Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model

Abstract

Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

Figure 1. Pipeline of screen for PA14 virulence-attenuated mutants in C. elegans.

The three screening steps for identification of P. aeruginosa PA14 virulence-attenuated mutants are outlined; details of the screens are presented in the Materials and Methods and the text. The number of mutants obtained after each round of screening, as well as those removed from the pool for various reasons, is shown. Note that the 313 mutants identified in the primary screen and the 180 from the secondary screen represent 294 and 170 unique genes respectively because some genes were represented by multiple mutants, and a small fraction of mutants were in intergenic regions (see text). In the tertiary screen a single mutant defined each gene.

Figure 2. 41 PA14 genes required for virulence in a C. elegans infection based killing model.

The ratio of nematode survival on mutant PA14 to that on wild-type PA14 (mutant LT₅₀/WT LT₅₀) is presented for 41 mutants identified after three rounds of screening as well as for the known virulence-attenuated mutants, lasR and pilA. The time to 50% death (LT₅₀) was calculated using a non-linear regression based on the Hill equation (Prism 5.0). 100–150 animals were tested in each experiment. Error bars represent the SEM of the ratios derived from at least two different experiments (lack of error bars indicates that the mutants for known virulence factors gacA, ptsP and vfr were tested only once). Red bars depict the ratio of the LT₅₀ of lasR or pilA to WT PA14. The lasR and pilA mutants were generated previously (see Materials and Methods); there are no alleles of lasR or pilA in the NR library. The number of alleles tested with an avirulent phenotype is indicated by a number below the graph: 1 indicates that a single allele was tested but that there exist multiple alleles in the master transposon library, S indicates only a single allele was available in the library. Genes that are predicted to be in operons are indicated (Y = yes, N = no). Genes in a single operon are represented in the same color and an underline designates that other genes within the same operon were tested for their role in virulence.

Figure 3. The catabolic arginine succinyltransferase (aru) operon is required for normal virulence in C. elegans.

A) aruFGDB is transcribed as a unit; the transcriptional regulator aruC is transcribed separately. The aruFGDB operon encodes enzymes for the major aerobic route of arginine utilization as an energy, carbon and nitrogen source , . In P. aeruginosa PA01, aruE belongs to a separate transcription unit . B) MAR2xT7 insertions in aruC, aruF, aruG, aruD and aruB all reduce the virulence of PA14. The single mutation in aruE has normal virulence.

Figure 4. Multiple transposon alleles of clpA, but not clpS, are virulence-attenuated.

ClpA is the chaperone subunit responsible for substrate recognition of the ClpAP ATP dependent protease common to Gram-negative proteobacteria . A) clpA (PA2620) is the second gene of a two gene operon; it is preceded by clpS (PA2621), encoding a ClpAP adaptor protein that has been shown to bind to the N-terminus of ClpA and inhibit ClpAP degradation of some substrates while enhancing the degradation of others . B) Four different MAR2xT7 transposon insertion alleles of clpA are decreased in virulence in C. elegans. C) Three of four MAR2xT7 insertion alleles of clpS exhibit wild-type levels of virulence. Only the clpS mutant (#34203) identified in the primary and secondary screens has a virulence-attenuated phenotype. A mutant in the ClpP proteolytic subunit (#52957) was also identified in our primary screen for virulence-attenuated mutants, but this mutant was defective in growth on minimal media and therefore was not analyzed further.

Figure 5. ΔexoU may be a sensitized background that can reveal virulence-associated genes.

Deletion of the Type III effector protein ExoU has no statistically significant impact on PA14 virulence in C. elegans (B and D and [25]). A) Hypothetical protein PA4016 and adjacent loci PA4017 and PA4015; PA4016 is most likely a single gene transcription unit. B) A PA4016 MAR2xT7 insertion mutant (#22683) in the ΔexoU strain background has attenuated virulence in C. elegans, but a second PA4016 insertion allele (#41856) in the WT strain does not. C) Glutathione synthetase, gshB (PA0407), is a single gene transcription unit. D) Multiple alleles of gshB exhibit reduced virulence in C. elegans but the gshB #6613 allele in the ΔexoU strain background is more attenuated.

