scholarly journals Impact of artificial sputum media formulation on Pseudomonas aeruginosa secondary metabolite production

2021 ◽  
Author(s):  
Rachel L. Neve ◽  
Brent D. Carrillo ◽  
Vanessa V. Phelan
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Culture Media ◽  
Nutrient Content ◽  
Opportunistic Pathogen ◽  
Defined Medium ◽  
Media Formulation ◽  
Porcine Gastric Mucin

In vitro culture media are being developed to understand how host site-specific nutrient profiles influence microbial pathogenicity and ecology. To mimic the cystic fibrosis (CF) lung environment, a variety of artificial sputum media (ASM) have been created. However, the composition of these ASM vary in the concentration of key nutrients, including amino acids, lipids, DNA, and mucin. In this work, we used feature-based molecular networking (FBMN) to perform comparative metabolomics of Pseudomonas aeruginosa , the predominant opportunistic pathogen infecting the lungs of people with CF, cultured in nine different ASM. We found that the concentration of aromatic amino acids and iron from mucin added to the media contribute to differences in the production of P. aeruginosa virulence-associated secondary metabolites. IMPORTANCE Different media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes.

scholarly journals Adjustment of Mineral Elements in the Culture Medium for the Micropropagation of Three Vriesea Bromeliads from the Brazilian Atlantic Forest: The Importance of Calcium

HortScience ◽  
2009 ◽  
Vol 44 (1) ◽  
pp. 106-112 ◽  
Author(s):  
Alice Noemí Aranda-Peres ◽  
Lázaro Eustáquio Pereira Peres ◽  
Edson Namita Higashi ◽  
Adriana Pinheiro Martinelli
Keyword(s):  
New Media ◽  
Mineral Composition ◽  
Culture Media ◽  
Dry Weight ◽  
Defined Medium ◽  
Ms Medium ◽  
Media Formulation ◽  
Half Strength

Many different species of Bromeliaceae are endangered and their conservation requires specific knowledge of their growth habits and propagation. In vitro culture of bromeliads is an important method for efficient clonal propagation and in vitro seed germination can be used to maintain genetic variability. The present work aims to evaluate the in vitro growth and nutrient concentration in leaves of the epiphyte bromeliads Vriesea friburguensis Mez, Vriesea hieroglyphica (Carrière) E. Morren, and Vriesea unilateralis Mez, which exhibit slow rates of growth in vivo and in vitro. Initially, we compared the endogenous mineral composition of bromeliad plantlets grown in half-strength Murashige and Skoog (MS) medium and the mineral composition considered adequate in the literature. This approach suggested that calcium (Ca) is a critical nutrient and this was considered for new media formulation. Three new culture media were defined in which the main changes to half-strength MS medium were an increase in Ca, magnesium, sulfur, copper, and chloride and a decrease in iron, maintaining the nitrate:ammonium rate at ≈2:1. The main difference among the three new media formulated was Ca concentration, which varied from 1.5 mm in half-strength MS to 3.0, 6.0, and 12 mm in M2, M3, and M4 media, respectively. Consistently, all three species exhibited significantly higher fresh and dry weight on M4, the newly defined medium with the highest level of Ca (12 mm). Leaf nitrogen, potassium, zinc, magnesium, and boron concentrations increased as Ca concentration in the medium increased from 1.5 to 12 mm.

scholarly journals Artificial Sputum Medium Formulation Impacts Pseudomonas aeruginosa Phenotype and Chemotype

2021 ◽  
Author(s):  
Rachel L. Neve ◽  
Vanessa V. Phelan
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Culture Media ◽  
Nutrient Concentrations ◽  
Disease States ◽  
Porcine Gastric Mucin ◽  
Nutritional Environment ◽  
Different Sources

