QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 12 August 2008
doi: 10.1242/jcs.026963

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Touz, M. C. Articles by Nash, T. E. Search for Related Content PubMed PubMed Citation Articles by Touz, M. C. Articles by Nash, T. E. Social Bookmarking                         What's this?

Arginine deiminase has multiple regulatory roles in the biology of Giardia lamblia

Maria Carolina Touz^1,*,, Andrea Silvana Rópolo^1,*, Maria Romina Rivero¹, Cecilia Veronica Vranych¹, John Thomas Conrad², Staffan Gunnar Svard³ and Theodore Elliott Nash²

¹ Instituto de Investigación Medica Mercedes y Martín Ferreyra, INIMEC – CONICET, Friuli 2434, Cordoba, Argentina
² Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD 20892, USA
³ Department of Cell and Molecular Biology, Uppsala University, SE-75 124 Uppsala, Sweden.

View larger version (21K): [in this window] [in a new window]   Fig. 1. G. lamblia ADI is associated with VSPs through their cytoplasmic tail. (A) Left: representation of the pull-down assays. Right: SDS-PAGE and Coomassie staining show an 85 kDa and an 13 kDa protein from both GS/H7 (GS) and WB/1267 (WB) isolates. Identification of these bands as being ADI was performed by LC/MS-MS. Controls without peptide fail to pull down any protein. (B) Protein-protein interactions were detected by the ability of yeast cells (AH109) to grow on selective plates. In the upper-left panel, the expression of the entire VSPH7 protein (H7-BD), the entire VSP1267 protein (1267-BD) and VSP1267 lacking its cytoplasmic tail (1267-tail-BD) with ADI (ADI-AD) is revealed by the presence of white colonies in minimal medium lacking leucine and tryptophan (–L/–T medium). Interaction of ADI-AD with both VSPH7-BD and VSP1267-BD is shown in the bottom-left panel by the growth of yeast colonies in plates lacking tryptophan, leucine and histidine [TDO (triple-dropout medium) plates]. No interaction between ADI and 1267-tail-BD is observed. Controls of the methodology include ESCP-BD–MuA-AD interaction (+) and emptyBD–ADI-AD vector (–). (C) Schematic representation of wild-type VSPH7 and transgenic VSPH7 proteins. The VSPH7 ORF contains a signal peptide, an extracellular domain, a transmembrane domain and a cytoplasmic tail. H7-HA has a HA epitope sequence at the C-terminus. H7-tail possesses the HA epitope right after the transmembrane domain of H7. R-H7 is VSPH7 containing a point mutation of the R residue of its cytoplasmic tail. (D) H7-HA and R-H7, but not H7-tail, co-immunoprecipitate with ADI. Antibody against ADI was used to immunoprecipitate comparable amounts of protein from WB transgenic cells. Western blotting of original lysate was stained with anti-HA mAb labeled with alkaline phosphatase (Lysate, bottom panel). UT, untransfected cells; STD, molecular weight standard.

View larger version (37K): [in this window] [in a new window]   Fig. 2. VSP citrullination is probably mediated by the PAD activity of ADI. (A) Top panels: confocal direct IFA was performed on permeabilized cells, showing a cytoplasmic distribution of ADI-HA (green) by using FITC-labeled anti-HA mAb and its partial colocalization (yellow in merge) with VSP9B10 (red) underneath the plasma membrane of the transgenic trophozoite. Nuclei are stained with DAPI (blue). Bottom panels: the same result was obtained using Alexa-Fluor-488–anti-ADI (green) in wild-type cells. Texas-Red–9B10 mAb was used to visualize VSP9B10 (red). Scale bars: 10 µm. (B) Western blotting of G. lamblia homogenates expressing different VSPs that reacted with anti-citrulline pAb (left panel). The same filter membranes were stripped, cut and reacted with 5C1, 9B10 and G10/4 mAbs against VSP1267, VSP9B10 and VSPH7, respectively (right panel), indicating that these VSPs are citrullinated. STD, molecular weight standard. (C) Dot-blotting to detect citrullination of the H6-CRGKA peptide after incubation with the purified recombinant ADI-HA (ADI-HA). A non-related purified enzyme ESCP-HA was used as a negative control. Dot-blotting to detect H6-CRGKA was performed using anti-H6 mAb. (D) Top panel: specific citrullination of the CRGKA tail is shown. Western blotting using anti-citrulline pAb performed after immunoprecipitation with anti-HA mAb of H7-transgenic trophozoites. Bottom panel: the presence of H7-HA, H7-tail and R-H7 after immunoprecipitation was analyzed using anti-HA mAb labeled with alkaline phosphatase. UT, untransfected cells.

