P. aeruginosa produces novel glycolipids in response to Pi stress
To determine changes in the membrane lipidome in response to P-stress, the model P. aeruginosa strain PAO1 was grown in minimal medium under high (1 mM) or low Pi (50 µM) conditions (Fig. 1a). The latter condition elicited strong alkaline phosphatase activity, measured through the liberation of para-nitrophenol (pNP) from pNPP (Fig. 1b), this being a strong indication that cells were P-stressed. Analysis of membrane lipid profiles using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) revealed the presence of several new lipids under Pi stress conditions (Fig. 1c). Thus, during Pi-replete growth (1 mM phosphate), the lipidome is dominated by two glycerophospholipids: PG (eluted at 6.8 min) and PE (eluted at 12.2 min). During Pi-stress a lipid species with mass to charge ratio (m/z) of 623 and 649 were also found, with MS fragmentation resulting in a 131 m/z peak, a diagnostic ion for the amino-acid containing ornithine lipid. This is consistent with previous reports of ornithine lipids in the P. aeruginosa membrane in response to Pi stress [29, 30].
a Growth of strain PAO1 WT in minimal medium A containing 1 mM phosphate (+Pi, blue) or 50 µM phosphate (−Pi, black) over 12 h. Data are the average of three independent replicates. b Liberation of para-nitrophenol (pNP) from para-nitrophenol phosphate (pNPP) through alkaline phosphatase activity, under Pi-replete (1 mM, black) and Pi-deplete (50 µM, yellow) conditions. Error bars represent the standard deviation of three independent replicates. c Representative chromatograms in negative ionisation mode of the P. aeruginosa lipidome when grown under phosphorus stress (−Pi, black) compared to growth under phosphorus sufficient conditions (+Pi, orange). PG phosphatidylglycerol, PE phosphatidylethanolamine, OL ornithine lipids. Lower panel: extracted ion chromatograms of three new glycolipid species in P. aeruginosa which are only produced during Pi-limitation (black, 1 mM; orange, 50 µM). MGDG monoglucosyldiacylglycerol, GADG glucuronic acid-diacylglycerol and UGL unconfirmed glycolipid. d Mass spectrometry fragmentation spectra of three glycolipid species present under Pi stress in P. aeruginosa, at retention times of 7.7 (m/z 774.7), 8.7 (m/z 786.7) and 9.8 (m/z 788.6) minutes, respectively. Each spectrum depicts an intact lipid mass with an ammonium (NH4+) adduct exhibiting neutral loss of a head group, yielding diacylglycerol (DAG) (595 m/z). Further fragmentation yields monoacylglycerols (MAG) with C16:0 or C18:1 fatty acyl chains.
Further to ornithine lipids, three unknown lipids eluting at 7.7, 8.7 and 9.8 min, were only present under Pi stress conditions (Fig. 1c). Using several rounds of MS fragmentation (MSn), with a quadrupole ion trap MS, fragmentation patterns characteristic of glycolipids were found for all three peaks. For each peak of interest, the most predominant lipid masses of 774.7, 786.8 and 788.6 m/z were analysed by MSn in positive ionisation mode (Fig. 1d). In each case, an initial head group was lost leaving a significant signal of 595.6 m/z, the mass of the glycolipid building block diacylglycerol (DAG). Further fragmentation leads to the loss of either fatty acyl chain from DAG, leaving monoacylglycerols of 313.2 and 339.3 m/z. Two monoacylglycerols with different masses are produced as a result of the original lipid containing 16:0 and 18:1 fatty acids (313.2 and 339.3 m/z monoacylglycerols, respectively). To further elucidate the identity of the peaks, a search for a neutral loss of a polar head group was carried out. Thus, the intact masses of 774.7 and 788.6 m/z in positive ionisation mode leads to the loss of a head group of −179 and −193 m/z, which corresponds to a hexose- and a glucuronate- group, respectively (Fig. 1d), suggesting the occurrence of novel monoglucosyldiacylglycerol (MGDG) and glucuronic acid diacylglycerol (GADG) glycolipids in P. aeruginosa. The third glycolipid peak at 8.7 min remains an unknown lipid with intact mass of 786.8 m/z (hereafter designated as a putative unknown glycolipid, UGL). Together, these data confirm the production of new glycolipids in P. aeruginosa in response to Pi stress.
