31 results on '"van Hoek, Monique L."'
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2. Diverse roles of low-molecular weight thiol GSH in Francisella’s virulence, location sensing and GSH-stealing from host
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van Hoek, Monique L., Marchesani, Alexander, and Rawat, Mamta
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- 2024
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3. Use of magnetic nanotrap particles in capturing Yersinia pestis virulence factors, nucleic acids and bacteria
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Ii, Alexandra N., Lin, Shih-Chao, Lepene, Benjamin, Zhou, Weidong, Kehn-Hall, Kylene, and van Hoek, Monique L.
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- 2021
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4. GATR-3, a Peptide That Eradicates Preformed Biofilms of Multidrug-Resistant Acinetobacter baumannii.
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van Hoek, Monique L., Alsaab, Fahad M., and Carpenter, Ashley M.
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PEPTIDES ,ACINETOBACTER baumannii ,MICROBIAL sensitivity tests ,PEPTIDE antibiotics ,ERYTHROCYTES ,BIOFILMS ,ANTIBIOTICS assay - Abstract
Acinetobacter baumannii is a gram-negative bacterium that causes hospital-acquired and opportunistic infections, resulting in pneumonia, sepsis, and severe wound infections that can be difficult to treat due to antimicrobial resistance and the formation of biofilms. There is an urgent need to develop novel antimicrobials to tackle the rapid increase in antimicrobial resistance, and antimicrobial peptides (AMPs) represent an additional class of potential agents with direct antimicrobial and/or host-defense activating activities. In this study, we present GATR-3, a synthetic, designed AMP that was modified from a cryptic peptide discovered in American alligator, as our lead peptide to target multidrug-resistant (MDR) A. baumannii. Antimicrobial susceptibility testing and antibiofilm assays were performed to assess GATR-3 against a panel of 8 MDR A. baumannii strains, including AB5075 and some clinical strains. The GATR-3 mechanism of action was determined to be via loss of membrane integrity as measured by DiSC
3 (5) and ethidium bromide assays. GATR-3 exhibited potent antimicrobial activity against all tested multidrug-resistant A. baumannii strains with rapid killing. Biofilms are difficult to treat and eradicate. Excitingly, GATR-3 inhibited biofilm formation and, more importantly, eradicated preformed biofilms of MDR A. baumannii AB5075, as evidenced by MBEC assays and scanning electron micrographs. GATR3 did not induce resistance in MDR A. baumannii, unlike colistin. Additionally, the toxicity of GATR-3 was evaluated using human red blood cells, HepG2 cells, and waxworms using hemolysis and MTT assays. GATR-3 demonstrated little to no cytotoxicity against HepG2 and red blood cells, even at 100 μg/mL. GATR-3 injection showed little toxicity in the waxworm model, resulting in a 90% survival rate. The therapeutic index of GATR-3 was estimated (based on the HC50 /MIC against human RBCs) to be 1250. Overall, GATR-3 is a promising candidate to advance to preclinical testing to potentially treat MDR A. baumannii infections. [ABSTRACT FROM AUTHOR]- Published
- 2024
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5. The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters
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van Hoek, Monique L., Prickett, M. Dennis, Settlage, Robert E., Kang, Lin, Michalak, Pawel, Vliet, Kent A., and Bishop, Barney M.
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- 2019
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6. Computationally Designed AMPs with Antibacterial and Antibiofilm Activity against MDR Acinetobacter baumannii.
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Alsaab, Fahad M., Dean, Scott N., Bobde, Shravani, Ascoli, Gabriel G., and van Hoek, Monique L.
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ACINETOBACTER baumannii ,ANTIMICROBIAL peptides ,ANTIBACTERIAL agents ,PEPTIDE antibiotics ,ERYTHROCYTES ,PEPTIDES - Abstract
The discovery of new antimicrobials is necessary to combat multidrug-resistant (MDR) bacteria, especially those that infect wounds and form prodigious biofilms, such as Acinetobacter baumannii. Antimicrobial peptides (AMPs) are a promising class of new therapeutics against drug-resistant bacteria, including gram-negatives. Here, we utilized a computational AMP design strategy combining database filtering technology plus positional analysis to design a series of novel peptides, named HRZN, designed to be active against A. baumannii. All of the HRZN peptides we synthesized exhibited antimicrobial activity against three MDR A. baumannii strains with HRZN-15 being the most active (MIC 4 µg/mL). This peptide also inhibited and eradicated biofilm of A. baumannii strain AB5075 at 8 and 16 µg/mL, which is highly effective. HRZN-15 permeabilized and depolarized the membrane of AB5075 rapidly, as demonstrated by the killing kinetics. HRZN 13 and 14 peptides had little to no hemolysis activity against human red blood cells, whereas HRZN-15, -16, and -17 peptides demonstrated more significant hemolytic activity. HRZN-15 also demonstrated toxicity to waxworms. Further modification of HRZN-15 could result in a new peptide with an improved toxicity profile. Overall, we successfully designed a set of new AMPs that demonstrated activity against MDR A. baumannii using a computational approach. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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7. Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound
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Chung, Ezra M. C., Dean, Scott N., Propst, Crystal N., Bishop, Barney M., and van Hoek, Monique L.
