Genome-Wide Identification of Pseudomonas aeruginosa Virulence-Related Genes Using a Caenorhabditis elegans Infection Model

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

Anti-quorum sensing agents from south Florida medicinal plants and their attenuation of Pseudomonas aeruginosa pathogenicity

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus . Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system

Inhibition of Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas aeruginosa by South Florida Plant Extracts▿

Quorum sensing (QS) is a key regulator of virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Aqueous extracts of six plants, Conocarpus erectus, Chamaesyce hypericifolia, Callistemon viminalis, Bucida buceras, Tetrazygia bicolor, and Quercus virginiana, were examined in this study for their effects on P. aeruginosa virulence factors and the QS system. C. erectus, B. buceras, and C. viminalis caused a significant inhibition of LasA protease, LasB elastase, pyoverdin production, and biofilm formation. Additionally, each plant presented a distinct effect profile on the las and rhl QS genes and their respective signaling molecules, suggesting that different mechanisms are responsible for efficacy. Extracts of all plants caused the inhibition of QS genes and QS-controlled factors, with marginal effects on bacterial growth, suggesting that the quorum-quenching mechanisms are unrelated to static or cidal effects

<i>P. aeruginosa</i> PA14 virulence-attenuated genes identified in the <i>C. elegans</i> infection model.

<p>Genes are listed in descending order of contribution to virulence (according to the ratio of mutant LT₅₀/wild-type LT₅₀) using the data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat-1002813-g002" target="_blank">Figure 2</a>. Genes previously identified as required for normal levels of <i>P. aeruginosa</i> virulence in various model systems are indicated. In some cases only <i>P. aeruginosa</i> strains other than PA14 were examined and the strain and mode of killing is indicated in parentheses. The other pathogens, in which orthologs of these genes have been implicated in virulence, are noted in the last column.</p

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

<p>Based on phylostratigraphic analysis, PA14 genes required for virulence in <i>C. elegans</i> were classified as either “<i>Pseudomonas</i>-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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813.s020" target="_blank">Table S7</a>.</p

Pipeline of screen for PA14 virulence-attenuated mutants in <i>C. elegans</i>.

<p>The three screening steps for identification of <i>P. aeruginosa</i> PA14 virulence-attenuated mutants are outlined; details of the screens are presented in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#s4" target="_blank">Materials and Methods</a> 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.</p

Pigment and motility phenotypes of seven novel virulence mutants.

<p>The average ratio of mutant to wild-type pyocyanin levels from four samples and the SEM is shown. The average ratio of mutant to wild-type pyoverdine levels from four samples and the SEM is shown. Twitching motility (1.5% LB agar) was measured as the radius of growth at the interface of the medium and the polystyrene plate and average radius and SEM from three inoculations is presented. Swimming motility was determined by the diameter of the turbid zone in semi-solid LB agar (0.35%) and average radius and SEM from three inoculations is presented. Swarming levels on the surface of 0.5% agar medium were qualitatively evaluated with number of and length of tendrils taken into account.</p