: SPC, sphingosylphosphorylcholine; Sph, sphingosine; Pyr, pyruvate; Std, standard. The sphingosine-sensitive reporter assay is an indirect method to assess sphingosine production, so to directly visualize the sphingosine formed upon SPC hydrolysis by PlcH we performed thin layer chromatography (TLC). Lipids extracted from P. aeruginosa Δ sphBCD supernatants exposed to SPC show sphingosine formation, while no sphingosine is seen in extracts from Δ plcHR exposed to SPC ( Figure 3B ). No sphingosine remains in the WT supernatants due to sphingosine metabolism which requires sphB and sphC , as we have recently shown 44 . The Clostridium perfringens alpha toxin, a hemolytic phospholipase C, also hydrolyzes SPC to generate sphingosine The alpha toxin from C. perfringens is a hemolytic phospholipase C with specificity for phosphocholine-containing lipids (abbreviated Cp PLC). As we have yet to purify active PlcH, we tested whether purified Cp PLC was capable of SPC hydrolysis. Pretreatment of SPC with purified Cp PLC led to reporter induction from the plcHR deletion strain carrying the chromosomal sphingosine-responsive reporter ( lacZ at the cerN locus), while incubation of SPC with the buffer control led to no induction in the plcHR deletion strain ( Figure 4A ). These data supported the ability of Cp PLC to hydrolyze SPC. Using thin-layer chromatography, we also demonstrate complete conversion of SPC to sphingosine within our limit of detection ( Figure 4B ). Thus, SPC is likely a general substrate for PLCs that prefer phosphorylcholine headgroups. Download figure Open in new tab Figure 4: The Clostridium perfringens alpha toxin hydrolyzes SPC to generate sphingosine. (A) Wild type with a chromosomal P cerN -lacZ reporter (same reporter as in Fig 2B , center panel) responds to SPC, while the same reporter in Δ plcHR does not. However, treatment of SPC with purified C. perfringens PLC enables responsiveness from the Δ plcHR reporter strain. (B) Using thin layer chromatography, when SPC is incubated with Cp PLC sphingosine is generated (see migration of the standards on either side of the TLC). TLC is stained with ninhydrin to detect the primary amine on the sphingoid base. Statistical significance for (A) noted as * (p < 0.05), *** (p < 0.001), and **** (p < 0.0001) using using 2-way ANOVA with Sidak’s post-test with pyruvate as the comparator within each strain. For (A), all collected data points are shown and are colored by experiment with white circles for all replicates from experiment #1, grey 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: Pyr, pyruvate (control); SPC, sphingosylphosphorylcholine; SPH or Sph, sphingosine; Cp PLC, Clostridium perfringens phospholipase C; Std, standard. The roles for SphR and GbdR in PlcH induction by SPC PlcH hydrolysis of SPC hydrolysis results in production of a sphingoid base ( Figure 3B ) and, very likely, phosphocholine, both of which could independently lead to induction of plcH transcription. We thus tested whether PlcH induction by SPC was regulated by SphR, GbdR, or a combination. Upon exposure to SPC, we measured PlcH activity by NPPC hydrolysis in WT, Δ sphR , Δ gbdR , and engineered strains with mutations in the SphR binding site of the cerN promoter, mutations in the GbdR binding site of the plcH promoter, and a strain containing both SphR and GbdR binding site mutations. While SPC induced PlcH enzyme activity in WT, the Δ sphR mutant and the strain with mutation of the SphR binding site showed no induction in the presence of SPC ( Figure 5 ). Interestingly, the Δ gbdR and GbdR binding site mutants showed increased PlcH activity compared to WT when exposed to sphingosine but not SPC ( Figure 5 ). These data indicate that PlcH induction by SPC is under SphR transcriptional control at this tested concentration of SPC. The SphR and GbdR binding site double mutant strain functioned as our negative control, since neither product of SPC hydrolysis would be capable of inducing PlcH, which also provides support that there is no way for SPC to induce PlcH in the absence of its hydrolysis to phosphocholine and sphingosine. Sphingosine and choline conditions were included