Detection of host-derived sphingosine by Pseudomonas aeruginosa is important for survival in the murine lung

Abstract

Pseudomonas aeruginosa is a common environmental bacterium that is also a significant opportunistic pathogen, particularly of the human lung. We must understand how P. aeruginosa responds to the lung environment in order to identify the regulatory changes that bacteria use to establish and maintain infections. The P. aeruginosa response to pulmonary surfactant was used as a model to identify transcripts likely induced during lung infection. The most highly induced transcript in pulmonary surfactant, PA5325 (sphA), is regulated by an AraC-family transcription factor, PA5324 (SphR). We found that sphA was specifically induced by sphingosine in an SphR-dependent manner, and also via metabolism of sphingomyelin, ceramide, or sphingoshine-1-phosphate to sphingosine. These sphingolipids not only play a structural role in lipid membranes, but some are also intracellular and intercellular signaling molecules important in normal eukaryotic cell functions as well as orchestrating immune responses. The members of the SphR transcriptome were identified by microarray analyses, and DNA binding assays showed specific interaction of these promoters with SphR, which enabled us to determine the consensus SphR binding site. SphR binding to DNA was modified by sphingosine and we used labeled sphingosine to demonstrate direct binding of sphingosine by SphR. Deletion of sphR resulted in reduced bacterial survival during mouse lung infection. In vitro experiments show that deletion of sphR increases sensitivity to the antimicrobial effects of sphingosine which could, in part, explain the in vivo phenotype. This is the first identification of a sphingosine-responsive transcription factor in bacteria. We predict that SphR transcriptional regulation may be important in response to many sites of infection in eukaryotes and the presence of homologous transcription factors in other pathogens suggests that sphingosine detection is not limited to P. aeruginosa.

Figure 1. Expression of sphA (PA5325) is induced by sphingosine.

(A) Arrangement of the sphA genomic region in P. aeruginosa PAO1. The genes in green are those discussed further in this study. Data from panels B through F all use a plasmid-borne sphA-lacZ reporter (pAL5) to assess regulation with pyruvate (pyr) used as the non-inducing control condition. Fold induction of sphA-lacZ is calculated compared to its pyruvate control. (B) sphA is induced in the presence of pulmonary surfactant (surf), sheep red blood cells (blood), and mouse fibroblasts (fibr). (C) The primary sphA-inducing component of fibroblasts is present in the organic fraction (org), compared to the aqueous fraction (aq) after the mouse fibroblasts (whole) were extracted with chloroform:methanol. (D) The sphingolipids sphingomyelin (sm), ceramide (cer), sphingosine-1-phosphate (S1P), and sphingosine (sph) induce sphA, while likely catabolic products palmitate (pm) and glycine (gly) do not cause induction. Sphingosine causes the highest level of induction. (E) Induction of sphA by sphingosine is dose dependent and occurs at physiologically relevant concentrations of sphingosine. Lines for statistical significance denote groups of samples with the same magnitude of significance, not grouped comparisons. (F) Induction of sphA by ceramide requires the neutral ceramidase gene, cerN, while sphingosine induction is independent of cerN. (G) Induction of sphA by S1P in a heterologous E. coli sphR-sphA-lacZ reporter system (pAL5) requires phosphatase treatment of S1P. Statistical significance determined using one way ANOVA with Dunnett's post-test for B–E & G with the uninduced condition being the comparator for all other data. In panel F, the wild type and ΔcerN data were compared by student t-test within each treatment condition. p-value summaries: n.s. = not significant; * for p<0.05; ** for p<0.01; *** for p<0.001; **** for p<0.0001. All experiments were performed at least three times and data shown is representative of both the scale and statistical significance levels of all experiments.

Figure 2. The transcription regulator SphR (PA5324) controls sphA induction in response to sphingosine.

(A) Sphingosine (sph) induces sphA-lacZ expression (pAL4) compared to the pyruvate (pyr) control in wild-type cells (wild type) but not in sphR mutant cells (ΔsphR). This regulation is restored by complementation (Comp) of sphR at the attTn7 locus. (B) In a heterologous E. coli system, the sphA-lacZ reporter (pAL4) is not responsive to sphingosine (second bar) unless the sphR gene is included in the system (pAL5) (fourth bar). Data for these panels were compared by student t-test comparing treatment conditions within each strain. p-value summaries: n.s. = not significant; **** for p<0.0001. All experiments were performed at least three times and data shown is representative of both the scale and statistical significance levels of all experiments.

Figure 3. Deletion of sphR reduces P. aeruginosa survival in the mouse lung.

Male C57Bl/6J mice were infected with 2×10⁷ CFU/mouse of each strain via oropharyngeal aspiration. Mice were euthanized and lungs harvested 24 hours after infection. Bacterial counts were determined by serial dilution onto Pseudomonas Isolation Agar (PIA). Deletion of sphR (ΔsphR) reduced P. aeruginosa survival 7.7-fold (p<0.001), an effect that was complemented by addition of sphR at the attTn7 site. Wild-type (WT) and ΔsphR cells carried the empty attTn7 insertion cassette as described in the methods section. Statistical significance determined using one way ANOVA with Tukey's post-test comparing all groups to each other. p-value summaries: n.s. = not significant; *** for p<0.001. Data shown is combined from two experiments. The same effect sizes and variance have been seen in at least one additional experiment for each strain, resulting in each strain having been compared to wild type in at least three independent experiments (described further in the Methods section).

