Giardial lipid rafts share virulence factors with secreted vesicles and participate in parasitic infection in mice

Brian I. Grajeda, Atasi De Chatterjee, Carmen M. Villalobos, Breanna C. Pence, Cameron C. Ellis, Vanessa Enriquez, Sourav Roy, Sukla Roychowdhury, Aaron K. Neumann, Igor C. Almeida, Steven E. Patterson, Siddhartha Das

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10 Scopus citations

Abstract

Giardia lamblia, a protozoan parasite, is a major cause of waterborne infection, worldwide. While the trophozoite form of this parasite induces pathological symptoms in the gut, the cyst form transmits the infection. Since Giardia is a noninvasive parasite, the actual mechanism by which it causes disease remains elusive. We have previously reported that Giardia assembles cholesterol and GM1 glycosphingolipid-enriched lipid rafts (LRs) that participate in encystation and cyst production. To further delineate the role of LRs in pathogenesis, we isolated LRs from Giardia and subjected them to proteomic analysis. Various cellular proteins including potential virulence factors—e.g., giardins, variant surface proteins, arginine deaminases, elongation factors, ornithine carbomyltransferases, and high cysteine-rich membrane proteins—were found to be present in LRs. Since Giardia secretes virulence factors encapsulated in extracellular vesicles (EVs) that induce proinflammatory responses in hosts, EVs released by the parasite were isolated and subjected to nanoparticle tracking and proteomic analysis. Two types of EV—i.e., small vesicles (SVs; <100 nm, exosome-like particles) and large vesicles (LVs; 100–400 nm, microvesicle-like particles)—were identified and found to contain a diverse group of proteins including above potential virulence factors. Although pretreatment of the parasite with two giardial lipid raft (gLR) disruptors, nystatin (27 μM) and oseltamivir (20 μM), altered the expression profiles of virulence factors in LVs and SVs, the effects were more robust in the case of SVs. To examine the potential role of rafts and vesicles in pathogenicity, Giardia-infected mice were treated with oseltamivir (1.5 and 3.0 mg/kg), and the shedding of cysts were monitored. We observed that this drug significantly reduced the parasite load in mice. Taken together, our results suggest that virulence factors partitioning in gLRs, released into the extracellular milieu via SVs and LVs, participate in spread of giardiasis and could be targeted for future drug development.

Original languageEnglish (US)
Article number974200
JournalFrontiers in Cellular and Infection Microbiology
Volume12
DOIs
StatePublished - Aug 23 2022

Bibliographical note

Funding Information:
This work was supported by a grant 1R21AI138061 from NIAID (NIH). The biochemical, molecular, and confocal microscopy experiments were conducted at the Biomolecule Analysis and Omics as well as Cellular Characterization and Biorepository Core Facilities at BBRC/UTEP supported by U54MD007592 (NIMHD/NIH). We also acknowledge support from the Building Scholar Grant (8RL5GM118969) to UTEP from NIGMS (NIH). TEM images were taken at the Molecular Microbiology Imaging Facility (Director: Dr. Wendy Beatty) of the Washington University, St. Louis, Mo. Drs. Brian Grajeda and Cameron Ellis were supported by BBRC/UTEP. Dr. Vanessa Enriquez was supported by a RISE grant from the NIGMS (5R25GM069621). dSTORM imaging facilities and analysis methods were developed under auspices of NIH/NIGMS 5P50GM085273-05. We acknowledge support for AN and CV from NIH/NIAID R01AI116894.

Publisher Copyright:
Copyright © 2022 Grajeda, De Chatterjee, Villalobos, Pence, Ellis, Enriquez, Roy, Roychowdhury, Neumann, Almeida, Patterson and Das.

Keywords

  • Giardia
  • extracellular vesicles
  • giardiasis
  • infection
  • lipid rafts
  • nystatin
  • oseltamivir
  • proteome

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