scholarly journals The Pseudomonas aeruginosa complement of lactate dehydrogenases enables use of D- and L-lactate and metabolic crossfeeding

2018 ◽  
Author(s):  
Yu-Cheng Lin ◽  
William-Cole Cornell ◽  
Alexa Price-Whelan ◽  
Lars E.P. Dietrich
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metabolic Pathways ◽  
Bacterial Colonization ◽  
Expression Patterns ◽  
Chronic Infections ◽  
Lactate Oxidation ◽  
Growth And Survival ◽  
Liquid Cultures ◽  
Pyruvate Fermentation ◽  
Lactate Dehydrogenases

ABSTRACTPseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to D-lactate during anaerobic survival, and LldE and LldD, which oxidize D-lactate and L-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant L-lactate oxidation during growth in aerobic cultures in both a defined MOPS-based medium and synthetic CF sputum medium. However, LldA differs from LldD in that its expression is induced specifically by the L-enantiomer of lactate. We also show that all four enzymes perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of D-lactate in the anaerobic zone that is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection.IMPORTANCELactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the L-form, generally produced by the host, and the D-form, which is usually produced by bacteria including the pulmonary pathogen Pseudomonas aeruginosa. Here, we characterize P. aeruginosa’s set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic D-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways could contribute to P. aeruginosa growth and survival in the lung.

scholarly journals The Pseudomonas aeruginosa Complement of Lactate Dehydrogenases Enables Use of D- and L-Lactate and Metabolic Cross-Feeding

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Yu-Cheng Lin ◽  
William Cole Cornell ◽  
Jeanyoung Jo ◽  
Alexa Price-Whelan ◽  
Lars E. P. Dietrich
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metabolic Pathways ◽  
Bacterial Colonization ◽  
Expression Patterns ◽  
Chronic Infections ◽  
Lactate Oxidation ◽  
Content Type ◽  
Growth And Survival ◽  
Liquid Cultures ◽  
Lactate Dehydrogenases

ABSTRACT Pseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to d-lactate during anaerobic survival, and LldE and LldD, which oxidize d-lactate and l-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant l-lactate oxidation during growth in aerobic cultures in both a defined MOPS (morpholinepropanesulfonic acid)-based medium and synthetic CF sputum media. However, LldA differs from LldD in that its expression is induced specifically by the l-enantiomer of lactate. We also show that the P. aeruginosa lactate dehydrogenases perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of d-lactate in the anaerobic zone, which is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection. IMPORTANCE Lactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the l-form, generally produced by the host, and the d-form, which is usually produced by bacteria, including the pulmonary pathogen Pseudomonas aeruginosa. Here, we characterize P. aeruginosa’s set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic d-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways might contribute to P. aeruginosa growth and survival in the lung.

scholarly journals Pseudomonas aeruginosa las and rhl quorum-sensing systems are important for infection and inflammation in a rat prostatitis model

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2612-2619 ◽  
Author(s):  
Lisa K. Nelson ◽  
Genevieve H. D'Amours ◽  
Kimberley M. Sproule-Willoughby ◽  
Douglas W. Morck ◽  
Howard Ceri
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Tissue Damage ◽  
Inflammatory Markers ◽  
Bacterial Colonization ◽  
Opportunistic Pathogen ◽  
Infection Model ◽  
Chronic Infections ◽  
Strain Pao1 ◽  
Rat Prostate

