Crystal structure and regulation mechanisms of the CyaB adenylyl cyclase from the human pathogen Pseudomonas aeruginosa.
ABSTRACT: Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a major cause of healthcare-associated infections. While the organism's intrinsic and acquired resistance to most antibiotics hinders treatment of P. aeruginosa infections, the regulatory networks controlling its virulence provide novel targets for drug development. CyaB, a key regulator of P. aeruginosa virulence, belongs to the Class III adenylyl cyclase (AC) family of enzymes that synthesize the second messenger cyclic adenosine 3',5'-monophosphate. These enzymes consist of a conserved catalytic domain fused to one or more regulatory domains. We describe here the biochemical and structural characterization of CyaB and its inhibition by small molecules. We show that CyaB belongs to the Class IIIb subfamily, and like other subfamily members, its activity is stimulated by inorganic carbon. CyaB is also regulated by its N-terminal MASE2 (membrane-associated sensor 2) domain, which acts as a membrane anchor. Using a genetic screen, we identified activating mutations in CyaB. By solving the crystal structure of the CyaB catalytic domain, we rationalized the effects of these mutations and propose that CyaB employs regulatory mechanisms similar to other Class III ACs. The CyaB structure further indicates subtle differences compared to other Class III ACs in both the active site and the inhibitor binding pocket. Consistent with these differences, we observed a unique inhibition profile, including identification of a CyaB selective compound. Overall, our results reveal mechanistic details of the physiological and pharmacological regulation of CyaB and provide the basis for its exploitation as a therapeutic drug target.
Project description:Infections caused by the opportunistic pathogen Pseudomonas aeruginosa involve the interplay of several bacterial virulence factors. It has recently been established that the delivery of toxic effector proteins by the type III secretion system is an important virulence mechanism in several animal models. Furthermore, the expression of the type III secretion system and its effectors has been correlated with a poor clinical outcome during human infections. A novel cyclic AMP (cAMP) regulatory network that controls the expression of virulence factors, including the type III secretion system, was examined to determine its contribution to P. aeruginosa colonization and dissemination in a mouse pneumonia model. Mutants lacking the two genome-encoded adenylate cyclases, CyaA and CyaB, and the cAMP-dependent regulator Vfr were examined. Based on the enumeration of bacteria in lungs, livers, and spleens, as well as the assessment of mouse lung pathology, mutations in the cyaB and vfr genes resulted in a more significantly attenuated phenotype than mutations in cyaA. Moreover, in this model, expression of the type III secretion system was essential for lung colonization and pathology. Strains with mutations in the exsA gene, which encodes a type III regulatory protein, or pscC, which encodes an essential component of the secretion apparatus, were also significantly attenuated. Finally, we demonstrate that virulence can be restored in an adenylate cyclase mutant by the overexpression of exsA, which specifically restores expression of the type III secretion system in the absence of a functional cAMP-dependent regulatory network.
Project description:The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [Keq] of ?2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers.IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.
Project description:The type III secretion system (T3SS) plays an important role in the pathogenesis of <i>Pseudomonas aeruginosa</i>. Expression of the T3SS is controlled under a complicate regulatory network. In this study, we demonstrate that NrtR (PA4916) is involved in the T3SS expression and pathogenesis of <i>P. aeruginosa</i> in a mouse acute pneumonia model. Overexpression of the T3SS central activator ExsA or exogenous supplementation of cAMP restored the expression of T3SS in the ?<i>nrtR</i> mutant, suggesting that NrtR might regulate T3SS through the cAMP-Vfr signaling pathway. Further experiments demonstrated that the decrease of cAMP content is not due to the expression change of adenylate cyclases or phosphodiesterase in the ?<i>nrtR</i> mutant. As it has been shown that <i>nadD2</i> is upregulated in the ?<i>nrtR</i> mutant, we overexpressed <i>nadD2</i> in wild type PAK, which reduced the intracellular cAMP level and the expression of the T3SS genes. Meanwhile, deletion of <i>nadD2</i> in the ?<i>nrtR</i> mutant restored the expression and secretion of the T3SS. Co-immunoprecipitation assay revealed an interaction between NadD2 and the catalytic domain of the adenylate cyclase CyaB. Further <i>in vitro</i> assay indicated that NadD2 repressed the enzymatic activity of CyaB. Therefore, we have identified a novel regulatory mechanism of T3SS in <i>P. aeruginosa</i>.
