Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo

T. C. Paredes-Santos, T. Tomita, M. Yan Fen, W. de Souza, M. Attias, R. C. Vommaro, L. M. Weiss

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

Original languageEnglish (US)
Pages (from-to)39-47
Number of pages9
JournalMicrobes and Infection
Volume18
Issue number1
DOIs
StatePublished - Jan 1 2016

Fingerprint

Toxoplasma
Cysts
Parasites
Developmental Biology
Felidae
Vacuoles
Infection
Genetic Promoter Regions
Birds
Fluorescent Antibody Technique
Reproduction
Mammals
Plasmids
Cell Culture Techniques
Polymerase Chain Reaction
In Vitro Techniques
Proteins

Keywords

  • Atypical strain
  • Bradyzoite
  • Conversion
  • Cyst
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Infectious Diseases

Cite this

Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo. / Paredes-Santos, T. C.; Tomita, T.; Yan Fen, M.; de Souza, W.; Attias, M.; Vommaro, R. C.; Weiss, L. M.

In: Microbes and Infection, Vol. 18, No. 1, 01.01.2016, p. 39-47.

Research output: Contribution to journalArticle

@article{32060d526b384cb8b8f76e522e3405a3,
title = "Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo",
abstract = "Toxoplasma gondii is a protozoan that infects 30{\%} of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.",
keywords = "Atypical strain, Bradyzoite, Conversion, Cyst, Toxoplasma gondii",
author = "Paredes-Santos, {T. C.} and T. Tomita and {Yan Fen}, M. and {de Souza}, W. and M. Attias and Vommaro, {R. C.} and Weiss, {L. M.}",
year = "2016",
month = "1",
day = "1",
doi = "10.1016/j.micinf.2015.09.016",
language = "English (US)",
volume = "18",
pages = "39--47",
journal = "Microbes and Infection",
issn = "1286-4579",
publisher = "Elsevier Masson SAS",
number = "1",

}

TY - JOUR

T1 - Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo

AU - Paredes-Santos, T. C.

AU - Tomita, T.

AU - Yan Fen, M.

AU - de Souza, W.

AU - Attias, M.

AU - Vommaro, R. C.

AU - Weiss, L. M.

PY - 2016/1/1

Y1 - 2016/1/1

N2 - Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

AB - Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

KW - Atypical strain

KW - Bradyzoite

KW - Conversion

KW - Cyst

KW - Toxoplasma gondii

UR - http://www.scopus.com/inward/record.url?scp=84953711636&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84953711636&partnerID=8YFLogxK

U2 - 10.1016/j.micinf.2015.09.016

DO - 10.1016/j.micinf.2015.09.016

M3 - Article

C2 - 26432517

AN - SCOPUS:84953711636

VL - 18

SP - 39

EP - 47

JO - Microbes and Infection

JF - Microbes and Infection

SN - 1286-4579

IS - 1

ER -