as positive controls for each transcriptional regulator system. Download figure Open in new tab Figure 5: Contributions of GbdR- and SphR-dependent regulation to SPC induction of secreted PlcH enzyme activity. Secreted PlcH enzyme activity measured by NPPC hydrolysis and normalized by fold induction to the pyruvate condition for each strain. Data shown as means of three independent experiments with standard error bars, as the number of groups made plotting all data points, as done for the other figures, impractical. Statistical significance noted as * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) using 2-way ANOVA with Sidak’s post-test showing comparisons within induction condition to the WT strain. Abbreviations: bs, binding site; mut, mutant; SPC, sphingosylphosphorylcholine; SPH, sphingosine; CHO, choline; PYR, pyruvate. Discussion Here, we show that the P. aeruginosa virulence factor PlcH can hydrolyze SPC resulting in sphingosine production. This finding suggests that, in addition to the classical substrates phosphatidylcholine and sphingomyelin considered during infection, P. aeruginosa may also be capable of perturbing host signaling via SPC hydrolysis. Since the C. perfringens PLC also shows SPC hydrolysis, this suggests that SPC may be a target for other phosphocholine-specific PLCs. SPC is structurally similar to sphingomyelin ( Fig. 1a ) and PlcH can hydrolyze a range of phosphocholine-containing compounds, therefore it is not surprising that SPC hydrolysis by P. aeruginosa is dependent on PlcH. The hydrolysis of SPC by both P. aeruginosa and C. perfringens PLCs, which are very different by sequence and structure 46 - 48 , supports the idea that phosphocholine recognition is the primary driver of hydrolysis by these enzymes. While this has been well studied for the phosphocholine-hydrolyzing PLCs of C. perfringens and B. cereus , substrate recognition by PlcH is not well-described. Apart from the active site threonine (T178), discovered by homology to Francisella tularensis AcpA and subsequently experimentally tested 7 , 9 , 19 , very little is known about PlcH recognition of the phosphocholine headgroup or the moiety attached to the phosphocholine. Since the affinity of PlcH for NPPC is much lower than for phosphatidylcholine or sphingomyelin 9 , 19 , some portion of the acyl moieties on these molecules are likely recognized. SPC can induce PlcH production, and it does so primarily through SphR-dependent induction, a pathway we have recently described 23 . The primacy of the sphingosine moiety for PlcH induction may have more to do with the SPC concentration used during these experiments than it does the comparative importance of the individual regulators per se. The responsiveness of GbdR to GB produced from exogenous choline is less sensitive than SphR detection of exogenous sphingosine. SphR can detect exogenous sphingosine as low as 2.5 µM with a maximal response at 200 µM 23 , 43 , 44 , whereas the lower limit for choline detection is ∼3 µM 49 , 50 , governed by the transport Kd, with a maximal response at 2 mM 24 . Thus, at the concentration used in these experiments and the experimental timing, the sphingosine released from SPC hydrolysis is more important for PlcH induction than the phosphocholine. This relationship might not be the same at all concentrations, time steps, or in vivo. The data presented here use induction conditions with different concentrations of SPC. To measure PlcH activity, P. aeruginosa strains were induced with 100 μM SPC, while for reporter assays, we used 20 μM SPC. Within the human body, SPC is commonly seen at a concentration estimated around 50 ± 15 nM, a concentration much lower than the concentrations tested in this study 30 , 51 . However, steady state levels in a whole compartment (like the blood) are often much lower than concentrations within local environments in which a product is being actively produced, such as in association with platelets. Thus, it remains an open question whether PlcH hydrolysis of SPC happens in vivo and whether P. aeruginosa SPC hydrolysis has any impact on the host during infection. Given SPC’s important roles in regulating