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BIOLOGICAL SCIENCES / MICROBIOLOGY
Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence
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Departments of *Microbiology and Molecular Genetics and Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; Department of Molecular Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114; ¶Shriners Hospital for Children, 3229 Burnet Avenue, Cincinnati, OH 45229; and ||Graduate School of Information Science/Human Informatics and Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Contributed by Frederick M. Ausubel, December 22, 2005
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Abstract |
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The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action.
cytotoxicity | biofilm
Recent advances in genome sequencing have also led to the identification of families of enzymes responsible for the turnover of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which appears to be a common chemical messenger present uniquely in bacteria (1). c-di-GMP was initially identified as an activator of a cellulose synthetase complex in Gluconacetobacter xylinus (3). In G. xylinus and other bacteria, c-di-GMP is synthesized by a family of enzymes called diguanyl cyclases (DGCs), which share a sequence motif (the so-called GGDEF domain) (4). The levels of c-di-GMP are also controlled by c-di-GMP phosphodiesterases (PDEs) that contain a conserved EAL domain (4). A number of proteins contain both of these domains, suggesting they may be bifunctional enzymes with opposing activities, although the EAL domains of these presumptive bifunctional proteins tend to be more divergent and perhaps inactive (5).
The significant fraction of proteins with GGDEF and/or EAL domains identified by the analysis of genome sequences show a multimodular arrangement, in which the DGC and PDE domains are fused to signal receiver or transmission domains, suggesting that the activities of many DGCs and PDEs are regulated by environmental signals (6). VieA and PvrR, regulators of virulence in Vibrio cholerae and in certain strains of Pseudomonas aeruginosa, are examples of modular proteins with a PDE domain fused to a response regulator domain (6, 7). WspR, a P. aeruginosa chemosensor with a DGC and a response regulator domain, directs formation of c-di-GMP when expressed in the absence of WspF (8). Finally, two related regulators of surface protein expression in Bordetella pertussis and in P. aeruginosa are chimeric proteins with a response regulator module fused to a PDE domain and appear to function in conjunction with a linked two-component signal transduction system (9, 10). The targets of the regulatory activities of c-di-GMP in a variety of species appear to be surface-localized adhesive structures, including exopolysaccharides or fimbrial organelles. A number of DGC and PDE proteins are anchored to the membrane via transmembrane segments (11), suggesting that this localization is important for their function.
A number of proteins with predicted or documented DGC or PDE activity have been shown to regulate virulence-related traits of diverse pathogenic bacteria, including biofilm formation. Late-stage biofilm formation by Burkholderia cepacia involves a protein with a DGC domain (12), and biofilm formation in Yersinia pestis is controlled by HmsT (DGC) and HmsP (PDE), containing the consensus DGC and PDE domains, respectively (13). In Salmonella species, GcpA, which contains a GGDEF domain, regulates the synthesis of the cellulose component in biofilm communities (14). Another Salmonella regulator of biofilm formation and motility, AdrA, has a DGC domain and is responsible for synthesis of c-di-GMP in vivo (15). In Vibrio species, DGCs or PDEs have been implicated in regulating capsular production, motility, biofilm formation, the rugose phenotype, and virulence (16–19). Finally, Arr, a P. aeruginosa PDE domain-containing protein regulates biofilm formation in response to aminoglycosides (20).
One of the remarkable features of proteins involved in the metabolism of c-di-GMP is the apparent redundancy of proteins with DGC and PDE domains (11). However, it is unclear what environmental conditions control expression of each of their respective genes, and how the activity of their products is regulated. Even more intriguing is how c-di-GMP concentrations can affect targets specific for one enzyme that produces or degrades c-di-GMP, considering that, as a small molecule, this dinucleotide is presumably freely diffusible in the cytoplasm.
Here we report the results of a systematic analysis of phenotypes of DGC and PDE domain-containing proteins in P. aeruginosa. In the annotated sequence of two P. aeruginosa strains, PAO1 and PA14, multiple coding sequences for proteins with GGDEF/EAL domains were identified. We examined a comprehensive set of mutants with transposon insertions in each of these genes generated by near-saturation mutagenesis of the genome of strain PA14, as well as of clones of the putative DGCs and PDEs overexpressed in wild-type PA14. We identified a number of mutants with distinct phenotypes: a block in type III secretion system (TTSS)-mediated cytotoxicity and inability to form biofilm or pellicle. We were also able to demonstrate formation or hydrolysis of c-di-GMP in P. aeruginosa expressing a number of DGCs and PDEs, respectively. Different PA14 mutants in DGCs and PDE genes displayed different degrees of attenuation of virulence in murine infection models, implicating c-di-GMP in virulence.
