The sensor kinase KinB regulates virulence in acute Pseudomonas aeruginosa infection

Two-component sensors are widely used by bacteria to sense and respond to the environment. Pseudomonas aeruginosa has one of the largest sets of two-component sensors known in bacteria, which likely contributes to its unique ability to adapt to multiple environments, including the human host. Several of these two-component sensors, such as GacS and RetS, have been shown to play roles in virulence in rodent infection models. However, the role and function of the majority of these two-component sensors remain unknown. Danio rerio is a recently characterized model host for pathogenesis-related studies that is amenable to higher-throughput analysis than mammalian models. Using zebrafish embryos as a model host, we have systematically tested the role of 60 two-component sensors and identified 6 sensors that are required for P. aeruginosa virulence. We found that KinB is required for acute infection in zebrafish embryos and regulates a number of virulence-associated phenotypes, including quorum sensing, biofilm formation, and motility. Its regulation of these phenotypes is independent of its kinase activity and its known response regulator AlgB, suggesting that it does not fit the canonical two-component sensor-response regulator model.     

The sensor kinase CbrA is a global regulator that modulates metabolism, virulence, and antibiotic resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that possesses a large arsenal of virulence factors enabling the pathogen to cause serious infections in immunocompromised patients, burn victims, and cystic fibrosis patients. CbrA is a sensor kinase that has previously been implied to play a role with its cognate response regulator CbrB in the metabolic regulation of carbon and nitrogen utilization in P. aeruginosa. Here it is demonstrated that CbrA and CbrB play an important role in various virulence and virulence-related processes of the bacteria, including swarming, biofilm formation, cytotoxicity, and antibiotic resistance. The cbrA deletion mutant was completely unable to swarm while exhibiting an increase in biofilm formation, supporting the inverse regulation of swarming and biofilm formation in P. aeruginosa. The cbrA mutant also exhibited increased cytotoxicity to human lung epithelial cells as early as 4 and 6 h postinfection. Furthermore, the cbrA mutant demonstrated increased resistance toward a variety of clinically important antibiotics, including polymyxin B, ciprofloxacin, and tobramycin. Microarray analysis revealed that under swarming conditions, CbrA regulated the expression of many genes, including phoPQ, pmrAB, arnBCADTEF, dnaK, and pvdQ, consistent with the antibiotic resistance and swarming impairment phenotypes of the cbrA mutant. Phenotypic and real-time quantitative PCR (RT-qPCR) analyses of a PA14 cbrB mutant suggested that CbrA may be modulating swarming, biofilm formation, and cytotoxicity via CbrB and that the CrcZ small RNA is likely downstream of this two-component regulator. However, as CbrB did not have a resistance phenotype, CbrA likely modulates antibiotic resistance in a manner independent of CbrB.     

Carbamate kinase from Pseudomonas aeruginosa: purification, characterization, physiological role, and regulation.

Pseudomonas aeruginosa PAO1 possessed a carbamate kinase (CKase) distinct from carbamoylphosphate synthetase as well as from a constitutive acetate kinase which also catalyzes the phosphorylation of ADP by carbamoylphosphate. CKase was purified to homogeneity. Polyacrylamide gel electrophoresis of cross-linked CKase in the presence of sodium dodecyl sulfate showed that the enzyme consists of two subunits with identical molecular weights (37,000). The optimal pH of enzyme activity is 7.0. The double-reciprocal plot for carbamoylphosphate was linear at 2 mM ADP, yielding an apparent Km of 5 mM. However, at 0.25 mM ADP, the plot was concave upward, and a Hill plot of the data yielded a coefficient of 1.4. This apparent cooperativity at low ADP concentrations might serve to reduce the extent of catabolism of carbamoylphosphate under growth conditions yielding high energy charge. Experiments on the regulation of synthesis under various growth conditions showed a response to three regulatory signals: CKase was induced to high levels by anaerobiosis, induced to moderate levels by arginine, and repressed by ammonia. Thus, CKase expression is regulated in a manner that allows the enzyme to function as a provider of ammonia under aerobic conditions and of ATP under anaerobic conditions. ATP was an effective inhibitor of CKase activity; this inhibition provides the cell with an effective mechanism for avoiding a futile cycle resulting from the simultaneous operation of CKase and carbamoylphosphate synthetase when cells are grown in the presence of exogenous arginine.     

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.     

