The Drosophila melanogaster Toll Pathway Participates in Resistance to Infection by the Gram-Negative Human Pathogen Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is a gram-negative pathogen that infects immunocompromised and cystic fibrosis patients. The molecular basis of the host-P. aeruginosa interaction and the effect of specific P. aeruginosa virulence factors on various components of the innate immunity pathways are largely unknown. We examine interactions between P. aeruginosa virulence factors and components of innate immunity response in the Drosophila melanogaster model system to reveal the importance of the Toll signaling pathway in resistance to infection by the P. aeruginosa human isolate PA14. Using the two PA14-isogenic mutants plcS and dsbA, we show that Drosophila loss-of-function mutants of Spatzle, the extracellular ligand of Toll, and Dorsal and Dif, two NF-κB-like transcription factors, allow increased P. aeruginosa infectivity within fly tissues. In contrast, a constitutively active Toll mutant and a loss-of-function mutant of Cactus, an IκB-like factor that inhibits the Toll signaling, reduce infectivity. Our finding that Dorsal activity is required to restrict P. aeruginosa infectivity in Drosophila provides direct in vivo evidence for Dorsal function in adult fly immunity. Additionally, our results provide the basis for future studies into interactions between P. aeruginosa virulence factors and components of the Toll signaling pathway, which is functionally conserved between flies and humans.

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miR-958 inhibits Toll signaling and Drosomycin expression via direct targeting of Toll and Dif in Drosophila melanogaster

AJP Cell Physiology ◽

10.1152/ajpcell.00251.2016 ◽

2017 ◽

Vol 312 (2) ◽

pp. C103-C110 ◽

Cited By ~ 9

Author(s):

Shengjie Li ◽

Yao Li ◽

Li Shen ◽

Ping Jin ◽

Liming Chen ◽

...

Keyword(s):

Innate Immunity ◽

Drosophila Melanogaster ◽

Signaling Pathway ◽

Posttranscriptional Regulation ◽

Molecular Mechanisms ◽

Toll Signaling ◽

Modulation Pattern ◽

Direct Targeting

Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well studied, the precise mechanisms of posttranscriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. In this study, we used an in silico strategy in combination with the Gal80ts-Gal4 driver system to identify microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila. We report that overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. We further demonstrate in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958 sponge rescued the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results, not only revealed a novel function and modulation pattern of miR-958, but also provided a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.

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Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides

Frontiers in Physiology ◽

10.3389/fphys.2021.758859 ◽

2021 ◽

Vol 12 ◽

Author(s):

Young Min Bae ◽

Yong Hun Jo ◽

Bharat Bhusan Patnaik ◽

Bo Bae Kim ◽

Ki Beom Park ◽

...

Keyword(s):

Innate Immunity ◽

Antimicrobial Peptides ◽

Signaling Pathway ◽

Tenebrio Molitor ◽

Functional Assay ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Reading Frame ◽

E Coli ◽

Toll Signaling

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

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Paeonol Attenuates Quorum-Sensing Regulated Virulence and Biofilm Formation in Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2021.692474 ◽

2021 ◽

Vol 12 ◽

Author(s):

Dan Yang ◽

Suqi Hao ◽

Ling Zhao ◽

Fei Shi ◽

Gang Ye ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Biofilm Formation ◽

Resistant Bacteria ◽

Infection Model ◽

Dependent Manner ◽

Gram Negative ◽

C Elegans

With the prevalence of multidrug-resistant bacteria and clinical -acquired pathogenic infections, the development of quorum-sensing (QS) interfering agents is one of the most potential strategies to combat bacterial infections and antibiotic resistance. Chinese herbal medicines constitute a valuable bank of resources for the identification of QS inhibitors. Accordingly, in this research, some compounds were tested for QS inhibition using indicator strains. Paeonol is a phenolic compound, which can effectively reduce the production of violacein without affecting its growth in Chromobacterium violaceum ATCC 12472, indicating its excellent anti-QS activity. This study assessed the anti-biofilm activity of paeonol against Gram-negative pathogens and investigated the effect of paeonol on QS-regulated virulence factors in Pseudomonas aeruginosa. A Caenorhabditis elegans infection model was used to explore the anti-infection ability of paeonol in vivo. Paeonol exhibited an effective anti-biofilm activity against Gram-negative bacteria. The ability of paeonol to interfere with the AHL-mediated quorum sensing systems of P. aeruginosa was determined, found that it could attenuate biofilm formation, and synthesis of pyocyanin, protease, elastase, motility, and AHL signaling molecule in a concentration- and time-dependent manner. Moreover, paeonol could significantly downregulate the transcription level of the QS-related genes of P. aeruginosa including lasI/R, rhlI/R, pqs/mvfR, as well as mediated its virulence factors, lasA, lasB, rhlA, rhlC, phzA, phzM, phzH, and phzS. In vivo studies revealed that paeonol could reduce the pathogenicity of P. aeruginosa and enhance the survival rate of C. elegans, showing a moderate protective effect on C. elegans. Collectively, these findings suggest that paeonol attenuates bacterial virulence and infection of P. aeruginosa and that further research elucidating the anti-QS mechanism of this compound in vivo is warranted.

