Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression.

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P. aeruginosa dominates over other pathogens to cause worsening disease. Here, we show that P. aeruginosa responds to dynamic changes in iron concentration, often associated with viral infection and pulmonary exacerbations, to become more competitive via expression of the TseT toxic effector. However, this behavior can be therapeutically targeted using the iron chelator deferiprone to block TseT expression and competition. Overall, we find that iron concentration and TseT expression significantly correlate with microbial diversity in the respiratory tract of people with CF. These findings improve our understanding of how P. aeruginosa becomes increasingly dominant with age in people with CF and provide a therapeutically targetable pathway to help prevent this shift.

Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.

Commentary on Variants in the ABCC6 Gene Implicated in Pseudoxanthoma Elasticum, a Heritable Ectopic Mineralization Disorder.

Significant progress has been made in understanding pseudoxanthoma elasticum (PXE), which results from mutations in ABCC6. The low prevalence of PXE and its heterotypic presentation confound genotype-phenotype correlations and the characterization of many identified variants. Kowal et al. (2022) present an in vivo model to characterize and annotate ABCC6 variants, establishing a novel system for allele annotation in the patient population.

Serralysin family metalloproteases protects Serratia marcescens from predation by the predatory bacteria Micavibrio aeruginosavorus.

Micavibrio aeruginosavorus is an obligate Gram-negative predatory bacterial species that feeds on other Gram-negative bacteria by attaching to the surface of its prey and feeding on the prey's cellular contents. In this study, Serratia marcescens with defined mutations in genes for extracellular cell structural components and secreted factors were used in predation experiments to identify structures that influence predation. No change was measured in the ability of the predator to prey on S. marcescens flagella, fimbria, surface layer, prodigiosin and phospholipase-A mutants. However, higher predation was measured on S. marcescens metalloprotease mutants. Complementation of the metalloprotease gene, prtS, into the protease mutant, as well as exogenous addition of purified serralysin metalloprotease, restored predation to wild type levels. Addition of purified serralysin also reduced the ability of M. aeruginosavorus to prey on Escherichia coli. Incubating M. aeruginosavorus with purified metalloprotease was found to not impact predator viability; however, pre-incubating prey, but not the predator, with purified metalloprotease was able to block predation. Finally, using flow cytometry and fluorescent microscopy, we were able to confirm that the ability of the predator to bind to the metalloprotease mutant was higher than that of the metalloprotease producing wild-type. The work presented in this study shows that metalloproteases from S. marcescens could offer elevated protection from predation.

Structural analysis reveals pathomechanisms associated with pseudoxanthoma elasticum-causing mutations in the ABCC6 transporter.

Mutations in ABC subfamily C member 6 (ABCC6) transporter are associated with pseudoxanthoma elasticum (PXE), a disease resulting in ectopic mineralization and affecting multiple tissues. A growing number of mutations have been identified in individuals with PXE. For most of these variants, no mechanistic information is available regarding their role in normal and pathophysiologies. To assess how PXE-associated mutations alter ABCC6 biosynthesis and structure, we biophysically and biochemically evaluated the N-terminal nucleotide-binding domain. A high-resolution X-ray structure of nucleotide-binding domain 1 (NBD1) of human ABCC6 was obtained at 2.3 Å that provided a template on which to evaluate PXE-causing mutations. Biochemical analysis of mutations in this domain indicated that multiple PXE-causing mutations altered its structural properties. Analyses of the full-length protein revealed a strong correlation between the alterations in NBD properties and the processing and expression of ABCC6. These results suggest that a significant fraction of PXE-associated mutations located in NBD1 causes changes in its structural properties and that these mutation-induced alterations directly affect the maturation of the full-length ABCC6 protein.

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF.

SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens.

Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS. This gene was named serralysin-like protease E, SlpE, and was found in the majority (67%) of tested clinical isolates, but was absent from most tested non-clinical isolates including the insect pathogen and reference S. marcescens strain Db11. Purified recombinant SlpE exhibited calcium-dependent protease activity similar to metalloproteases PrtS and SlpB. Induction of slpE in the low-protease-producing S. marcescens strain PIC3611 highly elevated extracellular protease activity, and extracellular secretion required the lipD type 1 secretion system gene. Transcription of slpE was highly reduced in an eepR transcription factor mutant. Mutation of the slpE gene in a highly proteolytic clinical isolate reduced its protease activity, and evidence suggests that SlpE confers cytotoxicity of S. marcescens to the A549 airway carcinoma cell line. Together, these data reveal SlpE to be an EepR-regulated cytotoxic metalloprotease associated with clinical isolates of an important opportunistic pathogen.