Secondary-site mutation restores the transport defect caused by the transmembrane domain mutation of the xenobiotic transporter MexB in Pseudomonas aeruginosa

It has been suggested that the MexB subunit of the MexAB-OprM efflux transporter of Pseudomonas aeruginosa exports xenobiotics in an energy-dependent manner. To investigate the role of the transmembrane segments (TMS) of MexB in the transporter activity, we isolated 24 spontaneous mutants showing hypersusceptibility to antibiotics. Among them, three mutations were located at TMS-3, TMS-4, and TMS-10 having amino acid substitution Leu376vPro, Gly397vVal, and Val928vGly, respectively. A secondary mutation, which suppressed the defect caused by the Val928vGly mutation in TMS-10, was found at the 403rd amino acid residue in TMS-4 with a change of glycine to serine, suggesting that TMS-4 and TMS-10 may be in close proximity. This result provided strong support for the recent notion that negatively charged residues in TMS-4 might form a salt-bridge with a positive charge in TMS-10 (Guan, L., and Nakae, T. (2001) J. Bacteriol. 183, 1734-1739). The transporter function impaired by the Gly397vVal mutation in TMS-4 was recovered by the secondary mutation, Gln998vHis, in the loop between TMS-11 and TMS-12, thereby suggesting that TMS-4 and TMS-11 or TMS-12 might also be in close proximity. Thus, it is most likely that TMS-4, TMS-10, and TMS-11 or TMS-12 are packed close three dimensionally.     

Adenylate kinase as a virulence factor of Pseudomonas aeruginosa

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.     

Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides 2.4.1.

Photosynthesis gene expression in Rhodobacter sphaeroides is controlled in part by the two-component (Prr) regulatory system composed of a membrane-bound sensor kinase (PrrB) and a response regulator (PrrA). Hydropathy profile-based computer analysis predicted that the PrrB polypeptide could contain six membrane-spanning domains at its amino terminus and a hydrophilic, cytoplasmic carboxyl terminus. Both the localization and the topology of the PrrB sensor kinase have been studied by generating a series of gene fusions with the Escherichia coli periplasmically localized alkaline phosphatase and the cytoplasmic beta-galactosidase. Eighteen prrB-phoA and five prrB-lacZ fusions were constructed and expressed in both E. coli and R. sphaeroides. Enzymatic activity assays and immunoblot analyses were performed to identify and to localize the different segments of PrrB in the membrane. The data obtained in E. coli generally correlated with the data obtained in R. sphaeroides and support the computer predictions. On the basis of the theoretical model and the results provided by these studies, a topological model for the membrane localization of the PrrB polypeptide is proposed.     

Animal protection and structural studies of a consensus sequence vaccine targeting the receptor binding domain of the type IV pilus of Pseudomonas aeruginosa

One of the main obstacles in the development of a vaccine against Pseudomonas aeruginosa is the requirement that it is protective against a wide range of virulent strains. We have developed a synthetic-peptide consensus-sequence vaccine (Cs1) that targets the host receptor-binding domain (RBD) of the type IV pilus of P. aeruginosa. Here, we show that this vaccine provides increased protection against challenge by the four piliated strains that we have examined (PAK, PAO, KB7 and P1) in the A.BY/SnJ mouse model of acute P. aeruginosa infection. To further characterize the consensus sequence, we engineered Cs1 into the PAK monomeric pilin protein and determined the crystal structure of the chimeric Cs1 pilin to 1.35 Å resolution. The substitutions (T130K and E135P) used to create Cs1 do not disrupt the conserved backbone conformation of the pilin RBD. In fact, based on the Cs1 pilin structure, we hypothesize that the E135P substitution bolsters the conserved backbone conformation and may partially explain the immunological activity of Cs1. Structural analysis of Cs1, PAK and K122-4 pilins reveal substitutions of non-conserved residues in the RBD are compensated for by complementary changes in the rest of the pilin monomer. Thus, the interactions between the RBD and the rest of the pilin can either be mediated by polar interactions of a hydrogen bond network in some strains or by hydrophobic interactions in others. Both configurations maintain a conserved backbone conformation of the RBD. Thus, the backbone conformation is critical in our consensus-sequence vaccine design and that cross-reactivity of the antibody response may be modulated by the composition of exposed side-chains on the surface of the RBD. This structure will guide our future vaccine design by focusing our investigation on the four variable residue positions that are exposed on the RBD surface.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Rer1, a putative transmembrane domain receptor responsible for targeting proteins to the endoplasmic reticulum

Summary Localization of resident proteins provides identity to subcellular compartments. Most proteins depend on a combination of both retention and retrieval to maintain their steady-state distribution. Rerl is a putative receptor protein mediating retrieval of membrane proteins of the endoplasmic reticulum. This retrieval relies on an unusual hydrophobic target sequence, the transmembrane domain. Apart from Rerl, coatomer is also required to retrieve escaped membrane proteins from the early Golgi region back to the endoplasmic reticulum. Current evidence suggests that the Rerl-mediated retrieval of membrane proteins is a general sorting pathway in eukaryotic cells contributing to the maintenance of compartmental identity in the early secretory pathway.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