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- 2017
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8. Ab initio Designed Antimicrobial Peptides Against Gram-Negative Bacteria.
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Bobde, Shravani S., Alsaab, Fahad M., Wang, Guangshuan, and Van Hoek, Monique L.
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ANTIBIOTICS ,ANTIMICROBIAL peptides ,GRAM-negative bacterial diseases ,ANTIBACTERIAL agents ,PEPTIDOMIMETICS ,ERYTHROCYTES ,GRAM-positive bacteria ,GRAM-negative bacteria - Abstract
Antimicrobial peptides (AMPs) are ubiquitous amongst living organisms and are part of the innate immune system with the ability to kill pathogens directly or indirectly by modulating the immune system. AMPs have potential as a novel therapeutic against bacteria due to their quick-acting mechanism of action that prevents bacteria from developing resistance. Additionally, there is a dire need for therapeutics with activity specifically against Gram-negative bacterial infections that are intrinsically difficult to treat, with or without acquired drug resistance. Development of new antibiotics has slowed in recent years and novel therapeutics (like AMPs) with a focus against Gram-negative bacteria are needed. We designed eight novel AMPs, termed PHNX peptides, using ab initio computational design (database filtering technology combined with the novel positional analysis on APD3 dataset of AMPs with activity against Gram-negative bacteria) and assessed their theoretical function using published machine learning algorithms, and finally, validated their activity in our laboratory. These AMPs were tested to establish their minimum inhibitory concentration (MIC) and half-maximal effective concentration (EC
50 ) under CLSI methodology against antibiotic resistant and antibiotic susceptible Escherichia coli and Staphylococcus aureus. Laboratory-based experimental results were compared to computationally predicted activities for each of the peptides to ascertain the accuracy of the computational tools used. PHNX-1 demonstrated antibacterial activity (under high and low-salt conditions) against antibiotic resistant and susceptible strains of Gram-positive and Gram-negative bacteria and PHNX-4 to -8 demonstrated low-salt antibacterial activity only. The AMPs were then evaluated for cytotoxicity using hemolysis against human red blood cells and demonstrated some hemolysis which needs to be further evaluated. In this study, we successfully developed a design methodology to create synthetic AMPs with a narrow spectrum of activity where the PHNX AMPs demonstrated higher antibacterial activity against Gram-negative bacteria compared to Gram-positive bacteria. Thus, these peptides present novel synthetic peptides with a potential for therapeutic use. Based on our findings, we propose upfront selection of the peptide dataset for analysis, an additional step of positional analysis to add to the ab initio database filtering technology (DFT) method, and we present laboratory data on the novel, synthetically designed AMPs to validate the results of the computational approach. We aim to conduct future in vivo studies which could establish these AMPs for clinical use. [ABSTRACT FROM AUTHOR]- Published
- 2021
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9. Genetic Determinants of Antibiotic Resistance in Francisella.
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Kassinger, Stephen J. and van Hoek, Monique L.
