Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa.

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

Development of a multiplex PCR assay for the detection of metallo-beta-lactamase genes in Pseudomonas aeruginosa / Desarrollo de un PCR múltiple para la detección de genes metalo-beta-lactamasa en Pseudomonas aeruginosa

SUMMARY: The appearance of Pseudomonas aeruginosa strains with multi-resistance to antibiotics is a clinical problem of great relevance. The methods for detecting these resistances are laborious and slow, which is a complication when treating patients promptly. In this work, we developed a simple method for simultaneous detection of several carbapenem resistance genes using a multiplex PCR assay. The PCR assay developed, followed by electrophoretic separation of fragments, allows to simultaneously identify the presence of 6 antibiotic resistance genes: bla-VIM (261 bp), bla-IMP (587 bp), bla-SPM (648 bp), bla-GIM-1 (753 bp), bla-NDM-1 (813 bp) and bla-KPC (882 bp). We analyzed 7 clinical isolates of P. aeruginosa obtained in Chile, finding the resistance genes bla-VIM, bla-IMP, bla-SPM, bla-GIM, and bla-NDM in 5 of them. We found a perfect correlation between the detection of various resistance genes by PCR and the results obtained by antibiograms. Interestingly, 2 of the strains possessed 3 different resistance genes simultaneously. Finally, in this work, we found the presence of 3 genes never described before in clinical isolates of P. aeruginosa in Chile (bla-IMP, bla-SPM, and bla-GIM-1). We developed a rapid multiplex PCR test for the simultaneous detection of up to 6 antibiotic resistance genes of the metallo-β-lactamase family in P. aeruginosa.

La aparición de cepas de Pseudomonas aeruginosa con resistencias a diversos antibióticos es un problema clínico de gran relevancia. Los métodos de detección de dichas resistencias son laboriosos y lentos, lo que genera una complicación al momento de tratar a los pacientes oportunamente. En este trabajo desarrollamos un método simple de detección simultánea de varios genes de resistencia a carbapenem, mediante un sistema de PCR múltiple. El ensayo de PCR desarrollado, seguido de una separación electroforética de los amplicones, permite distinguir simultáneamente la presencia de 6 genes de resistencia a antibióticos: bla-VIM (261 pb), bla-IMP (587 pb), bla-SPM (648 pb), bla-GIM-1 (753 pb), bla-NDM-1 (813 pb) y bla-KPC (882 pb). Analizamos 7 aislados clínicos obtenidos en Chile, encontrando en 5 de ellos los genes de resistencia bla-VIM, bla-IMP, bla-SPM, bla-GIM y bla-NDM. Encontramos una perfecta correlación entre la detección de diversos genes de resistencia y los resultados obtenidos mediante antibiogramas. Interesantemente, 2 de las cepas mostraron poseer simultáneamente 3 genes de resistencia distintos. Por último, en este trabajo encontramos la presencia de 3 genes nunca antes descritos en aislados clínicos de P. aeruginosa en Chile (bla-IMP, bla-SPM y bla-GIM-1). Hemos desarrollado un test rápido de PCR múltiple, para la detección simultánea de hasta 6 genes de resistencia a antibióticos de la familia.a de las metallo-b-lactamases en P. aeruginosa.

Measurement of neutral ceramidase activity in vitro and in vivo.

Neutral ceramidase is a hydrolase of ceramide that has been implicated in multiple biologic processes, including inflammation and oncogenesis. Ceramides and other sphingolipids, belong to a family of N-acyl linked lipids that are biologically active in signaling, despite their limited structural functions. Ceramides are generally pro-apoptotic, while sphingosine and sphingosine-1-phosphate (S1P) exert proliferative and pro-oncogenic effects. Ceramidases are important regulators of ceramide levels that hydrolyze ceramide to sphingosine. Thus, ceramidase inhibition significantly increases the quantities of ceramide and its associated signaling. To better understand the function of ceramide, biochemical and cellular assays for enzymatic activity were developed and validated to identify inhibitors of human neutral ceramidase (nCDase). Here we review the measurement of nCDase activity both in vitro and in vivo.

