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.
Pseudomonas aeruginosa, an opportunistic Gram-negative human pathogen, is one of the leading causes of hospital-acquired infections. In the context of a breakdown in host defenses, it is capable of infecting a plethora of tissue types, causing both acute and chronic infections. Burn victims as well as immunocompromised, mechanically ventilated, and cystic fibrosis (CF) patients are particularly susceptible to P. aeruginosa infection [1]. Over the last few decades, a steady increase in drug resistant P. aeruginosa strains has made antibiotic treatment more difficult [2]. In part because no new antibiotics effective against P. aeruginosa are imminently available as treatment options, the pressing need for drugs to fight this pathogen has focused study on its virulence factors as potential drug targets, and more generally energized a search for novel anti-infectives [3]–[5]. One likely reason that P. aeruginosa is a common nosocomial pathogen is because it is capable of thriving in a wide variety of environmental niches, including surfaces in hospital rooms, water, soil and plants [6]. Consistent with its ecological ubiquity, P. aeruginosa has a relatively large genome that presumably promotes survival in diverse habitats. In addition to inhabiting a wide variety of ecological niches, P. aeruginosa is also a multi-host pathogen, capable of infecting hosts as divergent as amoebae, plants, insects, flies, nematodes, and mice [7]–[13]. Progress in fighting P. aeruginosa infections will be aided by a fundamental understanding of the myriad ways that P. aeruginosa can survive in different environments and cause disease in diverse hosts. Our laboratory has developed a Pseudomonas aeruginosa - Caenorhabditis elegans infection-based model for studying host-pathogen interactions that is genetically tractable from both the perspectives of the host and the pathogen. This model (referred to as “slow-killing” or SK), which primarily utilizes P. aeruginosa strain PA14 [8], requires live bacteria and a set of bacterial virulence factors that distinguish it from rapid toxin-mediated PA14 killing of C. elegans (“fast killing” or FK) that occurs on high osmolarity media [12], [14]. Under standard laboratory conditions [15], C. elegans animals have a normal lifespan of approximately two weeks when feeding on non-pathogenic Escherichia coli strain OP50, and OP50 does not accumulate in the C. elegans intestine during the first few days of life. In contrast, when C. elegans are transferred at the L4 larval stage from E. coli OP50 to a lawn of P. aeruginosa strain PA14, the animals die in two-three days [12]. A few PA14 cells initially accumulate in the anterior and posterior portions of the nematode intestine, then over the course of one to two days bacteria spread throughout the intestine and the intestinal lumen becomes severely distended with a corresponding reduction in volume of the intestinal epithelial cells. Ultimately, PA14 cells move across the intestinal epithelial barrier destroying tissue, but it is not known whether tissue invasion is required for killing [16]. C. elegans rapidly responds to the presence of pathogenic PA14 by enhancing the transcription of hundreds of genes including a number of predicted secreted proteins (C-type lectins, CUB-domain containing proteins, ShK toxins) that may have antimicrobial or detoxifying activity [17], [18]. Two of the major classes of PA14 response genes, C-type lectins and CUB-like domain containing proteins, also play a role in mammalian innate immunity [19]–[21]. In C. elegans, many immune response genes are regulated by the PMK-1 p38 mitogen-activated protein kinase (MAPK), the terminal MAPK in an evolutionary conserved signaling cassette required for defense against pathogens in both nematodes and mammals. Approximately 25% of the C. elegans genes regulated by PMK-1 are also induced in response to P. aeruginosa PA14 [18] and C. elegans p38 MAPK pathway mutants exhibit enhanced sensitivity to PA14 as well as a variety of other bacterial and fungal pathogens [22]–[24]. Several hundred genes have been implicated in P. aeruginosa virulence based on data obtained from a wide variety of host infection models (Pseudomonas. com). Many of the well-studied P. aeruginosa virulence-related factors participate directly or indirectly in physical interactions with the host cell and/or host proteins, including secretion systems (type II, type III, type VI) and associated effectors (including ExoT, ExoU, ExoS, ToxA, phospholipase C, and alkaline protease), flagella, and structures involved in attachment to host cells such as type IV pili. Other recognized virulence factors include those involved in quorum sensing (AHL and PQS systems), iron acquisition (pyochelin, pyoverdine), small molecule/toxin synthesis (phenazines, hydrogen cyanide), alginate, LPS, and biofilm. Not all of these classes of virulence-related factors play a significant role in P. aeruginosa strain PA14 virulence in C. elegans; for example, the Type III secretion system and its associated virulence effectors have been shown to play no detectable role in nematode killing, in contrast to playing key roles in pathogenesis in mammals and insects [25]. However, a variety of P. aeruginosa PA14 virulence factors required for killing C. elegans in the SK infection model are also required for full pathogenesis in mammalian models, including the quorum sensing regulators LasR and RhlR, the two component regulator GacA, the alternate sigma factor RpoN, the periplasmic protease MucD, and the phosphoenolpyruvate-protein phosphotransferase and transcriptional regulator PtsP [13], [26]–[29]. Additionally P. aeruginosa virulence-related factors involved in LPS biogenesis and type IV pilus assembly and function also play a role in both mammalian and C. elegans hosts [30]–[33]. A common theme that has emerged from the study of bacterial virulence in a wide variety of pathogens and hosts is an association linking virulence-related genes with regions of genomic plasticity, including genomic pathogenicity islands (PAIs) [34], so-called “replacement islands” harboring the pyoverdine [35] and O-antigen biosynthetic loci [36], and plasmids [37]. These findings indicate that horizontal gene transfer has played an important role in the evolution of virulence. For example, phylogenetic analysis of three sub-families