Comparative proteomics uncover the lipid renovation pathway in P. aeruginosa
To determine the proteomic response of P. aeruginosa to phosphorus limitation, and identify the genes involved in glycolipid formation, strain PAO1 was cultivated under high and low Pi conditions for 8 h and the cellular proteome then analysed. A total of 2844 proteins were detected, 175 of which were found to be differentially regulated by Pi availability (Fig. 2a, Table S1). In line with previous transcriptomic studies of strain PAO1 [18], major phosphorus acquisition mechanisms were highly expressed under Pi stress conditions, e.g. the Pi-specific transporter PstSCAB, the two-component regulator PhoBR (Table S1) [31].
a Volcano plot depicting differentially expressed proteins when comparing Pi-replete and Pi-deplete conditions. Significantly upregulated proteins when under Pi stress are shown in red (left), and those that are significantly upregulated when Pi is sufficient are in green (right). Significance was accepted when the false discovery rate (FDR) was <0.05, and a fold change ≥2. b The proposed pathway for lipid remodelling through the PlcP-Agt pathway. PlcP degrades membrane phospholipids such as PG, to generate diacylglycerol (DAG) intermediates for the formation of MGDG and GADG through the activity of glycosyltransferases, using either UDP-glucose or UDP-glucuronate as the co-substrate [41]. c Genomic organisation of predicted lipid remodelling genes in P. aeruginosa. Glycosyltransferases (orange) PA3218 (Agt1) and PA0842 (Agt2) are predicted to be involved in glycolipid synthesis. PA3219 is predicted to be PlcP in P. aeruginosa. Predicted Pho box sequences in the promoter regions (represented in blue boxes) of each glycosyltransferase operon from P. aeruginosa strains representing the PAO1 clade, the PA7 clade and the PA14 clade are shown. The black dots represent residues which are conserved in the Pho box consensus CTGTCATNNNNCTGTCAT [42]. d Metatranscriptomic analysis of PlcP-Agt lipid remodelling genes in sputum samples from a cystic fibrosis patient 7-days (CF_D-7) and 8-days (CF_D-8) before death [26] and a Danish CF patient (CF_Person G) [27] as well as a wound sample from a burns patient from the USA (Burn patient) [27]. Relative abundance is expressed as RPKM (reads per kilobase of transcript, per million mapped reads). PhoA (PA3296) encodes an alkaline phosphatase [38]. The list of RPKM abundance of individual genes of P. aeruginosa PAO1 is shown in Table S4.
Comparative proteomics also identified several genes which are likely important for membrane lipid remodelling (Fig. 2b) including PA3219 (4.6-fold increase under Pi-deplete conditions, FDR < 0.01), encoding a putative phospholipase C protein, and PA0842 (4-fold increase under Pi-deplete conditions, FDR < 0.01), encoding a putative glycosyltransferase (Fig. 2a). PA3219 has 47% protein sequence identity to PlcP from Phaeobacter sp. MED193 and 46% identity to PlcP from Sinorhizobium meliloti [26,27,28]. In these bacteria, PlcP is essential in the lipid remodelling pathway for the formation of the diacylglycerol (DAG) backbone, representing the essential intermediate for the production of glycolipids [32, 33]. In P. aeruginosa PAO1, PA3219 appears to form an operon with PA3218, a putative glycosyltransferase likely under the control of the PhoBR two-component system, as a highly conserved Pho box sequence was recognisable in the promoter region (Fig. 2c). PA3218 (hereafter referred to as Agt1) has 41% protein sequence identity to the Agt of Phaeobacter sp. MED193. PA0842 showed 35% identity to the Agt of Phaeobacter sp. MED193 and a Pho box sequence is also found in its promoter region. This corroborates the finding that the PA0842 protein (hereafter referred to as Agt2) was significantly upregulated under Pi-deplete conditions (Fig. 2a). In summary, comparative proteomic analysis suggests that P. aeruginosa PAO1 adopts this PlcP-Agt lipid remodelling pathway for the production of glycolipids in response to Pi-stress (Fig. 2b).
