Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosacan be achieved without iron provision by maintaining local phosphate abundance: role of pH

Bacterial strains

Studies were performed with P. aeruginosa PAO1 strains obtained from two laboratories, MPAO1 (B. Iglewski, the original strain used to create the transposon mutant library at the University of Washington), and CorPAO1 (P. Cornelis), as well as with the CorPAO1 derivative mutant ΔPvdD/ΔPchEF.

Mouse model of lethal gut-derived sepsis

Animal experiments were approved by the Animal Care and Use Committee at the University of Chicago (IACUC protocol 71744). Male C57BL6/HSD mice weighing 18 to 22 g were used for all experiments. Gut-derived sepsis was modeled by performing a 30% surgical left lateral hepatectomy with simultaneous injection of 10⁷ CFU P. aeruginosa into cecum of mice pre-fasted 18 hours prior to surgery as previously described [16]. Mice were allowed access to either tap water, or 25 mM potassium phosphate-buffer (PB) pH 7.5, or 25 mM PB pH 6.0 through over the course of the experimental period.

Measurement of intestinal mucosal pH

Intestinal mucosa (overlying mucus and intestinal epithelial cells) pH was measured with phenol red. Following 24 hrs after surgery, mice were sacrificed, and distal intestine of mice was harvested from rectum to jejunum, gently washed with water to remove loose luminal contents and then stained by flashing 5 times with 0.4% phenol red in buffer (0.145 M NaCl, 0.002 M KH₂PO₄, 0.003 M Na₂HPO₄). The intestine was opened longitudinally and mucosal pH measured semi-quantitatively using pH standards stained with phenol red.

C. elegansmodel

C. elegans killing assays were performed as we previously reported [9] with modifications. Briefly, P. aeruginosa PAO1 grown on solid TSB was collected and suspended in either 25 mM potassium phosphate buffer (PB), pH 6.0 or PB pH 7.5 to a 30 μl volume that was poured on NGM agarized media (peptone, 2.5 g/L; NaCl, 3 g/L; MgSO₄, 1 mM; CaCl₂, 1 mM; agar 17 g/L) supplemented with 25 mM PB pH 6.0 or pH 7.5, respectively. PAO1 lawns were grown during 24 hrs at 37°C following overnight incubation at room temperature, and then were used for feeding C. elegans. As a control of phosphate limitation, P. aeruginosa PAO1 lawns were prepared on NGM containing 0.1 mM PB, pH6.0. Pre-fasted worms were transferred onto lawns and mortality followed for up to 60 hrs.

Genome-wide transcriptional analysis

All samples for gene expression analysis were prepared in triplicate. P. aeruginosa MPAO1 cells collected from lawns grown on NGM/[Pi]25 mM, pH 6.0 or NGM/[Pi]25, pH 7.5 were used for RNA isolation as previously described. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility and data were analyzed as previously described [9]. Microarray data were deposited in GEO database, accession number GSE29789.

QRT-PCR analysis

Multiplex qRT-PCR was performed to simultaneously analyze the expression of selected genes in P. aeruginosa MPAO1 grown under pH 6.0 and pH 7.5 in NGM-Pi 25 mM. Gene clusters for the analysis were chosen as representatives of phosphate signaling and acquisition, quorum sensing, and iron acquisition. Overnight P. aeruginosa MPAO1 culture was diluted 1:50 in triplicate in 25 mM phosphate NGM media at pH 6.0 and 7.5, and grown for 9 hrs at 37°C. RNA was isolated and reversed to cDNA as previously described [7]. QRT-PCR analysis was performed as previously described [9]. Briefly, gene specific primers (Tm = 60°C) to amplify 100 bp fragments of target mRNA were designed based on in silica analysis for amplification specificity by BLAST search against the database of P. aeruginosa PAO1 genome. Gene expression was normalized to tpiA (PA4748) whose expression was not influenced by pH in microarray analysis, and which was used in our previous QRT-PCR analyses [9]. Fold changes of expression levels were determined by normalization to expression at pH 6.0.