Figure 4. Determination of the probable SphR binding site from SphR-regulated promoters.

(A) Fold-induction of ß-galactosidase activity from each reporter and the truncations compared to a pyruvate non-induced control (not shown). Promoter deletion mapping demonstrated a minimal region required for each SphR-controlled transcript. The negative numbers below each panel refer to the position relative to the translational start of each gene. (B) KALIGN showing the nucleotide alignment of the conserved region from the minimal regulatory regions defined in (A). The black bars denote the two direct repeat half-sites typical of AraC-family transcription factors. Below the alignments is the MEME-generated logo showing the strength of conservation based on the six half-sites from these three promoters. (C) ß-galactosidase assay for wt sphA-lacZ and the sphA-lacZ** promoter mutant (TG at the beginning of the first half-site was changed to AA) to demonstrate the importance of the conserved region for induction. Statistical significance determined using one way ANOVA with Dunnett's post-test with the uninduced pyruvate condition being the comparator for all other data. p-value summaries: n.s. = not significant; **** for p<0.0001. All experiments were performed at least three times and data shown is representative of both the scale and statistical significance levels of all experiments.

Figure 5. SphR directly binds to its target regulatory regions and binding is stimulated by sphingosine.

The binding of MBP-SphR to target DNA was measured using electrophoretic mobility shift assays (EMSAs). (A) Sphingosine (sph) is required for robust MBP-SphR binding to target DNA and sphingosine stimulates SphR DNA binding in a dose-dependent manner. Based on this data all subsequent EMSAs were conducted with 10 µM sphingosine. (B) MBP-SphR (SphR) binds in a dose-dependent manner to sphA, sphB, and cerN upstream regulatory regions, but not to the sphR-independent plcH regulatory region. The MBP-SphR concentration (µM) is shown below the lanes. Specificity is shown by lack of MBP-SphR binding to the plcH promoter and by the ability to compete the bulk of the shift with unlabelled competitor DNA (UC) denoted by the + sign under the lanes. (C) MBP-SphR binds a 59-bp oligonucleotide probe containing the predicted SphR binding site upstream of sphA. The extra shifts seen in panels A & B are absent from the EMSAs with these minimal probes and likely are due to additional interaction sites. Mutation of two conserved residues in half-site #1 (TG to AA) (sphA**) results in substantial reduction in MBP-SphR binding. All experiments were performed at least three times and data shown is representative of all experiments.

Figure 6. SphR directly binds to sphingosine.

The association of MBP-SphR was determined by measuring binding of ³H-sphingosine and reporting counts per minute (CPM). Sphingosine minimally associates with amylose beads alone (beads) or an un-related AraC-family transcription factor (MBP-CdhR), but approximately 11-fold more ³H-sphingosine binds to MBP-SphR (p<0.0001). Statistical significance determined using one way ANOVA with Dunnett's post-test with the beads alone condition being the comparator for all other data. p-value summaries: n.s. = not significant; **** for p<0.0001. This experiment was performed at least three times and data shown is representative of both the scale and statistical significance levels of all experiments.

Figure 7. Deletion of the SphR-regulon member, sphA, reduces P. aeruginosa survival in the mouse lung.

Male C57Bl/6J mice were infected with 2×10⁷ CFU/mouse of each strain via oropharyngeal aspiration. Mice were euthanized and lungs harvested 24 hours after infection. Bacterial counts were determined by serial dilution onto PIA. Deletion of sphA (ΔsphA) reduced P. aeruginosa survival 9-fold (p<0.001). Deletion of the other SphR-regulon members cerN and sphC (as an sphBCD operon representative) resulted in no survival defect. Statistical analysis determined using one way ANOVA with Dunnett's post-test with wild type as the comparator. Data shown is combined from two experiments. The same effect sizes and variance have been seen in at least one additional experiment for each strain, resulting in each strain having been compared to wild type in at least three independent experiments (described further in the Methods section).

Figure 8. Deletion of sphA or sphR renders P. aeruginosa susceptible to killing by sphingosine.

Strains with deletions in sphA (red), sphR (black), and sphC (green) were compared to wild type (blue) using a sphingosine killing assay in neopeptone as described in the Methods section. Cells were exposed to vehicle alone (0) or one of two concentrations of sphingosine for 30 minutes followed by serial dilution and plating. The mean of the vehicle treatment samples for each strain was set as 100% survival for that strain. Statistical significance determined using one way ANOVA with Dunnett's post-test with wild type at each concentration being the comparator for the mutant strain data at the same concentration. p-value summaries: n.s. = not significant; ** for p<0.01; *** for p<0.001; **** for p<0.0001. This experiment was performed more than three times and data shown is representative of both the scale and statistical significance levels of all experiments.