endothelial cell and barrier function, future studies should investigate alterations in these responses and their alteration in response to WT and plcH mutant strains. Materials and Methods Strains and growth conditions Pseudomonas aeruginosa PA14 and isogenic mutant strains ( Table 1 ) were maintained on Lysogeny Broth-Lennox formulation (LB) or Pseudomonas isolation agar (PIA) plates with 50 µg/mL gentamicin added when appropriate. Escherichia coli strains used in this study were maintained on LB plates or liquid LB supplemented with 10 µg/mL or 7 µg/mL gentamicin, respectively. During genetic manipulations, P. aeruginosa was selected for, and E. coli selected against, using PIA plates supplemented with 50 µg/mL gentamicin. Prior to transcriptional and enzyme induction studies, P. aeruginosa was grown at 37°C overnight in morpholinepropanesulfonic acid (MOPS) medium 52 modified as previously described 53 , and supplemented with 20 mM pyruvate and 5 mM glucose. View this table: View inline View popup Download powerpoint Table 1: Strains, plasmids, and oligonucleotides used in this study General Allelic Exchange, Chromosomal Alterations, and Electroshock Transformations All allelic exchange constructs were generated using the pMQ30 non-replicative, counter-selectable vector 54 . Briefly, after constructs were cloned into the pMQ30 backbone, they were transformed into chemically competent S17 λ pir E. coli . For conjugation, donor E. coli were mixed with respective recipient P. aeruginosa strains, pelleted via centrifugation, resuspended in a small volume of LB, and spotted onto LB plates, and incubated overnight at 30 °C. Single-crossover integrants were selected by plating on PIA with 50 μg/ml gentamicin following incubation at 37 °C for 24 hours. Selected single crossover integrants were inoculated into LB, incubated at 37 °C for 3-4 hours with shaking, and plated onto LB and LB with no NaCl containing 5 % sucrose and incubated overnight at 30 °C. Sucrose resistant colonies were screened for loss of gentamicin resistance prior to PCR screening to determine whether each double-crossover colony was a mutant or WT revertant. Briefly, the allelic exchange vector for mutation of the GbdR binding site in the plcH promoter used HiFi assembly (NEB) of two PCR products generated from PA14 genomic DNA (using primers 2916 & 2917 for the upstream side and 2918 & 2919 for the downstream side), a synthetic fragment containing the GbdR binding site mutation region “GbdR bs mut”, and HindIII+KpnI cut pMQ30. Sequence verified plasmids were transformed into chemically competent S17λ pir E. coli and allelic exchange using recipient PA14 strains was completed as described above, resulting in strains PD183 (GbdR bs mutant) and PD187 (SphR bs & GbdR bs double mutant). Generation of the SphR binding site mutant in the cerN promoter was previously described 23 . The allelic exchange vector for generation of the lacZcerN synthetic operon at the chromosomal cerN locus in PA14 was built using HiFi assembly (NEB) from three PCR products (amplifying cerN upstream and downstream fragments and lacZ fragment) and HindIII+KpnI cut pMQ30. The cerN upstream fragment was amplified with primers #2888 & #2892 and the downstream region using #2893 & #2891, both using PA14 genomic DNA as the template. The lacZ gene was amplified from pMW5 20 with primers #2894 and #2895. Sequence verified plasmids were transformed into chemically competent S17λ pir E . coli and allelic exchange was completed as described above, resulting in strain PD171 ( lacZ-cerN ). The allelic exchange vector for P sphB -lacZYA incorporation into the PA14 chromosome was built by amplifying the region upstream of the sphBCD operon from P. aeruginosa PAO1 using primers #2082 and #2286, digestion of the product with HindIII and KpnI, and ligation into similarly cut pGW78, resulting in interim plasmid 1. The downstream region of the sphBCD operon was amplified with primers #2287 and #2288, digestion with enzymes NheI and SphI and ligation into similarly cut interim plasmid 1 at the 3’ end of lacZYA , yielding interim plasmid 2. The lacZYA with sphBCD flanking regions was cut from