Project description:Pseudomonas aeruginosa, a ubiquitous bacteria found in diverse ecological niches, is an important cause of acute infections in immunocompromised individuals and chronic infections in patients with Cystic Fibrosis. One signaling molecule required for the coordinate regulation of virulence factors associated with acute infections is 3', 5'-cyclic adenosine monophosphate, (cAMP), which binds to and activates a catabolite repressor homolog, Vfr. Vfr controls the transcription of many virulence factors, including those associated with Type IV pili (TFP), the Type III secretion system (T3SS), the Type II secretion system, flagellar-mediated motility, and quorum sensing systems. We previously identified FimL, a protein with histidine phosphotransfer-like domains, as a regulator of Vfr-dependent processes, including TFP-dependent motility and T3SS function. In this study, we carried out genetic and physiologic studies to further define the mechanism of action of FimL. Through a genetic screen designed to identify suppressors of FimL, we found a putative cAMP-specific phosphodiesterase (CpdA), suggesting that FimL regulates cAMP levels. Inactivation of CpdA increases cAMP levels and restores TFP-dependent motility and T3SS function to fimL mutants, consistent with in vivo phosphodiesterase activity. By constructing combinations of double and triple mutants in the two adenylate cyclase genes (cyaA and cyaB), fimL, and cpdA, we show that ?fimL mutants resemble ?cyaB mutants in TM defects, decreased T3SS transcription, and decreased cAMP levels. Similar to some of the virulence factors that they regulate, we demonstrate that CyaB and FimL are polarly localized. These results reveal new complexities in the regulation of diverse virulence pathways associated with acute P. aeruginosa infections.
Project description:Complementation of an Escherichia coli cya mutant with a genomic library from Aeromonas hydrophila allowed isolation of clones containing two different cya genes. Whereas one of these genes (cyaA) coded for an adenylyl cyclase (AC1) belonging to the previously described class I adenylyl cyclases (ACs), the second one (cyaB) coded for a protein (AC2) that did not match any previously characterized protein when compared to protein sequence databases. In particular, it did not align with any of members of the three known classes of ACs. The purified AC2 enzyme exhibited remarkable biochemical characteristics, namely, an optimum activity at a high temperature (65 degrees C) and at an alkalinic pH (9.5). In order to investigate the functions of both cyclases in A. hydrophila, each gene was inactivated in the chromosome and the resulting mutant strains were examined for physiological alterations. It was shown that, in contrast to cyaA, the cyaB gene was not expressed under usual laboratory growth conditions. However, introduction of a plasmid harboring the cyaB gene in a cyaA mutant, as well as in a cyaA cyaB mutant, allowed cyclic AMP production. AC2 is the first member of a new class of previously unrecognized ACs, and to date, no functional counterpart has been demonstrated in other organisms. However, scanning databases revealed a significant similarity between AC2 and the gene product of three hyperthermophilic archaebacteria: Methanobacterium thermoautotrophicum, Archaeglobus fulgidus, and Methanococcus jannaschii. The possibility of a gene transfer between such phylogenetically divergent bacteria is discussed.
Project description:Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3', 5'-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems.
Project description:Pseudomonas aeruginosa is an important human pathogen which uses the type III secretion system (T3SS) as a primary virulence factor to establish infections in humans. The results presented in this report revealed that the ATP-binding protein PA4595 (named ArtR, a Regulator that is an ATP-activated Repressor of T3SS) represses T3SS expression in P. aeruginosa. The expression of T3SS genes, including exoS, exoY, exoT, exsCEBA, and exsD-pscB-L, increased significantly when artR was knockout. The effect of ArtR on ExsA is at the transcriptional level, not at the translational level. The regulatory role and cytoplasm localization of ArtR suggest it belongs to the REG sub-family of ATP-binding cassette (ABC) family. Purified GST-tagged ArtR showed ATPase activity in vitro. The conserved aspartate residues in the dual Walker B motifs prove to be essential for the regulatory function of ArtR. The regulation of T3SS by ArtR is unique, which does not involve the known GacS/A-RsmY/Z-RsmA-ExsA pathway or Vfr. This is the first REG subfamily of ATP-binding cassette that is reported to regulate T3SS genes in bacteria. The results specify a novel player in the regulatory networks of T3SS in P. aeruginosa.
Project description:UNLABELLED:FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE:FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.
Project description:FimV is a <i>Pseudomonas aeruginosa</i> inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds-lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB-<i>pilG</i> and <i>fimV</i> mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in <i>P. aeruginosa</i> twitching motility.<b>IMPORTANCE</b><i>Pseudomonas aeruginosa</i> is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements-including the PilSR two-component system-to cell poles for optimal function.
Project description:Pseudomonas aeruginosa is an important human pathogen which uses the type III secretion system 21 (T3SS) as a primary virulence factor to establish infections in humans. The results presented in this 22 report revealed that the ATP-binding protein PA4595 (named ArtR, a Regulator that is an ATP-activated 23 Repressor of T3SS) represses T3SS expression in P. aeruginosa. The expression of T3SS genes, 24 including exoS, exoY, exoT, exsCEBA and exsD-pscB-L, increased significantly when artR was 25 knockout. The effect of ArtR on ExsA is at the transcriptional level, not at the translational level. The 26 regulatory role and cytoplasm localization of ArtR suggest it belongs to the REG sub-family of 27 ATP-binding cassette (ABC) family. Purified GST-tagged ArtR showed ATPase activity in vitro. The 28 conserved aspartate residues in the dual Walker B motifs prove to be essential for the regulatory 29 function of ArtR. The regulation of T3SS by ArtR is unique, which does not involve the known 30 GacS/A-RsmY/Z-RsmA-ExsA pathway or Vfr. This is the first REG subfamily of ATP-binding 31 cassette that is reported to regulate T3SS genes in bacteria. The results specify a novel player in the 32 regulatory networks of T3SS in P. aeruginosa. Overall design: RNA-seq for PA4595