of the type III effector HopZ in the plant pathogen Pseudomonas syringae, suggested that at least two were acquired by P. syringae from disparate donors [38]. Analysis of the occurrence of virulence factors across many pathogen genomes has suggested that there is an overrepresentation of virulence factors on genomic islands [39], and two virulence factor- containing pathogenicity islands, PAPI-I and PAPI-II, have been identified in P. aeruginosa [40]. Although there are many published examples linking virulence-related factors to putative pathogenicity islands, a preliminary study from our laboratory showed that the presence of genes occurring in the highly virulent strain PA14, but not in the less virulent strain PAO1, could not be correlated with increased virulence across a wider sampling of strains, suggesting that virulence is a combinatorial and multifactorial product of the interactions of many potential virulence factors [32]. These data were seemingly at odds with the expected over-representation of virulence factors in strain-specific regions such as genomic islands, but were not definitive because only a limited set of virulence factors were available for analysis. Further, comparison of the sequences of five pathogenic P. aeruginosa strains suggested that virulence was primarily encoded by a core P. aeruginosa genome [41], a set of genes shared by all strains, and not the auxiliary genome defined by regions of genomic plasticity that are strain-specific. An unbiased comprehensive list of P. aeruginosa virulence factors required to cause disease in C. elegans would allow us to better understand what genes are the major contributors to virulence and whether these genes are primarily located in regions of genome plasticity or not. We considered this question worthy of investigation because it seemed likely to us that the virulence factors of an opportunistic multi-host pathogen might as a group be distinct from the virulence factors of host-specific pathogens. We report here the results of a genome-wide screen using a previously constructed non-redundant PA14 transposon library consisting of 5850 members that represents insertions in approximately 80% of the non-essential ORFs in P. aeruginosa strain PA14 [42]. Previous studies to identify P. aeruginosa virulence factors in vivo using a number of different technologies and infection models have been limited by the complexity and redundancy of mutant collections or screening procedures [13], [26], [27], [43]–[51]. We examined the genes identified in this genome-wide screen for their functions, presence on putative and characterized genomic islands, and their phylogenetic distribution across prokaryotes. We demonstrate that the major genes contributing to PA14 virulence in C. elegans are not enriched on genomic islands, are not PA14 or P. aeruginosa specific genes, and may in fact be biased for ancient genes common to many other prokaryotic species.
Seven putative virulence-related factors, cold shock domain protein PA0456, ABC transporters PchH and PchI, aminopeptidase PepP, putative enoyl-CoA hydratase/isomerase PA0745, ATPase/molecular chaperone ClpA, and putative transcriptional regulator PA14_27700 were chosen for further study. Mutants corresponding to these factors (clpA, Figure 4B; pchH and pchI, Figure 6B; pepP, Figure S5B; PA0456, Figure S6B; PA14_27700, Figure S8B; and PA0745, Figure S9B, C) all have a strong avirulent phenotype in C. elegans, exhibit normal growth kinetics in vitro (Figure S12), and represent genes whose role in P. aeruginosa virulence has not been previously characterized. The avirulent phenotype of all these mutants was confirmed with multiple transposon alleles except for pchH for which there is only a single allele available. In addition, in the case of PA0745, an in-frame deletion mutant was generated that was severely impaired in virulence, similar to the transposon allele #37629 isolated in the screen (Figure S9C). Many of the genes previously identified as necessary for virulence of PA14 in C. elegans, for example those coding for the quorum sensing regulators RhlR and LasR, are known regulators of multiple virulence factors or virulence associated pathways [58], [59]. Both lasR and rhlR mutants have a spectrum of pigment and motility defects. lasR and rhlR mutants produce reduced levels of the blue-green pigment pyocyanin, rhlR produces no pyocyanin, and lasR mutants produce varying amounts dependent on conditions and growth phase [60]. Under certain growth conditions, rhlR and lasR mutants have been reported to produce less of the fluorescent siderophore pyoverdine [61]. lasR and rhlR mutants also exhibit dramatically reduced swarming motility [62], which is dependent on both the type IV pilus and the flagella and regulated by quorum sensing and a host of transcription factors [63]. We tested the seven selected virulence-related mutants for defects in motility (twitching, swimming and swarming assays) as well as for pyocyanin and pyoverdine production in comparison to lasR and rhlR mutants to determine whether they had a similar spectrum of defects and/or could be classified into groups based on common pigment or motility phenotypes (Table 2). It should be noted that of these phenotypes, only mutants in which type IV pilus function is affected have been shown to exhibit reduced virulence in the C. elegans infection model; pyocyanin does not appear to be necessary for virulence in the SK model and the roles of pyoverdine production, swimming and swarming have not been directly tested [12], [14]. Significantly, with the exception of PA14_27700, all of the mutants exhibited defects in some aspect of motility or pigment production. Mutation of putative cold shock protein PA0456 diminished pyocyanin production as did the quorum sensing regulators lasR and rhlR, whereas a pepP mutant had elevated pyocyanin levels. The putative enoyl-CoA hydratase/isomerase PA0745 produced reduced levels of pyoverdine. Among the tested mutants, 4/7 had clear swarming defects, but exhibited normal levels of swimming and twitching motility, suggesting that neither flagella nor type IV pili function was compromised. The clpA mutant was slightly attenuated for swarming and twitching, implying that there might be a type IV pilus defect in this mutant. The list of PA14 genes identified as being required for full virulence in C. elegans from the genome-wide