interim plasmid 2 with HindIII and SphI and ligated into similarly cut pMQ30 yielding plasmid p lacZYA::sphBCD . Conjugation and allelic exchange were conducted as described above, resulting in strain LAH118. Chemicals and notes on sphingolipid stability, solubility, and handling All media, media components, and standard chemicals were purchased from Thermofisher or Sigma. Sphingolipids such as sphingosine, phytosphingosine, sphinganine, sphingosylphosphocholine, and ceramide were purchased from either Cayman Chemicals or Avanti Polar Lipids. All sphingolipids were dissolved in 95% ethanol (with sonication when necessary) and stored as 50 mM stocks at -20°C. The storage of sphingolipids in aliquot form is critical, as multiple freeze-thaw cycles lead to decrease in sphingolipid potency and function (i.e., antimicrobial activity for sphingoid bases and the ability to stimulate gene induction via SphR 43 , 44 ). Sphingolipids were delivered to culture vessel in ethanol and ethanol was evaporated either by air drying or a gentle stream of nitrogen gas. Phospholipase C activity assays (NPPC assays) As a readout for PlcH activity (i.e., phospholipase C activity), we measured hydrolysis of the synthetic substrate p -nitrophenylphosphorylcholine (NPPC) based on the methodology of Kurioka and Mastuda 44 and modified as previously described 20 . Briefly, P. aeruginosa strains were grown overnight shaking at 37°C in MOPS media with 25 mM pyruvate and 5 mM glucose, collected by centrifugation, and washed in MOPS media prior to resuspension in MOPS with 25 mM pyruvate. Culture density was adjusted to OD 600 = 0.5 with MOPS media with 25 mM pyruvate and with or without 100 μM SPC, sphingosine, or 2 mM choline. Cultures were incubated for 4 hours shaking at 37°C. To measure NPPC hydrolysis, one volume of culture was mixed with one volume of 2 X NPPC reaction buffer (200 mM Tris pH 7.2, 50% glycerol, 20 mM NPPC). NPPC hydrolysis was then measured by quantifying absorbance at 410 nm every five minutes for thirty minutes. Prior to normalization, phospholipase C activity was first calculated to determine micromoles of p -nitrophenol generated per minute of reaction per optical density (OD 600 ), using the nitrophenol extinction coefficient of 17,700 M-1 cm-1 55 . Clostridium perfringens alpha toxin reaction To assess if Clostridium perfringens alpha toxin hydrolyzes SPC to sphingosine, a bioassay and thin layer chromatography, 2 mg/mL C. perfringens alpha toxin (in molecular biology grade water) was incubated with 100 µM SPC for 4 hours at 37°C, shaking. After incubations, lipids were extracted using the Bligh and Dyer method 56 and were prepared as described in the TLC section. Thin layer chromatography (TLC) To visually assess SPC hydrolysis to sphingosine, we used thin later chromatography. P. aeruginosa strains were grown overnight at 37 °C, shaking in MOPS media with 25 mM pyruvate and 5 mM glucose. Cells were collected via centrifugation, washed in MOPS media, and cell pellets resuspended in MOPS media with 25 mM pyruvate. Cultures were adjusted to OD 600 = 0.5 in MOPS media with 25 mM pyruvate in a mutli-well plate and choline was added to a concentration of 2 mM. Cultures were incubated shaking for 4 hours at 37°C. After inductions, supernatants were moved to 13 x 100 mm borosilicate glass tubes and incubated with 100 μM SPC for 4 hours, shaking at 37°C. After induction period, lipids were extracted using the Bligh and Dyer method 56 . 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 N 2 gas before final resuspension in 20 ml of ethanol. TLC silica gel 60 F254 plates (Sigma Aldrich) were pre-run with acetone and allowed to dry. Lipid extracts or standards at 100 µM were spotted onto the silica plates. After all samples were dried, plates were run with a chloroform:methanol:water (65:25:4) mobile phase. After the mobile phase approached the top of the plate, the plate was removed, dried, and sprayed with 0.2% ninhydrin solution (Acros Organics) to detect the sphingolipids by the primary amine group. sphAlacZ, sphB-lacZ , and lacZ-cerN reporter assays To investigate sphingosine product formation upon SPC hydrolysis, sphA, sphB , and cerN transcriptional induction was measure using P sphA-lacZ (previously described 43 , 44 ), sphBCD::lacZYA , and lacZcerN reporter strains and constructs, the latter two constructed as described above. P. aeruginosa strains were grown overnight at 37°C, shaking, in MOPS media with 25 mM pyruvate, 5 mM glucose, and 20 μg/mL gentamicin when necessary. Cells were collected via centrifugation, washed in MOPS media, and cell pellets were resuspended in MOPS media with 25 mM pyruvate (supplemented with 20 μg/mL gentamicin if necessary). Culture densities were adjusted to OD 600 = 0.5 in MOPS media with 25 mM pyruvate in multi-well plates with or without 20 μM SPC, sphingosine, sphinganine, phytosphingosine, or ceramide. Cultures were incubated with shaking at 37°C for 4 hours. β-galactosidase assays were performed as previously described 43 , 44 using Miller’s method 57 . Funding NIH NIAID R56AI173006 (MJW) CYSTIC FIBROSIS FOUNDATION WARGO24G0 (MJW) NIH NHLBI T32 HL076122 (PD) NIH NIAID T32 AI055402 (LAH) Acknowledgements We’d like to thank Jacob Mackinder, Jon Boyson, John Barlow, Bruno Martorelli di Genova, and Markus Thali for helpful discussions. We’d also like to thank Rob Hondal for assistance with ChemDraw. References 1. ↵ Berka , R.M. , Gray , G.L. , and Vasil , M.L. ( 1981 ). Studies of phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa . Infect Immun 34 , 1071 – 1074 . OpenUrl Abstract / FREE Full Text 2. Lanotte , P. , Mereghetti , L. , Lejeune , B. , Massicot , P. , and Quentin , R. ( 2003 ). Pseudomonas aeruginosa and cystic fibrosis: correlation between exoenzyme production and patient’s clinical state . Pediatr Pulmonol 36 , 405 – 412 . OpenUrl CrossRef PubMed 3. Nguyen , D. , Emond , M.J. , Mayer-Hamblett , N. , Saiman , L. , Marshall , B.C. , and Burns , J.L. ( 2007 ). Clinical response to azithromycin in cystic fibrosis correlates with in vitro effects on Pseudomonas aeruginosa phenotypes . Pediatr Pulmonol 42 , 533 – 541 . OpenUrl CrossRef PubMed 4. ↵ Woods , D.E. , Lam , J.S. , Paranchych , W. , Speert , D.P. , Campbell , M. , and Godfrey , A.J. ( 1997 ). Correlation of Pseudomonas aeruginosa virulence factors from clinical and environmental isolates with pathogenicity in the neutropenic mouse . Can J Microbiol 43 , 541 – 551 . OpenUrl CrossRef PubMed 5. ↵ Bergan , T. ( 1981 ). Pathogenetic factors of Pseudomonas aeruginosa . Scand J Infect Dis Suppl 29 , 7 – 12 . OpenUrl PubMed 6. ↵ Lopez , D.J. , Collado , M.I. , Ibarguren , M. , Vasil , A.I. , Vasil , M.L. , Goni , F.M. , and Alonso , A. ( 2011 ). Multiple phospholipid substrates of phospholipase C/sphingomyelinase HR2 from Pseudomonas aeruginosa . Chem Phys Lipids 164 , 78 - 82 . doi: 10.1016/j.chemphyslip.2010.11.001 . OpenUrl CrossRef PubMed 7. ↵ Stonehouse , M.J. , Cota-Gomez , A. , Parker , S.K. , Martin , W.E. , Hankin , J.A. , Murphy , R.C. , Chen , W. , Lim , K.B. , Hackett , M. , Vasil , A.I. , and Vasil , M.L. ( 2002 ). A novel class of microbial phosphocholine-specific phospholipases C . Mol Microbiol 46 , 661 – 676 . OpenUrl CrossRef PubMed Web of Science 8. Cota-Gomez , A. , Vasil , A.I. , Kadurugamuwa , J. , Beveridge , T.J. , Schweizer , H.P. , and Vasil , M.L. ( 1997 ). PlcR1 and PlcR2 are putative calcium-binding proteins required for secretion of the hemolytic phospholipase C of Pseudomonas aeruginosa . Infect Immun 65 , 2904 – 2913 . OpenUrl Abstract / FREE Full Text 9. ↵ Truan , D. , Vasil , A. , Stonehouse , M. , Vasil , M.L. , and Pohl , E. ( 2013 ). High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa . Protein Expr Purif 90 , 40 – 46 . doi: 10.1016/j.pep.2012.11.005 . OpenUrl CrossRef PubMed 10. ↵ Pritchard , A.E. , and Vasil , M.L. ( 1986 ). Nucleotide sequence and expression of a phosphate-regulated gene encoding a secreted hemolysin of Pseudomonas aeruginosa . J Bacteriol 167 , 291 – 298 . doi: 10.1128/jb.167.1.291-298.1986 . OpenUrl Abstract / FREE Full Text 11. ↵ Wiener-Kronish , J.P. , Sakuma , T. , Kudoh , I. , Pittet , J.F. , Frank , D. , Dobbs , L. , Vasil , M.L. , and Matthay , M.A. ( 1993 ). Alveolar epithelial injury and pleural empyema in acute P. aeruginosa pneumonia in anesthetized rabbits . J Appl Physiol (1985) 75 , 1661 – 1669 . doi: 10.1152/jappl.1993.75.4.1661 . OpenUrl CrossRef PubMed Web of Science 12. Meyers , D.J. , Palmer , K.C. , Bale , L.A. , Kernacki , K. , Preston , M. , Brown , T. , and Berk , R.S. ( 1992 ). In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa . Toxicon 30 , 161 – 169 . OpenUrl CrossRef PubMed 13. Terada , L.S. , Johansen , K.A. , Nowbar , S. , Vasil , A.I. , and Vasil , M.L. ( 1999 ). Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity . Infect Immun 67 , 2371 – 2376 . OpenUrl Abstract / FREE Full Text 14. Wieland , C.W. , Siegmund , B. , Senaldi , G. , Vasil , M.L. , Dinarello , C.A. , and Fantuzzi , G. ( 2002 ). Pulmonary inflammation induced by Pseudomonas aeruginosa lipopolysaccharide, phospholipase C, and exotoxin A: role of interferon regulatory factor 1 . Infect Immun 70 , 1352 – 1358 . OpenUrl Abstract / FREE Full Text 15. Berk , R.S. , Brown , D. , Coutinho , I. , and Meyers , D. ( 1987 ). In vivo studies with two phospholipase C fractions from Pseudomonas aeruginosa . Infect Immun 55 , 1728 – 1730 . OpenUrl Abstract / FREE Full Text 16. Wargo , M.J. , Gross , M.J. , Rajamani , S. , Allard , J.L. , Lundblad , L.K. , Allen , G.B. , Vasil , M.L. , Leclair , L.W. , and Hogan , D.A. ( 2011 ). Hemolytic phospholipase C inhibition protects lung function during Pseudomonas aeruginosa infection . Am J Respir Crit Care Med 184 , 345 – 354 . doi: 10.1164/rccm.201103-0374OC . OpenUrl CrossRef PubMed Web of Science 17. Holm , B.A. , Keicher , L. , Liu , M.Y. , Sokolowski , J. , and Enhorning , G. ( 1991 ). Inhibition of pulmonary surfactant function by phospholipases . J Appl Physiol (1985) 71 , 317 – 321 . doi: 10.1152/jappl.1991.71.1.317 . OpenUrl CrossRef PubMed Web of Science 18. Konig , B. , Vasil , M.L. , and Konig , W. ( 1997 ). Role of haemolytic and non-haemolytic phospholipase C from Pseudomonas aeruginosa in interleukin-8 release from human monocytes . J Med Microbiol 46 , 471 – 478 . doi: 10.1099/00222615-46-6-471 . OpenUrl CrossRef PubMed 19. ↵ Vasil , M.L. , Stonehouse , M.J. , Vasil , A.I. , Wadsworth , S.J. , Goldfine , H. , Bolcome , R.E. , 3rd . , and Chan , J. ( 2009 ). A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells . PLoS Pathog 5 , e1000420 . OpenUrl CrossRef PubMed 20. ↵ Wargo , M.J. , Ho , T.C. , Gross , M.J. , Whittaker , L.A. , and Hogan , D.A. ( 2009 ). GbdR regulates Pseudomonas aeruginosa plcH and pchP transcription in response to choline catabolites . Infect Immun 77 , 1103 – 1111 . OpenUrl Abstract / FREE Full Text 21. ↵ Hampel , K.J. , Labauve , A.E. , Meadows , J.A. , Fitzsimmons , L.F. , Nock , A.M. , and Wargo , M.J. ( 2014 ). Characterization of the GbdR Regulon in Pseudomonas aeruginosa . J Bacteriol 196 , 7 – 15 . doi: 10.1128/JB.01055-13 . OpenUrl Abstract / FREE Full Text 22. ↵ Fitzsimmons , L.F. , Hampel , K.J. , and Wargo , M.J. ( 2012 ). Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa . J Bacteriol 194 , 4718 – 4726 . JB.00596-12 [pii] doi: 10.1128/JB.00596-12 . OpenUrl Abstract / FREE Full Text 23. ↵ Mackinder , J.R. , Hinkel , L.A. , Schutz , K. , Eckstrom , K. , Fisher , K. , and Wargo , M.J. ( 2024 ). Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH) . J Bacteriol 206 , e0038223 . doi: 10.1128/jb.00382-23 . OpenUrl CrossRef PubMed 24. ↵ Shortridge , V.D. , Lazdunski , A. , and Vasil , M.L. ( 1992 ). Osmoprotectants and phosphate regulate expression of phospholipase C in Pseudomonas aeruginosa . Mol Microbiol 6 , 863 – 871 . OpenUrl CrossRef PubMed Web of Science 25. ↵ Sage , A.E. , Vasil , A.I. , and Vasil , M.L. ( 1997 ). Molecular characterization of mutants affected in the osmoprotectant-dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1 . Mol Microbiol 23 , 43 – 56 . OpenUrl CrossRef PubMed Web of Science 26. ↵ Kurioka , S. , and Matsuda , M. ( 1976 ). Phospholipase C assay using p-nitrophenylphosphoryl-choline together with sorbitol and its application to studying the metal and detergent requirement of the enzyme . Anal Biochem 75 , 281 – 289 . OpenUrl CrossRef PubMed 27. ↵ Ge , D. , Yue , H.W. , Liu , H.H. , and Zhao , J. ( 2018 ). Emerging roles of sphingosylphosphorylcholine