scholarly journals Lectin Activity of the TcdA and TcdB Toxins ofClostridium difficile

2018 ◽  
Vol 87 (3) ◽  
Author(s):  
Lauren E. Hartley-Tassell ◽  
Milena M. Awad ◽  
Kate L. Seib ◽  
Maria Scarselli ◽  
Silvana Savino ◽  
...  
Keyword(s):  
Blood Group ◽  
Small Gtpases ◽  
Lewis Antigens ◽  
Target Cells ◽  
Ofclostridium Difficile ◽  
Content Type ◽  
Affinity Ligand ◽  
Wide Range ◽  
Hospital Acquired

ABSTRACTClostridium difficileis a major cause of hospital-acquired antibiotic-associated diarrhea.C. difficileproduces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both α- and β-linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal α- and β-linked galactose, including blood groups, Lewis antigens,N-acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissuesin vivo.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals Neurons Are Host Cells for Mycobacterium tuberculosis

2014 ◽  
Vol 82 (5) ◽  
pp. 1880-1890 ◽  
Author(s):  
Philippa J. Randall ◽  
Nai-Jen Hsu ◽  
Dirk Lang ◽  
Susan Cooper ◽  
Boipelo Sebesho ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Mycobacterium Tuberculosis ◽  
Host Cells ◽  
Productive Infection ◽  
Target Cells ◽  
Dependent Manner ◽  
Content Type ◽  
The Central Nervous System

ABSTRACTMycobacterium tuberculosisinfection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions ofM. tuberculosiswith neuronsin vitroandin vivowere investigated. The data obtained demonstrate that neurons can act as host cells forM. tuberculosis.M. tuberculosisbacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization ofM. tuberculosisbacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalizeM. tuberculosis. InternalizedM. tuberculosisbacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h.M. tuberculosisbacillus infection of neurons was confirmedin vivoin the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells forM. tuberculosiswithin the central nervous system.

scholarly journals USE OF MICRONUCLEUS EXPERIMENTS FOR THE DETECTION OF HUMAN CANCER RISKS: A BRIEF OVERVIEW

2021 ◽  
Vol 65 (2) ◽  
Author(s):  
Armen Nersesyan ◽  
◽  
Miroslav Mišík ◽  
Andriy Cherkas ◽  
Viktoria Serhiyenko ◽  
...  
Keyword(s):  
Blood Cells ◽  
Environmental Control ◽  
Human Cancer ◽  
Occupational Exposures ◽  
Human Biomonitoring ◽  
Target Cells ◽  
Cancer Risks ◽  
Wide Range

Introduction. Micronuclei (MN) are small extranuclear DNA-containing structures that are formed as a consequence of structural and numerical chromosomal aberrations. The advantage of MN experiments compared to conventional chromosomal analyses in metaphase cells is that the scoring is by far less time consuming and laborious. MN experiments are currently widely used for the routine screening of chemicals in vitro and in vivo but also for environmental control and human biomonitoring Objectives. The purpose of this review was to collect data on the use of MN experiments for the detection of increased cancer risks as a consequence of environmental, lifestyle and occupational exposures and the detection/diagnosis of different forms of cancer. Methods. Analysis of the literature on methods for MN experiments with humans; as well as the use of this technique in different areas of research. Results. To date, a wide range of protocols for human biomonitoring studies has been developed for the measurement of MN formation in peripheral blood cells and in epithelial from different organs (buccal and nasal cavity, cervix and bladder). In addition to MN, other nuclear anomalies can be scored which reflect genetic instability as well as acute toxicity and the division of target cells. Conclusions. The evidence is accumulating that MN can be used as a diagnostic tool for the detection of increased cancer risks as well as for the early diagnosis of cervical and bladder cancer

scholarly journals An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

2021 ◽  
Author(s):  
Darcy S.O. Mora ◽  
Madeline Cox ◽  
Forgivemore Magunda ◽  
Ashley B. Williams ◽  
Lyndsey Linke
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Nucleic Acids ◽  
Endosomal Escape ◽  
Therapeutic Window ◽  
Target Cells ◽  
Full Potential ◽  
Wide Range ◽  
Delivery Platform

There is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza- targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.

scholarly journals Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo

2019 ◽  
Vol 93 (18) ◽  
Author(s):  
Artem Baidaliuk ◽  
Elliott F. Miot ◽  
Sebastian Lequime ◽  
Isabelle Moltini-Conclois ◽  
Fanny Delaigue ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Line ◽  
Content Type ◽  
Cells In Culture ◽  
Mosquito Cells ◽  
Wide Range ◽  
Natural Hosts

ABSTRACT Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses. IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

scholarly journals New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ren-Yi Lu ◽  
Ting-Jun-Hong Ni ◽  
Jing Wu ◽  
Lan Yan ◽  
Quan-Zhen Lv ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Pathogenic Fungi ◽  
Control Group ◽  
Survival Times ◽  
Content Type ◽  
Fungal Burden ◽  
Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

scholarly journals A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

scholarly journals Quantification of the Adenylate Cyclase Toxin of Bordetella pertussisIn Vitroand during Respiratory Infection

2013 ◽  
Vol 81 (5) ◽  
pp. 1390-1398 ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Jason M. Warfel ◽  
Christopher D. Paddock ◽  
Tara F. Jones ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Respiratory Tract ◽  
Whooping Cough ◽  
Extensive Study ◽  
Target Cells ◽  
Human Infants ◽  
Content Type ◽  
Adenylate Cyclase Toxin

ABSTRACTWhooping cough results from infection of the respiratory tract withBordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrationsin vitro, ACT has not been observed or quantifiedin vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human andin vitrodata, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108CFU/mlB. pertussisand 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108CFU/ml of a laboratory strain ofB. pertussiswas culturedin vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

scholarly journals In VivoProgrammed Gene Expression Based on Artificial Quorum Networks

2015 ◽  
Vol 81 (15) ◽  
pp. 4984-4992 ◽  
Author(s):  
Teng Chu ◽  
Yajun Huang ◽  
Mingyu Hou ◽  
Qiyao Wang ◽  
Jingfan Xiao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Cell Density ◽  
Protective Antigen ◽  
Expression System ◽  
Edwardsiella Tarda ◽  
Content Type ◽  
Genetic Components ◽  
Wide Range

ABSTRACTThe quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of theVibrio fischeri luxI-luxRquorum sensing system. In order to achievein vivoprogrammed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter ParaBAD, into the QS system.In vitroexpression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density.In vivoexpression assays confirmed that the araQS system presented anin vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applicationsin vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuatedEdwardsiella tardastrain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expressionin vivoand might have potential uses, including, but not limited to, bacterial vector vaccines.

scholarly journals Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

2014 ◽  
Vol 83 (2) ◽  
pp. 502-513 ◽  
Author(s):  
Shan Li ◽  
Lianfa Shi ◽  
Zhiyong Yang ◽  
Yongrong Zhang ◽  
Gregorio Perez-Cordon ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cysteine Protease ◽  
Lethal Dose ◽  
Enzymatic Activities ◽  
Target Cells ◽  
Content Type ◽  
Cytopathic Effects

TcdB is one of the key virulence factors ofClostridium difficilethat is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (VHH) library raised against TcdB, we identified two VHH antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB inin vivotoxicity using a sensitive systemic challenge model in mice. Consistent with thesein vitroresults, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

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