screen provided the opportunity to examine the distribution within a species and conservation across bacterial species of a large set of genes required for virulence in a single host. We determined whether this set of virulence associated genes was biased towards Pseudomonas core or strain-specific (auxiliary) regions of the P. aeruginosa genome (as defined by Mathee et al. [41] and/or whether these virulence genes were preferentially located on genomic islands, as previously suggested for Pseudomonas virulence factors [39], [40]. In addition, we examined whether the PA14 virulence genes had a narrow phylogenetic distribution (unique to PA14, P. aeruginosa, Pseudomonas, or closely-related organisms) or were broadly distributed across prokaryotic phylogeny. We used four sets of genes identified in our screen and a set of previously defined PA14 virulence genes downloaded from the Virulence Factor Database (VFDB) for all analyses. The sets of unique genes identified in the primary (294), secondary (170) and tertiary (41) virulence-attenuated screens outlined above and the auxotrophic genes identified in the primary screen but subsequently discarded (76) were used and for simplicity are referred to below as primary, secondary, tertiary, and auxotroph sets. All statistical analyses of the virulence genes identified in the C. elegans screen were done in comparison to the genes represented in the non-redundant (NR) library, as opposed to the entire PA14 genome, because this was the starting set for the screen. We used all four sets of genes in our comparisons to gain statistical power because the final set of 41 verified virulence-related genes was so small that most analyses did not make statistical cutoffs. In addition, using sets of genes from subsequent rounds of screening allowed us to look for enrichment that correlated with the refinement of the screen. To compare the virulence-related genes identified in our screen to previously identified P. aeruginosa genes, we made use of a set of 241 P. aeruginosa strain PA14 virulence genes downloaded from the Virulence Factor Database (VFDB) [52].
We set out to define the spectrum of genes required for P. aeruginosa PA14 infection in a single host organism with the ultimate goal of elucidating the mechanisms underlying pathogenesis in this multi-host opportunistic pathogen. A genome-wide unbiased screen for P. aeruginosa strain PA14 mutants defective in killing C. elegans identified a set of 180 putative virulence-related mutants (corresponding to 170 genes) after two rounds of screening. The screen was validated by the isolation of mutants previously shown to be required for P. aeruginosa virulence in both nematodes and mammals or known to regulate processes or pathways linked to pathogenesis including, but not limited to, genes involved in quorum sensing, two component regulators of virulence, transcriptional regulators, genes involved in type IV pilus production, and O-antigen biosynthesis. Twenty genes in the 170 gene set overlapped with a set of previously defined virulence factors in VFDB, a database of Pseudomonas-related virulence factors. Overall, the PA14 genes identified in the genome-wide screen have an overrepresentation of highly conserved genes present in many bacterial phyla and are part of the stable P. aeruginosa genome, rather than being located on pathogenicity islands. The set of 170 virulence-related genes is broadly distributed across 27 defined functional classes and DAVID GO term analysis and mapping onto KEGG pathways did not reveal any interpretable enrichment for particular functions or pathways. This breadth of functional classes parallels the virulence-attenuated mutants identified in an independent unbiased screen using signature tagged mutagenesis carried out by Potvin and coworkers in a rat chronic infection model [46]. Unlike the genes identified in our screen and by Potvin et al. , virulence-related factors in the VFDB are enriched for secretion- and adherence-related proteins. A major difference between the virulence-related genes in VFDB and the genes identified in our unbiased screen is that many of the genes in VFDB were included because they encode secreted toxins, secretion systems, or cell surface structures [52], [67]. However, the sensitivity of our screen favored identification of mutants with strongly attenuated virulence. This was expected given the nature of the primary screen that required that both the parent nematodes live long enough to produce a significant brood and that the nematode brood mature on the mutant bacterial lawn. It is possible, therefore that mutants with a weak virulence-attenuated phenotype were not detected and this could potentially skew the collective analysis of virulence factors. One explanation for why so few mutants identified in our screen correspond to secretion pathways or to secreted effectors is that many of P. aeruginosa virulence effectors appear to function redundantly in the C. elegans killing assay. In support of this conclusion, disruption of the ExoU cytotoxic phospholipase had no statistically significant impact on virulence, but appeared to create a sensitized background that allowed the detection of other relatively weak virulence factors. Further, McEwan et al. have shown that whereas PA14 exotoxin A (toxA) mutants have no or little defect in virulence, overexpression of ToxA in E. coli activates the worm immune system and ultimately kills an immune-compromised animal, suggesting that ToxA may play an active, but to date undetected, role in PA14 pathogenesis in the nematode host [68]. Similarly, Dunbar et al. [69] have shown that ToxA inhibits protein synthesis in C. elegans intestinal cells during an infection. ExoU and ToxA are secreted by distinct systems and the weak virulence attenuation of secretion system mutants in C. elegans implies that multiple secretion systems and effectors may contribute to virulence in C. elegans with no single system being paramount. An alternative explanation for the identification of a limited number of secretion-related mutants in our screen may be linked to the fitness costs of maintaining a large set of effectors targeting a wide range of potential hosts. Only four Type III effectors have been identified in P. aeruginosa, whereas 46 families of effector proteins have been identified in various strains of the related plant pathogen P. syringae [70] and