Pseudomonas aeruginosa frequently acts as an opportunistic pathogen of mucosal surfaces; yet, despite causing aggressive prostatitis in some men, its role as a pathogen in the prostate has not been investigated. Consequently, we developed a Ps. aeruginosa infection model in the rat prostate by instilling wild-type (WT) Ps. aeruginosa strain PAO1 into the rat prostate. It was found that Ps. aeruginosa produced acute and chronic infections in this mucosal tissue as determined by bacterial colonization, gross morphology, tissue damage and inflammatory markers. WT strain PAO1 and its isogenic mutant PAO-JP2, in which both the lasI and rhlI quorum-sensing signal systems have been silenced, were compared during both acute and chronic prostate infections. In acute infections, bacterial numbers and inflammatory markers were comparable between WT PA01 and PAO-JP2; however, considerably less tissue damage occurred in infections with PAO-JP2. Chronic infections with PAO-JP2 resulted in reduced bacterial colonization, tissue damage and inflammation as compared to WT PAO1 infections. Therefore, the quorum-sensing lasI and rhlI genes in Ps. aeruginosa affect acute prostate infections, but play a considerably more important role in maintaining chronic infections. We have thus developed a highly reproducible model for the study of Ps. aeruginosa virulence in the prostate.

scholarly journals PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

2014 ◽  
Vol 82 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Ziyu Sun ◽  
Jing Shi ◽  
Chang Liu ◽  
Yongxin Jin ◽  
Kewei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Colonization ◽  
Mrna Level ◽  
Sos Response ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Chronic Infections ◽  
Mobility Shift ◽  
Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

Impact of glycerol-3-phosphate dehydrogenase on virulence factor production byPseudomonas aeruginosa

2014 ◽  
Vol 60 (12) ◽  
pp. 857-863 ◽  
Author(s):  
Jonathan B. Daniels ◽  
Jessica Scoffield ◽  
Jessica L. Woolnough ◽  
Laura Silo-Suh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metabolic Pathways ◽  
Lung Surfactant ◽  
Carbon Sources ◽  
Chronic Infections ◽  
Available Nutrients ◽  
Therapeutic Benefits ◽  
Glycerol Utilization ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Virulence Factor Production

Pseudomonas aeruginosa establishes life-long chronic infections in the cystic fibrosis (CF) lung by utilizing various adaptation strategies. Some of these strategies include altering metabolic pathways to utilize readily available nutrients present in the host environment. The airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphatidylcholine, a major component of lung surfactant. Pseudomonas aeruginosa can degrade phosphatidylcholine to glycerol and fatty acids to increase the availability of usable carbon sources in the CF lung. In this study, we show that some CF-adapted P. aeruginosa isolates utilize glycerol more efficiently as a carbon source than nonadapted isolates. Furthermore, a mutation in a gene required for glycerol utilization impacts the production of several virulence factors in both acute and chronic isolates of P. aeruginosa. Taken together, the results suggest that interference with this metabolic pathway may have potential therapeutic benefits.

scholarly journals Pseudomonas aeruginosa Forms Biofilms in Acute Infection Independent of Cell-to-Cell Signaling

2007 ◽  
Vol 75 (8) ◽  
pp. 3715-3721 ◽  
Author(s):  
J. Andy Schaber ◽  
W. Jeffrey Triffo ◽  
Sang Jin Suh ◽  
Jeffrey W. Oliver ◽  
Mary Catherine Hastert ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Colonization ◽  
Acute Infection ◽  
Opportunistic Pathogen ◽  
Confocal Scanning Laser Microscopy ◽  
Chronic Infections ◽  
Signaling Mechanism ◽  
Confocal Scanning Laser

ABSTRACT Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

scholarly journals Extensive Genomic Plasticity in Pseudomonas aeruginosa Revealed by Identification and Distribution Studies of Novel Genes among Clinical Isolates

2006 ◽  
Vol 74 (9) ◽  
pp. 5272-5283 ◽  
Author(s):  
Kai Shen ◽  
Sameera Sayeed ◽  
Patricia Antalis ◽  
John Gladitz ◽  
Azad Ahmed ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genomic Library ◽  
Bacterial Species ◽  
Expression Patterns ◽  
Bacterial Pathogen ◽  
Prototype Strain ◽  
Population Based ◽  
Clinical Isolates ◽  
Chronic Infections ◽  
Insert Size

ABSTRACT The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, ∼1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.

scholarly journals Pseudomonas aeruginosa PA14 Enhances the Efficacy of Norfloxacin against Staphylococcus aureus Newman Biofilms

