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International Journal of Molecular... Oct 2023PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient...
PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD while it lost activity towards NADP, and the Km value (7770.67 μM) of the mutant PaIDH2-L589 I600 for NADP was higher than that observed for NAD (5824.33 μM), indicating a shift in coenzyme specificity from NADP to NAD for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium .
Topics: Coenzymes; Pseudomonas aeruginosa; NADP; NAD; Amino Acid Sequence; Isocitrate Dehydrogenase; Isocitrates; Kinetics
PubMed: 37834433
DOI: 10.3390/ijms241914985 -
Antimicrobial Agents and Chemotherapy Jul 2023The impact of broad-spectrum β-lactamases on the susceptibility to novel β-lactamase/β-lactamase inhibitor combinations was evaluated both in Pseudomonas aeruginosa...
Impact of Acquired Broad Spectrum β-Lactamases on Susceptibility to Novel Combinations Made of β-Lactams (Aztreonam, Cefepime, Meropenem, and Imipenem) and Novel β-Lactamase Inhibitors in Escherichia coli and Pseudomonas aeruginosa.
The impact of broad-spectrum β-lactamases on the susceptibility to novel β-lactamase/β-lactamase inhibitor combinations was evaluated both in Pseudomonas aeruginosa and Escherichia coli using isogenic backgrounds. Cefepime-zidebactam displayed low MICs, mainly due to the significant intrinsic antibacterial activity of zidebactam. Cefepime-taniborbactam showed excellent activity against recombinant E. coli strains, including metallo-β-lactamase producers, whereas aztreonam-avibactam remained the best therapeutic option against class B β-lactamase-producing P. aeruginosa.
Topics: Cefepime; beta-Lactamase Inhibitors; Meropenem; beta-Lactams; Aztreonam; Imipenem; Pseudomonas aeruginosa; Escherichia coli; beta-Lactamases; Anti-Bacterial Agents; Azabicyclo Compounds; Microbial Sensitivity Tests
PubMed: 37255469
DOI: 10.1128/aac.00339-23 -
Journal of Infection in Developing... Jul 2023Pseudomonas aeruginosa (PA) has emerged as a significant cause of Gram-negative infections, particularly in patients with impaired host defenses. It is one of the six... (Review)
Review
Pseudomonas aeruginosa (PA) has emerged as a significant cause of Gram-negative infections, particularly in patients with impaired host defenses. It is one of the six ESKAPE pathogens that majorly cause severe nosocomial infections. In addition to biofilm formation, PA possesses various virulence factors. It can be life-threatening due to his remarkable capacity to resist antibiotics, either intrinsically, developing adaptative resistance, or following the acquisition of resistance genes. The situation worsens when these mechanisms co-exist, conferring worrying multi-resistant phenotypes. Therapeutic options are becoming limited, which has led to the development of new antibiotics and novel alternative therapeutic strategies that require the exploration of other therapeutic avenues. Although mostly at the preclinical stages, many recent studies have reported several innovative therapeutic technologies that have demonstrated pronounced effectiveness in fighting against drug-resistant Pa strains. This literature review aims to discuss the mechanism of pyocyanic bacillus resistance to antibiotics, highlight the current state of some novel antibiotics and combination therapies, and the new alternative therapeutic approaches for treating PA infections.
Topics: Humans; Virulence Factors; Pseudomonas aeruginosa; Anti-Bacterial Agents; Phenotype; Pseudomonas Infections
PubMed: 37515798
DOI: 10.3855/jidc.17739 -
Journal of Bacteriology Oct 2023is one of the most common biofilm-forming pathogens responsible for lung infections of individuals with cystic fibrosis (CF). becomes tolerant to antimicrobials in the...
is one of the most common biofilm-forming pathogens responsible for lung infections of individuals with cystic fibrosis (CF). becomes tolerant to antimicrobials in the biofilm state and is difficult to treat. Production of extracellular polymeric substances (EPS), such as alginate and extracellular DNA (eDNA), can allow adherence to abiotic and biotic surfaces, antimicrobial evasion, and resilience to environmental pressures. Alginate-producing mucoid variants of are frequently isolated from CF airway samples and are associated with worsening patient outcomes. While eDNA is a major structural component of nonmucoid biofilms, the potential role of eDNA in mucoid biofilms is unclear. Here, we investigate how eDNA contributes to clinical mucoid biofilm physiology and integrity. We predicted that eDNA plays a structural and mechanical role in mucoid biofilms. To test this, we quantified biofilm eDNA in mucoid biofilms and used microscopy and rheology to visualize eDNA and detect changes in biofilm structure and mechanics upon DNaseI treatment. We showed that biofilm eDNA abundance is diverse across clinical mucoid strains and observed a temporal increase in foci of eDNA within intact mucoid biofilms. Increased cell dispersal and reduced biomass were also observed following DNaseI treatment of mucoid biofilms. Degradation of eDNA also impacted the mechanical integrity of mucoid biofilms by increasing the stiffness and decreasing the cohesion of the biofilm. These findings advance our understanding of clinical mucoid biofilms and facilitate the development of new approaches to target biofilms by exploiting the functions of EPS components. IMPORTANCE Understanding the role of eDNA in mucoid biofilms will lead to therapeutic strategies that combat the biophysical and structural function of EPS for the eradication of bacteria in mucoid biofilms during chronic infections. This knowledge can be used to further identify unknown matrix component interactions within pathogenic biofilm-forming clinical isolates.