a typical P. syringae strain has 20 to 30 effectors [71]. In P. syringae, which has a much more limited host range than P. aeruginosa, type III effectors mostly target host defense signaling pathways and both enhance virulence in particular host plants, while eliciting a strong immune response in others [72]. This fact, combined with the observations that the genes encoding secreted effectors are often under diversifying selection [38], [73] and are typically located in plastic regions of the genome [70], [74], suggests that P. syringae strains actively co-evolve with a limited number of hosts. In contrast, from first principles, it seems highly unlikely that a broad host-range pathogen like P. aeruginosa PA14 can be simultaneously co-evolving with all of its multiple hosts. Therefore P. aeruginosa might employ a broader set of strategies to ensure survival in diverse hosts instead of maintaining large sets of host-specific virulence-related effectors. In this context, a number of the strongly virulence-attenuated PA14 mutants identified in our screen may correspond to factors that enable survival of P. aeruginosa in the hostile environment of the C. elegans intestinal tract, which is acidic and filled with enzymes such as proteases, lipases, and DNAse that potentially disrupt bacteria [75]. Moreover, in response to pathogens, the nematode specifically upregulates transcription of many putative antimicrobial genes [18]. In order for P. aeruginosa to proliferate in the intestine and cause disease, it first has to survive. Both the two component potassium sensor KpdD and the nitrogen assimilation regulatory protein GlnK, identified in our screen, have recently been shown to play a role in the persistence of S. typhimurium in the C. elegans intestine and defects in outer membrane integrity may reduce the survival of kdpD and glnK mutants in the host [76]. In addition, the identification of two PA14 genes required for biosynthesis of glutathione, gshA and gshB, may be related to the role of glutathione as a protectant against stresses encountered in the worm intestine including reactive oxygen species and low pH [77]. Cold shock domain proteins, like PA0465 identified in our screen are another class of molecule that are induced by environmental stress and are generally thought to play a protective role in the cell [78]. Identification of a number of PA14 mutants corresponding to metabolic genes illustrates the importance of nutrient acquisition in virulence. Without specific biosynthetic or metabolic capabilities a pathogen may be unable to colonize or grow within a host. For example, P. aeruginosa mutants defective in purine biosynthesis are unable to replicate in neutropenic mice, presumably because the in vivo environment is deficient in purine [48]. In the cases of intercellular pathogens such as Listeria monocytogenes and Mycobacterium tuberculosis, specific amino acid and nucleotide auxotrophs are reduced in growth in vivo [79], [80]. We identified a number of metabolic genes including prpB and prpC and several aru genes that may be important for bacterial metabolism and growth under the nutrient conditions within the nematode intestine. Further, some of the putative virulence-attenuated mutants identified in the primary screen that were set aside for further study because they were determined to be auxotrophs (a typical step in many screens) might specifically reflect nutrient availability in the nematode intestine. In this regard it is notable that mutations in nine purine, five pyrimidine, and six tryptophan biosynthetic genes were identified in the primary screen for virulence-attenuated mutants, and although some of these mutants exhibited reduced growth on the killing assay medium, many did not have any observable difference from wild-type PA14, suggesting that the reduction in virulence might be due to aberrant growth of these auxotrophs in vivo. The predominant contributors to PA14 virulence in our C. elegans infection based assay appear not to be individual effectors, but genes that regulate numerous effectors (like the quorum sensing regulators lasR and rhlR), genes that are vital for protecting the bacteria from the host defense onslaught, and genes that help P. aeruginosa obtain the necessary nutrients to survive in the host. Therefore the strategy of a broad host range opportunistic pathogen might fundamentally differ from a pathogen that targets specific hosts, relying more on multiple partially redundant secretion systems and their cognate effectors and strategies for survival under a wide-variety of metabolic and environmental conditions. The long-recognized association between virulence genes and regions of genomic plasticity, particularly genomic islands acquired by lateral transfer of genetic material [81], has been attributed to the competitive advantage conferred by horizontally-acquired virulence factors in an ongoing co-evolutionary struggle between a host and pathogen. Two pathogenicity islands carrying plant and animal virulence-related genes have been identified and characterized in P. aeruginosa PA14 (PAPI-1 and PAPI-2). Of the 11 genes located on PAPI-1 previously shown to be required for normal levels of virulence in plants and mice [40], only one (rcsC) was identified in our secondary screen as a weak mutant and it did not re-test in the tertiary screen. Overall our results showed no enrichment of virulence-associated genes on predicted genomic islands, or on the known genomic islands PAPI-1, PAPI-2, or PAGI-1. More generally, whether or not virulence genes in aggregate are preponderantly associated with genomic islands in P. aeruginosa has not been experimentally demonstrated. The statistical power of analysis with respect to the genomic locations of genes is limited in part by the fact that not all genes are expected to segregate independently. In this regard, it is worth noting that the apparent enrichment of P. aeruginosa VFDB genes on predicted genomic islands is primarily due to the cluster of functionally interdependent Type III secretion apparatus genes located between gene loci PA14_42440 and PA14_42660. In summary, our data and analysis of existing data suggest that neither P. aeruginosa VFDB genes nor the virulence-attenuated genes identified in our screen are preferentially found on genomic islands. Roughly paralleling our observations with genomic islands, our analysis of