Stabilization of Nucleotide Binding Domain Dimers Rescues ABCC6 Mutants Associated with Pseudoxanthoma Elasticum.

ABC transporters are polytopic membrane proteins that utilize ATP binding and hydrolysis to facilitate transport across biological membranes. Forty-eight human ABC transporters have been identified in the genome, and the majority of these are linked to heritable disease. Mutations in the ABCC6 (ATP binding cassette transporter C6) ABC transporter are associated with pseudoxanthoma elasticum, a disease of altered elastic properties in multiple tissues. Although ∼200 mutations have been identified in pseudoxanthoma elasticum patients, the underlying structural defects associated with the majority of these are poorly understood. To evaluate the structural consequences of these missense mutations, a combination of biophysical and cell biological approaches were applied to evaluate the local and global folding and assembly of the ABCC6 protein. Structural and bioinformatic analyses suggested that a cluster of mutations, representing roughly 20% of the patient population with identified missense mutations, are located in the interface between the transmembrane domain and the C-terminal nucleotide binding domain. Biochemical and cell biological analyses demonstrate these mutations influence multiple steps in the biosynthetic pathway, minimally altering local domain structure but adversely impacting ABCC6 assembly and trafficking. The differential impacts on local and global protein structure are consistent with hierarchical folding and assembly of ABCC6. Stabilization of specific domain-domain interactions via targeted amino acid substitution in the catalytic site of the C-terminal nucleotide binding domain restored proper protein trafficking and cell surface localization of multiple biosynthetic mutants. This rescue provides a specific mechanism by which chemical chaperones could be developed for the correction of ABCC6 biosynthetic defects.

Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation.

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys(447), obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.

Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens.

The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens.

Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions.

The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end, the ssa1-45 mutant has proved invaluable. However, the biochemical defects associated with the corresponding Ssa1-45 protein (P417L) are unknown. Consequently, we characterized Ssa1 P417L, as well as a P417S variant, which corresponds to a mutation in the gene encoding the yeast mitochondrial Hsp70. We discovered that the P417L and P417S proteins exhibit accelerated ATPase activity that was similar to the Hsp40-stimulated rate of ATP hydrolysis of wild-type Ssa1. We also found that the mutant proteins were compromised for peptide binding. These data are consistent with defects in peptide-stimulated ATPase activity and with results from limited proteolysis experiments, which indicated that the mutants' substrate binding domains were highly vulnerable to digestion. Defects in the reactivation of heat-denatured luciferase were also evident. Correspondingly, yeast expressing P417L or P417S as the only copy of Ssa were temperature sensitive and exhibited defects in Ssa1-dependent protein translocation and misfolded protein degradation. Together, our studies suggest that the structure of the substrate binding domain is altered and that coupling between this domain and the nucleotide binding domain is disabled when the conserved P417 residue is mutated. Our data also provide new insights into the nature of the many cellular defects associated with the ssa1-45 allele.

Inducible polymerization and two-dimensional assembly of the repeats-in-toxin (RTX) domain from the Pseudomonas aeruginosa alkaline protease.

Self-assembling proteins represent potential scaffolds for the organization of enzymatic activities. The alkaline protease repeats-in-toxin (RTX) domain from Pseudomonas aeruginosa undergoes multiple structural transitions in the presence and absence of calcium, a native structural cofactor. In the absence of calcium, this domain is capable of spontaneous, ordered polymerization, producing amyloid-like fibrils and large two-dimensional protein sheets. This polymerization occurs under near-physiological conditions, is rapid, and can be controlled by regulating calcium in solution. Fusion of the RTX domain to a soluble protein results in the incorporation of engineered protein function into these macromolecular assemblies. Applications of this protein sequence in bacterial adherence and colonization and the generation of biomaterials are discussed.

Modulation of the epithelial sodium channel (ENaC) by bacterial metalloproteases and protease inhibitors.

The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence.

Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.

Revertant mutants modify, but do not rescue, the gating defect of the cystic fibrosis mutant G551D-CFTR.

