scholarly journals Perforin-2 Permeabilizes the Envelope of Phagocytosed Bacteria

2018 ◽  
Author(s):  
Fangfang Bai ◽  
Ryan M. McCormack ◽  
Suzanne Hower ◽  
Gregory V. Plano ◽  
Mathias G. Lichtenheld ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Surface Antigen ◽  
Bactericidal Activity ◽  
Bacterial Pathogens ◽  
Intracellular Pathogen ◽  
Wild Type

AbstractPerforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study we used Salmonella typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2‘s bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of antigens contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface antigen was found to be independent of Perforin-2. Consistent with our in vitro results a protease sensitive, periplasmic superoxide disumutase (SodCII) contributed to the virulence of S. typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial envelope.

scholarly journals Reg4 defenses the intestine against Salmonella infection via binding the flagellin

2021 ◽  
Author(s):  
Yongtao Xiao ◽  
weipeng wang ◽  
Ying lu ◽  
xinbei tian ◽  
shanshan chen ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Bactericidal Activity ◽  
Intestinal Inflammation ◽  
Bactericidal Effect ◽  
Intestinal Epithelia ◽  
Food Borne ◽  
Negative Impacts ◽  
First Time

Salmonella Typhimurium is gram-negative flagellated bacteria that can cause food-borne gastroenteritis and diarrhea in humans and animals. The regenerating islet-derived family member 4 (Reg4) is overexpressed in the gastrointestinal tract during intestinal inflammation. However, the role of Reg4 in the intestinal inflammation induced by Salmonella Typhimurium is largely unknown. In this study, we reported for the first time that Reg4 has bactericidal activity against intestinal infection caused by Salmonella Typhimurium. In vivo, Reg4 could reduce the colonization of Salmonella Typhimurium and attenuate intestinal inflammation in the Salmonella Typhimurium-infected model. Additionally, the mice with the epithelial cell specific deletion of Reg4 (Reg4ΔIEC) exhibited more severe intestinal inflammation and more colonization of Salmonella Typhimurium. However, the administration of Reg4 could reverse these negative impacts. In vitro, Reg4 protein was showed to inhibit the growth of Salmonella Typhimurium. We further investigate the function motif of Reg4 and find that the "HDPQK" motif in Reg4 is essential to its bactericidal activity. Reg4 exerted the bactericidal effect by binding to the flagellin of Salmonella Typhimurium and suppressing its motility, adhesion, and invasion to the intestinal epithelia. In conclusion, our findings identify Reg4 as a novel antimicrobial peptide against infection by Salmonella Typhimurium and explore its possible mechanism, which may be of great significance for developing novel agents against flagellated micro pathogens.

scholarly journals Analysis of the Behavior of eryC Mutants of Brucella suis Attenuated in Macrophages

2005 ◽  
Vol 73 (10) ◽  
pp. 6782-6790 ◽  
Author(s):  
Sonja Burkhardt ◽  
Maria P. Jiménez de Bagüés ◽  
Jean-Pierre Liautard ◽  
Stephan Köhler
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Allelic Exchange ◽  
Brucella Suis ◽  
Lower Extent ◽  
Intracellular Multiplication

ABSTRACT The facultatively intracellular pathogen Brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in eryC of Brucella suis were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the eryC mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly, eryC mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the eryC gene significantly reduced the intramacrophagic and intramurine fitness of B. suis.

scholarly journals Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2530
Author(s):  
Bijean D. Ford ◽  
Diego Moncada Giraldo ◽  
Camilla Margaroli ◽  
Vincent D. Giacalone ◽  
Milton R. Brown ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Bactericidal Activity ◽  
Scavenger Receptor ◽  
Receptor Expression ◽  
Blood Monocytes ◽  
Bacterial Killing ◽  
Blood Neutrophils ◽  
Vitro Model

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats

2021 ◽  
pp. 1-24
Author(s):  
Juho-Matti Renko ◽  
Arun Kumar Mahato ◽  
Tanel Visnapuu ◽  
Konsta Valkonen ◽  
Mati Karelson ◽  
...  
Keyword(s):  
Mapk Signaling ◽  
Dopamine Neurons ◽  
Dopamine Depletion ◽  
Wild Type ◽  
Disease Modifying Therapy ◽  
Immortalized Cells ◽  
Midbrain Dopamine ◽  
6 Hydroxydopamine

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP +-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and could have protected dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying therapy for PD.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach ◽  
Telomeric Protein ◽  
Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

scholarly journals Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 320
Author(s):  
Thaís Pereira da Silva ◽  
Fernando Jacomini de Castro ◽  
Larissa Vuitika ◽  
Nayanne Louise Costacurta Polli ◽  
Bruno César Antunes ◽  
...  
Keyword(s):  
Structural Changes ◽  
Capillary Permeability ◽  
Structural Data ◽  
Histopathological Analysis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Antigenic Properties ◽  
Harmful Effects

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

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