the frequency of PA14 virulence genes in the core and auxiliary genomes (both from our screen and the VFDB) showed no statistically significant over or underrepresentation. The main difference between the core versus auxiliary genome distinction, and that of genomic islands, is that auxiliary genes include both genes that are lost from the genomes of some isolates, as well as those genes that are newly acquired. Genomic islands by contrast, specifically include only newly acquired genes. Taken together, these results suggest that there is no specific enrichment of P. aeruginosa virulence-related genes on islands or in strain-specific regions of the P. aeruginosa genome, at least with respect to those that are involved in the C. elegans slow killing assay. To further test the hypothesis that the arsenal of P. aeruginosa virulence factors includes newly evolved and or newly acquired genes we investigated the phylogenetic breadth of distribution of the PA14 virulence genes, as well as the degree to which they belong to a set of putative old conserved genes, the so-called high-frequency-broad-phylogeny (HFBP) set, and probable newer genes, the Pseudomonas-genus-specific (PGS) set. We found that consistent with the result that virulence genes are not located on islands, that PA14 virulence genes involved in C. elegans killing are enriched in HFBP genes, which as a group are likely to be the most ancient and conserved prokaryotic genes. The vast majority of PA14 virulence genes appear not to be specific to the Pseudomonas genus, and in fact, such recently acquired or novel genes are underrepresented among our putative virulence genes. These observations underscore the point that many virulence factors with the most significant contribution to virulence can be old, highly conserved genes. Although the C. elegans model, in which nematodes are “force-fed” a monoculture of P. aeruginosa [13], [43], [44], is somewhat artificial, so are other laboratory models of P. aeruginosa infection that require pricking of the body of a fly [45], lung inoculation with agarose beads in rats [46] and mice [47], and non-lethal cutaneous burns in mice [82]. The artificiality of these models and the need for a compromised host is in part dictated by the opportunistic nature of the pathogen. The fact that conserved immune defense pathways are activated in the C. elegans host by P. aeruginosa strongly supports the view that the nematode is responding to P. aeruginosa as a pathogen [83]. Although we don' t yet know whether P. aeruginosa is a natural pathogen of C. elegans, since both organisms live in the soil, it is likely that they encounter one another, and it is not inconceivable that the pathogenicity interactions that we observe may approximate the interactions of P. aeruginosa with weakened individual C. elegans animals in the wild. Although some putative P. aeruginosa virulence factors are common to both mammalian and C. elegans host models, the degree to which P. aeruginosa virulence factors are shared, and implicitly, the degree to which P. aeruginosa virulence strategies are common to C. elegans and mammalian hosts is not yet clear. That only a single putative virulence-related gene is shared between the 170 virulence-attenuated genes from our secondary set, the rat chronic infection set defined by Potvin et al. , and the VFDB set, argues against the idea of a core set of virulence factors common to all infection models. The spectrum of virulence factors that play a role in a given host model is likely to depend on a wide variety of factors including the characteristics of the site of infection, such as pH, ionic strength, nutrient availability, and temperature, the type of immune compromise, the phase of infection, and the particulars of the immune response, including the presence of host factors, and even host behavior. Indeed, two additional PA14 pathogenicity assays in C. elegans have been developed in our laboratory, a toxin-mediated killing model [12], [14] and a liquid killing assay (N. Kirienko and F. Ausubel, unpublished). PA14 appears to utilize distinct mostly non-overlapping sets of virulence-related genes to kill nematodes in the three different models. Nevertheless, all three of these assays have identified virulence factors that play important roles in various aspects of mammalian pathogenesis. The implication for human pathology is that the predominant virulence factors that play a role in different types of P. aeruginosa infection in humans may be somewhat distinct. In summary, the data from our unbiased genome-wide screen for P. aeruginosa virulence factors involved in C. elegans killing in a specific infection model, suggest that in comparison to host-specific pathogens, P. aeruginosa may employ a smaller arsenal of host-specific effectors, and rely more on conserved, generic virulence factors and on its ability to endure host defense responses. While it is not yet clear that this same strategy is employed by multi-host pathogens beyond P. aeruginosa that are capable of infecting organisms from multiple phylogenetic kingdoms, this may explain why the major genes contributing to PA14 virulence in C. elegans are not overrepresented on genomic islands, are not PA14 or P. aeruginosa specific genes, and may in fact be biased for ancient genes common to many other prokaryotic species. These observations are consistent with the view of P. aeruginosa PA14 as a generalist pathogen for which the relationship with C. elegans is opportunistic rather than co-evolved. Indeed it is likely that no significant co-evolution occurs between P. aeruginosa and any of the hosts for which it is an opportunistic pathogen, both because of the rarity of pathogenic interaction, and because of the likelihood that co-evolution with multiple hosts would necessitate balancing opposing evolutionary pressures from those hosts. From a clinical perspective, the multiplicity and apparent combinatorial nature of P. aeruginosa' s virulence factors may pose a challenge for the development of new therapeutics to fight Pseudomonas infection. This work does not settle the question of whether the profile of virulence factors of a multi-host pathogen is likely to be different from that of host-specific pathogens in terms of the reliance on conserved effectors that target highly conserved features of eukaryotic biology, but it is a question that deserves further inquiry.