The PlcP-Agt mediated lipid renovation pathway is strictly conserved in P. aeruginosa and actively transcribed in the metatranscriptomes of cystic fibrosis patients
To uncover how widespread this predicted PlcP-Agt lipid remodelling pathway is amongst the genus Pseudomonas, including P. aeruginosa strains, we conducted a thorough comparative genomics analysis of these lipid renovating loci. PlcP-Agt is strictly conserved in all 770 genome-sequenced P. aeruginosa strains in the IMG/M database, including all three-previously recognised P. aeruginosa lineages [34, 35], group 1 represented by strain PAO1, group 2 represented by strain PA14 and group 3 represented by strain PA7 (Fig. 3, Table S2). Indeed, this remodelling pathway is prevalent in many Pseudomonas groups, including the plant pathogen P. syringae. To investigate whether the PlcP-Agt lipid remodelling pathway is involved in host-pathogen interactions, we analysed metatranscriptomic datasets from CF patients, where P. aeruginosa is known to be prevalent in the fatal exacerbation period before patient death [26]. To the best of our knowledge, only two studies have reported the metatranscriptome of the bacterial community present in CF sputum [26, 27]. Indeed, phoBR and pstS are amongst the most highly expressed genes, confirming previous observations that P. aeruginosa is Pi-limited during human airway epithelia infection [36, 37]. Interestingly, the alkaline phosphatase phoA [38] was highly expressed in sputum from CF patients but not from wound samples which was also dominated by P. aeruginosa. Importantly, the transcripts of P. aeruginosa agt1/plcP/agt2 are highly expressed in CF sputum during the fatal exacerbation period before death (Fig. 2d). Therefore, our phylogenomic and metatranscriptomic analyses suggest that not only is the PlcP-Agt lipid remodelling pathway strictly conserved and prevalent in P. aeruginosa, but also the corresponding genes are also highly expressed during CF patient infection, suggesting a potential role for lipid renovation in host-pathogen interactions.
The phylogeny of Pseudomonas clades was determined using the nucleotide sequences of six housekeeping genes (rpoB, rpoD, dnaE, recA, atpD, gyrB) retrieved from each genome using IQ-Tree [43]. The filled colour indicates the presence of the genes in the genomes whereas a blank indicates the absence of the corresponding gene in the genomes. The two-component system PhoBR (black circles) is found in all genomes and the PlcP-Agt1/Agt2 are strictly conserved in all 770 genome-sequenced P. aeruginosa strains that form three clades represented by strain PA14, PA01 and PA7, respectively. Bootstrap values >75% are shown.
Experimental validation of the lipid renovation pathway for glycolipid formation in P. aeruginosa
To validate the function of these two putative glycosyltransferases (Agt1, Agt2) in the formation of glycolipids, we synthesised the codon-optimised genes (PA3218 and PA0842, respectively) for recombinant expression in E. coli. The total lipidomes from the recombinant E. coli strains were then analysed by HPLC-MS to determine the presence of glycolipids in a gain-of-function assay. Expression of P. aeruginosa Agt1 (PA3218) was sufficient for the production of MGDG (eluted at 7.7 min) in E. coli, confirmed through MSn fragmentation (Fig. 4a). No UGL nor GADG was observed in the lipidome of this Agt1-overexpressing E. coli strain. Expressing Agt2 (PA0842) from P. aeruginosa in E. coli was sufficient for the accumulation of the GADG glycolipid (eluted at 9.8 min), also confirmed through the MSn fragmentation pattern (Fig. 4b). Equally, no UGL nor MGDG was observed in the Agt2-overexpressing E. coli strain. Production of these glycolipids was not observed in the same strain of E. coli transformed with an empty vector control (pET28a). It is therefore likely that UGL production is carried out by another glycosyltransferase, the identity of which remains to be discovered.
