scholarly journals Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones

Parasitology ◽  
2013 ◽  
Vol 140 (4) ◽  
pp. 499-508 ◽  
Author(s):  
J. DE GRAEF ◽  
J. DEMELER ◽  
P. SKUCE ◽  
M. MITREVA ◽  
G. VON SAMSON-HIMMELSTJERNA ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Anthelmintic Resistance ◽  
Life Stage ◽  
Resistant Isolate ◽  
Gene Transcript ◽  
Macrocyclic Lactones ◽  
Transcript Levels ◽  
Cooperia Oncophora

SUMMARYMembers of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

scholarly journals ABC transporters in gills of rainbow trout (Oncorhynchus mykiss)

2020 ◽  
Vol 223 (15) ◽  
pp. jeb221069
Author(s):  
Christian Kropf ◽  
Karl Fent ◽  
Stephan Fischer ◽  
Ayako Casanova ◽  
Helmut Segner
Keyword(s):  
Rainbow Trout ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Functional Activity ◽  
Organic Molecules ◽  
Expression Patterns ◽  
Primary Cultures ◽  
Gene Transcript ◽  
Rainbow Trout Oncorhynchus Mykiss

ABSTRACTFish gills are a structurally and functionally complex organ at the interface between the organism and the aquatic environment. Gill functions include the transfer of organic molecules, both natural ones and xenobiotic compounds. Whether the branchial exchange of organic molecules involves active transporters is currently not known. Here, we investigated the presence, diversity and functional activity of ATP-binding cassette (ABC) transporters in gills of juvenile rainbow trout. By means of RT-qPCR, gene transcripts of members from the abcb, abcc and abcg subfamilies were identified. Comparisons with mRNA profiles from trout liver and kidney revealed that ABC transporters known to have an apical localization in polarized epithelia, especially abcc2 and abcb1, were under-represented in the gills. In contrast, ABC transporters with mainly basolateral localization showed comparable gene transcript levels in the three organs. The most prominent ABC transporter in gills was an abcb subfamily member, which was annotated as abcb5 based on the synteny and phylogeny. Functional in vivo assays pointed to a role of branchial ABC transporters in branchial solute exchange. We further assessed the utility of primary gill cell cultures to characterize transporter-mediated branchial exchange of organic molecules, by examining ABC transporter gene transcript patterns and functional activity in primary cultures. The gill cultures displayed functional transport activity, but the ABC mRNA expression patterns were different to those of the intact gills. Overall, the findings of this study provide evidence for the presence of functional ABC transporter activity in gills of fish.

scholarly journals Chorioamnionitis Induces a Specific Signature of Placental ABC Transporters Associated with an Increase of miR-331-5p in the Human Preterm Placenta

2018 ◽  
Vol 45 (2) ◽  
pp. 591-604 ◽  
Author(s):  
Guinever Eustaquio do Imperio ◽  
Enrrico Bloise ◽  
Mohsen Javam ◽  
Phetcharawan Lye ◽  
Andrea Constantinof ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Staining Intensity ◽  
Distinct Pattern ◽  
Mrna Levels ◽  
Cancer Resistance ◽  
Glycoprotein P ◽  
Immunological Responses

Background/Aims: The ATP-binding cassette (ABC) transporters mediate drug biodisposition and immunological responses in the placental barrier. In vitro infective challenges alter expression of specific placental ABC transporters. We hypothesized that chorioamnionitis induces a distinct pattern of ABC transporter expression. Methods: Gene expression of 50 ABC transporters was assessed using TaqMan® Human ABC Transporter Array, in preterm human placentas without (PTD; n=6) or with histological chorioamnionitis (PTDC; n=6). Validation was performed using qPCR, immunohistochemistry and Western blot. MicroRNAs known to regulate P-glycoprotein (P-gp) were examined by qPCR. Results: Up-regulation of ABCB9, ABCC2 and ABCF2 mRNA was detected in chorioamnionitis (p<0.05), whereas placental ABCB1 (P-gp; p=0.051) and ABCG2 (breast cancer resistance protein-BCRP) mRNA levels (p=0.055) approached near significant up-regulation. In most cases, the magnitude of the effect significantly correlated to the severity of inflammation. Upon validation, increased placental ABCB1 and ABCG2 mRNA levels (p<0.05) were observed. At the level of immunohistochemistry, while BCRP was increased (p<0.05), P-gp staining intensity was significantly decreased (p<0.05) in PTDC. miR-331-5p, involved in P-gp suppression, was upregulated in PTDC (p<0.01) and correlated to the grade of chorioamnionitis (p<0.01). Conclusions: Alterations in the expression of ABC transporters will likely lead to modified transport of clinically relevant compounds at the inflamed placenta. A better understanding of the potential role of these transporters in the events surrounding PTD may also enable new strategies to be developed for prevention and treatment of PTD.