P. aeruginosa strain PA14 mutants are gentamycin resistant MAR2xT7 transposon insertion mutants unless otherwise stated [42]. The lasR mutant used as a control was a deletion that removes the lasR ATG and carries a gentamycin cassette [84]. Similarly, the rhlR mutant used as a control in the pigment and motility assays was a deletion mutant that carries a gentamycin cassette [84]. These mutants have an identical avirulent phenotype to subsequently generated clean in-frame PA14 ΔlasR and ΔrhlR mutants (data not shown). The pilA mutant is a tetracycline resistant Tn5-B30 transposon insertion mutant provided by G. O' Toole [85]. ΔPA0745 is a complete in-frame deletion of the ORF with a concomitant insertion of a PacI restriction site generated by the method previously described in Chand et. al. [86]. Unless otherwise indicated, MAR2xT7 mutant bacteria were streaked from frozen stocks onto LB agar containing 15 µg/ml gentamycin to isolate single colonies used for inoculation. WT PA14 (as well as lasR and pilA mutants) was streaked on LB agar containing 100 µg/ml rifampicin. N2 Bristol L4 animals were used for the primary and secondary screens. Tertiary tests were done with CF512 fer-15 (b26) II; fem-1 (hc17) IV temperature sensitive sterile nematodes. CF512 worms were propagated at 15°C, egg-prepped and hatched overnight at 20°C in M9 liquid media in the absence of food. Starved L1 animals were dropped onto NGM plates seeded with standard E. coli OP50 and grown for approximately 20 hours at 15°C and then 20 more hours at 25°C to generate staged L4 sterile animals for experimentation. We used the non-redundant PA14 MAR2xT7 transposon library of ordered mutants to screen for avirulent mutants [42]. The entire library was screened twice with the exception of a single 96-well plate of mutants (plate 14. 3) that was not screened because it contained mostly known slow-growing mutants. Bacteria were inoculated from the frozen 96-well stock plates into 150 µl of LB in 0. 5 ml 96-well Masterblocks (Greiner) and grown overnight with shaking for 16 hours at 37°C. 10 µl of each culture was spotted onto slow killing (SK) agar (standard nematode growth media, NGM, containing 0. 35% instead of 0. 25% peptone) in each of two wells of a 6-well culture plate (Falcon) [12]. Two previously identified virulence-attenuated mutants, quorum sensing regulator lasR (highly attenuated) and typeIV pilin pilA (moderately attenuated) were included as positive controls in the screen. Plates were scored qualitatively after 4 days at 25°C and designated as either “strong”, indicating large numbers of gravid animals (generally on lasR mutant bacteria hundreds of gravid nematodes were present and the bacterial lawn was mostly or completely consumed) or “weak”, indicating an observable increase in the number of worms or gravid adults present compared to PA14 (pilA usually resulted in an increase in the total brood size with a bias towards older stage larvae and gravid adults; the pilA control wells exhibited variability in the number of progeny alive and were near the limit of sensitivity in the screen). Each mutant was screened blind on two separate days for a total of four wells screened per mutant. Mutants that were scored as strong in either one or both repetitions or weak in both were retained for a total of 399 primary mutants. Approximately half way through the primary screen it became clear that lawn growth phenotype was relevant to our study, and from then on we scored for lawn growth. We therefore have a crude assessment of growth for about 50% of the mutants identified in the primary screen. All 399 of the mutants from the primary screen were streaked from the frozen library stock onto LB agar plates containing 15 µg/ml gentamycin and grown overnight at 37°C. To quickly test for potential auxotrophs, a single colony from each mutant was picked and streaked on 1) Neidhardt supplemented MOPS defined media without amino acids or nucleotides (TEKNOVA EZ Rich Media), 2) Neidhardt supplemented MOPS defined media plus amino acids and nucleotides, and 3) LB containing 15 µg/ml gentamycin agar plates. Plates were incubated overnight at 37°C. 86 mutants that either exhibited no growth on minimal media minus supplements or significantly reduced growth were removed from the pool of putative avirulent mutants and not examined in further rounds of screening (Table S2). 15/56 of the auxotroph mutants for which a plate growth phenotype had been annotated (see above) had obvious growth defects on SK agar plates. The 313 mutants from the primary set that exhibited normal growth on minimal media without addition of amino acids and nucleotides (Table S3) were screened for attenuation of virulence in standard SK assays and scored for both killing of parental (P0) animals and the number and maturity of worm progeny produced [13]. A single bacterial colony was inoculated into 5 ml of LB media and grown for 14. 5 hours at 37°C with aeration on a rotating wheel. 10 µl of this overnight culture was spread onto each of two SK agar plates (3. 5 cm culture plates (Falcon), plates were incubated for 24 hours at 37°C, and then 24 hours at 25°C. 30–40 N2 L4 animals (raised on standard NGM plates with E. coli OP50 as food) were picked to each SK plate and the plates were incubated at 25°C for a total of 60–80 animals per assay. Live and dead animals were counted 2–3 times over 1–3 days after transfer to the pathogen. Animals were considered dead if they did not respond to a gentle touch and were removed from the plate. After 4 days at 25°C, the number and age of the nematode progeny on each plate was qualitatively assessed as compared to those on PA14 WT. Secondary positives were ranked as “strong” (strongly virulence-attenuated similar to the lasR mutant), “moderate” (less attenuated than lasR but greater than or equal to pilA) and “weak” (less attenuated than pilA for parental killing but clearly exhibiting an increase in progeny number and age over WT PA14). 