Pyoverdin assay

Pyoverdin production was measured by fluorescence at 400 ± 10/460 ± 10 excitation/emission, and measurements of relative fluorescence units (RFU) were normalized to cell density units as absorbance at 600 nm in bacterial cultures growing in black, clear bottom 96-well plates (Corning Incorporated, Corning, NY, Costar 3603) using a 96-well Microplate Fluorimeter Plate Reader (Synergy HT, Biotek Inc., Winooski, VT). In the experiments with iron supplementation, pyoverdin was measured in supernatants by absorbance at 405 nm as previously described [17], and normalized to initial cell density.

Iron concentration assay

Iron was measured using a Roche/Hitachi MOD P automated clinical chemistry analyzer using the FerroZine method (the minimum detection limit is 0.1 μg/ml). Results were reproduced in 3 biological replicates.

Bioinformatics

Microarray data were analyzed using gene annotations provided by the SEED database http://www.theSEED.org/ and Pseudomonas Genome Database http://www.pseudomonas.com/.

Statistical analysis

Statistical analysis of the data was performed with Student t-test using Sigma plot software, and Kaplan-Maier survival graphs using SPSS 18 software.

Surgical injury (30% hepatectomy) increases the distal intestinal mucosal pH that can be maintained by pH adjusted oral phosphate supplementation

In order to determine whether the pH of the intestinal mucosa, the major colonization site of microbial pathogens, is affected by surgical injury, mucosal pH was measured using phenol red staining of intestinal segments of control and surgically injured mice. The pH of proximal colon segments, the densest region of microbial adherence, was measured in mice 22 hours following sham laparotomy or 30% hepatectomy. Results demonstrated pH shift from ~6.0 in sham mice to ~ 7.0-7.5 in mice subjected to 30% hepatectomy (Figure 1A). In mice drinking an oral ad libitum solution of 25 mM phosphate buffer adjusted to pH 6.0 or 7.5, intestinal mucosal pH in the proximal colon stabilized to the corresponding pH suggesting that, in mice, distal intestinal pH can be manipulated by oral pH adjustment (Figure 1B).

Oral phosphate protects against the lethal effect of intestinal P. aeruginosafollowing surgical injury in a pH dependent manner

We next determined the effect of pH on the expression of a lethal phenotype in intestinal P. aeruginosa using a model developed by our laboratory [16, 18]. In this model, mice are subjected to an otherwise fully recoverable surgical injury (30% hepatectomy) with simultaneous injection of P. aeruginosa into the cecum which consistently results in > 60% mortality in 48 hr. In the present study, to generate negative controls, groups of mice were subjected to hepatectomy without injection of P. aeruginosa and drank either water, or 25 mM [Pi], pH 6.0, or 25 mM [Pi], pH 7.5 ad libitum (n = 16/group). No mice in any of these groups developed signs of sepsis or mortality at 48 hours and appeared completely healthy. In contrast, and consistent with our previous studies in this model [7–9], mice drinking water ad libitum and intestinally inoculated with P. aeruginosa PAO1 following surgical hepatectomy developed gross signs of sepsis (chromodacctyrrhea, ruffled fur, lethary, scant diarrhea) and a ~60% mortality rate at 48 hours. Mortality in mice intestinally inoculated with P. aeruginosa PAO1 following 30% hepatectomy and drinking 25 mM [Pi], pH 7.5 ad libitum was significantly attenuated (from 60% to 30%) with an even further mortality attenuation down to ~ 10% when mice drank 25 mM [Pi], pH 6.0 (Figure 2A).