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Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Molecules ◽

10.3390/molecules26061497 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1497

Author(s):

Pansong Zhang ◽

Qiao Guo ◽

Zhihua Wei ◽

Qin Yang ◽

Zisheng Guo ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Mammalian Cells ◽

Secretion System ◽

Chinese Herbs ◽

Infection Model ◽

Lung Pathology ◽

Promising Alternative ◽

The Impact

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.

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Tackling Virulence of Pseudomonas aeruginosa by the Natural Furanone Sotolon

Antibiotics ◽

10.3390/antibiotics10070871 ◽

2021 ◽

Vol 10 (7) ◽

pp. 871

Author(s):

Mohammed F. Aldawsari ◽

El-Sayed Khafagy ◽

Ahmed Al Saqr ◽

Ahmed Alalaiwe ◽

Hisham A. Abbas ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Bacterial Infections ◽

Therapeutic Agent ◽

Bacterial Resistance ◽

Inhibitory Concentration ◽

Bacterial Biofilm ◽

Resistant Bacteria ◽

Resistance Development

The bacterial resistance development due to the incessant administration of antibiotics has led to difficulty in their treatment. Natural adjuvant compounds can be co-administered to hinder the pathogenesis of resistant bacteria. Sotolon is the prevailing aromatic compound that gives fenugreek its typical smell. In the current work, the anti-virulence activities of sotolon on Pseudomonas aeruginosa have been evaluated. P. aeruginosa has been treated with sotolon at sub-minimum inhibitory concentration (MIC), and production of biofilm and other virulence factors were assessed. Moreover, the anti-quorum sensing (QS) activity of sotolon was in-silico evaluated by evaluating the affinity of sotolon to bind to QS receptors, and the expression of QS genes was measured in the presence of sotolon sub-MIC. Furthermore, the sotolon in-vivo capability to protect mice against P. aeruginosa was assessed. Significantly, sotolon decreased the production of bacterial biofilm and virulence factors, the expression of QS genes, and protected mice from P. aeruginosa. Conclusively, the plant natural substance sotolon attenuated the pathogenicity of P. aeruginosa, locating it as a plausible potential therapeutic agent for the treatment of its infections. Sotolon can be used in the treatment of bacterial infections as an alternative or adjuvant to antibiotics to combat their high resistance to antibiotics.

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Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽

10.1534/genetics.109.106567 ◽

2009 ◽

Vol 183 (3) ◽

pp. 1165-1173 ◽

Cited By ~ 34

Author(s):

Shu Kondo ◽

Matthew Booker ◽

Norbert Perrimon

Keyword(s):

Cell Culture ◽

Drosophila Melanogaster ◽

Large Scale ◽

Transgenic Animals ◽

Selection Marker ◽

Bacterial Artificial Chromosomes ◽

Loss Of Function ◽

Artificial Chromosomes ◽

Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

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Efficacy of Humanized Cefiderocol Exposures over 72 Hours against a Diverse Group of Gram-Negative Isolates in the Neutropenic Murine Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01040-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01040-18 ◽

Cited By ~ 3

Author(s):

Sean M. Stainton ◽

Marguerite L. Monogue ◽

Masakatsu Tsuji ◽

Yoshinori Yamano ◽

Roger Echols ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Infection Model ◽

Diverse Group ◽

Gram Negative ◽

Content Type ◽

Adaptive Resistance

ABSTRACT Herein, we evaluated sustainability of humanized exposures of cefiderocol in vivo over 72 h against pathogens with cefiderocol MICs of 0.5 to 16 μg/ml in the neutropenic murine thigh model. In Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae displaying MICs of 0.5 to 8 μg/ml (n = 11), sustained kill was observed at 72 h among 9 isolates. Postexposure MICs revealed a single 2-dilution increase in one animal compared with controls (1/54 samples, 1.8%) at 72 h. Adaptive resistance during therapy was not observed.