180 MAR2xT7 mutants exhibited either attenuation of P0 parental killing or allowed increased production and development of nematode progeny (Table S4). The identity of the MAR2xT7 mutants was confirmed by sequencing of arbitrary PCR products as previously described [42], see Table S4. 58 MAR2xT7 mutants were re-screened in standard SK assays using sterile fer-15 (b26) I: fem-1 (hc17) IV animals (Table S5). Bacteria were grown as indicated above for the secondary screen. Three 3. 5 cm SK agar plates were seeded with each bacterial strain and 35–50 L4 worms were transferred to each plate for a total of 100–150 animals per assay. Live and dead animals were counted every day over approximately 7 days. PA14 WT and lasR were included in each assay as controls. The resulting 41 virulence-attenuated mutants shown in Figure 2 were all tested in at least two separate experiments unless otherwise indicated. Statistical analysis of the curves summarized in Figure 2 was done using Prism 5 Log-rank (Mantel-Cox) test and the difference between the mutant and wild-type curves was highly significant in all cases with a p-value<0. 0001. Time to 50% survival was calculated (Prism 5. 0 linear regression Hill equation LogEC50). All killing assays presented in the manuscript (Figures 3,4, 5,6, S5, S6, S7, S8, S9, S10, and S11) are a representative example of two or more experiments that resulted in the summary shown in Figure 2. Growth of bacterial mutants was evaluated by four methods: 1) Overnight cultures inoculated from a single colony into 5 ml of LB and grown for 14. 5 hours at 37°C with shaking were visually compared to wild-type PA14 grown under the same conditions. Mutants that were observably less turbid were considered slow growers (in the case of PA14_45650 mutant #54246 the cells appeared to be lysed). 2) Slow killing agar plates spread with each mutant were examined prior to transfer of worms. Bacterial lawns that were thin or exhibited other aberrant phenotypes (nusA mutant #55834 had large colonies that emerged on top of the lawn) were removed from the mutant pool. 3) Many of the secondary and tertiary positive mutants were grown overnight in LB and M63 minimal media in a 96-well plate without shaking and the OD600 was measured every 15 minutes for 15 hours at 37°C in a Molecular Devices Spectra Max M5. The rate of growth during two hours of maximal growth was compared to WT (Table S5). 4) The growth of nine mutants (pchH, pchI, PA14_27700, PA2550, PA0456, PA0745, clpA, PA1216, vqsR) in 5 ml of M63 minimal in a standard culture tube on a rotation wheel at 37°C was measured by counting colony forming units. All nine of these mutants grew as well as WT (Figure S12). A pyocyanin assay was modified from Essar et. al. 1990 [87]. A single colony of WT or mutant bacteria was inoculated into 5 ml of LB media and grown for 16 hours at 37°C on a rotating wheel. 1 ml of saturated overnight culture was transferred to a microfuge tube and cells were pelleted by centrifugation at 14,000 RPM for 2 minutes. 800 µl of the supernatant was transferred to a new tube, extracted with 600 µl of chloroform and the phases were separated by centrifugation for 5 minutes at 14,000 RPM. The chloroform phase was then re-extracted with 0. 3 ml of 0. 2 N hydrochloric acid. The pyocyanin content of 100 µl of the aqueous acidic phase was quantitated based on absorbance at 520 nm. The A520 was normalized to cell number (A600 of the original overnight culture). A ΔphzA1-G1/ΔphzA2-G2 [88] mutant that does not produce any phenazines had no detectable A520. The pyocyanin produced by each bacterial strain was measured from the growth of two individual colonies on two separate days. For each of the four cultures, the ratio of A520 (normalized to cell number as measured by A600) of mutant to WT was calculated and the average of these four ratios is shown in Table 2. The error presented is the SEM of the four mutant/WT ratios. A single colony of WT or mutant bacteria was inoculated into 5 ml of M9 media and grown overnight for 18 hours at 37°C on a rotating wheel. Cells were pelleted by centrifugation at 14,000 RPM for 2 minutes in a microfuge tube and the supernatant was diluted 10 fold in 10 mM Tris pH 7. 4. Pyoverdine content was determined by measurement of fluorescence (400 nm excitation, 460 nm emission) [89], [90]. No fluorescence above background was detected in pyoverdine biosynthetic mutants, pvdD (#40342) and pvdA (#30448). The pyoverdine produced by each bacterial strain was measured from the outgrowth of two individual colonies on two separate days. For each of the four cultures, the ratio of fluorescence (normalized to cell number as measured by A600) of mutant to WT was calculated and the average of these four ratios is shown in Table 2. The error presented is the SEM of the four mutant/WT ratios. The swarming assay was modified from Overhage (2008) [91]. A single colony of WT or mutant bacteria was inoculated into 5 ml of LB media and grown for 15 hours at 37°C with aeration. 2 µl of each overnight culture was spotted onto the surface of LB 0. 5% agar and SK 0. 