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We determined the evolutionary relationships among the DGC (GGDEF domain, pfam PF00990) and PDE (EAL domain, pfam PF0053) (Fig. 6 A and B, which is published as supporting information on the PNAS web site). The phylogenetic trees show that the DGC modules can be grouped into three families (I–III). The proteins in group I contain almost exclusively DGC domains with a conserved GGEEF motif and are not fused to a PDE module (Fig. 6 A and C). All but two members in groups II and III are DGC-PDE proteins containing both DGC and PDE modules. This phylogenetic relationship suggests that proteins with only a DGC domain have evolved independently from those in which a DGC domain is linked to a PDE domain. Groups II and III can be distinguished based on their diminishing similarity to the consensus DGC sequence, with members of group III being most distant (Fig. 6D). When known DGCs are included in this analysis [PleD of Caulobacter crescentus (24) and the G. xylinus DGC (3, 25)], both of these proteins cluster in group I. The poor correlation of group III GGDEF domains with the consensus sequence suggests they may be enzymatically inactive.
A similar alignment and phylogenic analysis of the PDE domains are shown in Fig. 6 B and D. Three of these putative PDE modules, in PA2200, PA2818, and PA3285, appear to be more closely related and are not linked to DGCs. The other two (PvrR and PA3947) are in a separate branch of the tree. PvrR shows a significant overall similarity to V. cholerae VieS, and this close relationship is reflected in their respective PDE domains.
Genomic Analysis of Genes Encoding DGC, PDE, and DGC-PDE. We examined the prevalence of genes encoding putative DGCs and PDEs among P. aeruginosa clinical and environmental isolates using the microarray analysis described previously (26). The results shown in Fig. 7A, which is published as supporting information on the PNAS web site, demonstrate that the majority of these genes, with the exception of PAO1 genes PA2771 and PA2818, are components of the P. aeruginosa core genome and are found in all strains tested, suggesting that the products of these genes are used by the bacteria in one or several environmental niches that this organism can occupy. The two exceptions, PA2771 and PA2818, are missing in a significant fraction of strains analyzed, including PA14. These genes are located in or near regions of high genomic plasticity and are probably components of genomic islands (26). We determined the occupancy of these sites by PCR-mediated amplification of their respective chromosomal regions using primers that were located in core genomic sequences flanking PA2771 and PA2818. All strains harboring PA2771 (13 of 20 examined) had sequences identical to those found at the corresponding location in PAO1, whereas the seven strains that lacked PA2771 matched the sequence in the PA14 genome (Fig. 7B). In the PA14 genome, the PA2771 location contains four ORFs, one encoding a putative acid phosphatase and three hypothetical proteins.
PA2818 is found only in a limited number (5 of 20 tested) of P. aeruginosa strains. In PAO1, it is located near a cluster of three t-RNA genes (PA2819.1–3), a region of the genome that is highly variable in P. aeruginosa strains of different origin. Analysis of this chromosomal region by PCR and sequencing showed that three distinct gene sequences can occupy this locus (Fig. 7C). Five strains contain a sequence corresponding to that found in the PAO1 genome, including PA2818 and PA2819. The second island is found in PA14 and 12 other strains harbor sequences that are identical to the flanks of a DNA segment specifically found in the so-called clone C strains, which is highly prevalent in European isolates (27). Last, three strains contain sequences at the 5' end that are homologous to the DNA of P. aeruginosa Clone C and a sequence at their 3' homologous to PAGI-2, a genomic island found in one of the P. aeruginosa clone C strains (28). Neither Clone C DNA nor PAGI-2 DNA harbors ORFs encoding DGC or PDE proteins. Thus, in addition to the sequence present in PAO1, this location can apparently be occupied by one of two different genomic islands that share similarity with the ends of genetic elements found in PA14 and certain CF strains (27, 29). Therefore, PA2818 and PA2771 are located near sites of integration for multiple genomic islands.
