Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from Pseudomonas aeruginosa

2019 ◽  
Author(s):  
Hajar Mohammadi-Barzelighi ◽  
Bahram Nasr-Esfahani ◽  
Bita Bakhshi ◽  
Bahram Daraei ◽  
Sharareh Moghim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Shigella Flexneri ◽  
Bacterial Species ◽  
Antimicrobial Effect ◽  
P Value ◽  
Antibiofilm Activity ◽  
Peripheral Blood Mononuclear ◽  
Biofilm Inhibition

Background and Objectives: The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from Pseudomonas aeruginosa against different bacterial species. Materials and Methods: The azurin gene was cloned in the pET21a vector. The pET21a-azurin construct was transformed into Escherichia coli BL21. The recombinant Azurin was expressed and purified using affinity chromatography and con- firmed by Western blotting. The cytotoxicity of rAzurin was assessed on peripheral blood mononuclear cells. Antibacterial and antibiofilm activity of rAzurin with different concentrations were determined by micro-broth dilution and crystal violet methods, respectively. The effect of rAzurin on bacterial species was statistically analyzed by t- test and spearman correla- tion. Results: The identity of purified protein was confirmed by blotting and distinguished as a 14 kDa band on 15% SDS-PAGE. The IC50 of rAzurin on Peripheral Blood Mononuclear Cell (PBMC) was determined as 377.91±0.5 µg/mL in 24 h. Vibrio cholerae and Campilobacter jejuni displayed the most sensitivity to rAzurin (27.5 and 55 μg/mL, respectively) and the highest resistance (220 μg/mL) was displayed by P. aeruginosa and E. coli. The MIC for other species was 110 μg/mL. The Minimum Biofilm Inhibition Concentration (MBIC) was determined as 220 μg/mL for Salmonella enterica and V. cholerae, 300 μg/mL for Shigella sonnei, Shigella flexneri and P. aeruginosa and 440 μg/mL for the other species. The antimicrobial effect of rAzurin on bacterial species were significant (p value<0.05) and correlation coefficient was negative. Conclusion: The rAzurin appears to be an appropriate choice and a new strategy for prevention of bacterial infection. It inhibits bacterial growth and biofilm formation and candidates as antimicrobial peptides.

scholarly journals Planktonic and Biofilm-Associated Pseudomonas aeruginosa and Staphylococcus epidermidis Elicit Differential Human Peripheral Blood Cell Responses

2021 ◽  
Vol 9 (9) ◽  
pp. 1846
Author(s):  
Esingül Kaya ◽  
Giovanna Batoni ◽  
Mariagrazia Di Luca ◽  
Eleonora Apolloni ◽  
Alessandro Mazzoni ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Staphylococcus Epidermidis ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Bacterial Species ◽  
Planktonic Cells ◽  
Peripheral Blood Mononuclear ◽  
Activation Markers

Despite the considerable progress made in recent years, our understanding of the human immune response to microbial biofilms is still poor. The aim of the present study was to compare the in vitro response of human peripheral blood mononuclear cells (PBMC) to biofilms and planktonic cells of Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacterial species particularly relevant in patients with cystic fibrosis or undergoing endovascular catheterization, respectively. PBMC isolated from healthy donors were co-cultured with 24 h-old biofilms or with exponentially growing cells of both species. Following 24 h of co-culture, the expression of early activation markers and the levels of cytokines in the culture supernatants were assessed by flow cytometry, while biofilm biomass and architecture were evaluated by crystal violet staining, CFU count, and confocal microscopy. Around 20% of PBMC was activated in response to both biofilms and planktonic cells of P. aeruginosa. In contrast, planktonic cells of S. epidermidis induced a statistically higher degree of activation than their biofilm counterpart (25% versus 15%; p < 0.01). P. aeruginosa biofilms stimulated pro-inflammatory (TNF-α, IL-1β, IFN-γ, and IL-6) and anti-inflammatory (IL-10) cytokine production at statistically significant levels higher than its planktonic counterpart, while an opposite trend was observed with S. epidermidis. Differences in the architecture of the biofilms and in the number of PBMC infiltrating the biofilms between the two bacterial species may at least partially explain these findings. Collectively, the results obtained highlighted marked differences in the host–cell response depending on the species and the mode of growth (biofilms versus planktonic cultures), allowing speculations on the different strategies adopted by P. aeruginosa and S. epidermidis to persist in the host during the course of chronic infections.

