Dual transcriptional analysis reveals adaptation of host and pathogen to intracellular survival of Pseudomonas aeruginosa associated with urinary tract infection

Abstract

Long-term survival of bacterial pathogens during persistent bacterial infections can be associated with antibiotic treatment failure and poses a serious public health problem. Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa, which can cause both acute and chronic infections, are particularly challenging due to its high intrinsic resistance to antibiotics. The ineffectiveness of antibiotics is exacerbated when bacteria reside intracellularly within host cells where they can adopt a drug tolerant state. While the early steps of adherence and entry of P. aeruginosa into mammalian cells have been described, the subsequent fate of internalized bacteria, as well as host and bacterial molecular pathways facilitating bacterial long-term survival, are not well defined. In particular, long-term survival within bladder epithelial cells has not been demonstrated and this may have important implications for the understanding and treatment of UTIs caused by P. aeruginosa. Here, we demonstrate and characterize the intracellular survival of wild type (WT) P. aeruginosa inside bladder epithelial cells and a mutant with a disruption in the bacterial two-component regulator AlgR that is unable to survive intracellularly. Using simultaneous dual RNA-seq transcriptional profiling, we define the transcriptional response of intracellular bacteria and their corresponding invaded host cells. The bacterial transcriptional response demonstrates that WT bacteria rapidly adapt to the stress encountered in the intracellular environment in contrast to ΔalgR bacteria. Analysis of the host transcriptional response to invasion suggests that the NF-κB signaling pathway, previously shown to be required for extracellular bacterial clearance, is paradoxically also required for intracellular bacterial survival. Lastly, we demonstrate that intracellular survival is important for pathogenesis of P. aeruginosa in vivo using a model of murine urinary tract infection. We propose that the unappreciated ability of P. aeruginosa to survive intracellularly may play an important role in contributing to the chronicity and recurrence of P. aeruginosa in urinary tract infections.

Fig 1. Pseudomonas aeruginosa survives intracellularly.

A gentamicin protection assay was used to quantify intracellular bacterial burden. (A) Cells were infected with WT PAO1 at MOI = 1 or 10. (B) Host cell survival at 3 and 24 hpi with WT bacteria at MOI = 1 or 10 relative to uninfected cells harvested at the same time point. (C) Cells were infected at MOI = 1 with two clinical strains (BWH005 and BWH039) which were isolated from urine. (D) Cells were infected at MOI = 10 for 2 hours and incubated in the absence, to measure adherent bacteria, or presence of gentamicin, to measure invading bacteria, and harvested at 3 hpi. (E-G) Cells were infected with WT GFP-expressing bacteria at MOI = 10, fixed at the indicated time points and stained with anti-LAMP-1 antibodies and DAPI. (E) Representative images of invaded cells. Maximum intensity projections of XY (middle), and orthogonal XZ (gray outline) and YZ (orange outline) projections of the serial confocal images are shown. Scale bar = 5μm. (F) Intracellular bacterial load as determined by microscopy. (G) Percent of invaded cells with LAMP-1-associated bacteria. Data is representative of 4–6 biological replicates with at least 50 cells per condition. Error bars represent standard deviation. Data were analyzed by one-way ANOVA (p = 0.02).

Fig 2. The bacterial two-component response regulator AlgR is required for intracellular survival of Pseudomonas aeruginosa.

(A) Cells were infected with increasing MOIs of ΔalgR bacteria and intracellular bacterial load was determined. (B) Percent of recovered bacteria at 48 hpi relative to 3 hpi with MOI = 10. Each data point represents an independent experiment. Data were analyzed by unpaired T-test. (C) Host cell survival was measured at 3 and 24 hpi with WT or ΔalgR bacteria at MOI = 10 relative to uninfected cells harvested at the same time point. (D) algR was complemented in the ΔalgR deletion strain and cells were infected and harvested as in A. (E-F) Cells were infected with ΔalgR GFP-expressing bacteria at MOI = 10, fixed at the indicated time points and stained with anti-LAMP-1 antibodies and DAPI. (E) Representative images of invaded cells. Maximum intensity projections of XY (middle), and orthogonal XZ (gray outline) and YZ (orange outline) projections of the serial confocal images are shown. Scale bar = 5μm. (F) Intracellular bacterial load as determined by microscopy. (G) Percent of invaded cells with LAMP-1-associated bacteria. Data is representative of 4–6 biological replicates with at least 50 cells per condition. Error bars represent standard deviation.

Fig 3. Phosphorylation-independent and -dependent roles of AlgR in intracellular survival and host cell invasion.

