scholarly journals The Actin Cross-linking Domain of the Vibrio cholerae RTX Toxin Directly Catalyzes the Covalent Cross-linking of Actin

2006 ◽  
Vol 281 (43) ◽  
pp. 32366-32374 ◽  
Author(s):  
Christina L. Cordero ◽  
Dmitry S. Kudryashov ◽  
Emil Reisler ◽  
Karla J. Fullner Satchell
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Tissue Transglutaminase ◽  
Lethal Factor ◽  
Host Cells ◽  
Cross Linking ◽  
Cell Extracts ◽  
Rtx Toxin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LFNACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LFNACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg2+ and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.

scholarly journals Cryptosporidium parvum Pyruvate Kinase Inhibitors With in vivo Anti-cryptosporidial Efficacy

2022 ◽  
Vol 12 ◽  
Author(s):  
Shahbaz M. Khan ◽  
Xuejin Zhang ◽  
William H. Witola
Keyword(s):  
Pyruvate Kinase ◽  
Cryptosporidium Parvum ◽  
Diarrheal Disease ◽  
Severe Disease ◽  
The United States ◽  
Metabolic Energy ◽  
Host Cells ◽  
Infection Model

Cryptosporidium parvum is a highly prevalent protozoan parasite that causes a diarrheal disease in humans and animals worldwide. Thus far, the moderately effective nitazoxanide is the only drug approved by the United States Food and Drug Administration for treating cryptosporidiosis in immunocompetent humans. However, no effective drug exists for the severe disease seen in young children, immunocompromised individuals and neonatal livestock. C. parvum lacks the Krebs cycle and the oxidative phosphorylation steps, making it dependent solely on glycolysis for metabolic energy production. Within its glycolytic pathway, C. parvum possesses two unique enzymes, the bacterial-type lactate dehydrogenase (CpLDH) and the plant-like pyruvate kinase (CpPyK), that catalyze two sequential steps for generation of essential metabolic energy. We have previously reported that inhibitors of CpLDH are effective against C. parvum, both in vitro and in vivo. Herein, we developed an in vitro assay for the enzymatic activity of recombinant CpPyK protein and used it to screen a chemical compound library for inhibitors of CpPyK’s activity. The identified inhibitors were tested (at non-toxic concentrations) for efficacy against C. parvum using in vitro assays, and an in vivo mouse infection model. We identified six CpPyK inhibitors that blocked in vitro growth and proliferation of C. parvum at low micromolar concentrations (EC50 values ranging from 10.29 to 86.01 μM) that were non-toxic to host cells. Among those six compounds, two (NSC252172 and NSC234945) were found to be highly efficacious against cryptosporidiosis in immunocompromised mice at a dose of 10 mg/kg body weight, with very significant reduction in parasite load and amelioration of intestinal pathologies. Together, these findings have unveiled inhibitors for an essential molecular target in C. parvum and demonstrated their efficacy against the parasite in vitro and in vivo. These inhibitors are, therefore, potential lead-compounds for developing efficacious treatments for cryptosporidiosis.

scholarly journals Aerobic metabolism in Vibrio cholerae is required for population expansion during infection

2020 ◽  
Author(s):  
Andrew J. Van Alst ◽  
Victor J. DiRita
Keyword(s):  
Vibrio Cholerae ◽  
Oxidative Metabolism ◽  
Metabolic Pathways ◽  
Diarrheal Disease ◽  
Population Expansion ◽  
Bacterial Disease ◽  
High Cell ◽  
Metabolic Processes

AbstractVibrio cholerae is a bacterial pathogen that replicates to high cell density in the small intestine of human hosts leading to the diarrheal disease cholera. During infection, V. cholerae senses and responds to environmental signals that govern cellular responses. Spatial localization of V. cholerae within the intestine affects nutrient availability and therefore the metabolic pathways required for the replicative success of the pathogen. Metabolic processes used by V. cholerae to reach such high cell densities are not fully known. Here we seek to better define the metabolic traits that contribute to high levels of V. cholerae during infection by investigating mutant strains in key carbohydrate metabolism pathways. By disrupting the pyruvate dehydrogenase (PDH) complex and pyruvate formate-lyase (PFL), we could differentiate aerobic and anaerobic metabolic pathway involvement in V. cholerae proliferation. We demonstrate that oxidative metabolism is a key contributor to the replicative success of V. cholerae in vivo using an infant mouse model where PDH mutants were attenuated 100-fold relative to wild type for colonization. Additionally, metabolism of host substrates such as mucin were determined to support V. cholerae growth in vitro as a sole carbon source primarily in aerobic growth conditions. Mucin likely contributes to population expansion during human infection as it is a ubiquitous source of carbohydrates. These data highlight the importance of oxidative metabolism in the intestinal environment and warrants further investigation of how oxygen and other host substrates shape the intestinal landscape that ultimately influences bacterial disease. We conclude from our results that oxidative metabolism of host substrates such as mucin is a key driver of V. cholerae growth and proliferation during infection, leading to the substantial bacterial burden exhibited in cholera patients.ImportanceVibrio cholerae remains a challenge in the developing world and incidence of the disease it causes, cholera, is anticipated to increase with rising global temperatures and with emergent, highly infectious strains. At present, the underlying metabolic processes that support V. cholerae growth during infection are less well understood than specific virulence traits such as production of a toxin or pilus. In this study we determined that oxidative metabolism of host substrates such as mucin contribute significantly to V. cholerae population expansion in vivo. Identifying metabolic pathways critical for growth can provide avenues for controlling V. cholerae infection and the knowledge may be translatable to other pathogens of the gastrointestinal tract.

scholarly journals Vibrio cholerae Requires rpoS for Efficient Intestinal Colonization