scholarly journals Screening of Streptococcus pneumoniae ABC Transporter Mutants Demonstrates that LivJHMGF, a Branched-Chain Amino Acid ABC Transporter, Is Necessary for Disease Pathogenesis

2009 ◽  
Vol 77 (8) ◽  
pp. 3412-3423 ◽  
Author(s):  
Shilpa Basavanna ◽  
Suneeta Khandavilli ◽  
Jose Yuste ◽  
Jonathan M. Cohen ◽  
Arthur H. F. Hosie ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Bacterial Pathogens ◽  
Tryptophan Fluorescence ◽  
Growth Defect ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACT Bacterial ABC transporters are an important class of transmembrane transporters that have a wide variety of substrates and are important for the virulence of several bacterial pathogens, including Streptococcus pneumoniae. However, many S. pneumoniae ABC transporters have yet to be investigated for their role in virulence. Using insertional duplication mutagenesis mutants, we investigated the effects on virulence and in vitro growth of disruption of 9 S. pneumoniae ABC transporters. Several were partially attenuated in virulence compared to the wild-type parental strain in mouse models of infection. For one ABC transporter, required for full virulence and termed LivJHMGF due to its similarity to branched-chain amino acid (BCAA) transporters, a deletion mutant (ΔlivHMGF) was constructed to investigate its phenotype in more detail. When tested by competitive infection, the ΔlivHMGF strain had reduced virulence in models of both pneumonia and septicemia but was fully virulent when tested using noncompetitive experiments. The ΔlivHMGF strain had no detectable growth defect in defined or complete laboratory media. Recombinant LivJ, the substrate binding component of the LivJHMGF, was shown by both radioactive binding experiments and tryptophan fluorescence spectroscopy to specifically bind to leucine, isoleucine, and valine, confirming that the LivJHMGF substrates are BCAAs. These data demonstrate a previously unsuspected role for BCAA transport during infection for S. pneumoniae and provide more evidence that functioning ABC transporters are required for the full virulence of bacterial pathogens.

scholarly journals ABC transporter research: going strong 40 years on

2015 ◽  
Vol 43 (5) ◽  
pp. 1033-1040 ◽  
Author(s):  
Frederica L. Theodoulou ◽  
Ian D. Kerr
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Industrial Biotechnology ◽  
Global Food Security ◽  
Physiological Processes ◽  
Causative Factors ◽  
Experimental Approaches ◽  
Global Food

In most organisms, ABC transporters constitute one of the largest families of membrane proteins. In humans, their functions are diverse and underpin numerous key physiological processes, as well as being causative factors in a number of clinically relevant pathologies. Advances in our understanding of these diseases have come about through combinations of genetic and protein biochemical investigations of these transporters and the power of in vitro and in vivo investigations is helping to develop genotype–phenotype understanding. However, the importance of ABC transporter research goes far beyond human biology; microbial ABC transporters are of great interest in terms of understanding virulence and drug resistance and industrial biotechnology researchers are exploring the potential of prokaryotic ABC exporters to increase the capacity of synthetic biology systems. Plant ABC transporters play important roles in transport of hormones, xenobiotics, metals and secondary metabolites, pathogen responses and numerous aspects of development, all of which are important in the global food security area. For 3 days in Chester, this Biochemical Society Focused Meeting brought together researchers with diverse experimental approaches and with different fundamental questions, all of which are linked by the commonality of ABC transporters.

scholarly journals The threat of reduced efficacy of anthelmintics against gastrointestinal nematodes in sheep from an area considered anthelmintic resistance-free

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Antonio Bosco ◽  
Jan Kießler ◽  
Alessandra Amadesi ◽  
Marian Varady ◽  
Barbara Hinney ◽  
...  
Keyword(s):  
Southern Italy ◽  
Anthelmintic Resistance ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Minor Role ◽  
Macrocyclic Lactones ◽  
On Farm ◽  
Pooled Samples