Cells were infected at MOI = 10 with the kinase deficient strain ΔalgZ, the phosphomimetic D54E or non-phosphorylatable D54N single point algR mutants, or the pilus deficient strain ΔpilA. (A, C, E) At indicated time points, cells were lysed and the intracellular bacterial load was determined. (B, D, F) Percent of recovered bacteria at 48 hpi relative to 3 hpi. Each data point represents an independent experiment. *p<0.05 **p<0.005 based on one-way ANOVA.

Fig 4. Bacterial adaptation to the host intracellular environment is rapid and is impaired in ΔalgR bacteria.

Dual RNA-seq libraries of WT- and ΔalgR-invaded cells were made from cells sorted 2 hpi and enriched using P. aeruginosa-specific probes using PatH-Cap. (A) Log₂ fold change in gene expression of intracellular versus planktonic WT bacteria. Differentially expressed genes (padj<0.01 for WT, 825 genes, dark gray) and those differentially expressed in both comparisons encoding genes belonging to pyoverdine biosynthesis (13, green), T3S secretion (28, red), arginine degradation (6, blue), and LPS biosynthesis (8, pink) GO terms are highlighted. (B) Log₂ fold change in gene expression of intracellular P. aeruginosa WT versus ΔalgR bacteria. Genes highlighted in blue (7) are expressed at higher levels by intracellular WT bacteria; genes highlighted in red (17) are expressed at higher levels by intracellular ΔalgR bacteria (padj<0.05). Unlabeled points (10) represented uncharacterized genes that encode hypothetical proteins. (C) Percent of reads from dual RNAseq pre-PatH-Cap libraries that align to P. aeruginosa genome. (D) Composition of bacterial fraction of dual RNA-seq pre-PatH-Cap libraries.

Fig 5. Activation of the host NF-ΚB signaling pathway is correlated with bacterial survival rather than clearance.

“Unexposed”, “exposed” but uninfected GFP negative and “invaded” GFP positive cells were infected with WT or ΔalgR PAO1 GFP-expressing bacteria and sorted 2 hpi. (A) Differential expression analysis of WT exposed and invaded cells. Differentially expressed genes (padj < 0.01) highlighted in blue; top 10 genes labeled. (B) Gene expression profiles of WT- and ΔalgR-invaded cells were compared to unexposed cells. Normalized enrichment score (NES) for gene sets significantly enriched (FDR qval<0.005) in each comparison are shown. (C) Heat map showing genes differentially expressed between WT-invaded and ΔalgR-invaded cells. Genes belonging to the TNFα via NF-κB gene set are highlighted. (D) Cells were pre-treated for 30 min with GSK 319347A or Wedelolactone before infection with WT P. aeruginosa at MOI = 10. At indicated time points, the number of intracellular bacteria was determined. (E) Cells were pre-treated with TNFα blocking monoclonal antibodies or recombinant TNFα 1 hour before infection with WT or ΔalgR P. aeruginosa, respectively, at MOI = 10. At indicated time points, the number of intracellular bacteria was determined.

Fig 6. Bacterial intracellular survival increases in vivo persistence in a mouse model of urinary tract infection and decreases antibiotic efficacy.

(A-C) C57BL/6 female mice were transurethrally inoculated with 1–6 x 10⁷ CFU P. aeruginosa. (A) At 3 hours or 2 days post-infection, bladders were harvested and incubated for 30 minutes in media alone or gentamicin to kill extracellular bacteria; the entire bladder was then homogenized to determine total (untreated) and intracellular (gentamicin treated) bacterial load. n = 5 per time point per condition. (B) Mice were infected with the lab-adapted strain PAO1 or two clinical strains isolated from urine (BWH005 and BWH039). Seven days post-infection, bladders were harvested, homogenized and bacterial load was determined. n = 4 or 5 per strain. (C) Mice were infected with WT, ΔalgR or ΔpilA PAO1 P. aeruginosa. Two days post-infection, bladders were harvested and homogenized to determine bacterial load. Adjusted P value of Dunn’s multiple comparisons test between groups displayed. n = 10–15 mice from 3 independent experiments. (D) Bladder epithelial cells were infected with WT bacteria for 2 hours and then incubated with gentamicin plus the cell permeable antibiotic ciprofloxacin at either 10 or 25 μg/mL. At the indicated time points intracellular bacterial load was determined. For comparison, the same inoculum of bacteria was treated with 25 μg/mL ciprofloxacin without host cells and the number of surviving bacteria was determined. (E) Cells were infected with WT bacteria and treated as in D with ciprofloxacin (10 μg/mL) and the lysate was plated in the absence of presence of ciprofloxacin (10 μg/mL). Minimum level of detection was 5 CFU/well.