2020 ◽  
Vol 202 (18) ◽  
Author(s):  
Giulia Orazi ◽  
Fabrice Jean-Pierre ◽  
George A. O’Toole
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agents ◽  
Respiratory Infections ◽  
Multiple Infection ◽  
Bacterial Biofilms ◽  
Interspecies Interactions ◽  
Chronic Infections ◽  
Content Type

ABSTRACT The thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both are among the most common etiologic agents of chronic wound infections. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa PA14 to shift S. aureus Newman norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus. We previously showed that P. aeruginosa supernatant dissipates the S. aureus membrane potential, and furthermore, depletion of the S. aureus proton motive force recapitulates the effect of the P. aeruginosa PA14 supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus Newman. From these results, we hypothesize that exposure to P. aeruginosa PA14 exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus Newman to efflux norfloxacin. Surprisingly, the effect observed here of P. aeruginosa PA14 exoproducts on S. aureus Newman susceptibility to norfloxacin seemed to be specific to these strains and this antibiotic. Our results illustrate that microbially derived products can alter the ability of antimicrobial agents to kill bacterial biofilms. IMPORTANCE Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and nonhealing diabetic foot ulcers. Coinfection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with either organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

scholarly journals In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis

2021 ◽  
Vol 9 (3) ◽  
pp. 478
Author(s):  
Ersilia Vita Fiscarelli ◽  
Martina Rossitto ◽  
Paola Rosati ◽  
Nour Essa ◽  
Valentina Crocetta ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
In Vitro Test ◽  
Chronic Infections ◽  
Hospital Environments ◽  
Vitro Test ◽  
General Reliability ◽  
Phage Activity

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children’s Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

scholarly journals The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa

2021 ◽  
Vol 22 (8) ◽  
pp. 3982
Author(s):  
Karolina Kotecka ◽  
Adam Kawalek ◽  
Kamil Kobylecki ◽  
Aneta Agnieszka Bartosik
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Transcriptional Profiling ◽  
Mrna Level ◽  
Transcriptional Regulators ◽  
Resistance Mechanisms ◽  
Chronic Infections ◽  
Environmental Signals ◽  
Regulatory Systems

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459–PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459–PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa.

scholarly journals Pseudomonas aeruginosa PAO 1 In Vitro Time–Kill Kinetics Using Single Phages and Phage Formulations—Modulating Death, Adaptation, and Resistance

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 877
Author(s):  
Ana Mafalda Pinto ◽  
Alberta Faustino ◽  
Lorenzo M. Pastrana ◽  
Manuel Bañobre-López ◽  
Sanna Sillankorva
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phage Therapy ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Chronic Infections ◽  
Swarming Motility ◽  
Resistance Development ◽  
Healthcare Settings ◽  
Time Kill

Pseudomonas aeruginosa is responsible for nosocomial and chronic infections in healthcare settings. The major challenge in treating P. aeruginosa-related diseases is its remarkable capacity for antibiotic resistance development. Bacteriophage (phage) therapy is regarded as a possible alternative that has, for years, attracted attention for fighting multidrug-resistant infections. In this work, we characterized five phages showing different lytic spectrums towards clinical isolates. Two of these phages were isolated from the Russian Microgen Sextaphage formulation and belong to the Phikmvviruses, while three Pbunaviruses were isolated from sewage. Different phage formulations for the treatment of P. aeruginosa PAO1 resulted in diversified time–kill outcomes. The best result was obtained with a formulation with all phages, prompting a lower frequency of resistant variants and considerable alterations in cell motility, resulting in a loss of 73.7% in swimming motility and a 79% change in swarming motility. These alterations diminished the virulence of the phage-resisting phenotypes but promoted their growth since most became insensitive to a single or even all phages. However, not all combinations drove to enhanced cell killings due to the competition and loss of receptors. This study highlights that more caution is needed when developing cocktail formulations to maximize phage therapy efficacy. Selecting phages for formulations should consider the emergence of phage-resistant bacteria and whether the formulations are intended for short-term or extended antibacterial application.

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