scholarly journals The Zoonotic Helminth Parasite Fasciola hepatica: Virulence-Associated Cathepsin B and Cathepsin L Cysteine Peptidases Secreted by Infective Newly Excysted Juveniles (NEJ)

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3495
Author(s):  
Tara Barbour ◽  
Krystyna Cwiklinski ◽  
Richard Lalor ◽  
John Pius Dalton ◽  
Carolina De Marco Verissimo
Keyword(s):  
Vaccine Development ◽  
Fasciola Hepatica ◽  
Cathepsin L ◽  
Intestinal Wall ◽  
Mammalian Host ◽  
Parasite Invasion ◽  
Potent Inhibition ◽  
Cysteine Peptidases ◽  
Future Drug

Fasciolosis caused by Fasciola hepatica is a major global disease of livestock and an important neglected helminthiasis of humans. Infection arises when encysted metacercariae are ingested by the mammalian host. Within the intestine, the parasite excysts as a newly excysted juvenile (NEJ) that penetrates the intestinal wall and migrates to the liver. NEJ excystment and tissue penetration are facilitated by the secretion of cysteine peptidases, namely, cathepsin B1 (FhCB1), cathepsin B2 (FhCB2), cathepsin B3 (FhCB3) and cathepsin L3 (FhCL3). While our knowledge of these peptidases is growing, we have yet to understand why multiple enzymes are required for parasite invasion. Here, we produced functional recombinant forms of these four peptidases and compared their physio-biochemical characteristics. Our studies show great variation of their pH optima for activity, substrate specificity and inhibitory profile. Carboxy-dipeptidase activity was exhibited exclusively by FhCB1. Our studies suggest that, combined, these peptidases create a powerful hydrolytic cocktail capable of digesting the various host tissues, cells and macromolecules. Although we found several inhibitors of these enzymes, they did not show potent inhibition of metacercarial excystment or NEJ viability in vitro. However, this does not exclude these peptidases as targets for future drug or vaccine development.

scholarly journals SARS-CoV-2 RBD in vitro evolution parrots and predicts contagious mutation spread

2021 ◽  
Author(s):  
Gideon Schreiber ◽  
Jiri Zahradník ◽  
Shir Marciano ◽  
Maya Shemesh ◽  
Eyal Zoler ◽  
...  
Keyword(s):  
South African ◽  
Convergent Evolution ◽  
Vaccine Development ◽  
Spike Protein ◽  
Structural Basis ◽  
Receptor Binding Domain ◽  
In Vitro Evolution ◽  
High Affinity ◽  
Future Drug

Abstract SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the “European” (20E-EU1), “British” (501.V1),”South African” (501.V2), and Brazilian variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9Å Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

scholarly journals Reference gene analysis and its use for kinase expression profiling in Fasciola hepatica

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hicham Houhou ◽  
Oliver Puckelwaldt ◽  
Christina Strube ◽  
Simone Haeberlein
Keyword(s):  
Reference Genes ◽  
Drug Targets ◽  
Fasciola Hepatica ◽  
Expression Patterns ◽  
Housekeeping Gene ◽  
Trna Synthetase ◽  
Parasite Development ◽  
Mammalian Host ◽  
Life Stages

Abstract The liver fluke Fasciola hepatica causes fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping gene gapdh which is used in most Fasciola studies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for future F. hepatica research, such as studies on drug targets or parasite development.

scholarly journals Antibodies against MAEBL Ligand Domains M1 and M2 Inhibit Sporozoite Development In Vitro

2004 ◽  
Vol 72 (6) ◽  
pp. 3604-3608 ◽  
Author(s):  
Peter Preiser ◽  
Laurent Rénia ◽  
Naresh Singh ◽  
Bharath Balu ◽  
William Jarra ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Salivary Glands ◽  
Vaccine Development ◽  
Molecular Form ◽  
Mammalian Host ◽  
Phenotypic Differences ◽  
Apical Membrane Antigen 1 ◽  
Erythrocyte Binding ◽  
Development In Vitro