Abstract Background The worldwide increased difficulty to combat gastrointestinal nematode (GIN) infection in sheep, due to progressing anthelmintic resistance (AR), calls for an enhanced and standardized implementation of early detection of AR. This study provides a snapshot of the current AR status against benzimidazoles and macrocyclic lactones in southern Italy, generated with standardized techniques. Methods On 10 sheep farms, the efficacy of albendazole (ALB) and either eprinomectin (EPR) or ivermectin (IVM) was evaluated based on the faecal egg count reduction test (FECRT) performed with the Mini-FLOTAC. For each tested drug, 40 sheep were rectally sampled at D0 and sampled again 14 days after the treatment (D14). The FECRT was calculated from individual samples and pooled samples which consist of 5 individual samples. Efficacy was classified as ‘reduced, ‘suspected’ and ‘normal’. Coprocultures were set for D0 and D14 faecal samples of each group. From farms with FECR < 95%, an in vitro egg hatch test (EHT) and a follow-up FECRT using fenbendazole (FBZ) were conducted. Results Based on the FECR, high efficacy (from 95.7% to 100%) was observed for ALB and IVM in eight farms (Farms 3–10). On Farm 1 and Farm 2, the efficacy for the macrocyclic lactones was classified as ‘normal’, but ‘reduced’ efficacy was observed for ALB on Farm 1 (FECR = 75%) and ‘suspected’ efficacy on Farm 2 (FECR = 93.3%) with the predominant GIN genus Trichostrongylus followed by Haemonchus at D14. The FEC results of pooled samples strongly correlated with those of individual samples, for FEC at D0 (rs = 0.984; P < 0.0001) and at D14 (rs = 0.913; P < 0.0001). The classifications of efficacy in Farm 1 (FECR = 86.0%) and Farm 2 (FECR = 93.0%) in the follow-up FECRT with FBZ coincide with the main FECRT trial. The in vitro EHT confirmed AR in both farms (Farm 1: 89%; Farm 2: 74%). Conclusions In regions like southern Italy, where the negative impacts from AR have played a minor role, efficient monitoring of AR is important in order to evaluate potential risks and being able to promptly respond with countermeasures.

scholarly journals Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

2020 ◽  
Author(s):  
Fernanda Sales Coelho ◽  
Rutchanee Rodpai ◽  
Andre Miller ◽  
Shannon E Karinshak ◽  
Victoria H Mann ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Cell Line ◽  
Biomphalaria Glabrata ◽  
Embryonic Cell ◽  
Gene Transcript ◽  
Inflammatory Factor ◽  
Wild Type ◽  
Transcript Levels ◽  
Embryonic Cell Line

Abstract Background: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.Methods: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.Results: Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01).Conclusions: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

scholarly journals Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

2020 ◽  
Author(s):  
Fernanda Sales Coelho ◽  
Rutchanee Rodpai ◽  
Andre Miller ◽  
Shannon E Karinshak ◽  
Victoria H Mann ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Cell Line ◽  
Biomphalaria Glabrata ◽  
Embryonic Cell ◽  
Gene Transcript ◽  
Inflammatory Factor ◽  
Wild Type ◽  
Transcript Levels ◽  
Embryonic Cell Line

Abstract Background: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.Methods: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by quantitative reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.Results: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5±20.2% S.D, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66±0.10, ΔBgAIF, 2.30±0.22 (P ≤ 0.01).Conclusion: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

scholarly journals Use of larval migration inhibition assay for the evaluation of anthelmintic resistance in dairy heifers in Puerto Rico

1969 ◽  
Vol 99 (2) ◽  
pp. 117-124
Author(s):  
Lorraine López-Soberal ◽  
Guillermo Ortiz-Colón ◽  
Melvin Pagán-Morales ◽  
Esbal Jiménez-Cabán
Keyword(s):  
Puerto Rico ◽  
Anthelmintic Resistance ◽  
Resistant Isolate ◽  
Inhibition Assay ◽  
Macrocyclic Lactones ◽  
Migration Inhibition ◽  
Larval Migration ◽  
Dairy Heifers ◽  
Field Samples ◽  
Third Stage

Ivermectin (IVM) resistance of Cooperia spp. in dairy heifers from Puerto Rico was evaluated using the larval migration inhibition assay (LMIA). In eight of the farms where anthelmintic resistance had previously been evaluated using a fecal egg count reduction test (FECRT), third stage larvae (L3) were recovered from fecal cultures prior to the administration of IVM treatment. The effective concentration at which 50% of larval migration is inhibited (EC50) was determined in susceptible and resistant Cooperia spp. isolates from one farm. The susceptible and resistant isolates had an EC50 of 0.7224 uM and an EC50 of 7.0778 uM, respectively. This indicates that on average, larvae from the resistant isolate are 10X more resistant than those from the susceptible isolates. However, the LMIA was unable to discriminate consistently between susceptible and resistant larvae obtained from dairy farms evaluated. In one case, an EC50 value of 0.7263 uM, which is close to that of the susceptible isolate, did not agree with the FECR result of -136.19% documented on this farm. In two additional instances the assay was unable to discriminate between susceptible and resistant isolates. Indeed LMIA EC50 values obtained from some resistant isolates were lower than the EC50 value from susceptible isolates, whereas their FECRT results indicated that resistance to IVM was present. Possibly, the FECRT could have given a false positive for macrocyclic lactones resistance at these two farms. Therefore, more studies are needed to validate the effectiveness of the LMIA to detect anthelmintic resistance to macrocyclic lactones in Cooperia spp. larvae isolated from bovine fecal field samples.

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