Cystic fibrosis (CF) is caused by dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). One strategy to restore function to CF mutants is to suppress defects in CFTR processing and function using revertant mutations. Here, we investigate the effects of the revertant mutations G550E and 4RK (the simultaneous disruption of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K and R766K) on the CF mutant G551D, which impairs severely channel gating without altering protein processing and which affects a residue in the same α-helix as G550 and R555. Both G550E and 4RK augmented strongly CFTR-mediated iodide efflux from BHK cells expressing G551D-CFTR. To learn how revertant mutations influence G551D-CFTR function, we studied protein processing and single-channel behaviour. Neither G550E nor 4RK altered the expression and maturation of G551D-CFTR protein. By contrast, both revertants had marked effects on G551D-CFTR channel gating, increasing strongly opening frequency, while 4RK also diminished noticeably the duration of channel openings. Because G551D-CFTR channel gating is ATP independent, we investigated whether revertant mutations restore ATP dependence to G551D-CFTR. Like wild-type CFTR, the activity of 4RK-G551D-CFTR varied with ATP concentration, suggesting that 4RK confers some ATP dependence on the G551D-CFTR channel. Thus, the revertant mutations G550E and 4RK alter the gating pattern and ATP dependence of G551D-CFTR without restoring single-channel activity to wild-type levels. Based on their impact on the CF mutants F508del and G551D, we conclude that G550E and 4RK have direct effects on CFTR structure, but that their action on CFTR processing and channel function is CF mutation specific.

Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier-dependent pathway.

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27's ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4's impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin-proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.

Activation of the epithelial sodium channel (ENaC) by the alkaline protease from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that significantly contributes to the mortality of patients with cystic fibrosis. Chronic infection by Pseudomonas induces sustained immune and inflammatory responses and damage to the airway. The ability of Pseudomonas to resist host defenses is aided, in part, by secreted proteases, which act as virulence factors in multiple modes of infection. Recent studies suggest that misregulation of protease activity in the cystic fibrosis lung may alter fluid secretion and pathogen clearance by proteolytic activation of the epithelial sodium channel (ENaC). To evaluate the possibility that proteolytic activation of ENaC may contribute to the virulence of Pseudomonas, primary human bronchial epithelial cells were exposed to P. aeruginosa and ENaC function was assessed by short circuit current measurements. Apical treatment with a strain known to express high levels of alkaline protease (AP) resulted in an increase in basal ENaC current and a loss of trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To further characterize this AP-induced ENaC activation, AP was purified, and its folding, activity, and ability to activate ENaC were assessed. AP folding was efficient under pH and calcium conditions thought to exist in the airway surface liquid of normal and cystic fibrosis (CF) lungs. Short circuit measurements of ENaC in polarized monolayers indicated that AP activated ENaC in immortalized cell lines as well as post-transplant, primary human bronchial epithelial cells from both CF and non-CF patients. This activation was mapped to the γ-subunit of ENaC. Based on these data, patho-mechanisms associated with AP in the CF lung are proposed wherein secretion of AP leads to decreased airway surface liquid volume and a corresponding decrease in mucocilliary clearance of pulmonary pathogens.

Calcium-induced folding and stabilization of the Pseudomonas aeruginosa alkaline protease.

Pseudomonas aeruginosa is an opportunistic pathogen that contributes to the mortality of immunocompromised individuals and patients with cystic fibrosis. Pseudomonas infection presents clinical challenges due to its ability to form biofilms and modulate host-pathogen interactions through the secretion of virulence factors. The calcium-regulated alkaline protease (AP), a member of the repeats in toxin (RTX) family of proteins, is implicated in multiple modes of infection. A series of full-length and truncation mutants were purified for structural and functional studies to evaluate the role of Ca(2+) in AP folding and activation. We find that Ca(2+) binding induces RTX folding, which serves to chaperone the folding of the protease domain. Subsequent association of the RTX domain with an N-terminal α-helix stabilizes AP. These results provide a basis for the Ca(2+)-mediated regulation of AP and suggest mechanisms by which Ca(2+) regulates the RTX family of virulence factors.

Molecular modeling tools and approaches for CFTR and cystic fibrosis.

Cystic fibrosis is a multi-faceted disease resulting from the dysfunction of the CFTR channel. Understanding the structural basis of channel function and the structural origin of the defect is imperative in the development of therapeutic strategies. Here, we describe molecular modeling tools that, in conjunction with complementary experimental tools, lead to significant findings on CFTR channel function and on the effect of the pathogenic mutant F508del.