Transfection With Plasmid Causing Stable Expression of a Foreign Gene Affects General Proteome Pattern in Giardia lamblia Trophozoites

Abstract

Giardia lamblia is an important causative agent of persistent diarrhea in humans, domestic animals, and cattle. Basic research is usually performed with the strain WBC6 and includes genetic manipulations such as transfections. Here, we investigate how transfection with a plasmid causing stable expression of a foreign gene affects the whole proteome pattern. Using shotgun mass spectrometry, we compare the proteomes of untransfected trophozoites to trophozoites transfected with Escherichia coli glucuronidase A (GusA). Besides GusA, which is detected in the transfected trophozoites only, the proteomes of untransfected and transfected trophozoites differ by 132 differentially expressed proteins. In particular, transfection induces antigenic variation. Since transfection causing stable expression affects the proteome pattern, transfection experiments should take into account this effect. Due to a unique peptide panel, GusA is an example for a suitable internal standard for experiments involving transfected cells. Data are available via ProteomeXchange with identifier PXD022565.

Comparative Proteomic Analysis of Toxoplasma gondii RH Wild-Type and Four SRS29B (SAG1) Knock-Out Clones Reveals Significant Differences between Individual Strains

“…Overall, upon the application of very strict criteria for determining differential expression levels, SRS29B KO clones and WT tachyzoites varied by 21 out of 3236 proteins, which corresponds to 0.65%. This variability is far below the nearly 10% of differentially expressed proteins detected in WT and transfected clones of the phylogenetically distant protist Giardia lamblia , for which the same strict criteria were applied [ 8 ]. However, as illustrated by the principal component analysis plots, the herein presented datasets on T. gondii WT and KO clones did not simply form two clusters of T. gondii WT proteome and KO proteome.…”

“…A suitable strategy to investigate such effects is the analysis of differentially expressed proteins in transformed clones and respective non-transformed wild-type (WT) parasites. For instance, in Giardia lamblia , nearly 10% of the overall proteome is altered upon transformation with a stable plasmid [ 8 ]. In order to investigate to what degree protein expression patterns other than the intended knockout (KO) are affected in T. gondii , a non-essential gene should be chosen whose KO has no impact on fitness, at least not during the in vitro culture of tachyzoites.…”

Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity

“…The label-free in solution proteomic analysis was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) by the Proteomics and Mass Spectrometry Core Facility of the University of Bern, Switzerland. Samples were digested (Braga-Lagache et al, 2016[ 3 ]) and analyzed by LC-MS/MS as described before (Heller et al, 2020[ 19 ]). The identified peptides were blasted against a database for T. spiralis ( https://parasite.wormbase.org/Trichinella_spiralis_prjna12603 ) and the UniProt database.…”

Comprehensive characterization of Cysteine-rich protein-coding genes of Giardia lamblia and their role during antigenic variation

“…Regarding proteomic data, earlier studies performed on the previous version of the Giardia genome and with parasite populations showed translational evidence for all groups of Cys-rich encoding genes [14,[63][64][65][66]. Therefore, the raw data from mass spectrometry experiments were downloaded from the PRIDE repository and re-analyzed with the MaxQuant software using the specific parameters described in each of these reports, but using the proteins fasta file of the GL2.1 genome.…”