in modulating cardiovascular functions and diseases . Acta Pharmacol Sin 39 , 1830 – 1836 . doi: 10.1038/s41401-018-0036-4 . OpenUrl CrossRef PubMed 28. ↵ Bruzik , K.S. ( 1988 ). Conformation of the polar headgroup of sphingomyelin and its analogues . Biochimica et Biophysica Acta (BBA) - Biomembranes 939 , 315 – 326 . OpenUrl CrossRef PubMed 29. ↵ Yatomi , Y. , Ruan , F. , Hakomori , S. , and Igarashi , Y. ( 1995 ). Sphingosine-1-phosphate: a platelet-activating sphingolipid released from agonist-stimulated human platelets . Blood 86 , 193 – 202 . OpenUrl Abstract / FREE Full Text 30. ↵ Liliom , K. , Sun , G. , Bunemann , M. , Virag , T. , Nusser , N. , Baker , D.L. , Wang , D.A. , Fabian , M.J. , Brandts , B. , Bender , K. , et al. ( 2001 ). Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors . Biochem J 355 , 189 – 197 . doi: 10.1042/0264-6021:3550189 . OpenUrl CrossRef PubMed Web of Science 31. ↵ Nixon , G.F. , Mathieson , F.A. , and Hunter , I. ( 2008 ). The multi-functional role of sphingosylphosphorylcholine . Prog Lipid Res 47 , 62 – 75 . doi: 10.1016/j.plipres.2007.11.001 . OpenUrl CrossRef PubMed Web of Science 32. ↵ Nofer , J.R. , Levkau , B. , Wolinska , I. , Junker , R. , Fobker , M. , von Eckardstein , A. , Seedorf , U. , and Assmann , G. ( 2001 ). Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids . J Biol Chem 276 , 34480 – 34485 . doi: 10.1074/jbc.M103782200 . OpenUrl Abstract / FREE Full Text 33. ↵ Meyer zu Heringdorf , D. , and Jakobs , K.H. ( 2007 ). Lysophospholipid receptors: signalling, pharmacology and regulation by lysophospholipid metabolism . Biochim Biophys Acta 1768 , 923 – 940 . doi: 10.1016/j.bbamem.2006.09.026 . OpenUrl CrossRef PubMed 34. ↵ Betto , R. , Teresi , A. , Turcato , F. , Salviati , G. , Sabbadini , R.A. , Krown , K. , Glembotski , C.C. , Kindman , L.A. , Dettbarn , C. , Pereon , Y. , et al. ( 1997 ). Sphingosylphosphocholine modulates the ryanodine receptor/calcium-release channel of cardiac sarcoplasmic reticulum membranes . Biochem J 322 ( Pt 1 ), 327 – 333 . doi: 10.1042/bj3220327 . OpenUrl Abstract / FREE Full Text 35. Yasukochi , M. , Uehara , A. , Kobayashi , S. , and Berlin , J.R. ( 2003 ). Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine . Pflugers Arch 445 , 665 – 673 . doi: 10.1007/s00424-002-0945-3 . OpenUrl CrossRef PubMed Web of Science 36. ↵ Mogami , K. , Mizukami , Y. , Todoroki-Ikeda , N. , Ohmura , M. , Yoshida , K. , Miwa , S. , Matsuzaki , M. , Matsuda , M. , and Kobayashi , S. ( 1999 ). Sphingosylphosphorylcholine induces cytosolic Ca(2+) elevation in endothelial cells in situ and causes endothelium-dependent relaxation through nitric oxide production in bovine coronary artery . FEBS Lett 457 , 375 – 380 . doi: 10.1016/s0014-5793(99)01076-5 . OpenUrl CrossRef PubMed Web of Science 37. ↵ Jeon , E.S. , Song , H.Y. , Kim , M.R. , Moon , H.J. , Bae , Y.C. , Jung , J.S. , and Kim , J.H. ( 2006 ). Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK . J Lipid Res 47 , 653 – 664 . doi: 10.1194/jlr.M500508-JLR200 . OpenUrl Abstract / FREE Full Text 38. ↵ Kim , K.H. , Kim , Y.M. , Lee , M.J. , Ko , H.C. , Kim , M.B. , and Kim , J.H. ( 2012 ). Simvastatin inhibits sphingosylphosphorylcholine-induced differentiation of human mesenchymal stem cells into smooth muscle cells . Exp Mol Med 44 , 159 – 166 . doi: 10.3858/emm.2012.44.2.011 . OpenUrl CrossRef PubMed 39. ↵ Jeon , E.S. , Lee , M.J. , Sung , S.M. , and Kim , J.H. ( 2007 ). Sphingosylphosphorylcholine induces apoptosis of endothelial cells through reactive oxygen species-mediated activation of ERK . J Cell Biochem 100 , 1536 – 1547 . doi: 10.1002/jcb.21141 . OpenUrl CrossRef PubMed Web of Science 40. ↵ Yamamoto , H. , Naito , Y. , Okano , M. , Kanazawa , T. , Takematsu , H. , and Kozutsumi , Y. ( 2011 ). Sphingosylphosphorylcholine and lysosulfatide have inverse regulatory functions in monocytic cell differentiation into macrophages . Arch Biochem Biophys 506 , 83 – 91 . doi: 10.1016/j.abb.2010.11.004 . OpenUrl CrossRef PubMed 41. ↵ Boguslawski , G. , Lyons , D. , Harvey , K.A. , Kovala , A.T. , and English , D. ( 2000 ). Sphingosylphosphorylcholine induces endothelial cell