Defining the core essential genome of Pseudomonas aeruginosa

Abstract

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains of Pseudomonas aeruginosa on five different media and developed a statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain-medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient in P. aeruginosa to converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

Keywords: ESKAPE; Tn-Seq; antibiotic discovery; gram-negative pathogens.

Fig. 1.

Tn-Seq of P. aeruginosa clinical isolates. (A) Phylogenetic dendrogram of 2,560 P. aeruginosa genomes (NCBI Genome database); PAO1 and strains selected for mutagenesis are indicated. (B) Variable sequencing reads that map to TA sites in an exemplary region of five genes in strain PA14 (including hemL, thiE, and thiD) under different growth conditions highlight the conditional essentiality of these genes. The gene arrow indicates gene direction from 5′ to 3′. (C) Normalized read counts mapping to the pilY1 gene (3,477 bp) in all nine strains in LB medium demonstrate the variable essentiality of pilY1 in different strains as an example of the genomic heterogeneity of P. aeruginosa isolates. Genes are shown 5′ (left) to 3′ (right); a black tick on the x axis indicates the TA insertion site location within the gene.

Fig. 2.

Validation of FiTnEss predictions on a set of conditionally essential gene deletions. (A) FiTnEss prediction for ilvC in urine. The gray histogram is the actual distribution of Tn-Seq reads for all genes with 18 insertion sites in PA14 grown in urine, the black line is the theoretical distribution calculated by FiTnEss, and the red line is where ilvC falls at the far left (i.e., essential) of the bimodal distribution. (Inset) Read numbers at useable (red) and removed (blue) TA sites are shown; a black tick on the x axis indicates the TA insertion site location within the gene, and the gene is presented 5′ (left) to 3′ (right). (B) FiTnEss essentiality predictions (Left; nonessential, high-stringency essential, and maximal stringency essential predictions are displayed as dark green circles, light green circles, and blank spaces, respectively) of five representative gene deletion mutants from PA14; actual mutant growth mirrored predicted growth on five media (Right). The red box identifies the absence of growth of ΔilvC (the deletion mutant highlighted in A), thus experimentally confirming its essentiality on urine. The full growth profiles of 23 gene deletions can be found in SI Appendix, Fig. S4. (C) Summary of FiTnEss performance based on actual deletion mutant growth profiles. Gene-medium instances are indicated in parentheses; and red and green boxes highlight false-positive and false-negative rates, respectively.

Fig. 3.

Core essential genome definition plateau. (A) Total of 10,000 random calculations (gray) of the trajectory of the number of core essential genes in all five media tested, determined upon the sequential introduction of additional strains, up to a total of nine strains. The 10th and 90th percentiles (black lines) and mean (red line) core essential genome sizes, as determined by the 10,000 calculations, are highlighted. (B) False-positive rate of core essential genes upon the introduction of strains as calculated in A. The dashed line represents a 5% false-positive rate, with the median (blue) number of strains to cross this threshold being four, and the 10th (red) and 90th (green) percentiles crossing the threshold at three and five strains, respectively.

Fig. 4.

Core and conditionally essential gene functions in P. aeruginosa. (A) Chord diagram of the 321 core essential genes showing the relationship between subcellular location (bottom) and general function (top), with the number of genes for each category indicated. (B) Venn diagram showing the number of essential genes in all strains across three infection-relevant media.

Fig. 5.

In vivo murine infection with gene deletion mutants of conditionally essential genes identified by FiTnEss. (A) Bacterial burden in the spleen of neutropenic mice infected i.v. with wild-type (WT) PA14 and ΔpyrC, ΔpurH, ΔtpiA, ΔargG, and ΔthiC deletion mutants; statistical significance was determined with a Kruskal–Wallis test (n = 9). (B) Growth of WT PA14 and the ΔargG deletion mutant in human, fetal bovine, and mouse sera. (C) Bacterial burden in the lungs from mice infected intranasally with PA14 or the ΔthiC deletion mutant; statistical significance was determined with a Mann–Whitney U test (n = 15). (A and C) Bacterial burden determined at 16 h postinfection. Each dot represents a single mouse, with a line indicating the median; significance is displayed at ***P < 0.001 or **P < 0.01; the dashed line indicates the limit of detection of the assay; and data are a combination of two biological replicates.