Figure 6. PchH and PchI but not pyochelin are required for normal virulence of PA14 in C. elegans.

A) pchH and pchI encode putative ABC transporters with potential export functions and are the two terminal genes in a pyochelin biosynthetic operon . B) Two MAR2xT7 transposon insertions in pchI and the single available MAR2xT7 allele of pchH are virulence-attenuated. C) Transposon insertion mutations in either the pyochelin biosynthetic genes pchE and pchF or the outer membrane transporter fptA, which transports pyochelin complexed with iron into the cell, have little effect on virulence. Mutations in pchH and pchI have been previously shown to produce wild-type levels of pyochelin in culture supernatant and exhibit attenuated virulence in a neutropenic mouse model .

Figure 7. The distribution of P. aeruginosa PA14 genes required for virulence in C. elegans in the Core vs. Auxiliary genome and on both predicted and known genomic islands.

A) The percentages of P. aeruginosa PA14 genomic genes, PA14-NR Set mutants, and primary, secondary, tertiary, auxotroph, and VFDB set genes that are part of the P. aeruginosa core and auxiliary genome as defined by Mathee et al. . The auxotroph set is disproportionately part of the core genome. Genes in the primary, secondary, tertiary, and VFDB sets have proportions in the core and auxiliary genes that are statistically indistinguishable from the PA14 NR set and from the genome as a whole. B) The percentages of genes from the PA14 genome, the PA14-NR Set, and from the primary, secondary, tertiary, auxotroph, and VFDB sets that are located on genomic islands predicted by IslandViewer. Representation of primary, secondary, and tertiary gene sets on predicted islands was statistically representative of the genome as a whole and of the NR-set, whereas VFDB genes had a statistical overrepresentation of genes located on predicted genomic islands (p = 0.0005). C) The percentages of genes from the PA14 genomic, the PA14-NR Set, and from the primary, secondary, tertiary, auxotroph, and VFDB sets located on the known PAPI-1, PAPI-2, and PAGI-1 genomic islands. Representation of primary, secondary, and tertiary set genes was statistically identical to the genome as a whole and the NR-set. VFDB genes were statistically underrepresented on the known islands (p = 0.007). Refer to Table S7 for statistics.

Figure 8. Among the PA14 genes required for virulence in C. elegans, “Pseudomonas-genus-specific” (PGS) genes are underrepresented, whereas “high-frequency-broad-phylogeny” (HFBP) genes are overrepresented.

Based on phylostratigraphic analysis, PA14 genes required for virulence in C. elegans were classified as either “Pseudomonas-genus-specific” (PGS), presumably representing the newest genes in PA14, “high-frequency-broad-phylogeny” (HFBP), representing the oldest, most conserved genes in PA14, or “all others”. The percentage of each gene set, including the PA14 genome genes, the PA14-NR, primary, secondary, tertiary, auxotroph, and VFDB gene sets that are classified as PGS genes, HFBP genes, or all others genes, are shown. HFBP genes comprise 10% of the PA14 genome, and about 7% of the NR set genes. Furthermore, HFBP genes are increasingly overrepresented with successive iterations of the screen accounting for 13% of the primary set (p-value = 0.00004), 14% of the secondary set (p-value = 0.0005) and 19% of the tertiary set (p-value = 0.006). HFBP genes make up greater than 50% of the auxotroph set with a (p-value = 5.47×10⁻²⁸) relative to the NR set. The PA14 VFDB set contains an underrepresentation of HFBP genes (1.6%, p-value = 0.0001). PGS genes make up 11% and 9.6% of the PA14 genome and NR set respectively. Over successive iterations of the screen, PGS genes become numerically more underrepresented relative to the NR set, comprising 5.7% of the primary set (5.7%, p-value = 0.01), 5.2% of the secondary set (p-value = 0.03, not statistically significant), and 2.4% of the tertiary set (p-value = 0.08, not statistically significant). Due to the small numbers of genes in the secondary and tertiary sets, only the underrepresentation in the primary set is significant after application of multiple comparison correction (FDR, q< = 0.05). PGS genes are underrepresented in the auxotroph set (0%, p-value = 0.0006). Statistical data for this figure are presented in supplemental Table S7.