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The rapid in vivo evolution of Pseudomonas aeruginosa in ventilator-associated pneumonia patients leads to attenuated virulence

Open Biology ◽

10.1098/rsob.170029 ◽

2017 ◽

Vol 7 (9) ◽

pp. 170029 ◽

Cited By ~ 17

Author(s):

Ke Wang ◽

Yi-qiang Chen ◽

May M. Salido ◽

Gurjeet S. Kohli ◽

Jin-liang Kong ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Ventilator Associated Pneumonia ◽

Loss Of Function ◽

Short Term ◽

Airway Infections ◽

Evolutionary Trajectories ◽

Human Airways ◽

Late Stages

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe airway infections in humans. These infections are usually difficult to treat and associated with high mortality rates. While colonizing the human airways, P. aeruginosa could accumulate genetic mutations that often lead to its better adaptability to the host environment. Understanding these evolutionary traits may provide important clues for the development of effective therapies to treat P. aeruginosa infections. In this study, 25 P. aeruginosa isolates were longitudinally sampled from the airways of four ventilator-associated pneumonia (VAP) patients. Pacbio and Illumina sequencing were used to analyse the in vivo evolutionary trajectories of these isolates. Our analysis showed that positive selection dominantly shaped P. aeruginosa genomes during VAP infections and led to three convergent evolution events, including loss-of-function mutations of lasR and mpl , and a pyoverdine-deficient phenotype. Specifically, lasR encodes one of the major transcriptional regulators in quorum sensing, whereas mpl encodes an enzyme responsible for recycling cell wall peptidoglycan. We also found that P. aeruginosa isolated at late stages of VAP infections produce less elastase and are less virulent in vivo than their earlier isolated counterparts, suggesting the short-term in vivo evolution of P. aeruginosa leads to attenuated virulence.

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MiRNA-143 mediates the proliferative signaling pathway of FSH and regulates estradiol production

Journal of Endocrinology ◽

10.1530/joe-16-0488 ◽

2017 ◽

Vol 234 (1) ◽

pp. 1-14 ◽

Cited By ~ 19

Author(s):

Li Zhang ◽

XiaoXin Zhang ◽

Xuejing Zhang ◽

Yu Lu ◽

Lei Li ◽

...

Keyword(s):

Signaling Pathway ◽

Granulosa Cell ◽

Granulosa Cells ◽

Loss Of Function ◽

Cellular Processes ◽

Cell Functions ◽

Genes Expression ◽

Underlying Mechanisms

MicroRNAs (MiRNAs) play important regulatory roles in many cellular processes. MiR-143 is highly enriched in the mouse ovary, but its roles and underlying mechanisms are not well understood. In the current study, we show that miR-143 is located in granulosa cells of primary, secondary and antral follicles. To explore the specific functions of miR-143, we transfected miR-143 inhibitor into primary cultured granulosa cells to study the loss of function of miR-143 and the results showed that miR-143 silencing significantly increased estradiol production and steroidogenesis-related gene expression. Moreover, our in vivo and in vitro studies showed that follicular stimulating hormone (FSH) significantly decreased miR-143 expression. This function of miR-143 is accomplished by its binding to the 3’-UTR of KRAS mRNA. Furthermore, our results demonstrated that miR-143 acts as a negative regulating molecule mediating the signaling pathway of FSH and affecting estradiol production by targeting KRAS. MiR-143 also negatively acts in regulating granulosa cells proliferation and cell cycle-related genes expression. These findings indicate that miR-143 plays vital roles in FSH-induced estradiol production and granulosa cell proliferation, providing a novel mechanism that involves miRNA in regulating granulosa cell functions.

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Drosophilaas a Model Host forPseudomonas aeruginosaInfection

Journal of Bacteriology ◽

10.1128/jb.183.4.1466-1471.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1466-1471 ◽

Cited By ~ 173

Author(s):

David A. D'Argenio ◽

Larry A. Gallagher ◽

Celeste A. Berg ◽

Colin Manoil

Keyword(s):

Signal Transduction ◽

Pseudomonas Aeruginosa ◽

Drosophila Melanogaster ◽

Gene Cluster ◽

Virulence Factors ◽

Fruit Fly ◽

Colony Morphology ◽

Twitching Motility ◽

Wild Type ◽

Signal Transduction System

ABSTRACT Using the fruit fly Drosophila melanogaster as model host, we have identified mutants of the bacterium Pseudomonas aeruginosa with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in pilGHIJKL chpABCDE, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The pil chp genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.

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