Phenotypes of Transposon Mutants of DGC and PDE-Encoding Genes. The contribution of genes encoding DGC, PDE, and DGC-PDE proteins to TTSS-mediated cytotoxicity and biofilm formation, two virulence-associated traits of P. aeruginosa, was assessed. A library of mariner transposon insertions has been generated in strain PA14 and a substantial number (24,089) of the insertions precisely mapped by sequencing the junction of the transposon and the chromosome (30). A set of mutants was selected from the PA14 mutant transposon bank that had the highest BLAST scores of the respective junction sequences to the target DGC and PDE coding genes and the proximity of the insertions to the first codon. Table 2, which is published as supporting information on the PNAS web site, provides additional information about the PA14 genes, the identifiers of the insertion mutants, and a summary of the phenotypes examined in this study. This set of mutants, corresponding to insertions in all of the DGC, PDE, and DGC-PDE genes in the PA14 genome, was tested for their ability to abrogate TTSS-cytotoxicity. Insertions in PA3947 (rocR), PA4959 (fimX), and pvrR were defective for cytotoxicity, and insertion in PA3702 (wspR) caused a partial defect (Fig. 1A). These genes have been implicated in TTSS (10, 28, 31, 32). With several mutants (rocR, fimX, and pvrR), we observed that the phenotypes of corresponding deletion mutants did not agree with those generated by transposon insertions. A more detailed analysis of the cytotoxicity phenotypes of fimX and rocR is described in Supporting Text and Fig. 8, which are published as supporting information on the PNAS web site.
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Analysis of Overexpression of Genes Encoding Proteins with DGC and PDE Domains.
Proteins with DGC and PDE domains were overexpressed in P. aeruginosa PA14 by using vectors with the inducible tac promoter to assess the consequences of alterations in c-di-GMP levels. After the growth of these strains in isopropyl 
-D-thiogalactoside-containing media, the ability to kill CHO cells and form biofilm was determined (Fig. 2A). A number of overexpressing clones showed a significant impairment of cytotoxicity, without interfering with the ability of these bacteria to form biofilm. PA14, overexpressing PA2133, PA2200, PA3947, PA4367, PA4396, PA5017, and PA5442, were significantly impaired in their ability to kill CHO cells. All of these genes, with the exception of PA4396, encode proteins with a single PDE domain or DGC-PDE. Thus, the reduced cytotoxicity of these overexpressing clones is most likely a consequence of reduced c-di-GMP levels.
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Enzymatic Activities of Proteins with DGC and PDE Domains. We used reverse-phase HPLC to assess whether proteins with predicted DGC and PDE domains specify enzymes capable of formation or hydrolysis of c-di-GMP, respectively (Fig. 3, Table 1). To detect DGC activity, cultures overexpressing genes that encode DGC-containing proteins were extracted and analyzed for c-di-GMP content (Fig. 3A). We detected c-di-GMP in extracts of 7 of 17 strains expressing predicted DGCs and in one strain expressing a DGC-PDE protein (PA1727) with both a DGC and a PDE domain. The ability of the PDE-containing proteins to hydrolyze c-di-GMP was tested by adding synthetic c-di-GMP to lysates of strains overexpressing these proteins and measuring the disappearance of the dinucleotide from the reaction mixture (Fig. 3B, Table 1). c-di-GMP hydrolysis was detected in all but one strain expressing genes encoding PDE enzymes and none in genes encoding DGC-PDE.
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Contribution to Virulence During Acute Infection. To assess whether mutations in genes encoding DGC and PDE influence virulence, we tested several of the mutants with biofilm or cytotoxicity phenotypes in a murine thermal injury model (34). For comparison, we also included mutants in various virulence factors (fliC, pscC, pilA, and xcpQ) and selected an additional transposon mutant (PA5295) that lacks a phenotype in the biofilm and cytotoxicity assays. When thermally injured mice were infected with wild-type PA14, almost 80% died by the second day after infection (Fig. 4). No attenuation of virulence was seen in the mutants that lacked the major subunit of the type IV pilus adhesin (pilA) or were defective in type II secretion (xcpQ). Insertions into PA3947 (rocR), pvrR, and fliC completely abolished the ability of PA14 to cause lethal infection. Similarly, the virulence phenotypes of mutants in PA5295 or the biofilm-defective PA5487 was identical to wild type. Mutations in PA4332, PA3311, and PA5017 all exhibited an intermediate virulence phenotype similar to the TTSS mutant in pscC, even in the presence of demonstrable CHO cell cytotoxicity. The virulence phenotypes of mutants in PA4959 (fimX) and in PA3702 (wspR) were identical to wild type, despite a strong or partial defect in CHO cell cytotoxicity, respectively. The lack of correlation between cytotoxicity and virulence in mice suggests that some of the genes encoding DGCs and PDEs play a broader role in acute infections that may not involve TTSS.
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Examination of phenotypes of DGC, PDE, and DGC-PDE transposon insertion mutants revealed that several insertions have biofilm-related phenotypes, either a loss of the ability to form biofilm on solid surfaces or a so-called hyperbiofilm phenotype (pellicle producing). Other transposon insertions altered the ability of P. aeruginosa to intoxicate mammalian cells via the TTSS pathway. Mutation of pvrR, encoding a putative PDE, or fimX, which encodes a protein with relatively degenerate DGC and PDE domains, also compromised biofilm formation and the ability to kill CHO cells, a phenotype similar to a pilA mutant (lacking type IV pili). FimX-mediated twitching motility appears to be required for biofilm formation but is dispensable for cytotoxicity (Figs. 1B and 8).