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DRUG resistance in bacteria ,FRANCISELLA tularensis ,COLISTIN ,DRUG resistance in microorganisms ,MOLECULAR cloning ,TREATMENT effectiveness ,TULAREMIA - Abstract
Tularemia, caused by Francisella tularensis , is endemic to the northern hemisphere. This zoonotic organism has historically been developed into a biological weapon. For this Tier 1, Category A select agent, it is important to expand our understanding of its mechanisms of antibiotic resistance (AMR). Francisella is unlike many Gram-negative organisms in that it does not have significant plasmid mobility, and does not express AMR mechanisms on plasmids; thus plasmid-mediated resistance does not occur naturally. It is possible to artificially introduce plasmids with AMR markers for cloning and gene expression purposes. In this review, we survey both the experimental research on AMR in Francisella and bioinformatic databases which contain genomic and proteomic data. We explore both the genetic determinants of intrinsic AMR and naturally acquired or engineered antimicrobial resistance as well as phenotypic resistance in Francisella. Herein we survey resistance to beta-lactams, monobactams, carbapenems, aminoglycosides, tetracycline, polymyxins, macrolides, rifampin, fosmidomycin, and fluoroquinolones. We also highlight research about the phenotypic AMR difference between planktonic and biofilm Francisella. We discuss newly developed methods of testing antibiotics against Francisella which involve the intracellular nature of Francisella infection and may better reflect the eventual clinical outcomes for new antibiotic compounds. Understanding the genetically encoded determinants of AMR in Francisella is key to optimizing the treatment of patients and potentially developing new antimicrobials for this dangerous intracellular pathogen. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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10. Francisella novicida Two-Component System Response Regulator BfpR Modulates iglC Gene Expression, Antimicrobial Peptide Resistance, and Biofilm Production.
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Dean, Scott N., Milton, Morgan E., Cavanagh, John, and van Hoek, Monique L.
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DRUG resistance in microorganisms ,CATHELICIDINS ,GENE expression ,GENETIC regulation ,BACTERIAL proteins ,X-ray crystallography ,PROTEOMICS - Abstract
Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida , FTN_1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN_1452). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg
2+ . In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida. [ABSTRACT FROM AUTHOR]- Published
- 2020
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11. Two-Component Systems in Francisella Species.
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van Hoek, Monique L., Hoang, Ky V., and Gunn, John S.
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GENE expression in bacteria ,FRANCISELLA tularensis ,DNA-binding proteins ,CELLULAR signal transduction ,SPECIES - Abstract
Bacteria alter gene expression in response to changes in their environment through various mechanisms that include signal transduction systems. These signal transduction systems use membrane histidine kinase with sensing domains to mediate phosphotransfer to DNA-binding proteins that alter the level of gene expression. Such regulators are called two-component systems (TCSs). TCSs integrate external signals and information from stress pathways, central metabolism and other global regulators, thus playing an important role as part of the overall regulatory network. This review will focus on the knowledge of TCSs in the Gram-negative bacterium, Francisella tularensis , a biothreat agent with a wide range of potential hosts and a significant ability to cause disease. While TCSs have been well-studied in several bacterial pathogens, they have not been well-studied in non-model organisms, such as F. tularensis and its subspecies, whose canonical TCS content surprisingly ranges from few to none. Additionally, of those TCS genes present, many are orphan components, including KdpDE, QseC, QseB/PmrA, and an unnamed two-component system (FTN_1452/FTN_1453). We discuss recent advances in this field related to the role of TCSs in Francisella physiology and pathogenesis and compare the TCS genes present in human virulent versus. environmental species and subspecies of Francisella. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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12. Cathelicidin peptide rescues G. mellonella infected with B. anthracis.
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Blower, Ryan J., Popov, Serguei G., and van Hoek, Monique L.
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BACILLUS anthracis ,CATHELICIDIN antimicrobial peptide ,NATURAL immunity ,GREATER wax moth ,CIPROFLOXACIN - Published
- 2018
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13. Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.
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Bishop, Barney M., Juba, Melanie L., Russo, Paul S., Devine, Megan, Barksdale, Stephanie M., Scott, Shaylyn, Settlage, Robert, Michalak, Pawel, Gupta, Kajal, Vliet, Kent, Schnur, Joel M., and van Hoek, Monique L.
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- 2017
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14. Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii.
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Barksdale, Stephanie M., Hrifko, Evelyn J., Chung, Ezra Myung-Chul, and van Hoek, Monique. L.