Discovery of Novel Inhibitors of Uridine Diphosphate-N-Acetylenolpyruvylglucosamine Reductase (MurB) from Pseudomonas aeruginosa, an Opportunistic Infectious Agent Causing Death in Cystic Fibrosis Patients.

Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of Kd = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with Kd = 3.57 µM (LE 0.35).

The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein's role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.

Pseudomonas aeruginosa cytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that is highly prevalent in individuals with cystic fibrosis (CF). A major problem in treating CF patients infected with P. aeruginosa is the development of antibiotic resistance. Therefore, the identification of novel P. aeruginosa antibiotic drug targets is of the utmost urgency. The genome of P. aeruginosa contains four putative cytochrome P450 enzymes (CYPs) of unknown function that have never before been characterized. Analogous to some of the CYPs from Mycobacterium tuberculosis, these P. aeruginosa CYPs may be important for growth and colonization of CF patients' lungs. In this study, we cloned, expressed, and characterized CYP168A1 from P. aeruginosa and identified it as a subterminal fatty acid hydroxylase. Spectral binding data and computational modeling of substrates and inhibitors suggest that CYP168A1 has a large, expansive active site and preferentially binds long chain fatty acids and large hydrophobic inhibitors. Furthermore, metabolic experiments confirm that the enzyme is capable of hydroxylating arachidonic acid, an important inflammatory signaling molecule present in abundance in the CF lung, to 19-hydroxyeicosatetraenoic acid (19-HETE; Km = 41 µM, Vmax = 220 pmol/min/nmol P450), a potent vasodilator, which may play a role in the pathogen's ability to colonize the lung. Additionally, we found that the in vitro metabolism of arachidonic acid is subject to substrate inhibition and is also inhibited by the presence of the antifungal agent ketoconazole. This study identifies a new metabolic pathway in this important human pathogen that may be of utility in treating P. aeruginosa infections.

Effect of calcium on Pseudomonas aeruginasa and Bacillus cereus metabolites / Efeito do cálcio nos metabólitos de Pseudomonas aeruginasa e Bacillus cereus

The effects of Calcium (Ca+²) on virulence and some parameters should be analyzed in this study. Pseudomonas aeruginosa Gram (-) and Bacillus cereus Gram (+) were used. Both bacteria are soil bacteria. In this study; the effect of Ca+² on protease, amylase, LasB elastolytic assay, H2O2, pyorubin and biofilm on metabolites of these bacteria were investigated during 24 hour time. In this study, the effect of Ca+² on the production of some secondary metabolites on P. aeruginosa and B. cereus was investigated and presented for the first time by us.

Os efeitos do cálcio (Ca+²) na virulência e alguns parâmetros devem ser analisados neste estudo. Pseudomonas aeruginosa Gram (-) e Bacillus cereus Gram (+) foram usados. Ambas as bactérias são bactérias do solo. Neste estudo, o efeito do Ca+² sobre a protease, amilase, ensaio elastolítico LasB, H2O2, piorubina e biofilme nos metabólitos dessas bactérias foram investigados durante 24 horas. Neste estudo, o efeito do Ca+² na produção de alguns metabólitos secundários em P. aeruginosa e B. cereus foi investigado e apresentado pela primeira vez por nós.

Specific interactions between the alkaline protease of P. aeruginosa and its natural peptide inhibitor: ab initio molecular simulations.