Topics: Humans; Pseudomonas aeruginosa; Polysaccharides, Bacterial; Biofilms; Anti-Infective Agents; Alginates; DNA; Pseudomonas Infections
PubMed: 37791754
DOI: 10.1128/jb.00238-23 -
Microbiology Spectrum Aug 2023Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the...
Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common organisms isolated from CF patient airways. P. aeruginosa establishes chronic infections that persist throughout a patient's lifetime and is a major cause of morbidity and mortality. Throughout the course of infection, P. aeruginosa must evolve and adapt from an initial state of early, transient colonization to chronic colonization of the airways. Here, we examined isolates of P. aeruginosa from children under the age of 3 years old with CF to determine genetic adaptations the bacterium undergoes during this early stage of colonization and infection. These isolates were collected when early aggressive antimicrobial therapy was not the standard of care and therefore highlight strain evolution under limited antibiotic pressure. Examination of specific phenotypic adaptations, such as lipid A palmitoylation, antibiotic resistance, and loss of quorum sensing, did not reveal a clear genetic basis for such changes. Additionally, we demonstrate that the geography of patient origin, within the United States or among other countries, does not appear to significantly influence genetic adaptation. In summary, our results support the long-standing model that patients acquire individual isolates of P. aeruginosa that subsequently become hyperadapted to the patient-specific airway environment. This study provides a multipatient genomic analysis of isolates from young CF patients in the United States and contributes data regarding early colonization and adaptation to the growing body of research about P. aeruginosa evolution in the context of CF airway disease. Chronic lung infection with Pseudomonas aeruginosa is of major concern for patients with cystic fibrosis (CF). During infection, P. aeruginosa undergoes genomic and functional adaptation to the hyperinflammatory CF airway, resulting in worsening lung function and pulmonary decline. All studies that describe these adaptations use P. aeruginosa obtained from older children or adults during late chronic lung infection; however, children with CF can be infected with P. aeruginosa as early as 3 months of age. Therefore, it is unclear when these genomic and functional adaptations occur over the course of CF lung infection, as access to P. aeruginosa isolates in children during early infection is limited. Here, we present a unique cohort of CF patients who were identified as being infected with P. aeruginosa at an early age prior to aggressive antibiotic therapy. Furthermore, we performed genomic and functional characterization of these isolates to address whether chronic CF P. aeruginosa phenotypes are present during early infection.
Topics: Humans; Cystic Fibrosis; Pseudomonas aeruginosa; Pseudomonas Infections; Lung; Genomics; Anti-Bacterial Agents
PubMed: 37358436
DOI: 10.1128/spectrum.01556-23 -
Nature Chemical Biology Sep 2023Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that...
Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.
Topics: Aminopeptidases; Pseudomonas aeruginosa; Peptides, Cyclic; Biofilms; Peptide Hydrolases; Bacterial Proteins
PubMed: 37386135
DOI: 10.1038/s41589-023-01373-8 -
Cell Reports Aug 2023Neutrophils play a critical role in the eradication of Pseudomonas aeruginosa, a major pathogen causing lung infection. However, the mechanisms used by the pathogen to...
Neutrophils play a critical role in the eradication of Pseudomonas aeruginosa, a major pathogen causing lung infection. However, the mechanisms used by the pathogen to evade neutrophil-mediated killing remain poorly understood. Using a high-density transposon screen, we find that P. aeruginosa colonization in the lung is promoted by pathogen nitrite reductase nirD. nirD is required for ammonia production from nitrite, a metabolite derived from nitrogen oxide (NO) generated by inducible NO synthetase (iNOS) in phagocytes. P. aeruginosa deficient in nirD exhibit reduced survival in wild-type neutrophils but not in iNOS-deficient neutrophils. Mechanistically, nirD enhances P. aeruginosa survival in neutrophils by inhibiting the localization of the pathogen in late phagosomes. P. aeruginosa deficient in nirD show impaired lung colonization after infection in wild-type mice but not in mice with selective iNos deficiency in neutrophils. Thus, P. aeruginosa uses neutrophil iNOS-mediated NO production to limit neutrophil pathogen killing and to promote its colonization in the lung.
Topics: Mice; Animals; Neutrophils; Pseudomonas aeruginosa; Nitric Oxide; Nitric Oxide Synthase Type II; Lung; Metabolic Networks and Pathways
PubMed: 37561628
DOI: 10.1016/j.celrep.2023.112973 -
Microbiology Spectrum Aug 2023Pseudomonas aeruginosa is a versatile pathogen that resists environmental stress, such as suboptimal pH. As a result of exposure to environmental stress, P. aeruginosa...