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PEPTIDE antibiotics ,AMERICAN alligator ,PSEUDOMONAS aeruginosa ,STAPHYLOCOCCUS aureus ,MULTIDRUG resistance in bacteria - Abstract
Background: Our group has developed a new process for isolating and identifying novel cationic antimicrobial peptides from small amounts of biological samples. Previously, we identified several active antimicrobial peptides from 100 µl of plasma from Alligator mississippiensis. These peptides were found to have in vitro antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In this work, we further characterize three of the novel peptides discovered using this process: Apo5, Apo6, and A1P. Results: We examined the activity of these peptides against multi-drug resistant strains and clinical isolates of common human pathogens. We investigated their structural characteristics using circular dichroism and tested for membrane disruption and DNA binding. These peptides were found to have strong in vitro activity against multi-drug resistant and clinically isolated strains of S. aureus, Escherichia coli, P. aeruginosa, and Acinetobacter baumannii. Apo5 and Apo6, peptides derived from alligator apolipoprotein C-1, depolarized the bacterial membrane. A1P, a peptide from the serpin proteinase inhibitor, did not permeabilize membranes. Performing circular dichroism analysis, Apo5 and Apo6 were found to be predominantly helical in SDS and TFE buffer, while A1P has significantly different structures in phosphate buffer, SDS, and TFE. None of these peptides were found to be hemolytic to sheep red blood cells or significantly cytotoxic up to 100 µg/ml after 24 h exposure. Conclusions: Overall, we suggest that Apo5 and Apo6 have a different mode of action than A1P, and that all three peptides make promising candidates for the treatment of drug-resistant bacteria, such as A. baumannii. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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15. Nanoaerosols reduce required effective dose of liposomal levofloxacin against pulmonary murine Francisella tularensis subsp. novicida infection.
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Propst, Crystal N., Nwabueze, Albert O., Kanev, Igor L., Pepin, Rachel E., Gutting, Bradford W., Morozov, Victor N., and van Hoek, Monique L.
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FRANCISELLA ,QUANTUM dots ,LIPOSOMES ,INTRAPERITONEAL injections ,LABORATORY mice - Abstract
Background: The Institute of Theoretical and Experimental Biophysics in Moscow recently developed a new nanoaerosol generator. This study evaluated this novel technology, which has the potential to enhance therapeutic delivery, with the goal of using the generator to treat pulmonary Francisella tularensis subsp. novicida (F. novicida) infections in BALB/c mice. Results: First, the analysis of quantum dots distribution in cryosections of murine lungs demonstrated that nanoaerosols penetrate the alveoli and spread more homogenously in the lungs than upon intranasal delivery. Second, the generator was used to aerosolize the antibiotic levofloxacin to determine the effectiveness of nanoaerosolized levofloxacin as treatment against F. novicida. The generator was capable of delivering a sufficient dose of nanoaerosolized liposome-encapsulated levofloxacin to rescue mice against 100LD
50 of F. novicida. Conclusions: The nanoaerosol-delivered dosage of liposome-encapsulated levofloxacin required to rescue mice is approximately 94× lower than the oral required dose and approximately 8× lower than the intraperitoneal dose required for rescue. In addition, treatment with nanoaerosols consumes less total volume of therapeutic solutions and is gentler on sprayed material than the aerosolization by a conventional three-jet Collison nebulizer as seen by the preservation of liposomes. This could represent a significant advance for the use of expensive therapeutics and lung directed therapies. [ABSTRACT FROM AUTHOR]- Published
- 2016
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16. Potentiation of Francisella resistance to conventional antibiotics through small molecule adjuvants.
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Stephens, Matthew D., Hubble, Veroncia B., Ernst, Robert K., van Hoek, Monique L., Melander, Roberta J., Cavanagh, John, and Melander, Christian
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- 2016
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17. A rapid real-time quantitative PCR assay to determine the minimal inhibitory extracellular concentration of antibiotics against an intracellular Francisella tularensis Live Vaccine Strain.
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Aloni-Grinstein, Ronit, Shifman, Ohad, Lazar, Shlomi, Steinberger-Levy, Ida, Maoz, Sharon, Ber, Raphael, Van Hoek, Monique L., Kubelkova, Klara, and Straskova, Adela
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FRANCISELLA tularensis ,ANTIBIOTICS ,TULAREMIA - Abstract
Francisella tularensis is a highly virulent facultative intracellular bacterium. The lack of a safe and efficient vaccine makes antibiotics the preferred treatment. F. tularensis antibiotic susceptibility tests are based on the in vitro standard CLSIapproved microdilution method for determining the MIC. However, limited data are available regarding the minimal inhibitory extracellular concentration (MIEC) needed to eradicate intracellular bacteria. Here, we evaluated the MIEC values of various WHOrecommended antibiotics and compared the MIEC values to the established MICs. We describe a rapid 3-h quantitative PCR (qPCR) intracellular antibiogram assay, which yields comparable MIEC values to those obtained by the classical 72-h cfu assay. This rapid qPCR assay is highly advantageous in light of the slow growth rates of F. tularensis. Our results showed that the MIECs obtained for doxycycline, chloramphenicol and ciprofloxacin were indicative of intracellular activity. Gentamicin was not effective against intracellular bacteria for at least 32 h post treatment, raising the question of whether slow-penetrating gentamicin should be used for certain stages of the disease. We suggest that the qPCR intracellular antibiogram assay may be used to screen for potentially active antibiotics against intracellular F. tularensis as well as to detect strains with acquired resistance to recommended antibiotics. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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18. Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry.