We have also performed a comprehensive analysis of phenotypes of P. aeruginosa overexpressing each PDE and DGC domain and attempted to correlate these with measurements of c-di-GMP synthesis or degradation. In many instances, we observed multilayer pellicles that were associated with overexpression of enzymes containing a DGC module but not a PDE domain, although overexpression of PA1727, containing both PDE and DGC domains, consistently showed an enhanced ability to form biofilm. In general, we were able to correlate pellicle formation with high levels of c-di-GMP. PA1727 was the only bimodular (DGC-PDE) enzyme that showed elevated levels of c-di-GMP. In all, these data suggest that surface changes associated with enhanced ability to form biofilm depend at least in part on an increase in levels of c-di-GMP. Moreover, the ability of P. aeruginosa to form pellicles, presumably a consequence of production of surface components that promote interbacterial adherence, does not interfere with cytotoxicity, which requires attachment via the type IV pili and TTSS injection of effectors.
One of the more striking outcomes of the comprehensive analysis of DGCs and PDEs encoded in the P. aeruginosa genome is that they do not appear to be redundant, because related mutants have different phenotypes when grown under identical conditions. Although all of these enzymes presumably regulate cellular levels of c-di-GMP, the phenotypes associated with mutating particular DGC or PDE genes do not strictly correlate with alterations in the levels of this dinucleotide. For example, P. aeruginosa expressing PA2870 and PA3343, two DGCs that produce high levels of c-di-GMP in our assay (Table 1), do not cause an alteration in the biofilm phenotype. Therefore, simple changes in c-di-GMP levels cannot explain the observed effect on biofilm, cytotoxicity, or any other biological function in which this dinucleotide participates. The distinct specificity of each enzyme may be manifested locally, where the production or hydrolysis of c-di-GMP is intimately related to its site of activity.
Finally, we determined that mutations in genes encoding DGC and PDE result in distinct phenotypes in a P. aeruginosa murine model of burn-wound infection. In this acute infection model, initiation of biofilm formation is apparently not important, as indicated by the lack of attenuation of the pilA mutant. In contrast, hyperbiofilm formation (pellicle) may reduce the virulence capacity of P. aeruginosa. It is also conceivable that, in tissues of infected animals, these proteins control novel virulence traits. The next challenge is to elucidate the precise sites of activity of c-di-GMP, which would provide clues about the function of DGCs and PDEs in bacterial physiology, adaptation to environmental niches, and virulence.
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CHO Cell Cytotoxicity Assay. The cytotoxic activity of P. aeruginosa transposon mutants was measured by release of lactate dehydrogenase (LDH) from CHO after exposure to bacteria, as described (35). Briefly, CHO cells were washed and covered with F-12 media containing 1% FBS and 2 mM glutamine. CHO cells were infected with mid-log P. aeruginosa at an initial multiplicity of infection of 10. Culture supernatants were collected at the indicated times, and LDH released from cells was measured by using a Roche Applied Science LDH kit, per the manufacturer’s instructions. Three independent assays were carried out for each strain.
Murine Model of Burn-Wound Infection. Thermal injury and infection were carried out as described (36, 37). Briefly, mice were infected with 2 x 106 wild-type and mutant P. aeruginosa, and the surviving mice were counted daily. At this dose, 10% of mice infected with wild-type P. aeruginosa PA14 survived at day 5.
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Abbreviations: c-di-GMP, bis-(3'-5')-cyclic dimeric GMP; DGC, diguanyl cyclase; PDE, phosphodiesterase; TTSS, type III secretion system; CHO, Chinese hamster ovary; LDH, lactate dehydrogenase; DGC-PDE, diguanyl cyclase-phosphoesterase fusion.
**To whom correspondence may be addressed. E-mail: ausubel{at}molbio.mgh.harvard.edu or stephen_lory{at}hms.harvard.edu
H.K. and V.L. contributed equally to this work. 
Author contributions: H.K., V.L., A.B., and S.L. designed research; H.K., V.L., A.B., and A.N.N. performed research; N.L., J.U., S.M., D.G.L., M.H., Y.H., and F.M.A. contributed new reagents/analytic tools; H.K., V.L., A.B., and S.L. analyzed data; and H.K., V.L., A.B., F.M.A., and S.L. wrote the paper.
Conflict of interest statement: No conflicts declared.
© 2006 by The National Academy of Sciences of the USA
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