Alkaline protease aeruginolysin (APR) is an important virulence factor in the evasion of the immune system by Pseudomonas aeruginosa (P. aeruginosa). The P. aeruginosa genome also encodes the highly potent and specific APR peptide inhibitor (APRin). However, the structural reason for the significant inhibition has not been revealed. Using ab initio molecular simulations, we here investigated the specific interactions between APR and APRin to elucidate which amino acid residues of APRin and APR contribute strongest to the inhibition. Since APR has a Zn2+ ion at the ligand-binding site and histidine and glutamic acid residues are coordinated with Zn2+, it is essential to precisely describe these coordination bonds to elucidate the specific interactions between APR and APRin. Therefore, we employed the ab initio fragment molecular orbital method to investigate the specific interactions at an electronic level. The results revealed that Ser1 and Ser2 at the N-terminus of APRin significantly contribute to the binding between APRin and APR. In particular, Ser1 binds strongly to Zn2+ as well as to the sidechains of His176(Hid), His180(Hid), and His186(Hid) in APR. This is the main reason for the strong interaction between APR and APRin. The results also elucidated significant contributions of the positively charged Arg83 and Arg90 residues of APRin to the binding with APR. These findings may provide information useful for the design of novel small agents as potent APR inhibitors.

Biosynthesis of fluopsin C, a copper-containing antibiotic from Pseudomonas aeruginosa.

Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a five-enzyme pathway in Pseudomonas aeruginosa synthesizes a small-molecule copper complex, fluopsin C, in response to elevated copper concentrations. Fluopsin C is a broad-spectrum antibiotic that contains a copper ion chelated by two minimal thiohydroxamates. Biosynthesis of the thiohydroxamate begins with cysteine and requires two lyases, two iron-dependent enzymes, and a methyltransferase. The iron-dependent enzymes remove the carboxyl group and the α carbon from cysteine through decarboxylation, N-hydroxylation, and methylene excision. Conservation of the pathway in P. aeruginosa and other bacteria suggests a common role for fluopsin C in the copper stress response, which involves fusing copper into an antibiotic against other microbes.

Alleviation of Pseudomonas aeruginosa Infection by Propeptide-Mediated Inhibition of Protease IV.

Pseudomonas aeruginosa, an opportunistic human pathogen, expresses protease IV (PIV) for infection. Since the PIV activity can be inhibited by its propeptide, we tried to alleviate the severity of P. aeruginosa infection using the purified PIV propeptide (PIVpp). The PIVpp treatment of P. aeruginosa could significantly inhibit the PIV activity and reduce the virulence of P. aeruginosa in multiple invertebrate infection models, such as nematodes, brine shrimp, and mealworms. The effectiveness of PIVpp was further confirmed using mouse skin infection and acute/chronic lung infection models. The amount of PIVpp that inhibited the PIV activity of P. aeruginosa by 65% could alleviate the severity of infection significantly in all of the skin and acute/chronic lung infections. In addition, the PIVpp treatment of P. aeruginosa facilitated the healing of the skin wound infections and repressed the growth of P. aeruginosa in the infected lung. The PIVpp itself did not cause the induction of inflammatory cytokines or have any harmful effects on host tissues and did not affect bacterial growth. Taken together, P. aeruginosa infections can be alleviated by PIVpp treatment. IMPORTANCE Pseudomonas aeruginosa is a highly antibiotic-resistant pathogen and is extremely difficult to treat. Instead of using conventional antibiotics, we attempted to alleviate P. aeruginosa infection using factors that P. aeruginosa itself produces naturally. Extracellular proteases are powerful virulence factors and important targets to control the P. aeruginosa infections. Propeptides are originally expressed as part of extracellular proteases, inhibiting their activity until they go out of the cell, preventing them from becoming toxic to the cells themselves. We confirmed, from multiple animal experiments, that treating P. aeruginosa with the purified propeptide can alleviate its infectivity. Propeptides specifically inhibit only their cognate protease without inhibiting other essential proteases of the host. The development of resistance can be avoided because the propeptide-mediated inhibition is an inherent mechanism of P. aeruginosa; hence, it will be difficult for P. aeruginosa to alter this mechanism. Since propeptides do not affect bacterial growth, there is no selective pressure to develop resistant cells.

Interplay of OpdP Porin and Chromosomal Carbapenemases in the Determination of Carbapenem Resistance/Susceptibility in Pseudomonas aeruginosa.

Carbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.

Structural basis for diguanylate cyclase activation by its binding partner in Pseudomonas aeruginosa.

Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in Pseudomonas aeruginosa. Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling.

Phosphopantetheinyl transferase binding and inhibition by amidino-urea and hydroxypyrimidinethione compounds.

Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.

Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance.

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.

Hexabromocyclododecanes Are Dehalogenated by CYP168A1 from Pseudomonas aeruginosa Strain HS9.

Hexabromocyclododecanes (HBCDs) are widely used brominated flame retardants that cause antidiuretic hormone syndrome and even induce cancer. However, little information is available about the degradation mechanisms of HBCDs. In this study, genomic and proteomic analyses, reverse transcription-quantitative PCR, and gene knockout assays reveal that a cytochrome P450-encoding gene is responsible for HBCD catabolism in Pseudomonas aeruginosa HS9. The CO difference spectrum of the enzyme CYP168A1 was matched to P450 characteristics via UV visibility. We demonstrate that the reactions of debromination and hydrogenation are carried out one after another based on detection of the metabolites pentabromocyclododecanols (PBCDOHs), tetrabromocyclododecadiols (TBCDDOHs), and bromide ion. In the 18O isotope experiments, PBCD18OHs were only detected in the H218O group, proving that the added oxygen is derived from H2O, not from O2. This study elucidates the degradation mechanism of HBCDs by Pseudomonas. IMPORTANCE Hexabromocyclododecanes (HBCDs) are environmental pollutants that are widely used in industry. In this study, we identified and characterized a novel key dehalogenase, CYP168A1, that is responsible for HBCD degradation from Pseudomonas aeruginosa strain HS9. This study provides new insights into understanding biodegradation of HBCDs.

A Dual-Specificity Inhibitor Targets Polyphosphate Kinase 1 and 2 Enzymes To Attenuate Virulence of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections, which are becoming increasingly difficult to treat due to antibiotic resistance. Polyphosphate (polyP) plays a key role in P. aeruginosa virulence, stress response, and antibiotic tolerance, suggesting an attractive drug target. Here, we show that the small molecule gallein disrupts polyphosphate homeostasis by inhibiting all members of both polyphosphate kinase (PPK) families (PPK1 and PPK2) encoded by P. aeruginosa, demonstrating dual-specificity PPK inhibition for the first time. Inhibitor treatment phenocopied ppk deletion to reduce cellular polyP accumulation and attenuate biofilm formation, motility, and pyoverdine and pyocyanin production. Most importantly, gallein attenuated P. aeruginosa virulence in a Caenorhabditis elegans infection model and synergized with antibiotics while exhibiting negligible toxicity toward the nematodes or HEK293T cells, suggesting our discovery of dual-specificity PPK inhibitors as a promising starting point for the development of new antivirulence therapeutics. IMPORTANCE Many priority bacterial pathogens such as P. aeruginosa encode both PPK1 and PPK2 enzymes to maintain polyphosphate homeostasis. While PPK1 and PPK2 have distinct structures and catalytic mechanisms, they are both capable of synthesizing and consuming polyphosphate; thus, PPK2 enzymes can compensate for the loss of PPK1 and vice versa. In this study, we identified the small molecule gallein as a dual-specificity inhibitor of both PPK1 and PPK2 enzyme families in P. aeruginosa. Inhibitor treatment reduced cellular polyP in wild-type (WT), Δppk1, and Δppk2 strains to levels that were on par with the Δppk1 Δppk2A Δppk2B Δppk2C knockout control. Treatment also attenuated biofilm formation, motility, toxin production, and virulence to a similar extent, thereby elucidating a hitherto-undocumented role of PPK2 enzymes in P. aeruginosa virulence phenotypes. This work therefore establishes PPK2s, in addition to PPK1, as valuable drug targets in P. aeruginosa and provides a favorable starting molecule for future inhibitor design efforts.