ABSTRACTArtificial sputum medium (ASM) is a class of in vitro bacterial culture medium intended to mimic the nutritional environment of cystic fibrosis (CF) pulmonary mucus. One of the most commonly studied microbes in ASM is Pseudomonas aeruginosa, a prevalent and dangerous pathogen of the CF pulmonary microbiome. Many ASM formulations have been reported in literature, with differing nutrient concentrations and availability. Here, we show that common formulations of ASM yield different phenotypes and chemotypes of P. aeruginosa. Further, we demonstrate that iron in commercial porcine gastric mucin (PGM) is sufficient to alter production of P. aeruginosa siderophores in the chemically defined ASM, synthetic CF medium 1 (SCFM1). These results highlight that the choice of ASM formulation for in vitro investigations of microbial pathogenicity, physiology, and interactions should be carefully considered.IMPORTANCEIn vitro culture media are being developed to resemble the in vivo nutritional environment more closely. These culture media are used to investigate microbial pathogenicity and ecology in environments that are more reflective of disease states. In cystic fibrosis (CF), a number of different artificial sputum media (ASM) formulations have been created to recapitulate the CF lung environment. However, these ASM have different sources and concentrations of nutrients. Here, we cultured Pseudomonas aeruginosa in nine different formulations of ASM. P. aeruginosa is the primary pathogen causing lung infection in CF.We show that different ASM formulations lead to different phenotypes and chemotypes by P. aeruginosa and one component of ASM, mucin, contains high levels of iron, which may affect P. aeruginosa physiology.

Some amino acids of the Pseudomonas aeruginosa MutL D(Q/M)HA(X)2E(X)4E conserved motif are essential for the in vivo function of the protein but not for the in vitro endonuclease activity

DNA Repair ◽  
2011 ◽  
Vol 10 (11) ◽  
pp. 1106-1113 ◽  
Author(s):  
Elisa M.E. Correa ◽  
Mariana A. Martina ◽  
Luisina De Tullio ◽  
Carlos E. Argaraña ◽  
José L. Barra
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Endonuclease Activity ◽  
Conserved Motif

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 Specific and Global RNA Regulators in Pseudomonas aeruginosa

2021 ◽  
Vol 22 (16) ◽  
pp. 8632
Author(s):  
Petra Pusic ◽  
Elisabeth Sonnleitner ◽  
Udo Bläsi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Binding ◽  
Gene Networks ◽  
Target Genes ◽  
Opportunistic Pathogen ◽  
Chronic Infections ◽  
Intrinsic Resistance ◽  
Large Gene

Pseudomonas aeruginosa (Pae) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae. Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.

scholarly journals A Novel Indole Compound That Inhibits Pseudomonas aeruginosa Growth by Targeting MreB Is a Substrate for MexAB-OprM

2007 ◽  
Vol 189 (19) ◽  
pp. 6870-6881 ◽  
Author(s):  
Gregory T. Robertson ◽  
Timothy B. Doyle ◽  
Qun Du ◽  
Leonard Duncan ◽  
Khisimuzi E. Mdluli ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agents ◽  
Opportunistic Pathogen ◽  
Binding Pocket ◽  
Cross Resistance ◽  
Dependent Manner ◽  
Intrinsic Resistance ◽  
Outer Membrane Channel

ABSTRACT Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.

scholarly journals Characterization of Alanine Catabolism in Pseudomonas aeruginosa and Its Importance for Proliferation In Vivo

2009 ◽  
Vol 191 (20) ◽  
pp. 6329-6334 ◽  
Author(s):  
Megan L. Boulette ◽  
Patricia J. Baynham ◽  
Peter A. Jorth ◽  
Irena Kukavica-Ibrulj ◽  
Aissa Longoria ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Carbon Sources ◽  
Opportunistic Pathogen ◽  
Lung Model ◽  
Host Infection ◽  
In Vivo Growth

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa causes a variety of infections in immunocompromised individuals, including individuals with the heritable disease cystic fibrosis. Like the carbon sources metabolized by many disease-causing bacteria, the carbon sources metabolized by P. aeruginosa at the host infection site are unknown. We recently reported that l-alanine is a preferred carbon source for P. aeruginosa and that two genes potentially involved in alanine catabolism (dadA and dadX) are induced during in vivo growth in the rat peritoneum and during in vitro growth in sputum (mucus) collected from the lungs of individuals with cystic fibrosis. The goals of this study were to characterize factors required for alanine catabolism in P. aeruginosa and to assess the importance of these factors for in vivo growth. Our results reveal that dadA and dadX are arranged in an operon and are required for catabolism of l-alanine. The dad operon is inducible by l-alanine, d-alanine, and l-valine, and induction is dependent on the transcriptional regulator Lrp. Finally, we show that a mutant unable to catabolize dl-alanine displays decreased competitiveness in a rat lung model of infection.

scholarly journals Comparative Amino Acids Studies on Phac Synthases and Proteases as Well as Establishing a New Trend in Experimental Design

