Comparative proteomics of threeGiardia lambliastrains: investigation of antigenic variation in the post-genomic era

Abstract

Giardia lamblia is a causative agent of persistent diarrhoea widespread in regions with low hygienic standards. Laboratory research is based on cloned lines issuing from various patient isolates typed in the late 1980s and 90s using restriction analysis and serology. In the present study, we compared the well-characterized strain WBC6 with another clone of the parent WB isolate termed WBA1 and with a clone from another isolate, GS/M-83-H7, using shotgun mass spectrometry proteomics. We identified 398 proteins differentially expressed between the GS and both WB isolates and 97 proteins differentially expressed between the two WB isolates. We investigated the expression levels of the predominant variant-specific surface proteins (VSPs) in each clone and matched the previously described major VSPs of each strain to the corresponding open reading frame sequences identified by whole-genome sequencing efforts. Furthermore, since the original WB isolate comes from a patient treated with metronidazole, we compared the susceptibilities of the strains to nitro compounds, as well the expression levels of enzymes involved in nitro reduction and on the corresponding enzyme activities and found distinct differences between the three strains.

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

“…The present dataset confirms our initial hypothesis that the transfection of G. lamblia by plasmids followed by an antibioticbased selection of transfected trophozoites affects the whole genome expression pattern thereby confirming and extending previous findings (Su et al, 2007). Furthermore, it induces antigenic variation like other drug-based selections (Emery et al, 2018;Müller et al, 2019) or in strains from different genetic backgrounds (Emery et al, 2014;Emery et al, 2015a;Müller et al, 2020a). For instance, we have found 17 DE surface antigens FIGURE 1 | Primary sequence of the transgene E. coli glucuronidase A with the identified peptides highlighted in bold red letters.…”

Nitroreductase Activites in Giardia lamblia: ORF 17150 Encodes a Quinone Reductase with Nitroreductase Activity

“…Malate dehydrogenase provides electrons from carbon sources different from glucose, such as amino acids, the predominant C sources for G. lamblia in axenical culture [ 31 ]. The NR1 homologous NR2 was not detected neither in the present study nor in previously published whole cell shotgun MS studies [ 27 , 30 ], nor by immunoblotting [ 27 ]. A recently published study on genetic polymorphisms in genes involved in resistance against metronidazole listed malate dehydrogenase as a potential resistance marker besides the “usual suspects”, such as PFORs, ferredoxins, and NRs [ 32 ] including the NR2 (GL50803_6175) orthologue DHA2_153380.…”

“…This is interesting, because in previously published proteome datasets [ 27 , 30 ], NR3 could not be detected in G. lamblia WBC6.…”

“…After digestion, the solution above the filter was transferred into a clean 1.5 mL polypropylene reaction vial and acidified with 7.5 μL of 20% ( v/v ) trifluoroacetic acid. After an incubation for 15 min at room temperature, the digest was spun for 1 min at 16,000× g , and the cleared supernatant was transferred to a HPLC vial for subsequent nano-liquid reversed phase chromatography coupled to tandem mass spectrometry, as described earlier [ 30 ].…”

Proteomic Insights into the Biology of the Most Important Foodborne Parasites in Europe

“…The latest research on the surface proteome of G. intestinalis used shotgun mass spectrometry to analyze the proteomes of trophozoites of the three different strains. This allowed the identification of 2368 proteins, among which, using monoclonal antibodies, the variant-specific surface proteins were identified [ 53 ].…”