Antibodies Against Pseudomonas aeruginosa Alkaline Protease Directly Enhance Disruption of Neutrophil Extracellular Traps Mediated by This Enzyme.

Extracellular traps released by neutrophils (NETs) are essential for the clearance of Pseudomonas aeruginosa. Alkaline protease (AprA) secreted by P. aeruginosa negatively correlates with clinical improvement. Moreover, anti-AprA in patients with cystic fibrosis (CF) can help identify patients with aggressive forms of chronic infection. However, the mechanism underlying the clinical outcomes remains unclear. We demonstrated that aprA deficiency in P. aeruginosa decreased the bacterial burden and reduced lung infection. AprA degraded NET components in vitro and in vivo but did not affect NET formation. Importantly, antibodies induced by AprA acted as an agonist and directly enhanced the degrading activities of AprA. Moreover, antisera from patients with P. aeruginosa infection exhibited antibody-dependent enhancement (ADE) similar to that of the antibodies we prepared. Our further investigations showed that the interaction between AprA and the specific antibodies might make the enzyme active sites better exposed, and subsequently enhance the recognition of substrates and accelerate the degradation. Our findings revealed that AprA secreted by P. aeruginosa may aggravate infection by destroying formed NETs, an effect that was further enhanced by its antibodies.

Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase.

Iron is an essential micronutrient for the survival and virulence of the bacterial pathogen Pseudomonas aeruginosa. To overcome iron withholding and successfully colonize a host, P. aeruginosa uses a variety of mechanisms to acquire iron, including the secretion of high-affinity iron chelators (siderophores) or the uptake and utilization of heme. P. aeruginosa heme oxygenase (HemO) plays pivotal roles in heme sensing, uptake, and utilization and has emerged as a therapeutic target for the development of antipseudomonal agents. Using a high-throughput fluorescence quenching assay combined with minimum inhibitory concentration measurements, we screened the Selleck Bioactive collection of 2100 compounds and identified acitretin, a Food and Drug Administration-approved oral retinoid, as a potent and selective inhibitor of HemO. Acitretin binds to HemO with a KD value of 0.10 ± 0.02 µM and inhibits the growth of P. aeruginosa PAO1 with an IC50 of 70 ± 18 µg/mL. In addition, acitretin showed good selectivity for HemO, which uniquely generates BVIXß/δ, over human heme oxygenase (hHO1) and other BVIXα-producing homologues such as the heme oxygenases from Neisseria meningitidis (nmHO) and Acinetobacter baumannii (abHO). The binding of acitretin within the HemO active site was confirmed by 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance, and molecular modeling provided further insight into potential interactions of acitretin with residues specific for orienting heme in the ß/δ selective HemO. Moreover, at 20 µM, acitretin inhibited the enzymatic activity of HemO in P. aeruginosa cells by >60% and effectively blocked the ability of P. aeruginosa to sense and acquire heme as demonstrated in the ß-galactosidase transcriptional reporter assay.

MetW regulates the enzymatic activity of MetX in Pseudomonas.

Methionine is a canonical amino acid. The protein MetX is a homoserine O-acyltransferase utilized in the methionine biosynthetic pathway. The metW gene is found adjacent to the metX gene in some bacteria, but its functions are unclear. In this study, I focused on the function of MetW and MetX from Pseudomonas aeruginosa (PaMetW and PaMetX). I demonstrated that PaMetW interacted with and activated the homoserine O-succinyltransferase (HST) activity of PaMetX. Furthermore, I elucidated that the HST activity of PaMetX in complex with PaMetW was inhibited by the addition of S-adenosyl-l-homocysteine (SAH), although PaMetX alone showed no feedback inhibition. Since PaMetW possesses a glycine-rich sequence annotated as a SAM/SAH binding site, I also investigated the relationship between this glycine-rich sequence and the inhibition caused by SAH. I revealed that alanine mutation of PaMetW Gly24 reduced the inhibitory effect of SAH. These results suggest that MetW is a regulatory protein of MetX.