Characteristics, isolation and role in the infectious process of Pseudomonas aeruginosa protease

Pseudomonas aeruginosa produces the extracellular enzyme protease, which plays an important role in the development of the infectious process caused by this microorganism. Protease is produced in three types, I, II and III, with protease II being responsible for 75% of the total proteolytic activity of protease. The molecular mass of protease II has been determined by different methods; the values obtained are 23000 and 39500. This discrepancy may be associated with an autodigestion of the enzyme or with the presence in the periplasm of its producer of a nonactive precursor whose activation may lead to a change in the molecular mass. Pseudomonas aeruginosa protease is capable of cleaving high-molecular proteins into low-molecular ones, which are taken up by the microbial cell and serve as a source of nutrition. When injected into the bloodstream of animals, purified protease produces haemorrhagic lesions in internal organs; its subcutaneous injection provokes haemorrhage in the skin and subcutaneous tissues. Manifestation of high P. aeruginosa virulence on a model of burnt mouse skin requires that not only exotoxin A but also protease be produced. The protease is immunogenic and has, in toxoid form, been used experimentally in a multicomponent vaccine.     

Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed beta-helix

The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of beta1-4-linked O-acetylated d-mannuronate (M) and l-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed beta-helix (RHbetaH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHbetaH of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RHbetaH from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RHbetaH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHbetaH protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second d-mannuronate residue to l-guluronate along the epimerase catalytic face.     

A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa.

A number of Gram-negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium. The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated. A series of exotoxin A truncates, fused to beta-lactamase, was constructed. Hybrid proteins, which carry at their N- termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium. Hybrid proteins which carry residues 1-30 and 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period. A hybrid with beta-lactamase was constructed which carried only the N-terminal residues 1-3 and region 60-120 of exotoxin A. It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane. The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A-murine interleukin 4 hybrid protein was also secreted. The extracellular targeting signal between amino acids 60 and 120 is rich in anti-parallel beta-sheets. It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells. In the three- dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery.     

Nuclear trafficking of FGFR1: a role for the transmembrane domain

Several members of the fibroblast growth factor (FGF) family lack signal peptide (SP) sequences and are present only in trace amounts outside the cell. However, these proteins contain nuclear localization signals (NLS) and accumulate in the cell nucleus. Our studies have shown that full length FGF receptor 1 (FGFR1) accumulates within the nuclear interior in parallel with FGF-2. We tested the hypothesis that an atypical transmembrane domain (TM) plays a role in FGFR1 trafficking into the nuclear interior. With FGFR1 destined for constitutive fusion with the plasma membrane due to its SP, how the receptor may enter the nucleus is unclear. Sequence analysis identified that FGFR1 has an atypical TM containing short stretches of hydrophobic amino acids (a.a.) interrupted by polar a.a. The beta-sheet is the predicted conformation of the FGFR1 TM, in contrast to the alpha-helical conformation of other single TM tyrosine kinase receptors, including FGFR4. Receptor trafficking in live cells was studied by confocal microscopy via C-terminal FGFR1 fusions to enhanced green fluorescent protein (EGFP) and confirmed by subcellular fractionation and Western immunoblotting. Nuclear entry of FGFR1-EGFP was independent of karyokinessis, and was observed in rapidly proliferating human TE671 cells, in slower proliferating glioma SF763 and post-mitotic bovine adrenal medullary cells (BAMC). In contrast, a chimeric FGFR1/R4-EGFP, where the TM of FGFR1 was replaced with that of FGFR4, was associated with membranes (golgi-ER, plasma, and nuclear), but was absent from the nucleus and cytosol. FGFR1delta-EGFP mutants, with hydrophobic TM a.a. replaced with polar a.a., showed reduced association with membranes and increased cytosolic/nuclear accumulation with an increase in TM hydrophilicity. FGFR1(TM-)-EGFP (TM deleted), was detected in the golgi-ER vesicles, cytosol, and nuclear interior; thus demonstrating that the FGFR1 TM does not function as a NLS. To test whether cytosolic FGFR1 provides a source of nuclear FGFR1, cells were transfected with FGFR1(SP-) (SP was deleted), resulting in cytosolic, non-membrane, protein accumulation in the cytosol and the cell nucleus. Our results indicate that an unstable association with cellular membranes is responsible for the release of FGFR1 into the cytosol and cytosolic FGFR1 constitutes the source of the nuclear receptor.