migration and morphogenesis . Biochem Biophys Res Commun 272 , 603 – 609 . doi: 10.1006/bbrc.2000.2822 . OpenUrl CrossRef PubMed Web of Science 42. ↵ Kim , K.S. , Ren , J. , Jiang , Y. , Ebrahem , Q. , Tipps , R. , Cristina , K. , Xiao , Y.J. , Qiao , J. , Taylor , K.L. , Lum , H. , et al. ( 2005 ). GPR4 plays a critical role in endothelial cell function and mediates the effects of sphingosylphosphorylcholine . FASEB J 19 , 819 – 821 . doi: 10.1096/fj.04-2988fje . OpenUrl CrossRef PubMed 43. ↵ LaBauve , A.E. , and Wargo , M.J. ( 2014 ). Detection of host-derived sphingosine by Pseudomonas aeruginosa is important for survival in the murine lung . PLoS Pathog 10 , e1003889 . doi: 10.1371/journal.ppat.1003889 . OpenUrl CrossRef PubMed 44. ↵ DiGianivittorio , P. , Hinkel , L.A. , Mackinder , J.R. , Schutz , K. , Klein , E.A. , and Wargo , M.J. ( 2025 ). The Pseudomonas aeruginosa sphBC genes are important for growth in the presence of sphingosine by promoting sphingosine metabolism . Microbiology (Reading) 171 . doi: 10.1099/mic.0.001520 . OpenUrl CrossRef 45. ↵ Okino , N. , and Ito , M. ( 2007 ). Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa . J Biol Chem 282 , 6021 – 6030 . OpenUrl Abstract / FREE Full Text 46. ↵ Macfarlane , M.G. ( 1948 ). The biochemistry of bacterial toxins: 2. The enzymic specificity of Clostridium welchii lecithinase . Biochem J 42 , 587 – 590 . OpenUrl FREE Full Text 47. Guillouard , I. , Garnier , T. , and Cole , S.T. ( 1996 ). Use of site-directed mutagenesis to probe structure-function relationships of alpha-toxin from Clostridium perfringens . Infect Immun 64 , 2440 – 2444 . doi: 10.1128/iai.64.7.2440-2444.1996 . OpenUrl Abstract / FREE Full Text 48. ↵ Monturiol-Gross , L. , Villalta-Romero , F. , Flores-Diaz , M. , and Alape-Giron , A. ( 2021 ). Bacterial phospholipases C with dual activity: phosphatidylcholinesterase and sphingomyelinase . FEBS Open Bio 11 , 3262 – 3275 . doi: 10.1002/2211-5463.13320 . OpenUrl CrossRef PubMed 49. ↵ Chen , C. , Malek , A.A. , Wargo , M.J. , Hogan , D.A. , and Beattie , G.A. ( 2010 ). The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds . Mol Microbiol 75 , 29 - 45 . MMI6962 [pii] doi: 10.1111/j.1365-2958.2009.06962.x . OpenUrl CrossRef PubMed 50. ↵ Malek , A.A. , Chen , C. , Wargo , M.J. , Beattie , G.A. , and Hogan , D.A. ( 2011 ). Roles of three transporters, CbcXWV, BetT1, and BetT3, in Pseudomonas aeruginosa choline uptake for catabolism . J Bacteriol 193 , 3033 – 3041 . JB.00160-11 [pii] doi: 10.1128/JB.00160-11 . OpenUrl Abstract / FREE Full Text 51. ↵ Scherer , M. , Bottcher , A. , Schmitz , G. , and Liebisch , G. ( 2011 ). Sphingolipid profiling of human plasma and FPLC-separated lipoprotein fractions by hydrophilic interaction chromatography tandem mass spectrometry . Biochim Biophys Acta 1811 , 68 – 75 . doi: 10.1016/j.bbalip.2010.11.003 . OpenUrl CrossRef PubMed 52. ↵ Neidhardt , F.C. , Bloch , P.L. , and Smith , D.F. ( 1974 ). Culture medium for enterobacteria . J Bacteriol 119 , 736 – 747 . OpenUrl Abstract / FREE Full Text 53. ↵ Labauve , A.E. , and Wargo , M.J. ( 2012 ). Growth and Laboratory Maintenance of Pseudomonas aeruginosa . Curr Protoc Microbiol Chapter 6, Unit6E 1. doi: 10.1002/9780471729259.mc06e01s25 . OpenUrl CrossRef 54. ↵ Shanks , R.M. , Caiazza , N.C. , Hinsa , S.M. , Toutain , C.M. , and O’Toole , G.A. ( 2006 ). Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria . Appl Environ Microbiol 72 , 5027 – 5036 . OpenUrl Abstract / FREE Full Text 55. ↵ Shikita , M. , Fahey , J.W. , Golden , T.R. , Holtzclaw , W.D. , and Talalay , P. ( 1999 ). An unusual case of ‘uncompetitive activation’ by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings . Biochem J 341 ( Pt 3 ), 725 – 732 . doi: 10.1042/0264-6021:3410725 . OpenUrl Abstract / FREE Full Text 56. ↵ Bligh , E.G. , and Dyer , W.J. ( 1959 ). A rapid method of total lipid extraction and purification . Can J Biochem Physiol 37 , 911 – 917 . doi: 10.1139/o59-099 . OpenUrl CrossRef PubMed 57. ↵ Miller , J.H. ( 1972 ). Experiments in molecular genetics ( Cold Spring Harbor Laboratory ). View the discussion thread. 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