Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

Abstract

Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureusin vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner.IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

Keywords: HQNO; Nanostring; PQS; Pseudomonas aeruginosa; Staphylococcus aureus; alginate; cystic fibrosis; mucoid; polymicrobial; pyochelin; pyoverdine; siderophores.

FIG 1

Exogenous alginate protects S. aureus JE2 from P. aeruginosa PAO1 in coculture. (A and B) S. aureus (Sa) JE2 (A) and P. aeruginosa (Pa) PAO1 (B) growth curves over 10 h in liquid coculture in TSB with or without 1% seaweed-derived or 1% P. aeruginosa PAO1-derived alginate. CFU per milliliter were enumerated at the indicated time points and log₁₀ transformed. The dashed line at 2 log₁₀ CFU/ml indicates the limit of detection. (C and D) Biofilm (C) and planktonic (D) S. aureus JE2 growth after 16 h of static coculture in 1/2× MEM plus l-Gln and l-Arg with or without 1% seaweed-derived alginate. CFU per milliliter were enumerated and log₁₀ transformed. Significance was determined by one-way ANOVA with Dunnett’s posttest. a, P < 0.05 with S. aureus JE2 as the reference; b, P < 0.0001 with P. aeruginosa PAO1 plus S. aureus JE2 as the reference; bd, below detection.

FIG 2

Culture with mucoid P. aeruginosa PAO1 mucA22 delays S. aureus JE2 killing by wild-type P. aeruginosa PAO1. Shown are data for biofilm triculture on plastic with S. aureus JE2, P. aeruginosa PAO1, and P. aeruginosa PAO1 mucA22 in MEM plus l-Gln and l-Arg. CFU per milliliter were enumerated on mannitol salt agar (MSA) and log₁₀ transformed. S. aureus JE2 viability in the biofilm (A) and planktonic (B) fractions after 12 h and S. aureus JE2 viability in the biofilm (C) and planktonic (D) fractions after 16 h of coculture were determined. Significance was determined by one-way ANOVA with Dunnett’s posttest. a, P < 0.05 with S. aureus JE2 as the reference; b, P < 0.05 with P. aeruginosa PAO1 plus S. aureus JE2 as the reference; *, P < 0.05; ns, not significant.

FIG 3

Alginate synthesis is not required for S. aureus protection. S. aureus JE2 was cocultured in 1/2× MEM plus l-Gln and l-Arg on plastic with P. aeruginosa PAO1 or P. aeruginosa PAO1 algD::FRT with or without 1% seaweed-derived alginate. (A and B) S. aureus JE2 viability in the biofilm (A) and planktonic (B) fractions after 16 h. (C and D) P. aeruginosa PAO1 viability in the biofilm (C) and planktonic (D) fractions after 16 h. Significance was determined by one-way ANOVA with Dunnett’s posttest. a, P < 0.05 with S. aureus JE2 as the reference; b, P < 0.05 with P. aeruginosa PAO1 plus S. aureus JE2 as the reference; bd, below detection.

FIG 4

Exogenous alginate decreases P. aeruginosa PAO1 siderophore production. (A and B) P. aeruginosa PAO1 was grown on plastic in 1/2× MEM plus l-Gln and l-Arg with or without 1% alginate for 16 h at 37°C with 5% CO₂. Supernatants were collected from the planktonic fraction. (A) Pyoverdine was quantified by measuring RFU of the supernatants at 400-nm excitation and 460-nm emission wavelengths and normalizing the values to CFU per milliliter of the planktonic fraction. (B) Supernatants were diluted 1/2-fold in DI water or 2% alginate (for a final concentration of 1% alginate) and incubated statically at 37°C with 5% CO₂ for 5 min or 24 h. Pyoverdine was quantified by measuring RFU of the supernatants at 400-nm excitation and 460-nm emission wavelengths. Significance was determined by a paired t test. (C and D) P. aeruginosa PAO1 was grown in 25 ml TSB with shaking for 8 h. (C) pvdA expression was quantified by qRT-PCR, and the ΔΔC_T value was calculated relative to P. aeruginosa PAO1 rpoD expression. (D) Pyochelin was quantified by LC-MS/MS as described in Materials and Methods. Significance determined by one-way ANOVA with Dunnett’s posttest comparison to P. aeruginosa PAO1 for each experiment unless otherwise indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (for all statistical tests).

