Surviving the host: Microbial metabolic genes required for growth of Pseudomonas aeruginosa in physiologically-relevant conditions

Abstract

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B₁₂) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.

Keywords: Pseudomonas aeruginosa; host-like media; human skin organoid model; in vivo survival; murine abscess model.

Figure 1

Genes predicted as conditionally essential for growth in murine abscess and human skin models and in physiologically-relevant media (RPMI and or RPMI/Serum) cf. MHB as determined using Tn-Seq. (A) Venn diagrams showing essential genes that are unique or shared between particular host-like conditions (RPMI or RPMI/Serum: pink; human skin organoid model: amber; murine abscess model: blue), as determined by either Tradis or Transit cf. MHB. (B) Functional classes of essential genes of Pseudomonas aeruginosa grown in vitro (in RPMI and/or RPMI/Serum), murine abscesses, or human skin cf. MHB.

Figure 2

Nucleotide metabolism genes were predicted as conditionally essential, enriched and differentially expressed in RPMI cf. MHB. Conditionally essential genes in RPMI and/or RPMI/serum were integrated with RNA-Seq from P. aeruginosa grown in RPMI, and mapped to PPI networks in P. aeruginosa. A network of enriched gene ontology (GO) terms for nucleotide metabolism and biosynthesis is visualized here. Genes indicated with a square were essential in RPMI and/or RPMI/serum but not MHB. Those genes that were also essential in abscesses cf. MHB and/or the human skin model cf. MHB are indicated with orange arrows.

Figure 3

Cobalamin metabolism genes were predicted as conditionally essential, enriched and differentially expressed in RPMI cf. MHB and identified as conditionally essential for growth in physiologically-relevant conditions. Conditionally essential genes in RPMI and/or RPMI/serum were integrated with RNA-Seq from P. aeruginosa grown in RPMI cf. MHB, and mapped to PPI networks in P. aeruginosa. A network of enriched GO terms for cobalamin metabolism and biosynthesis is visualized here. Those genes that were also essential in abscesses cf. MHB and/or the human skin model cf. MHB are indicated with orange arrows.

Figure 4

Growth defects of Tn-insertion mutants from the ordered PAO1 Tn mutant library in genes identified as important for survival in physiologically-relevant media and in murine abscess or the human skin model. Mutants harbouring Tn5 IS50L derivative Tn insertions ISlacZ/hah or ISphoA/hah (indicated as Tn5ISlacZ or Tn5ISphoA) were grown in both RPMI/Serum and MHB for 16 h in 96-well plates and OD₆₀₀ was measured every 30 min. Growth curves in RPMI/serum for mutants that showed a growth defect in RPMI/serum but not in MHB (indicated by the green boxes on the left) are shown on the right and compared to WT PAO1 grown in the same media.

Figure 5

Deficiencies of mutants identified as important for growth in RPMI/Serum, murine abscess and/or a human skin model. (A) Growth defect compared to WT when grown in RPMI/Serum. *p < 0.05, **p < 0.01, ***p < 0.001 indicates significantly different from WT using two-way ANOVA. (B) Survival and (C) abscess formation in the murine abscess model. *p < 0.05, **p < 0.01, ***p < 0.001 indicates significantly different from WT using one-way ANOVA. (D) Competitive fitness of mutants in the human skin organoid model, cf. WT, measured as competitive index after inoculation with equal numbers of WT and mutant on the skin. *p < 0.05, **p < 0.001 indicated significantly different than 1 using 1-sample t-test.