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Juba, Melanie L., Russo, Paul S., Devine, Megan, Barksdale, Stephanie, Rodriguez, Carlos, Vliet, Kent A., Schnur, Joel M., van Hoek, Monique L., and Bishop, Barney M.
- Published
- 2015
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19. Extracellular vesicles from infected cells: potential for direct pathogenesis.
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Schwab, Angela, Meyering, Shabana S., Lepene, Ben, Iordanskiy, Sergey, van Hoek, Monique L., Hakami, Ramin M., Kashanchi, Fatah, Paiardini, Mirko, and Byeongwoon Song
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VESICLES (Cytology) ,CELLULAR pathology ,EXTRACELLULAR matrix proteins - Abstract
Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogenassociated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFkB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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20. Acyl carrier protein is a bacterial cytoplasmic target of cationic antimicrobial peptide LL-37.
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Myung-Chul Chung, Dean, Scott N., and van Hoek, Monique L.
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ANTIMICROBIAL peptides ,ACYL carrier protein ,CYTOPLASM ,FATTY acid-binding proteins ,MEMBRANE permeability (Biology) - Abstract
In addition tomembrane disruption, the cathelicidin antimicrobial peptide (AMP) LL-37 translocates through the bacterial inner membrane to target intracellular molecules. The present study aims to identify an alternate mechanism and a cytoplasmic target of LL-37 in Francisella. LL-37 binding proteins from Francisella novicida U112 bacterial lysates were precipitated by using biotinylated LL-37 (B-LL-37) and NeutrAvidin-agarose beads. Bound proteins were identified by LC-MS/MS, validated and characterized by bead pull-down assays and differential scanning fluorimetry (DSF). The cationic AMP (CAMP) LL- 37 was able to interact with Francisella cytoplasmic acyl carrier protein (AcpP; FTN1340/FTT1376). Further study confirmed that LL-37 peptide could bind to AcpP and that the sheep cathelicidin SMAP-29 (Sheep Myeloid Antimicrobial Peptide 29) further increased LL-37 binding to AcpP, suggesting a synergistic effect of SMAP-29 on the binding. LL-37 could also bind to both AcpP of Escherichia coli and Bacillus anthracis, implying amechanism of broad action of LL-37-AcpP binding. Overexpression of the acpP gene in F. novicida led to an increase in LL-37 susceptibility. LL-37 binding to AcpP changed the fatty acid composition profiles. Taken together, we identified a novel cytoplasmic target of LL-37 in Francisella, suggesting a mechanism of action of this peptide beyond membrane permeabilization. Our findings highlight a novel mechanism of antimicrobial activity of this peptide and document a previously unexplored target of α-helical CAMPs. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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21. Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics.
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Gupta, Kajal, Singh, Sameer, and van Hoek, Monique L.
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PATHOGENIC bacteria ,ANTI-infective agents ,PEPTIDE synthesis ,MYCOBACTERIUM smegmatis ,CELL membranes ,ANTIBIOTICS - Abstract
Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC
50 ranging from 0.05 to 1.88 µg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 µg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 µg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism with LL-37, ATRA-1A, and hBD3-Pep4. In further studies, polymyxin B synergized with LL-37, ATRA-1A, and hBD3-Pep4 while Rifampicin synergized with LL-37 and mCRAMP for intracellular killing of mycobacteria residing inside macrophages. These studies provide the foundation for the potential development of synthetic cationic antimicrobial peptides with activity against mycobacteria. [ABSTRACT FROM AUTHOR]- Published
- 2015
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22. Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis.
- Author
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Blower, Ryan J., Barksdale, Stephanie M., and van Hoek, Monique L.
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CATHELICIDIN antimicrobial peptide ,BURKHOLDERIA thailandensis ,SOIL microbiology ,MELIOIDOSIS ,BURKHOLDERIA pseudomallei ,CYCLIC peptides ,THERAPEUTICS - Abstract
Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly “highly resistant” to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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23. Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida.