a Extracted ion chromatogram of the MGDG lipid from recombinant E. coli expressing Agt1. An empty vector control is also shown (red line). The identity of MGDG is further validated using mass spectrometry fragmentation showing the neutral loss of 179 corresponding to the loss of glucose and the formation of monoacylglycerols (MAG) with C16:0 or C18:1 (m/z 313.2, 339.3). b Extracted ion chromatogram of the GADG lipid from recombinant E. coli expressing Agt2. An empty vector control is also shown (red line). The identity of GADG is further validated using mass spectrometry fragmentation showing the neutral loss of 193 corresponding to the loss of glucose and the formation of monoacylglycerols (MAG) with C16:0 or C18:1 (m/z 313.2, 339.2). c Purified Agt1 and Agt2 protein from recombinant E. coli (left panel) and Michaelis Menten kinetics of Agt1 towards UDP-glucose (middle panel) and Agt2 towards UDP-glucuronic acid (right panel) as substrate, respectively. d Mass spectrometry identification of MGDG and GADG produced from purified Agt1 and Agt2 using DAG and UDP-glucose and UDP-glucuronic acid as the substrate, respectively.
To confirm the role of Agt1 and Agt2 in the production of MGDG and GADG, we purified Agt1 and Agt2 from recombinant E. coli (Fig. 4c) and carried out enzyme assays using UDP-glucose and UDP-glucuronic acid as the sugar donor and DAG as the acceptor. Agt1 can only accept UDP-glucose as the substrate with an affinity of Km = 298.1 ± 9.5 µM (Fig. 4c, middle panel) and produced MGDG as expected (Fig. 4d, left panel). Similarly, Agt2 can use UDP-glucuronic acid as the substrate (Km = 373.0 ± 12.9 µM (Fig. 4c, right panel), producing the GADG lipid (Fig. 4d, right panel). Interestingly, the purified Agt2 enzyme can also use UDP-glucose to some extent with a Km of 480 µM (data not shown) although the corresponding lipid MGDG was not observed in the lipid extract from the lipidome of the recombinant host E. coli (Fig. 4b).
To further confirm the role of these genes in P. aeruginosa glycolipid biosynthesis we analysed the lipidomes of mutants in ΔplcP, Δagt1 and Δagt2 in strain PAO1 (Fig. 5a, b). Differences were analysed by searching for the intact masses of the glycolipids MGDG and GADG: 774.6 and 788.6 m/z in positive ionisation mode with an ammonium adduct, respectively. As expected, under Pi stress MGDG is no longer produced in the Δagt1 mutant and similarly GADG is no longer produced in the Δagt2 mutant (Fig. 5a). In the ΔplcP mutant, no MGDG was found and the GADG lipid was significantly reduced but not entirely abolished (Fig. 5b). The small amount of GADG produced in the ΔplcP mutant suggests that an alternative supply of DAG (independent of the degradation of phospholipids by PlcP) is available in this mutant. Nevertheless, lipidome analyses of the ΔplcP, Δagt1 and Δagt2 mutants strongly supports the key role of this PlcP-Agt pathway (Fig. 2a) in lipid renovation in P. aeruginosa.
Relative abundance of the glycolipid MGDG (a) and GADG (b) in P. aeruginosa mutants plcP (purple trace), agt1 (blue trace) and agt2 (green, trace) compared to the wild type (WT). Cells were cultivated under low Pi conditions (50 µM) and a representative extracted ion chromatogram of MGDG/GADG is shown between the WT, (black trace) and each mutant. The right most panel shows the abundance of MGDG or GADG calculated relative to an internal lipid standard d17:1/12:0 sphingosylphosphoethanolamine (Sigma-Aldrich) in the wild-type and mutant strains of P. aeruginosa. Values are calculated from three biological replicates and the error bars denote standard deviation. MGDG, monoglucosyldiacylglycerol, GADG, glucuronic acid-diacylglycerol. c Survival of glycolipid remodelling mutants ΔplcP (purple), Δagt1 (green) and Δagt2 (blue) when challenged with 4 µg mL−1 polymyxin B compared to WT under Pi stress (black). All experiments were conducted under Pi stress conditions and the results are the average of three biological replicates; error bars denote standard deviation. d Survival of glycolipid producing Escherichia coli when challenged with 20 µg mL−1 polymyxin B. All experiments were conducted in three replicates and error bars denote standard deviation. Black, E. coli containing the empty vector pET28a; green, E. coli containing plasmid pET28a-Agt1; blue, E. coli containing plasmid pET28a-Agt2.