FIG 5

Rhamnolipid production is posttranscriptionally altered by exogenous alginate. P. aeruginosa PAO1 was grown in TSB liquid culture for 8 h with or without 1% alginate. Supernatants were collected by centrifugation to remove cell debris and sterile filtering. Cell pellets were snap-frozen for subsequent expression analyses. (A) Representative drop collapse assay image. P. aeruginosa PAO1 supernatants were prepared from mucoid and nonmucoid P. aeruginosa PAO1 strains and serially diluted 1:2 in PBS. Surfactant activity was assessed by placing a 20-μl droplet of each supernatant dilution on plastic, placing the droplet at a 90° angle for 10 s, and assessing migration (PBS was supplemented with 0.01% crystal violet to aid visualization). Surfactant activity was quantified as the reciprocal of the highest dilution at which the drop migrates. (B) Rhamnolipid production by P. aeruginosa PAO1 quantified by drop collapse and normalized to CFU per milliliter to determine the surfactant score. (C and D) Rhamnolipid quantification by LC-MS/MS for total rhamnolipids (C) and mono- and dirhamnolipids (D). (E) Expression of the rhlA gene was quantified by qRT-PCR, and the ΔΔC_T value was calculated relative to P. aeruginosa PAO1 rpoD. Significance was determined for each experiment by one-way ANOVA with Dunnett’s posttest comparison to P. aeruginosa PAO1. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

Exogenous alginate downregulates PQS quorum sensing. (A) The PQS quorum sensing regulon. PQS synthesis is dependent on the pqsABCD genes located in the pqs operon. Both HHQ and HQNO have direct antimicrobial properties, while PQS is the ligand for PqsR/MvfR. When the PQS level is high, this ligand interacts with PqsR/MvfR to positively regulate many downstream virulence factors. PQS has a direct iron-chelating function and promotes the expression of siderophore-encoding genes. The downstream effects listed here focus on effects relevant to this study and are not exhaustive. (B to F) LC-MS/MS was used to quantify HQNO (B), HHQ (C), PQS (D), PCA (E), and pyocyanin (F) produced by P. aeruginosa. Panel B includes the key for all graphs in this figure. Significance was determined for each experiment by one-way ANOVA with Dunnett’s posttest comparison to P. aeruginosa PAO1. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 7

Alginate alters the expression of P. aeruginosa genes essential for S. aureus killing. (A to D) Raw Nanostring counts were normalized to values for the positive controls and three housekeeping genes (rpoD, ppiD, and fbp) and log₂ transformed. Three biological replicates were performed under each condition. Significantly differentially expressed genes were determined by an unpaired t test followed by the two-stage linear step-up procedure of Benjamini et al. (57) (with a q value of 1% for false discovery) and are marked by blue squares and labeled with the gene name. Genes significantly differentially expressed by mucoid P. aeruginosa in a previous study (32) but not by P. aeruginosa in the presence of exogenous alginate are marked by red triangles. (A and B) P. aeruginosa PAO1 (A) and P. aeruginosa PA14 (B) subcultured into TSB or TSB plus 1% alginate during the mid-log growth phase for 45 min. For clarity, only significantly downregulated genes are labeled in panel B. (C and D) P. aeruginosa PA14 monocultured with 0.25% alginate for 8 h (C) and cocultured with S. aureus JE2 in TSB with 0.25% alginate for 8 h (D). (E and F) S. aureus JE2 survival in the biofilm (E) and planktonic (F) fractions after 16 h of coculture with the indicated P. aeruginosa PA14 mutants corresponding to genes downregulated in one or more Nanostring experiments. Significance was determined by one-way ANOVA with Dunnett’s posttest comparison to S. aureus plus P. aeruginosa PA14. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (G and H) S. aureus JE2 survival in the biofilm (G) and planktonic (H) fractions after 16 h of coculture with P. aeruginosa PA14 motility mutants. Significance was determined by one-way ANOVA with Dunnett’s posttest. a, P < 0.05 with S. aureus JE2 as the reference; b, P < 0.05 with P. aeruginosa PAO1 plus S. aureus JE2 as the reference. WT, wild type.

FIG 8

Mucoid clinical isolates have various effects on S. aureus in coculture. (A) S. aureus JE2 viable counts after 16 and 24 h of coculture in flasks with shaking at 225 rpm in TSB with P. aeruginosa clinical isolates. nm, nonmucoid. (B and C) Log₂ transformation of Nanostring counts normalized to values for positive controls and three housekeeping genes (rpoD, ppiD, and fbp) for the indicated transcripts for the clinical isolate P. aeruginosa CFBRPA32 (a “mucoid killer”), mucoid laboratory strain P. aeruginosa PAO1 mucA22, and wild-type laboratory strain P. aeruginosa PAO1 after 24 h of culture in flasks with shaking at 225 rpm in TSB. Data are from two biological replicates per strain. Gene expression was analyzed by two-way ANOVA followed by Tukey’s multiple-comparison test. (B) Heat map and dendrogram of all genes significantly differentially regulated between any two strains. Expression values are displayed as within-row z-scores. Yellow indicates mucoid strains, and gray indicates nonmucoid strains. (C) All genes significantly differentially regulated between P. aeruginosa CFBRPA32 and P. aeruginosa PAO1 mucA22. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.