scholarly journals A Human Anti-Pseudomonas aeruginosa Serotype O6ad Immunoglobulin G1 Expressed in Transgenic Tobacco Is Capable of Recruiting Immune System Effector Function In Vitro

2007 ◽  
Vol 51 (9) ◽  
pp. 3322-3328 ◽  
Author(s):  
Michael D. McLean ◽  
Kurt C. Almquist ◽  
Yongfing Niu ◽  
Rhonda Kimmel ◽  
Zengzu Lai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune System ◽  
Transgenic Tobacco ◽  
Plant Transformation ◽  
Bacterial Pathogen ◽  
Effector Function ◽  
Transformation Vector ◽  
Plant Transformation Vector ◽  
Human Igg1

ABSTRACT The production of a recombinant human IgG1 in transgenic tobacco was examined to determine whether a plant-derived antibody could recruit immune system effector function against a bacterial pathogen. A plant transformation vector was engineered to contain genes for a human kappa light chain and a human gamma-1 heavy chain with VH and VL sequences from a previously identified human IgG2 monoclonal antibody (MAb) that specifically binds to and opsonizes Pseudomonas aeruginosa serotype O6ad. Unique NcoI and NotI restriction sites were incorporated to flank these variable sequences, resulting in a plant transformation vector that could be engineered for expression of any other human IgG1 antibody, requiring only the substitution of other VH and VL antigen-binding coding sequences. The plant-produced IgG1 was determined to have high-mannose glycan content and to be capable of mediating opsonophagocytosis of P. aeruginosa serotype O6ad in vitro using human complement and human polymorphonuclear leukocytes. Thus, MAbs produced in plants from this vector could provide human IgG1 MAbs for targeting other pathogens that require the recruitment of immune system effector functions.

scholarly journals Phage steering of antibiotic-resistance evolution in the bacterial pathogen, Pseudomonas aeruginosa

2020 ◽  
Vol 2020 (1) ◽  
pp. 148-157 ◽  
Author(s):  
James Gurney ◽  
Léa Pradier ◽  
Joanne S Griffin ◽  
Claire Gougat-Barbera ◽  
Benjamin K Chan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Bacterial Pathogen ◽  
Antibiotic Sensitivity ◽  
Resistant Bacteria ◽  
Phage Resistance ◽  
Antibiotic Resistant ◽  
Trade Off

Abstract Background and objectives Antimicrobial resistance is a growing global concern and has spurred increasing efforts to find alternative therapeutics. Bacteriophage therapy has seen near constant use in Eastern Europe since its discovery over a century ago. One promising approach is to use phages that not only reduce bacterial pathogen loads but also select for phage resistance mechanisms that trade-off with antibiotic resistance—so called ‘phage steering’. Methodology Recent work has shown that the phage OMKO1 can interact with efflux pumps and in so doing select for both phage resistance and antibiotic sensitivity of the pathogenic bacterium Pseudomonas aeruginosa. We tested the robustness of this approach to three different antibiotics in vitro (tetracycline, erythromycin and ciprofloxacin) and one in vivo (erythromycin). Results We show that in vitro OMKO1 can reduce antibiotic resistance of P. aeruginosa (Washington PAO1) even in the presence of antibiotics, an effect still detectable after ca.70 bacterial generations in continuous culture with phage. Our in vivo experiment showed that phage both increased the survival times of wax moth larvae (Galleria mellonella) and increased bacterial sensitivity to erythromycin. This increased antibiotic sensitivity occurred both in lines with and without the antibiotic. Conclusions and implications Our study supports a trade-off between antibiotic resistance and phage sensitivity. This trade-off was maintained over co-evolutionary time scales even under combined phage and antibiotic pressure. Similarly, OMKO1 maintained this trade-off in vivo, again under dual phage/antibiotic pressure. Our findings have implications for the future clinical use of steering in phage therapies. Lay Summary: Given the rise of antibiotic-resistant bacterial infection, new approaches to treatment are urgently needed. Bacteriophages (phages) are bacterial viruses. The use of such viruses to treat infections has been in near-continuous use in several countries since the early 1900s. Recent developments have shown that these viruses are not only effective against routine infections but can also target antibiotic resistant bacteria in a novel, unexpected way. Similar to other lytic phages, these so-called ‘steering phages’ kill the majority of bacteria directly. However, steering phages also leave behind bacterial variants that resist the phages, but are now sensitive to antibiotics. Treatment combinations of these phages and antibiotics can now be used to greater effect than either one independently. We evaluated the impact of steering using phage OMKO1 and a panel of three antibiotics on Pseudomonas aeruginosa, an important pathogen in hospital settings and in people with cystic fibrosis. Our findings indicate that OMKO1, either alone or in combination with antibiotics, maintains antibiotic sensitivity both in vitro and in vivo, giving hope that phage steering will be an effective treatment option against antibiotic-resistant bacteria.