Peripheral Blood CD14+ Cells From Healthy Subjects Carry a Circular Conformation of Latent Cytomegalovirus Genome

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 394-398 ◽  
Author(s):  
Cynthia A. Bolovan-Fritts ◽  
Edward S. Mocarski ◽  
Jean A. Wiedeman
Keyword(s):  
Peripheral Blood ◽  
Viral Genome ◽  
Mononuclear Cells ◽  
Shigella Flexneri ◽  
Polymerase Chain Reaction Detection ◽  
Chain Reaction ◽  
Peripheral Blood Mononuclear ◽  
Circular Plasmid ◽  
Host Detection ◽  
Polymerase Chain

Abstract The majority of the human population harbors latent cytomegalovirus. Although CD14+ peripheral blood mononuclear cells have been implicated as sites of latency, the conformation of the latent viral genome in these cells is unknown. In this study, the conformation of viral genomic DNA was assessed in CD14+ cells from healthy virus seropositive carriers using an electrophoretic separation on native agarose gels in combination with polymerase chain reaction detection. Here we show that the viral genome migrates as a circular plasmid with a mobility equivalent to a circular 230-kb Shigella flexneri megaplasmid marker. Neither linear nor complex or integrated forms of the viral genome were detected. This report provides further evidence that the CD14+ cell population is an important site of viral latency in the naturally infected human host. Detection of the viral genome as a circular plasmid during latency suggests that this virus maintains its genome in a manner analogous to other herpesviruses where latent viral genome conformation has been studied.

Cytokine Production by Human Peripheral Blood Mononuclear Cells Stimulated by a Pseudomonas Aeruginosa Culture Filtrate: Role of Plasma and Polymyxin B

1996 ◽  
Vol 19 (5) ◽  
pp. 276-283 ◽  
Author(s):  
S. Sundaram ◽  
T.W. Barrett ◽  
N.K. Butt ◽  
R. Porat ◽  
A.J. King ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Peripheral Blood Mononuclear Cells ◽  
Culture Filtrate ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Polymyxin B ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Bacterial Products

The lack of consensus regarding the significance of transmembrane passage of bacterial products across hemodialysis membranes can be related to several methodological differences in the various studies, including the choice of circulating fluid in the blood compartment of the model, nature and concentration of the bacterial products employed to challenge the dialysate compartment and whether cytokine production by PMBC or the limulus amebocyte lysate (LAL) assay was used as the index of transfer and the cytokine used as the read-out. In this study, we examined the production of interleukin-1 alpha (IL-1α), interieukin-1 receptor antagonist (IL-1Ra) and interleukin-8 (IL-8) by peripheral blood mononuclear cells (PBMC) incubated with a Pseudomonas aeruginosa culture filtrate. Further, the effects of 10% autologous human plasma and Polymyxin B sulfate (PmB) on cytokine production by PBMC were also characterized. The results of our study indicate that the Ps. aeruginosa culture filtrate had both PmB suppressible and PmB non-suppressible components and that the addition of 10% human plasma significantly enhanced cytokine production by both PmB suppressible and PmB non-suppressible components. The enhancing effect of plasma was most evident at low concentrations of the filtrate. The inhibitory effect of PmB was most evident in samples cultured in the presence of 10% plasma. There was a direct correlation between the production of IL-1α and IL-1Ra suggesting that both pro-inflammatory cytokines and cytokine-specific inhibitory proteins are concurrently produced. There results have direct relevance to selection of study conditions for in vitro models used to study the transmembrane passage of bacterial products across hemodialysis membranes

scholarly journals Effects of exercise on gene expression in human peripheral blood mononuclear cells

2004 ◽  
Vol 97 (4) ◽  
pp. 1461-1469 ◽  
Author(s):  
Peter H. Connolly ◽  
Vincent J. Caiozzo ◽  
Frank Zaldivar ◽  
Dan Nemet ◽  
Jennifer Larson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Interleukin 1 ◽  
P Value ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Anti Inflammatory