2000 ◽  
Vol 68 (12) ◽  
pp. 6691-6696 ◽  
Author(s):  
D. Scott Merrell ◽  
Anna D. Tischler ◽  
Sang Ho Lee ◽  
Andrew Camilli
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Detectable Effect ◽  
Intestinal Colonization ◽  
Insertional Inactivation ◽  
Intestinal Pathogen ◽  
Colonization Ability ◽  
Mutant Phenotypes

ABSTRACT Vibrio cholerae is a facultative intestinal pathogen that lives in aquatic environments, often in association with planktonic species. In the suckling mouse, oral inoculation withV. cholerae leads to intestinal colonization and symptoms of diarrheal disease. Results reported here indicate a role for the alternative sigma factor, RpoS, in intestinal colonization in this model of cholera. We constructed within rpoS multiple independent mutations which consistently resulted in a fivefold decrease in colonization ability as assessed by competition assays. These mutations had no detectable effect on the in vitro growth ofV. cholerae in a rich medium. The occurrence of spontaneous suppressor mutations potentially required for viability ofrpoS strains was ruled out by determination of the frequency of insertional inactivation of rpoS in comparison to two other nonessential loci. Finally, both the in vitro and in vivo mutant phenotypes of rpoS strains were fully complemented by providing rpoS in trans or by allelic reversion, indicating that the observed decrease in colonization fitness was indeed due to the loss of functional RpoS.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

2020 ◽  
Author(s):  
Avik Sotira Scientific
Keyword(s):  
Social Life ◽  
Plaque Assay ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Crystallographic Structure ◽  
Therapeutic Implications ◽  
Biochemical Assays ◽  
Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

scholarly journals Effective decellularisation of human saphenous veins for biocompatible arterial tissue engineering applications: Bench optimisation and feasibility in vivo testing

2021 ◽  
Vol 12 ◽  
pp. 204173142098752
Author(s):  
Nadiah S Sulaiman ◽  
Andrew R Bond ◽  
Vito D Bruno ◽  
John Joseph ◽  
Jason L Johnson ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Mechanical Strength ◽  
Vascular Tissue ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Replacement Model ◽  
In Vivo Testing ◽  
Vascular Scaffolds

Human saphenous vein (hSV) and synthetic grafts are commonly used conduits in vascular grafting, despite high failure rates. Decellularising hSVs (D-hSVs) to produce vascular scaffolds might be an effective alternative. We assessed the effectiveness of a detergent-based method using 0% to 1% sodium dodecyl sulphate (SDS) to decellularise hSV. Decellularisation effectiveness was measured in vitro by nuclear counting, DNA content, residual cell viability, extracellular matrix integrity and mechanical strength. Cytotoxicity was assessed on human and porcine cells. The most effective SDS concentration was used to prepare D-hSV grafts that underwent preliminary in vivo testing using a porcine carotid artery replacement model. Effective decellularisation was achieved with 0.01% SDS, and D-hSVs were biocompatible after seeding. In vivo xeno-transplantation confirmed excellent mechanical strength and biocompatibility with recruitment of host cells without mechanical failure, and a 50% patency rate at 4-weeks. We have developed a simple biocompatible methodology to effectively decellularise hSVs. This could enhance vascular tissue engineering toward future clinical applications.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

scholarly journals Lankesterella (Apicomplexa, Lankesterellidae) Blood Parasites of Passeriform Birds: Prevalence, Molecular and Morphological Characterization, with Notes on Sporozoite Persistence In Vivo and Development In Vitro

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1451
Author(s):  
Carolina Romeiro Fernandes Chagas ◽  
Josef Harl ◽  
Vytautas Preikša ◽  
Dovilė Bukauskaitė ◽  
Mikas Ilgūnas ◽  
...  
Keyword(s):  
Morphological Characterization ◽  
Life Cycles ◽  
Diagnostic Methods ◽  
Host Cells ◽  
Blood Parasites ◽  
Serinus Canaria ◽  
Long Time ◽  
Development In Vitro

Recent studies confirmed that some Hepatozoon-like blood parasites (Apicomplexa) of birds are closely related to the amphibian parasite Lankesterella minima. Little is known about the biology of these pathogens in birds, including their distribution, life cycles, specificity, vectors, and molecular characterization. Using blood samples of 641 birds from 16 species, we (i) determined the prevalence and molecular diversity of Lankesterella parasites in naturally infected birds; (ii) investigated the development of Lankesterella kabeeni in laboratory-reared mosquitoes, Culex pipiens forma molestus and Aedes aegypti; and (iii) tested experimentally the susceptibility of domestic canaries, Serinus canaria, to this parasite. This study combined molecular and morphological diagnostic methods and determined 11% prevalence of Lankesterella parasites in Acrocephalidae birds; 16 Lankesterella lineages with a certain degree of host specificity and two new species (Lankesterella vacuolata n. sp. and Lankesterella macrovacuolata n. sp.) were found and characterized. Lankesterella kabeeni (formerly Hepatozoon kabeeni) was re-described. Serinus canaria were resistant after various experimental exposures. Lankesterella sporozoites rapidly escaped from host cells in vitro. Sporozoites persisted for a long time in infected mosquitoes (up to 42 days post exposure). Our study demonstrated a high diversity of Lankesterella parasites in birds, and showed that several avian Hepatozoon-like parasites, in fact, belong to Lankesterella genus.

Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽  
2006 ◽  
Vol 133 (3) ◽  
pp. 261-278 ◽  
Author(s):  
A. HEMPHILL ◽  
N. VONLAUFEN ◽  
A. NAGULESWARAN
Keyword(s):  
Host Cell ◽  
Cell Biology ◽  
Neospora Caninum ◽  
Host Cells ◽  
Term Survival ◽  
Prevention Of Infection ◽  
Parasite Relationship ◽  
Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

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