- Author
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Dean, Scott N and van Hoek, Monique L
- Subjects
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FRANCISELLA novicida , *GRAM-negative bacteria , *BIOTERRORISM research , *MICROBIAL virulence , *MAPROTILINE , *THERAPEUTICS - Abstract
Development of new therapeutics against Select Agents such as Francisella is critical preparation in the event of bioterrorism. Testing FDA-approved drugs for this purpose may yield new activities unrelated to their intended purpose and may hasten the discovery of new therapeutics. A library of 420 FDA-approved drugs was screened for antibiofilm activity against a model organism for human tularemia, Francisella (F.) novicida, excluding drugs that significantly inhibited growth. The initial screen was based on the 2-component system (TCS) dependent biofilm effect, thus, the QseC dependence of maprotiline anti-biofilm action was demonstrated. By comparing their FDA-approved uses, chemical structures, and other properties of active drugs, toremifene and polycyclic antidepressants maprotiline and chlorpromazine were identified as being highly active against F. novicida biofilm formation. Further down-selection excluded toremifene for its membrane active activity and chlorpromazine for its high antimicrobial activity. The mode of action of maprotiline against F. novicida was sought. It was demonstrated that maprotiline was able to significantly down-regulate the expression of the virulence factor IglC, encoded on the Francisella Pathogenicity Island (FPI), suggesting that maprotiline is exerting an effect on bacterial virulence. Further studies showed that maprotiline significantly rescued F. novicida infected wax worm larvae. In vivo studies demonstrated that maprotiline treatment could prolong time to disease onset and survival in F. novicida infected mice. These results suggest that an FDA-approved drug such as maprotiline has the potential to combat Francisella infection as an antivirulence agent, and may have utility in combination with antibiotics. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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24. Analysis of mixed biofilm ( Staphylococcus aureus and Pseudomonas aeruginosa) by laser ablation electrospray ionization mass spectrometry.
- Author
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Dean, Scott N., Walsh, Callee, Goodman, Haddon, and van Hoek, Monique L.
- Subjects
BIOFILMS ,LASER ablation ,ELECTROSPRAY ionization mass spectrometry ,PSEUDOMONAS aeruginosa ,STAPHYLOCOCCUS aureus ,ANTI-infective agents - Abstract
Pseudomonas aeruginosaandStaphylococcus aureusare ubiquitous pathogens often found together in polymicrobial, biofilm-associated infections. This study is the first to use laser ablation electrospray ionization mass spectrometry (LAESI-MS) to rapidly study bacteria within a mixed biofilm. Fast, direct, non-invasive LAESI-MS analysis of biofilm could significantly accelerate biofilm studies and provide previously unavailable information on both biofilm composition and the effects of antibiofilm treatment. LAESI-MS was applied directly to a polymicrobial biofilm and analyzed with respect to whetherP. aeruginosaandS. aureuswere co-localized or self-segregated within the mixed biofilm. LAESI-MS was also used to analyze ions following LL-37 antimicrobial peptide treatment of the biofilm. This ambient ionization method holds promise for future biofilm studies. The use of this innovative technique has profound implications for the study of biofilms, as LAESI-MS eliminates the need for lengthy and disruptive sample preparation while permitting rapid analysis of unfixed and wet biofilms. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
25. Bioprospecting the American Alligator (Alligator mississippiensis) Host Defense Peptidome.
- Author
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Bishop, Barney M., Juba, Melanie L., Devine, Megan C., Barksdale, Stephanie M., Rodriguez, Carlos Alberto, Chung, Myung C., Russo, Paul S., Vliet, Kent A., Schnur, Joel M., and van Hoek, Monique L.
- Subjects
AMERICAN alligator ,BIOPROSPECTING ,PEPTIDE antibiotics ,ANIMAL defenses ,ANTI-infective agents ,CATIONS - Abstract
Cationic antimicrobial peptides and their therapeutic potential have garnered growing interest because of the proliferation of bacterial resistance. However, the discovery of new antimicrobial peptides from animals has proven challenging due to the limitations associated with conventional biochemical purification and difficulties in predicting active peptides from genomic sequences, if known. As an example, no antimicrobial peptides have been identified from the American alligator, Alligator mississippiensis, although their serum is antimicrobial. We have developed a novel approach for the discovery of new antimicrobial peptides from these animals, one that capitalizes on their fundamental and conserved physico-chemical properties. This sample-agnostic process employs custom-made functionalized hydrogel microparticles to harvest cationic peptides from biological samples, followed by de novo sequencing of captured peptides, eliminating the need to isolate individual peptides. After evaluation of the peptide sequences using a combination of rational and web-based bioinformatic analyses, forty-five potential antimicrobial peptides were identified, and eight of these peptides were selected to be chemically synthesized and evaluated. The successful identification of multiple novel peptides, exhibiting antibacterial properties, from Alligator mississippiensis plasma demonstrates the potential of this innovative discovery process in identifying potential new host defense peptides. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
26. Cranberry proanthocyanidins have anti-biofilm properties against Pseudomonas aeruginosa.
- Author
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Ulrey, Robert K., Barksdale, Stephanie M., Weidong Zhou, and van Hoek, Monique L.