scholarly journals Gene Stacking for Fungal Resistance in Plant Transformation Vector

2017 ◽  
Vol 14 (3) ◽  
pp. 1211-1219
Author(s):  
Sonia Sharma ◽  
Gurtej Singh ◽  
Sadiq Pasha Shaik ◽  
Gagandeep Singh ◽  
Sumangala Bhat ◽  
...  
Keyword(s):  
Plant Transformation ◽  
Fruit Production ◽  
Fungal Diseases ◽  
Fungal Resistance ◽  
Hindiii Site ◽  
Transformation Vector ◽  
Fungal Disease Resistance ◽  
Genes Encoding ◽  
Production Form ◽  
Plant Transformation Vector

ABSTRACT: Fungal diseases like early blight, late blight, fusarium wilt cause 30-40 per cent loss in fruit production. Form past decade many transgenic plants had been developed using genes encoding chitinases and glucanases with the objective of imparting fungal disease resistance. Since the genes encoding chitinase and glucanase act synergistically. The study was performed to construct plant transformation vector pRAGS carrying both ech42 and bgn under single T-DNA region. Initially, HindIII site at 5' end of earlier cloned bgn (T. harzianum) was removed using primers during reamplification of the gene. The amplicons were cloned into pTZ57R/T containing T overhangs at Eco321 site and transferred to E. coli DH5a and further to plant transformation vector pBI121 which was named as pRA121. In order to clone another gene (ech42) into pRA121, expression cassette from iHP vector was transferred to pRA121 and named as pRAG121. Further in order to gain XhoI site for cloning ech42 gene into pRAG121, ech42 (pSUM1) was cloned into pYES2/CT, named as pSAG1, ech42 from pSAG1 cloned with KpnI and XhoI in pRAG121 and named as pRAGS121. The vector constructed in the present study can be used to transform important crop plants to have enhanced resistance to fungal diseases.

Construction of a new type of multi-gene plant transformation vector and genetic transformation of tobacco

2017 ◽  
Vol 61 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Y. Dong ◽  
Y. C. Ren ◽  
M. S. Yang ◽  
J. Zhang ◽  
T. Qiu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Plant Transformation ◽  
Transformation Vector ◽  
New Type ◽  
Plant Transformation Vector

scholarly journals Phospholipid peroxidation fuels ExoU phospholipase-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology

2021 ◽  
Author(s):  
Salimata Bagayoko ◽  
Stephen Leon Icaza ◽  
Miriam Pinilla ◽  
Audrey Hessel ◽  
Karin Santoni ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Pathogen ◽  
Organ Damage ◽  
The Body ◽  
Cell Necrosis ◽  
Cellular Mechanisms ◽  
Dependent Cell

SummaryRegulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

scholarly journals Flagellar Motility Is a Key Determinant of the Magnitude of the Inflammasome Response to Pseudomonas aeruginosa

2013 ◽  
Vol 81 (6) ◽  
pp. 2043-2052 ◽  
Author(s):  
Yash R. Patankar ◽  
Rustin R. Lovewell ◽  
Matthew E. Poynter ◽  
Jeevan Jyot ◽  
Barbara I. Kazmierczak ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune System ◽  
Innate Immune System ◽  
Innate Immune ◽  
Bacterial Motility ◽  
Inflammasome Activation ◽  
Flagellar Motility ◽  
Content Type

ABSTRACTWe previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronicPseudomonas aeruginosainfections, enables bacteria to evade association and ingestion ofP. aeruginosaby phagocytes bothin vitroandin vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation byP. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotileP. aeruginosa. NonmotileP. aeruginosaelicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cellsin vitro. Importantly, nonmotileP. aeruginosaalso elicits reduced IL-1β levelsin vivoin comparison to those elicited by wild-typeP. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system.

scholarly journals Acquisition of Expression of the Pseudomonas aeruginosa ExoU Cytotoxin Leads to Increased Bacterial Virulence in a Murine Model of Acute Pneumonia and Systemic Spread