In order to define the effect of pH on the lethality of P. aeruginosa, we used a more ordered host model system of C. elegans where worms feed on P. aeruginosa lawns grown at varying levels of phosphate and pH. Briefly, nematodes fed on P. aeruginosa lawns grown on agarized Nematode Growth Media (NGM) in which 25 mM potassium-phosphate buffer was adjusted to pH 6.0 or pH 7.5. Suspension of P. aeruginosa PAO1 to create the bacterial lawns was also prepared in 25 mM [Pi] at pH6.0 or 7.5 respectively to maintain consistency throughout the experimental period. As positive controls, parallel experiments were performed where worms fed on lawns of P. aeruginosa grown on low phosphate medium (0.1 mM) similar to our previously published experiments [9]. Results demonstrated that the killing effect of P. aeruginosa against C. elegans at high phosphate concentration was enhanced at pH 7.5 compared to 6.0 (Figure 2B). Importantly, low phosphate conditions induced the highest lethality rate consistent with our previous findings and demonstrated that extracellular phosphate is a major cue that activates virulence [9]. Previous work from our laboratory demonstrated that red material accumulated in the digestive tube of dying of C. elegans worms feeding on P. aeruginosa at low phosphate that consisted of the P. aeruginosa virulence-related quinolone signal PQS complexed with iron (PQS-Fe ³⁺). This complex was determined to be toxic to C. elegans especially when combined with rhamnolipids [9]. In the current study, the red material was not observed when C. elegans fed on P. aeruginosa PAO1 lawns grown at [Pi] 25 mM, pH 7.5 suggesting a lack of either PQS or pyoverdin production. The observation of yellow-green coloration suggested however that pyoverdin was produced at [Pi] 25 mM pH 7.5. It is important to note that the adjustment of pH did not affect the intense green coloration under low phosphate conditions suggesting that phosphate limitation is still a major factor for green pigment production (Figure 2C). Furthermore, enhanced pyoverdin production under conditions of phosphate limitation was not affected if pH is stabilized using 25 mM HEPES, pH7.5 or 25 mM MOPS, pH 6.0 (Figure 2D).

A pH of 7.5 at high phosphate concentration (25 mM) induces the expression of iron starvation (IS) and ferrous uptake regulated (FUR) genes but not MvfR-PQS and results in expression of siderophore-mediated virulence in P. aeruginosa

While pyoverdin production is considered to be a quorum sensing related exoproduct of P. aeruginosa [19], our microarray results suggest that pH dependent expression of pyoverdin-related genes is not related to quorum sensing. To verify this, we dynamically measured P. aeruginosa PAO1 pyoverdin production during growth in liquid NGM media containing 25 mM [Pi] at pH 7.5 versus pH6.0. Results demonstrated that pyoverdin production was developed at 3 hrs of growth (Figure 3A) at 25 mM Pi, pH 7.5, and was partially suppressed by the addition of 100 μM Fe³⁺. Most notably, suppression of pyoverdin production at [Pi] 25 mM, pH 6.0 was significantly higher compared to that provided by iron supplementation at [Pi] 25 mM pH 7.5. The concentration of iron in both liquid media NGM Pi25 mM, pH 6.0 and NGM Pi25 mM, pH 7.5 was measured and found to be very low (< 0.1 μg/ml (< 1.78 μM)). Given that the concentration of iron needed to partially attenuate pyoverdin production in NGM Pi25 mM, pH 7.5 is as high as 100 μM (Figure 3A), we are confident that the pH, not the extracellular iron concentration, was a major factor leading to the triggering of pyoverdin production under conditions of similar extracellular iron concentration. Since iron binding to pyoverdin quenches its fluorescence, the pyoverdin production in these experiments was measured in supernatants by absorbance at 405 nm as previously described [17], and measurements were normalized to initial cell density. Results demonstrated that the expression of pyoverdin can be prevented without providing iron by maintaining local phosphate abundance at pH 6.0.

The effect of pH on pyoverdin production measured by fluorescence as previously described [9] was verified in the range of 4.0 to 8.5 (Figure 3B). Results demonstrated that the pyoverdin production is similar between pH4.0 and 6.0 (low level of pyoverdin), and between pH7.5 and 8.5 (high level of pyoverdin). We noticed however that the growth of P. aeruginosa at pH 4.0 was greatly delayed up to 4 hrs (Figure 3B, inserted panel). At this point, the pH of bacterial culture changed on its own from 4.0 to 5.5 and further changed to pH ~ 6.0 at 9 hrs. Bacteria significantly increased their growth rate at 9 hours. Alternatively, bacteria grew very well at pH 8.5, produced pyoverdin, and there was no change from the initial pH. This finding supports our hypothesis that P. aeruginosa can regulate its environmental pH to facilitate its colonization.