Pseudomonas aeruginosa is a versatile pathogen that resists environmental stress, such as suboptimal pH. As a result of exposure to environmental stress, P. aeruginosa shows an altered virulence-related phenotype. This study investigated the modifications that P. aeruginosa undertakes at a mildly low pH (pH 5.0) compared with the bacteria grown in a neutral medium (pH 7.2). Results indicated that in a mildly acidic environment, expression of two-component system genes (/ and /), lipid A remodeling genes such as and and virulence genes, i.e., and , were induced. Moreover, lipid A of the bacteria grown at a mildly low pH is modified by adding 4-amino-arabinose (l-Ara4N). Additionally, the production of virulence factors such as rhamnolipid, alginate, and membrane vesicles is significantly higher in a mildly low-pH environment than in a neutral medium. Interestingly, at a mildly low pH, P. aeruginosa produces a thicker biofilm with higher biofilm biomass. Furthermore, studies on inner membrane viscosity and permeability showed that a mildly low pH causes a decrease in the inner membrane permeability and increases its viscosity. Besides, despite the importance of PhoP, PhoQ, PmrA, and PmrB in Gram-negative bacteria for responding to low pH stress, we observed that the absence of each of these two-component systems does not meaningfully impact the remodeling of the P. aeruginosa envelope. Given that P. aeruginosa is likely to encounter mildly acidic environments during infection in its host, the alterations that the bacterium undertakes under such conditions must be considered in designing antibacterial strategies against P. aeruginosa. P. aeruginosa encounters environments with acidic pH when establishing infections in hosts. The bacterium develops an altered phenotype to tolerate a moderate decrease in the environmental pH. At the level of the bacterial envelope, modified lipid A composition and a reduction of the bacterial inner membrane permeability and fluidity are among the changes P. aeruginosa undergoes at a mildly low pH. Also, the bacterium is more likely to form biofilm in a mildly acidic environment. Overall, these alterations in the P. aeruginosa phenotype put obstacles in the way of antibacterial activities. Thus, considering physiological changes in the bacterium at low pH helps design and implement antimicrobial approaches against this hostile microorganism.
Topics: Virulence; Pseudomonas aeruginosa; Lipid A; Bacterial Proteins; Anti-Bacterial Agents; Gene Expression Regulation, Bacterial
PubMed: 37278652
DOI: 10.1128/spectrum.04832-22 -
Journal of Bacteriology Jun 2023Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These...
Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials.
Topics: Humans; Pyocins; Pseudomonas aeruginosa; Bacteriocins; Anti-Bacterial Agents; Bacteriophages
PubMed: 37260386
DOI: 10.1128/jb.00029-23 -
Frontiers in Cellular and Infection... 2023Chronic infections are a major clinical challenge in hard-to-heal wounds and implanted devices. is a common causative pathogen that produces numerous virulence factors....
INTRODUCTION
Chronic infections are a major clinical challenge in hard-to-heal wounds and implanted devices. is a common causative pathogen that produces numerous virulence factors. Due to the increasing problem of antibiotic resistance, new alternative treatment strategies are needed. Quorum sensing (QS) is a bacterial communication system that regulates virulence and dampens inflammation, promoting bacterial survival. QS inhibition is a potent strategy to reduce bacterial virulence and alleviate the negative impact on host immune response.
AIM
This study investigates how secreted factors from PAO1, cultured in the presence or absence of the QS inhibitor sodium salicylate (NaSa), influence host immune response.
MATERIAL AND METHODS
, THP-1 macrophages and neutrophil-like HL-60 cells were used. , discs of titanium were implanted in a subcutaneous rat model with local administration of culture supernatants. The host immune response to virulence factors contained in culture supernatants (+/-NaSa) was characterized through cell viability, migration, phagocytosis, gene expression, cytokine secretion, and histology.
RESULTS
, supernatants from NaSa-containing cultures significantly increased THP-1 phagocytosis and HL-60 cell migration compared with untreated supernatants (-NaSa). Stimulation with NaSa-treated supernatants resulted in: (i) significantly increased immune cell infiltration and cell attachment to titanium discs; (ii) increased gene expression of IL-8, IL-10, ARG1, and iNOS, and (iii) increased GRO-α protein secretion and decreased IL-1β, IL-6, and IL-1α secretion, as compared with untreated supernatants.
CONCLUSION
In conclusion, treating with NaSa reduces the production of virulence factors and modulates major immune events, such as promoting phagocytosis and cell migration, and decreasing the secretion of several pro-inflammatory cytokines.
Topics: Animals; Rats; Pseudomonas aeruginosa; Quorum Sensing; Sodium Salicylate; Titanium; Biological Transport
PubMed: 37614559
DOI: 10.3389/fcimb.2023.1183959