- Subjects
ANIMAL experimentation ,BIOFILMS ,CELL lines ,CRANBERRIES ,DRUG interactions ,FLAVONOIDS ,GENTAMICIN ,MASS spectrometry ,MICE ,PSEUDOMONAS ,T-test (Statistics) ,TANNINS ,PROTEOMICS ,DESCRIPTIVE statistics - Abstract
Background Bacteria within a biofilm are phenotypically more resistant to antibiotics, desiccation, and the host immune system, making it an important virulence factor for many microbes. Cranberry juice has long been used to prevent infections of the urinary tract, which are often related to biofilm formation. Recent studies have found that the A-type proanthocyanidins from cranberries have anti-biofilm properties against Escherichia coli. Methods Using crystal violet biofilm staining, resazurin metabolism assays, and confocal imaging, we examined the ability of A-type proanthocyanidins (PACs) to disrupt the biofilm formation of Pseudomonas aeruginosa. We used mass spectrometry to analyze the proteomic effects of PAC treatment. We also performed synergy assays and in vitro and in vivo infections to determine whether PACs, alone and in combination with gentamicin, could contribute to the killing of P. aeruginosa and the survival of cell lines and G. mellonella. Results Cranberry PACs reduced P. aeruginosa swarming motility. Cranberry PACs significantly disrupted the biofilm formation of P. aeruginosa. Proteomics analysis revealed significantly different proteins expressed following PAC treatment. In addition, we found that PACs potentiated the antibiotic activity of gentamicin in an in vivo model of infection using G.. mellonella. Conclusions Results suggest that A-type proanthocyanidins may be a useful therapeutic against the biofilm-mediated infections caused by P. aeruginosa and should be further tested. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
27. Cathelicidin antimicrobial peptide from Alligator mississippiensis has antibacterial activity against multi-drug resistant Acinetobacter baumanii and Klebsiella pneumoniae.
- Author
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Barksdale, Stephanie M., Hrifko, Evelyn J., and van Hoek, Monique L.
- Subjects
- *
AMERICAN alligator , *CATHELICIDIN antimicrobial peptide , *ANTIBACTERIAL agents , *MULTIDRUG resistance in bacteria , *KLEBSIELLA pneumoniae , *ACINETOBACTER baumannii - Abstract
Alligator mississippiensis (American alligator), a member of order Crocodilia, lives in bacteria-laden environments but is not often known to succumb to bacterial infections. Their serum has been shown to have antibacterial activity beyond that of human serum, and it is believed that this activity is partially due to cationic antimicrobial peptides (CAMPs). CAMPs are produced by many organisms as part of the innate immune system. CAMPs are attractive possible therapies against multi-drug resistant bacteria, such as those found in biofilm-infected war wounds, because they seldom cause genetic resistance in bacteria and are effective against antibiotic resistant bacteria. In this work, we identified, synthesized, and characterized a cathelicidin and two shorter fragments from the American alligator. We discovered the cathelicidin using Basic Local Alignment Search Tool (BLAST) alignment and by comparing A. mississippiensis expressed sequence tags (ESTs) with propeptide cathelicidins of other reptiles. We analyzed the structure using bioinformatics tools and circular dichroism and predicted that the full-length cathelicidin peptide has a mixed structure, with an N-terminal α-helix and a center Pro hinge. In minimal inhibitory concentration (MIC) assays, it was determined that the cathelicidin and the two shorter fragments have strong activity against multiple Gram-negative bacteria, including clinical isolates of multi-drug resistant (MDR) Acinetobacter baumannii and carbapenem-resistant Klebsiella pneumoniae. Using the ethidium bromide uptake assay, it was found that these peptides permeabilize the bacterial membrane and are less sensitive to salt inhibition than many other known CAMPs. The alligator cathelicidin peptides were not hemolytic against sheep red blood cells at 300 μg/ml and were not significantly cytotoxic against A549 human lung epithelial cells after 24 h exposure in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. These alligator cathelicidin peptides have activity similar to other CAMPs from reptiles such as NA-CATH. It is possible that the alligator cathelicidins play an important role in the innate immune response of A. mississippiensis , similar to LL-37 in humans. In addition, due to their activities against MDR bacteria and lack of cytotoxicity, the AM-CATH peptides could be an attractive platform for further development as a potential therapeutic. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
28. Characterization of Cimex lectularius (bedbug) defensin peptide and its antimicrobial activity against human skin microflora.