2000 ◽  
Vol 68 (7) ◽  
pp. 3998-4004 ◽  
Author(s):  
Markus Allewelt ◽  
Fadie T. Coleman ◽  
Martha Grout ◽  
Gregory P. Priebe ◽  
Gerald B. Pier
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Animal Models ◽  
Epithelial Cells ◽  
Murine Model ◽  
Lethal Dose ◽  
Bacterial Pathogen ◽  
Systemic Spread ◽  
Acute Pneumonia ◽  
Cultured Epithelial Cells

ABSTRACT Pseudomonas aeruginosa is the nosocomial bacterial pathogen most commonly isolated from the respiratory tract. Animal models of this infection are extremely valuable for studies of virulence and immunity. We thus evaluated the utility of a simple model of acute pneumonia for analyzing P. aeruginosa virulence by characterizing the course of bacterial infection in BALB/c mice following application of bacteria to the nares of anesthetized animals. Bacterial aspiration into the lungs was rapid, and 67 to 100% of the inoculum could be recovered within minutes from the lungs, with 0.1 to 1% of the inoculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin exclusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by P. aeruginosa is associated with a cytotoxic effect on epithelial cells in vitro and virulence in animal models. Insertional mutations in the exoU gene confer a noncytotoxic phenotype on mutant strains and decrease virulence for animals. We used the model of acute pneumonia to determine whether introduction of the exoUgene into noncytotoxic strains of P. aeruginosa lacking this gene affected virulence. Seven phenotypically noncytotoxicP. aeruginosa strains were transformed with pUCP19exoUspcU which carries the exoU gene and its associated chaperone. Three of these strains became cytotoxic to cultured epithelial cells in vitro. These strains all secreted ExoU, as confirmed by detection of the ExoU protein with specific antisera. The 50% lethal dose of exoU-expressing strains was significantly lower for all three P. aeruginosa isolates carrying plasmid pUCP19exoUspcU than for the isogenicexoU-negative strains. mRNA specific for ExoU was readily detected in the lungs of animals infected with the transformed P. aeruginosa strains. Introduction of the exoU gene confers a cytotoxic phenotype on some, but not all, otherwise-noncytotoxic P. aeruginosa strains and, for recombinant strains that could express ExoU, there was markedly increased virulence in a murine model of acute pneumonia and systemic spread.

Mutation of lasA and lasB reduces Pseudomonas aeruginosa invasion of epithelial cells

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2291-2299 ◽  
Author(s):  
Brigitte A. Cowell ◽  
Sally S. Twining ◽  
Jeffrey A. Hobden ◽  
Mary S. F. Kwong ◽  
Suzanne M. J. Fleiszig
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Culture Medium ◽  
Bacterial Pathogen ◽  
Regulatory Elements ◽  
Effector Proteins ◽  
Sensing Systems ◽  
Steady State Levels ◽  
Tight Junction Disruption

Pseudomonas aeruginosa is an opportunistic bacterial pathogen implicated in a variety of devastating conditions. Its flexibility as a pathogen is attributed to a myriad of virulence factors and regulatory elements that respond to prevailing environmental conditions. ExoS and ExoT are type III secreted effector proteins, regulated by the transcriptional activator ExsA, that can inhibit invasion of epithelial cells by cytotoxic strains of P. aeruginosa. This study sought to understand why invasive strains, which can secrete both ExoS and ExoT, still invade epithelial cells. The results showed that LasA and elastase (LasB), which are regulated by the Las and Rhl quorum-sensing systems, modulated P. aeruginosa invasion. Mutation of lasA and/or lasB reduced P. aeruginosa invasion, which was not fully restored by extracellularly added LasB, P. aeruginosa conditioned medium containing LasA and LasB, or EGTA pretreatment of cells. This indicated that protease effects on invasion involved factors additional to tight junction disruption and subsequent alterations to cell polarity. Upon mutation of lasA and/or lasB, steady-state levels of ExoS and ExoT were increased in culture medium of P. aeruginosa grown under conditions stimulatory for these toxins. The increase in ExoS was significantly correlated with reduced invasion. In vitro experiments showed that purified LasB degraded recombinant ExoS. Taken together, these studies suggest a mechanism by which invasive strains can synthesize inhibitors of invasion, ExoS and ExoT, yet still invade epithelial cells. By this mechanism, LasA and LasB decrease the levels of the toxins directly or indirectly, and thus reduce inhibition of invasion.

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