The protective role of glycolipids to antibiotic resistance in Pseudomonas aeruginosa
To assess whether growth of the glycolipid-deficient mutants (ΔplcP, Δagt1, Δagt2) was affected by Pi stress, we grew the mutants in the defined minimal medium under high and low Pi conditions. However, no significant difference in growth rates was found (Fig. S1). The presence of glycolipids in the membrane may, however, have a profound impact on the functioning of the membrane during Pi stress. For example, PG is an anionic lipid with net negative charges whereas MGDG has a neutral charged sugar group. Although a PG-to-GADG substitution may not necessarily change membrane charge [23], it may affect membrane curvature and the packing density of lipids. Thus, subsequent knock-on effects in membrane function might be expected [10]. We therefore set out to investigate whether membrane lipid composition may have an impact on antibiotic resistance in P. aeruginosa. As cationic antimicrobial peptides directly interact with bacterial cell membranes, we focused on the impact of lipid remodelling on the killing activity of polymyxin B. We conducted the analyses under P-deplete conditions, since Pi-stress is clinically important, already known to induce the expression of virulence factors [15, 17, 18, 30], and our own analysis confirmed that an array of genes involved in phosphate acquisition and lipid remodelling in P. aeruginosa are indeed highly expressed in sputum samples from the lung microbiome of CF patients (Fig. 2d). Polymyxins represent the drug-of-last resort for effectively treating carbapenem-resistant P. aeruginosa infections [3, 39].
To test the sensitivity of the mutants in the PlcP-Agt pathway to polymyxin B, we compared WT and mutants using kill curve analyses as the typically used disk diffusion method does not work efficiently for cationic antimicrobials [40]. Indeed, there was a significant decrease in the survival of all three PAO1 glycolipid synthesis mutants (ΔplcP, Δagt1 and Δagt2) compared to the wild type when challenged with polymyxin B, suggesting a protective role of glycolipids in polymyxin B resistance (Fig. 5c). Such a protective role of glycolipids in polymyxin B resistance was not observed for other antibiotics, including ciprofloxacin, gentamicin, ceftazidime and meropenem (data not shown). P. aeruginosa is known to enhance its resistance to polymyxins through decoration of its LPS layer using either 4-amino-4-deoxy-L-arabinose (L-Ara4N) by arnB [5], or the addition of phosphoethanolamine (pEtN) by eptA [6]. It is thought that these changes perturb the electrostatic interaction between the cationic polymyxin B and the normally negatively charged LPS. To investigate whether these mechanisms play a role in the glycolipid-deficient mutants, we conducted a comparative proteomics analysis of the ΔplcP mutant and WT under Pi-deplete conditions, which revealed only a small number of differentially expressed proteins (Table S3). The majority of these differentially expressed proteins are uncharacterised. However, importantly, LPS modification enzymes previously found to confer antimicrobial peptide resistance, such as ArnB and EptA, were not differentially expressed between the WT and ΔplcP mutant. Therefore, our data suggests that it is the glycolipids that are the major contributor to increased polymyxin B resistance, which constitutes a new biological mechanism for polymyxin resistance. To this end, we tested the resistance to polymyxin B of recombinant E. coli strains overexpressing P. aeruginosa Agt1 and Agt2, that produce MGDG and GADG, respectively (Fig. 4a, b). Indeed, in this gain-of-function assay, both Agt1 and Agt2-overexpressing E. coli strains had enhanced resistance to polymyxin B compared to the empty vector control (Fig. 5d), supporting the protective role of these glycolipids to antimicrobial peptides.
To conclude, we present here the discovery of novel glycolipids produced in P. aeruginosa during adapation to phosphorus stress. This lipid renovation pathway is strictly conserved in all P. aeruginosa isolates to date and highly expressed in the metatranscriptome of CF patients, suggesting a key role of lipid remodelling in the ecophysiology of this bacterium. Interestingly, lipid remodelling as a response to survive phosphorus stress in turn comes with trade-offs in terms of antibiotic resistance; these glycolipids may protect the bacterium from insult by cationic antimicrobial peptides, highlighting a new resistance mechanism to polymyxin B which has been previously overlooked. It remains to be seen whether the altered susceptibility to polymyxin B is the sole trade-off following lipid remodelling of phospholipids to glycolipids. After all, evolution appears to have selected phospholipids as the dominant lipids in the last universal common ancestor [12].
Source: Ecology - nature.com