2012 ◽  
Vol 13 (1) ◽  
Author(s):  
Amro Abd al fattah Amara ◽  
Saleh M. Matar ◽  
Ehab Serour
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Experimental Design ◽  
Castor Oil ◽  
Bacillus Pumilus ◽  
Geobacillus Stearothermophilus ◽  
Protein Database ◽  
Lipase Enzyme

ABSTRACT: A question addressed in this study is: why similar enzymes are classified into different subclasses? As an example, PhaC synthases are classified according to four different classes (I, II, III and IV). To answer this question we proposed that besides the catalytic residues, the overall amino acids (AAs) present are responsible for the differences observed. The AAs’ composition affects the structure/function/substrate specificity (SFS) of these enzymes. The differences between the classes in various PhaC synthases and proteases were analysed to support our argument. Homology and phylogenic tree of some selected PhaC synthases of different strains (representing the four classes) were demonstrated. The properties of a specific class of enzyme could not be changed into those of another by changing the catalytic residues. Moreover, these differences could not be detected from the proteins’ 3D structures, despite clear differences at the AAs level. Another question was also addressed: could we benefit from the various existing protein databases in the field of biotechnology? To answer this, we introduced a model for an Experimental Design based on the information in the protein database (for strains available in our lab) regarding their ability to degrade castor oil. Two enzymes in the phenol degradation pathway, phenol 2-monooxygenase and catechol 1,2-dioxygenase, and a lipase enzyme were analysed. These enzymes were screened and analysed according to the BLAST-protein database and BRENDA. The comprehensive enzyme information system compared six strains against each other, including: Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis, and Geobacillus stearothermophilus. Only P. aeruginosa proved to have the three required enzymes and was suitable for the production of lipases from castor oil (crude castor oil is usually contaminated with phenol) as indicated by the databases. In addition, in vivo castor oil degradation and in vitro lipase enzyme activity were analysed. The apparent lipase activity was 1070 Units/ml. Therefore, this new strategy is recommended to better understand the SFS as well as for using protein database in an Experimental Design.ABSTRAK: Kajian ini berkisar mengenai soalan: mengapa enzim yang sama diklassifikasikan kepada subkumpulan yang berbeza? Contohnya sintasis PhaC dikelaskan kepada empat kumpulan berbeza (I, II, III dan IV). Bagi menjawab soalan ini selain hipotesis katalitik residue diperkenalkan, faktor yang bertanggungjawab adalah asid amino (AAs) secara keseluruhannya. Komposisi AAs memberi kesan kepada struktur/fungsi/spesifikasi substrat (SFS) enzim-enzim ini. Perbezaan di antara kumpulan dalam pelbagai sintasis PhaC dan proteases telah dianalisis bagi menyokong hujah ini. Homologi dan asas phylogenic bagi sintasis PhaC tertentu yang berbeza strains (mewakili empat kumpulan) telah ditunjukkan. Sifat-sifat tidak boleh ditukarkan dengan menukarkan katalitik residue dari satu kepada yang lain. Tambahan pula, kelainannya tidak boleh dikesan dari struktur 3D protein, walaupun perbezaan yang nyata pada peringkat AAs. Soalan lain yang berkisar adalah: Adakah kita mendapat faedah dari pelbagai pengkalan data dalam bidang bioteknologi? Bagi menjawab soalan ini, model Rekabentuk Eksperimen telah diperkenalkan yang berasaskan maklumat pengkalan data protein bagi strains di makmal kami yang boleh degradasi minyak castor. Dua enzim dalam degradasi fenol telah dianalisis, fenol 2-monooxigenas dan catechol 1,2-dioxygenas serta enzim lipas. Enzim-enzim ini telah disaringkan dan dianalisis dengan merujuk kepada pengkalan data protein-BLAST dan BRENDA. Sistem maklumat enzim secara komprehensif terhadap enam strains termasuk Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis dan Geobacillus stearothermophilus. Hanya P. Aeruginosa terbukti mempunyai tiga enzim dan sesuai bagi penghasilan lipase dari minyak kastor (minyak kastor mentah selalunya tercemar dengan fenol) seperti yang ditunjukkan dari pengkalan data. Degradasi minyak kastor in vivo dan aktiviti enzim lipase in vitro telah dilaksanakan. Aktiviti lipase jelas adalah 1070 Units/ml. Kami mencadangkan menggunakan strategi ini bagi memahami SFS serta pengkalan data protein dalam Rekabentuk Eksperimen.KEYWORDS: amino acids; model; PhaC synthase; protease; lipase; experimental design

scholarly journals Comparison of the effects of metformin on MDA-MB-231 breast cancer cells in a monolayer culture and in tumour spheroids as a function of nutrient concentrations