ABSTRACT MAEBL is a type 1 membrane protein that is implicated in the merozoite invasion of erythrocytes and sporozoite invasion of mosquito salivary glands. This apical organelle protein is structurally similar to the ebl erythrocyte binding proteins, such as EBA-175, except that the tandem ligand domains of MAEBL are similar to part of the extracellular domain of apical membrane antigen 1 and not the Duffy binding-like domain. Although midgut and salivary gland sporozoites are morphologically similar, salivary gland sporozoites undergo a period of new gene expression after infecting the salivary glands, display distinct phenotypic differences, and are more infectious for the mammalian host. The objectives of this project were to determine the molecular form of MAEBL in the infectious salivary gland sporozoites and whether the ligand has a role in the sporozoite development to exoerythrocytic stages in hepatocytes. We determined that MAEBL is newly expressed in salivary gland sporozoites and in a form distinct from what is present in the midgut sporozoites or present in erythrocytic stages. Both ligand domains (M1 and M2) were expressed as part of a full-length membrane form of MAEBL in the salivary gland sporozoites in contrast to the other stages that retain only the M2 ligand domain as part of the membrane form of the protein. Antisera developed against the cysteine-rich regions of the extracellular portion of MAEBL inhibited sporozoite development to exoerythrocytic forms in vitro. Together these data indicate that MAEBL has a role in this third developmental stage in the life cycle of the malaria parasite. Thus, MAEBL is another target for pre-erythrocytic-stage vaccine development against malaria parasites.

scholarly journals Cloning and Expression of the Major SecretedCathepsin B-Like Protein from Juvenile Fasciola hepatica andAnalysis of Immunogenicity following Liver FlukeInfection

2003 ◽  
Vol 71 (12) ◽  
pp. 6921-6932 ◽  
Author(s):  
Ruby H. P. Law ◽  
Peter M. Smooker ◽  
James A. Irving ◽  
David Piedrafita ◽  
Rebecca Ponting ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cathepsin B ◽  
Fasciola Hepatica ◽  
Cathepsin L ◽  
Cloning And Expression ◽  
Significant Similarity ◽  
Peptide Substrates ◽  
Liver Flukes ◽  
Kinetics Of

ABSTRACT The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.

scholarly journals Major Secretory Antigens of the Helminth Fasciola hepatica Activate a Suppressive Dendritic Cell Phenotype That Attenuates Th17 Cells but Fails To Activate Th2 Immune Responses

2009 ◽  
Vol 78 (2) ◽  
pp. 793-801 ◽  
Author(s):  
David J. Dowling ◽  
Clare M. Hamilton ◽  
Sheila Donnelly ◽  
James La Course ◽  
Peter M. Brophy ◽  
...  
Keyword(s):  
Immune Responses ◽  
Intracellular Signaling ◽  
Glutathione Transferase ◽  
Fasciola Hepatica ◽  
Cathepsin L ◽  
Cell Phenotype ◽  
Mammalian Host ◽  
Toll Like Receptor 4 ◽  
Inflammatory Protein

ABSTRACT Fasciola hepatica is a helminth pathogen that drives Th2/Treg immune responses in its mammalian host. The parasite releases a large number of molecules that are critical to inducing this type of immune response. Here we have selected recombinant forms of two major F. hepatica secreted molecules, the protease cathepsin L (rFhCL1) and an antioxidant, sigma class glutathione transferase (rFhGST-si), to examine their interactions with dendritic cells (DCs). Despite enzymatic and functional differences between these molecules, both induced interleukin-6 (IL-6), IL-12p40, and macrophage inflammatory protein 2 (MIP-2) secretion from DCs and enhanced CD40 expression. While this induction was mediated by Toll-like receptor 4 (TLR4), the subsequent intracellular signaling pathways differed; rFhCL1 signaled through p38, and rFhGST-si mediated its effect via c-Jun N-terminal kinase (JNK), p38, p-NF-κBp65, and IRF5. Neither rFhCL1 nor rFhGST-si enhanced DC phagocytosis or induced Th2 immune responses in vivo. However, DCs matured in the presence of either enzyme attenuated IL-17 production from OVA peptide-specific T cells in vivo. In addition, DCs exposed to either antigen secreted reduced levels of IL-23. Therefore, both F. hepatica FhCL1 and FhGST-si modulate host immunity by suppressing responses associated with chronic inflammation—an immune modulatory mechanism that may benefit the parasite's survival within the host.

scholarly journals Complex and dynamic transcriptional changes allow the helminth Fasciola gigantica to adjust to its intermediate snail and definitive mammalian hosts

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiao-Xuan Zhang ◽  
Krystyna Cwiklinski ◽  
Rui-Si Hu ◽  
Wen-Bin Zheng ◽  
Zhao-An Sheng ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Liver Fluke ◽  
Fasciola Hepatica ◽  
Expression Profiles ◽  
Cathepsin L ◽  
Fasciola Gigantica ◽  
Mammalian Host ◽  
Specific Gene ◽  
Transcription Analysis ◽  
Transcriptional Changes