Exercise leads to increases in circulating levels of peripheral blood mononuclear cells (PBMCs) and to a simultaneous, seemingly paradoxical increase in both pro- and anti-inflammatory mediators. Whether this is paralleled by changes in gene expression within the circulating population of PBMCs is not fully understood. Fifteen healthy men (18–30 yr old) performed 30 min of constant work rate cycle ergometry (∼80% peak O2 uptake). Blood samples were obtained preexercise (Pre), end-exercise (End-Ex), and 60 min into recovery (Recovery), and gene expression was measured using microarray analysis (Affymetrix GeneChips). Significant differential gene expression was defined with a posterior probability of differential expression of 0.99 and a Bayesian P value of 0.005. Significant changes were observed from Pre to End-Ex in 311 genes, from End-Ex to Recovery in 552 genes, and from Pre to Recovery in 293 genes. Pre to End-Ex upregulation of PBMC genes related to stress and inflammation [e.g., heat shock protein 70 (3.70-fold) and dual-specificity phosphatase-1 (4.45-fold)] was followed by a return of these genes to baseline by Recovery. The gene for interleukin-1 receptor antagonist (an anti-inflammatory mediator) increased between End-Ex and Recovery (1.52-fold). Chemokine genes associated with inflammatory diseases [macrophage inflammatory protein-1α (1.84-fold) and -1β (2.88-fold), and regulation-on-activation, normal T cell expressed and secreted (1.34-fold)] were upregulated but returned to baseline by Recovery. Exercise also upregulated growth and repair genes such as epiregulin (3.50-fold), platelet-derived growth factor (1.55-fold), and hypoxia-inducible factor-I (2.40-fold). A single bout of heavy exercise substantially alters PBMC gene expression characterized in many cases by a brisk activation and deactivation of genes associated with stress, inflammation, and tissue repair.

Peripheral Blood CD14+ Cells From Healthy Subjects Carry a Circular Conformation of Latent Cytomegalovirus Genome

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 394-398 ◽  
Author(s):  
Cynthia A. Bolovan-Fritts ◽  
Edward S. Mocarski ◽  
Jean A. Wiedeman
Keyword(s):  
Peripheral Blood ◽  
Viral Genome ◽  
Mononuclear Cells ◽  
Shigella Flexneri ◽  
Polymerase Chain Reaction Detection ◽  
Chain Reaction ◽  
Peripheral Blood Mononuclear ◽  
Circular Plasmid ◽  
Host Detection ◽  
Polymerase Chain

The majority of the human population harbors latent cytomegalovirus. Although CD14+ peripheral blood mononuclear cells have been implicated as sites of latency, the conformation of the latent viral genome in these cells is unknown. In this study, the conformation of viral genomic DNA was assessed in CD14+ cells from healthy virus seropositive carriers using an electrophoretic separation on native agarose gels in combination with polymerase chain reaction detection. Here we show that the viral genome migrates as a circular plasmid with a mobility equivalent to a circular 230-kb Shigella flexneri megaplasmid marker. Neither linear nor complex or integrated forms of the viral genome were detected. This report provides further evidence that the CD14+ cell population is an important site of viral latency in the naturally infected human host. Detection of the viral genome as a circular plasmid during latency suggests that this virus maintains its genome in a manner analogous to other herpesviruses where latent viral genome conformation has been studied.

scholarly journals Monocytes, Peripheral Blood Mononuclear Cells, and THP-1 Cells Exhibit Different Cytokine Expression Patterns following Stimulation with Lipopolysaccharide

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Anita Schildberger ◽  
Eva Rossmanith ◽  
Tanja Eichhorn ◽  
Katharina Strassl ◽  
Viktoria Weber
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Peripheral Blood Mononuclear Cells ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Umbilical Vein ◽  
Expression Patterns ◽  
Cytokine Release ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

THP-1 cells are widely applied to mimic monocytes in cell culture models. In this study, we compared the cytokine release from THP-1, peripheral blood mononuclear cells (PBMC), monocytes, or whole blood after stimulation with lipopolysaccharide (LPS) and investigated the consequences of different cytokine profiles on human umbilical vein endothelial cell (HUVEC) activation. WhilePseudomonas aeruginosa-stimulated (10 ng/mL) THP-1 secreted similar amounts of tumor necrosis factor alpha (TNF-α) as monocytes and PBMC, they produced lower amounts of interleukin(IL)-8 and no IL-6 and IL-10. Whole blood required a higher concentration ofPseudomonas aeruginosa(1000 ng/mL) to induce cytokine release than isolated monocytes or PBMC (10 ng/mL). HUVEC secreted more IL-6 and IL-8 after stimulation with conditioned medium derived from whole blood than from THP-1, despite equal concentrations of TNF-αin both media. Specific adsorption of TNF-αor selective cytokine adsorption from the conditioned media prior to HUVEC stimulation significantly reduced HUVEC activation. Our findings show that THP-1 differ from monocytes, PBMC, and whole blood with respect to cytokine release after stimulation with LPS. Additionally, we could demonstrate that adsorption of inflammatory mediators results in reduced endothelial activation, which supports the concept of extracorporeal mediator modulation as supportive therapy for sepsis.

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