Next, we measured the expression of QS- and iron- related genes by qRT-PCR in P. aeruginosa PAO1 grown for 9 hrs in liquid NGM media at pH 7.5 versus 6.0. Gene expression was normalized to tpiA (PA4748) expression and then fold change was determined using expression of PAO1 measured in NGM at pH 6.0 as 100%. Results demonstrated increased expression of iron related genes and decreased expression of both quorum sensing and low phosphate- related genes at pH 7.5 versus 6.0 (Figure 3C). These data may confirm that pH-mediated expression of iron- regulated genes is not dependent on quorum sensing. However, we found significant down-regulation (10 fold) of the qscR gene encoding LuxR-type "orphan" receptor QscR, a potent QS repressor [20]. As down- regulation of qscR may modulate LasRI activity at the same level of lasR expression [21–23], we cannot completely exclude the role of quorum sensing in pyoverdin regulation. QscR shares affinity for lactone QS molecules with LasR and can form inactive heterodimers with LasR and RhlR monomers to negatively regulate QS. Therefore attenuation of QscR production could lead to LasRI-mediated expression of pyoverdin-related genes. Results from our microarray analysis performed on high cell density cells demonstrate that qscR was down-regulated (-1.55) while lasR (1.6 fold) was upregulated (GEO database, accession number GSE29789). Such subtle changes in the expression of transcriptional regulators LasR and QscR may have profound downstream effects and therefore we cannot reject or confirm a regulatory role of QS in pyoverdin production at pH 7.5.

Finally to confirm the critical role of siderophores on P. aeruginosa lethality induced at pH7.5, we performed reiterative experiments using the double mutant ΔPvdDΔPchEF in mice. Intestinal inoculation with ΔPvdDΔPchEF resulted in attenuated lethality in mice exposed to surgical injury suggesting that iron acquisition factors (i.e pyoverdin and pyochelin) play an important role in P. aeruginosa mortality when mice are orally supplemented with phosphate (Pi 25 mM) at pH 7.5 (Figure 3D).

P. aeruginosatends to alkalize medium at pH 6.0

Comparative analysis of iron-related subsystems during phosphate limitation and a pH shift from 6.0 to 7.5 reveals the significant protective effect of phosphate supplementation

We have previously shown that phosphate limitation induces three global virulence subsystems in P. aeruginosa PAO1 that include 1.) phosphate signaling/acquisition, 2.) MvfR-PQS of the core quorum sensing pathway and downstream regulated genes such as those involved in the biosynthesis of pyocyanin, and 3.) pyoverdin-related genes (Figure 4A, A') (Microarray data for phosphate limitation are deposited in GEO database, GEO accession number GSE30967). The upregulation of pyoverdin by phosphate limitation was surprising given that the expression of pyoverdin genes is regulated by the transcriptional regulator PvdS that by itself is part of the FUR regulon, and as such the expression of PvdS and its regulated genes strongly depends on iron concentration. One would assume that there is going to be more iron available at lower concentrations of phosphate since phosphate causes precipitation of iron, thereby decreasing its effective concentration. Indeed, the absence of activation of FUR-regulated genes (normally suppressed at high concentration of iron) suggested that iron was available for P. aeruginosa (Figure 4A) indicating that the response of P. aeruginosa at differing levels of Pi is not simply a matter of the interaction of iron and phosphate, but rather involves more complex yet- to- be elucidated mechanisms. Alternatively, the expression of pyoverdin genes and FUR regulon in high phosphate media at pH 7.5 (Figure 4B) demonstrated that P. aeruginosa was exposed to iron limiting conditions. Comparison of the signature of iron related genes during pH shift to 7.5 to that induced by iron limitation as reported by Ochsner et. al. [33] (Figure 4C) confirmed that P. aeruginosa experiences iron limitation at pH 7.5. Importantly, providing phosphate at pH 6.0 suppressed the expression of iron-related genes indicating a significant protective effect of phosphate supplementation at pH6.0.