5% agar plates and then incubated at 37°C overnight. Each mutant was tested in triplicate on two separate days. As expected, the rhlR mutant (deficient in rhamnolipid production) and pilA mutant (typeIV pilin) were defective in swarming [62]. Mutants were visually compared to WT PA14 and were qualitatively evaluated for swarming radius and number of tendrils (Table 2). The swimming motility assay was based on Darzins (1993) [92]. A single bacterial colony was picked with a straight end loop and inoculated into LB swim agar (0. 35% agar). Plates were incubated 8–12 hours at 37°C. The diameter of the flagellum-mediated motility generated turbid zone was measured. Each mutant was tested in triplicate and the average with SEM is presented in Table 2. The twitching motility assay was based on O' Toole 1998 [85]. A portion of a single bacterial colony was picked with a straight end inoculation loop and stabbed to the bottom of a LB agar plate (1. 5% agar). Plates were incubated overnight at 37°C and then 2 days at room temperature. The growth at the interface between the agar and the polystyrene plate (radius from the inoculation point) was measured. The pilA mutant exhibited no twitching motility. Each mutant was tested in triplicate and the average with SEM is presented in Table 2. A database of orthologs of P. aeruginosa strain PA14 genes across 727 sequenced prokaryotic genomes (including PA14) was created. Finished microbial genome sequences were obtained as downloaded packages from the NCBI ftp site (ftp: //ftp. ncbi. nlm. nih. gov/genomes/Bacteria/) on August 25,2008. PA14 proteins were used as BLASTP queries against each bacterial genome. Putative orthologs were reciprocal best hits against the corresponding proteins in the subject genomes. Blast results against subject genomes were required to have an e-value equal to or less than 0. 0001. Reciprocal blasts against the PA14 genome were required to have e-values of 0. 001 or less. Putative orthologs were required to align for at least 80 percent of their length and have less than 20% difference in protein sequence lengths, thereby conserving overall domain structure. The e-value constraint was permissive to allow detection of distant orthologs, but the requirement for alignment length was fairly stringent. Breadth of phylogenetic distribution, also called phylostratum, was a measure of the maximal phylogenetic distance at which an ortholog occurs. Breadth was defined as: 0 for proteins specific to PA14,1 for proteins that occur in multiple strains of Pseudomonas aeruginosa, but not in other species, 2 for proteins that occur in multiple Pseudomonas species but not in other genera, 3 if across gamma and beta proteobacteria, 4 if across proteobacteria, 5 if across eubacteria, and 6 if across eubacteria and archaea (Figure S13). P-values for overrepresentation and underrepresentation were calculated as Fisher Exact Test right and left probabilities, respectively, using version 1. 21 of the Text: : NSP: : Measures: : 2D: : Fisher Perl module, available from CPAN. org [93]. Multiple comparison correction using False Discovery Rate (FDR) was performed where indicated [94]. The maximal value of q was 0. 05. Genes of P. aeruginosa strain PA14 were mapped to KEGG pathways using the KEGG Mapper program (http: //www. genome. jp/kegg/tool/map_pathway1. html). The total number of mutants in each pathway was summed for those genes in the PA14 NR set and for each mutant set analyzed. P-values for overrepresentation of each pathway were calculated using Fisher exact test right-probabilities, and their significance was assessed using the Bonferroni correction [94], [95]. GO term analysis was performed using DAVID (http: //david. abcc. ncifcrf. gov/home. jsp) using the genes in the NR set as a background, and the genes in the sets of analyzed positives as gene-lists. The DAVID software package calculates p-values for each GO term automatically, and also gives an EASE score, which was used to assess the significance of the overrepresentation of any given GO term.
Pseudomonas aeruginosa is an opportunistic human pathogen that can also infect a wide range of model organisms, including the nematode Caenorhabditis elegans. To identify P. aeruginosa genes that play key roles in the pathogenic process, we performed a screen for mutants that exhibited reduced ability to kill C. elegans using a previously constructed non-redundant library representing approximately 80% of the non-essential P. aeruginosa PA14 genes. We defined a functionally diverse set of 180 P. aeruginosa mutants (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. The major contributors to P. aeruginosa virulence in the C. elegans infection model were not secretion systems or their corresponding effectors, but rather regulators (particularly ones that are involved in quorum sensing) and genes likely to play key roles in survival of P. aeruginosa within the host intestine. Moreover, these putative P. aeruginosa virulence genes are neither overrepresented in strain-specific regions nor in horizontally acquired genomic islands and furthermore tend to have orthologs that are widely distributed across sequenced prokaryotic species. These data underscore the diversity of pathways involved in virulence, and especially the importance of highly conserved genes for P. aeruginosa virulence in the C. elegans host model.
bacteriology
microbiology
host-pathogen interaction
animal models
caenorhabditis elegans
prokaryotic models
model organisms
microbial evolution
bacterial pathogens
microbial pathogens
biology
pathogenesis
gram negative
genetic screens
genetics
evolutionary biology
bacterial evolution
genetics and genomics
Genome-Wide Identification of Pseudomonas aeruginosa Virulence-Related Genes Using a Caenorhabditis elegans Infection Model