Abstract Background The tropical liver fluke, Fasciola gigantica causes fasciolosis, an important disease of humans and livestock. We characterized dynamic transcriptional changes associated with the development of the parasite in its two hosts, the snail intermediate host and the mammalian definitive host. Results Differential gene transcription analysis revealed 7445 unigenes transcribed by all F. gigantica lifecycle stages, while the majority (n = 50,977) exhibited stage-specific expression. Miracidia that hatch from eggs are highly transcriptionally active, expressing a myriad of genes involved in pheromone activity and metallopeptidase activity, consistent with snail host finding and invasion. Clonal expansion of rediae within the snail correlates with increased expression of genes associated with transcription, translation and repair. All intra-snail stages (miracidia, rediae and cercariae) require abundant cathepsin L peptidases for migration and feeding and, as indicated by their annotation, express genes putatively involved in the manipulation of snail innate immune responses. Cercariae emerge from the snail, settle on vegetation and become encysted metacercariae that are infectious to mammals; these remain metabolically active, transcribing genes involved in regulation of metabolism, synthesis of nucleotides, pH and endopeptidase activity to assure their longevity and survival on pasture. Dramatic growth and development following infection of the mammalian host are associated with high gene transcription of cell motility pathways, and transport and catabolism pathways. The intra-mammalian stages temporally regulate key families of genes including the cathepsin L and B proteases and their trans-activating peptidases, the legumains, during intense feeding and migration through the intestine, liver and bile ducts. While 70% of the F. gigantica transcripts share homology with genes expressed by the temperate liver fluke Fasciola hepatica, gene expression profiles of the most abundantly expressed transcripts within the comparable lifecycle stages implies significant species-specific gene regulation. Conclusions Transcriptional profiling of the F. gigantica lifecycle identified key metabolic, growth and developmental processes the parasite undergoes as it encounters vastly different environments within two very different hosts. Comparative analysis with F. hepatica provides insight into the similarities and differences of these parasites that diverged > 20 million years ago, crucial for the future development of novel control strategies against both species.

scholarly journals NDP-rhamnose biosynthesis and rhamnosyltransferases: building diverse glycoconjugates in nature

2021 ◽  
Vol 478 (4) ◽  
pp. 685-701
Author(s):  
Ben A. Wagstaff ◽  
Azul Zorzoli ◽  
Helge C. Dorfmueller
Keyword(s):  
Natural Products ◽  
Drug Discovery ◽  
Scientific Community ◽  
Pathogenic Bacteria ◽  
Vaccine Development ◽  
Ongoing Study ◽  
Deoxy Sugar ◽  
Future Drug

Rhamnose is an important 6-deoxy sugar present in many natural products, glycoproteins, and structural polysaccharides. Whilst predominantly found as the l-enantiomer, instances of d-rhamnose are also found in nature, particularly in the Pseudomonads bacteria. Interestingly, rhamnose is notably absent from humans and other animals, which poses unique opportunities for drug discovery targeted towards rhamnose utilizing enzymes from pathogenic bacteria. Whilst the biosynthesis of nucleotide-activated rhamnose (NDP-rhamnose) is well studied, the study of rhamnosyltransferases that synthesize rhamnose-containing glycoconjugates is the current focus amongst the scientific community. In this review, we describe where rhamnose has been found in nature, as well as what is known about TDP-β-l-rhamnose, UDP-β-l-rhamnose, and GDP-α-d-rhamnose biosynthesis. We then focus on examples of rhamnosyltransferases that have been characterized using both in vivo and in vitro approaches from plants and bacteria, highlighting enzymes where 3D structures have been obtained. The ongoing study of rhamnose and rhamnosyltransferases, in particular in pathogenic organisms, is important to inform future drug discovery projects and vaccine development.

scholarly journals SARS-CoV-2 RBD in vitro evolution follows contagious mutation spread, yet generates an able infection inhibitor

2021 ◽  
Author(s):  
Jiri Zahradnik ◽  
Shir Marciano ◽  
Maya Shemesh ◽  
Eyal Zoler ◽  
Jeanne Chiaravalli ◽  
...  
Keyword(s):  
South African ◽  
Receptor Binding ◽  
Vaccine Development ◽  
Spike Protein ◽  
Structural Basis ◽  
Receptor Binding Domain ◽  
In Vitro Evolution ◽  
High Affinity ◽  
Future Drug

SARS-CoV-2 is constantly evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2, resulted in the more contagious mutations, S477N, E484K, and N501Y to be among the first selected. This includes the British and South African variants. Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R in combination with N501Y. This said, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9A Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations provides structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

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