2018 ◽  
Author(s):  
Maruša Bizjak ◽  
Petra Malavašič ◽  
Sergej Pirkmajer ◽  
Mojca Pavlin
Keyword(s):  
Amino Acids ◽  
Cancer Cells ◽  
Propidium Iodide ◽  
Monolayer Culture ◽  
Culture Media ◽  
Cell Model ◽  
Nutrient Concentrations ◽  
Media Formulation ◽  
Tumour Spheroids

AbstractMetabolic pathways of cancer cells depend on the concentrations of nutrients in their micro-environment. However, they can vary also between monolayer cultures of cancer cells and tumour spheroids. Here we examined whether the absence of glucose, pyruvate and glutamine increases the sensitivity of MDA-MB-231 cells to metabolic drug metformin using twoin vitrocell models (monolayer culture and tumour spheroids). To evaluate the effects of nutrient depletion in more detail, we tested the effects of metformin in commonly used media (DMEM, MEM and RPMI-1640) that differ mainly in the concentrations of amino acids. We used MTS, Hoechst and propidium iodide assay to determine cell number, viability and survival, respectively. We evaluated the effects of metformin on the size of tumour spheroids and determined cell survival by calcein and propidium iodide staining. Finally, we observed the effects of metformin in nutrient depleted conditions on the phosphorylation of AMP-activated protein kinase using Western blotting. Our main finding is that the effects of metformin on MDA-MB-231 cells depend onin vitrocell model used (monolayer culture vs. tumour spheroids). While metformin did not have any major effect on proliferation of MDA-MB-231 cells grown in complete cell culture media in a monolayer culture, it disintegrated tumour spheroids in MEM and RPMI-1640 medium. The effects of metformin on tumour spheroids were most pronounced in MEM, which is deficient of several non-essential amino acids. Glutamine depletion had no effect on the sensitivity of MDA-MB-231 cells to metformin in all tested conditions, whereas pyruvate depletion sensitized MDA-MB-231 cells to metformin in a monolayer culture only in MEM. Taken together, our results show that media formulation as well asin vitrocell model (monolayer culture vs. tumour spheroids) must be considered, when we evaluate the effects of metformin on MDA-MB-231 cells as a function of nutrient availability.

scholarly journals Cochaperone Interactions in Export of the Type III Needle Component PscF of Pseudomonas aeruginosa

2010 ◽  
Vol 192 (14) ◽  
pp. 3801-3808 ◽  
Author(s):  
Sophie Plé ◽  
Viviana Job ◽  
Andréa Dessen ◽  
Ina Attree
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Complex Formation ◽  
Hot Spot ◽  
Protein Stabilization ◽  
Site Directed Mutagenesis ◽  
Surface Stability ◽  
Type Iii

ABSTRACT Type III secretion (T3S) systems allow the export and translocation of bacterial effectors into the host cell cytoplasm. Secretion is accomplished by an 80-nm-long needle-like structure composed, in Pseudomonas aeruginosa, of the polymerized form of a 7-kDa protein, PscF. Two proteins, PscG and PscE, stabilize PscF within the bacterial cell before its export and polymerization. In this work we screened the 1,320-Å2 interface between the two chaperones, PscE and PscG, by site-directed mutagenesis and determined hot spot regions that are important for T3S function in vivo and complex formation in vitro. Three amino acids in PscE and five amino acids in PscG, found to be relevant for complex formation, map to the central part of the interacting surface. Stability assays on selected mutants performed both in vitro on purified PscE-PscG complexes and in vivo on P. aeruginosa revealed that PscE is a cochaperone that is essential for the stability of the main chaperone, PscG. Notably, when overexpressed from a bicistronic construct, PscG and PscF compensate for the absence of PscE in cytotoxic P. aeruginosa. These results show that all of the information needed for needle protein stabilization and folding, its presentation to the T3 secreton, and its export is present within the sequence of the PscG chaperone.

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