- Author
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Kaushal, Akanksha, Gupta, Kajal, and van Hoek, Monique L.
- Subjects
- *
BEDBUGS , *DEFENSINS , *PEPTIDES , *ANTI-infective agents , *NATURAL immunity , *BACTERIAL antigens , *BACTERIAL cell membranes , *CIRCULAR dichroism - Abstract
Antimicrobial peptides are components of both vertebrate and invertebrate innate immune systems that are expressed in response to exposure to bacterial antigens. Naturally occurring antimicrobial peptides from evolutionarily ancient species have been extensively studied and are being developed as potential therapeutics against antibiotic resistant microorganisms. In this study, a putative Cimex lectularius (bedbug, CL) defensin is characterized for its effectiveness against human skin flora including Gram-negative and Gram-positive bacteria. The bedbug defensin (CL-defensin), belonging to family of insect defensins, is predicted to have a characteristic N-terminal loop, an α-helix, and an antiparallel β-sheet, which was supported by circular dichroism spectroscopy. The defensin was shown to be antimicrobial against Gram-positive bacteria commonly found on human skin ( Micrococcus luteus, Corynebacterium renale, Staphylococcus aureus and Staphylococcus epidermidis ); however, it was ineffective against common skin Gram-negative bacteria ( Pseudomonas aeruginosa and Acinetobacter baumannii ) under low-salt conditions. CL-defensin was also effective against M. luteus and C. renale in high-salt (MIC) conditions. Our studies indicate that CL-defensin functions by depolarization and pore-formation in the bacterial cytoplasmic membrane. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
29. Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production.
- Author
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Dean, Scott N., Myung-Chul Chung, and van Hoek, Monique L.
- Subjects
- *
BURKHOLDERIA infections , *BURKHOLDERIA , *GRAM-negative bacteria , *BDELLOVIBRIO , *AZOTOBACTERACEAE - Abstract
In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
30. EGR1 upregulation following Venezuelan equine encephalitis virus infection is regulated by ERK and PERK pathways contributing to cell death.
- Author
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Dahal, Bibha, Lin, Shih-Chao, Carey, Brian D., Jacobs, Jonathan L., Dinman, Jonathan D., van Hoek, Monique L., Adams, Andre A., and Kehn-Hall, Kylene
- Subjects
- *
VENEZUELAN equine encephalomyelitis , *ENCEPHALITIS viruses , *CELL death , *VIRUS diseases , *PROTEIN kinases - Abstract
Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
31. Antimicrobial activity of mosquito cecropin peptides against Francisella.
- Author
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Kaushal, Akanksha, Gupta, Kajal, Shah, Ruhee, and van Hoek, Monique L.
- Subjects
- *
FRANCISELLA tularensis , *ZOONOSES , *TULAREMIA , *PEPTIDE antibiotics , *NATURAL immunity , *PREVENTION of infectious disease transmission - Abstract
Francisella tularensis is the cause of the zoonotic disease tularemia. In Sweden and Scandinavia, epidemiological studies have implicated mosquitoes as a vector. Prior research has demonstrated the presence of Francisella DNA in infected mosquitoes but has not shown definitive transmission of tularemia from a mosquito to a mammalian host. We hypothesized that antimicrobial peptides, an important component of the innate immune system of higher organisms, may play a role in mosquito host-defense to Francisella . We established that Francisella sp. are susceptible to two cecropin antimicrobial peptides derived from the mosquito Aedes albopictus as well as Culex pipiens . We also demonstrated induced expression of Aedes albopictus antimicrobial peptide genes by Francisella infection C6/36 mosquito cell line. We demonstrate that mosquito antimicrobial peptides act against Francisella by disrupting the cellular membrane of the bacteria. Thus, it is possible that antimicrobial peptides may play a role in the inability of mosquitoes to establish an effective natural transmission of tularemia. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
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