View larger version (38K): [in this window] [in a new window]   Fig. 3. ADI participates in the control of cell death, probably by altering VSP switching. (A) Cytotoxicity assays of G. lamblia trophozoites WB/1267, GS/H7, WB/1267 transfected with H7-HA or WB/1267 transfected with R-H7 (from left to right on x axis) analyzed after 24 hours post-addition of the anti-VSPH7-specific antibodies G10-4 and anti-GS-VSPs pAb by estimating the number of adherent viable parasites. Controls include cells without treatment (w/o mAb) and the use of a non-related mAb (8G8 mAb). Data represent the means ± s.d. for n=2 of three independent experiments. (B) Progenies were analyzed by addition of goat anti-mouse FITC-conjugated antibody in IFA. Positive cells correspond to trophozoites expressing VSPH7. DIC, differential interference contrast. Scale bars: 10 µm. (C) VSP expression is established in WB/9B10 wild-type and WB/9B10-ADI+ transgenic trophozoites after a short time exposure to specific VSP9B10 mAb. Controls had no exposure to the mAb (–). Data represent the means ± s.d. for n=3 of two independent experiments. (D) VSP9B10 citrullination is analyzed by western blotting in WB/9B10 and WB/9B10-ADI+ trophozoites after a short time exposure to the anti-VSP9B10 mAb. The membrane labeled with anti-citrulline pAb (right) was stripped and re-blotted with anti-VSP9B10 mAb (left). A similar amount of loaded VSP9B10 is observed. Molecular mass of VSP9B10 is indicated on the right.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Post-translational modifications of ADI. (A) Western blotting using anti-HA mAb shows ADI-HA in ADI-transgenic trophozoites. Multiple bands are also obtained using specific anti-ADI pAb in wild-type cells. The band of 64-66 kDa corresponding to the predicted protein sequences is observed in both cases (arrowhead), together with degradation products (gray arrows) including the low-weight band (gray arrow with asterisk) found in the peptide pull-down (see Fig. 1). Also, for both HA-tagged and native ADI, an increase in molecular mass from a 64-66 kDa to a 85 kDa band is shown (black arrow), suggesting that ADI undergoes post-translational modification. STD, molecular weight standard. (B) Western blot assays using anti-SUMO1 mAb recognize an 85 kDa band (arrow) in both WB/1267 (WB) and GS/H7 (GS) G. lamblia clones. The same filter membrane was stripped and re-blotted with anti-ADI pAb, showing a perfect match with the higher band that is recognized by the anti-SUMO1 mAb. To confirm the lack of residual primary antibodies after stripping, only the secondary antibody was added to the stripped blots, showing no signal (Control). (C) Western blotting using biotin-conjugated anti-ADI pAb was performed to detect ADI bands after immunoprecipitation with anti-SUMO1 mAb. (a) ADI in lysate before IPP; (b) ADI after immunoprecipitation by using 0.1 µg of anti-SUMO1 mAb (arrow); (c) ADI after immunoprecipitation by using 1 µg of anti-SUMO1 mAb (arrow); (d) control using a non-related anti-HA mAb; and (e) supernatant of sample c.

View larger version (27K): [in this window] [in a new window]   Fig. 5. ADI expression increases during G. lamblia differentiation. (A) IFA and confocal microscopy show the G. lamblia encystation process. CWP2 (red) is synthesized and transported in ESVs in encysting trophozoites (ET, arrowhead). At the end of the encystation process, CWP2 is found in mature cyst walls (Cyst). Nuclei are stained with DAPI (blue). Scale bars: 10 µm. (B) Slot-blotting qualitatively shows gdh, adi and cwp2 gene expression at 0, 6, 24 and 48 hours of encystation in wild-type cells (WB/1267wt) and transgenic trophozoites (WB/1267-ADI+). The assay was performed in triplicate. (C) Analysis of gdh, adi and cwp2 gene expression by RT-PCR and optical density quantified by densiometry comparing both wild-type (WB/1267wt) and ADI-transgenic (WB/1267-ADI+) trophozoites. Data represent the means ± s.d. for n=4 of two independent experiments. (D) Western blotting using specific antibodies shows ADI, modified citrulline (Cit), VSP1267 and CWP2 protein expression in both wild-type (WB/1267wt) and ADI-transgenic (WB/1267-ADI+) trophozoites. Non-encysting trophozoites (NT, 10 µg) and 24-hour-encysting trophozoites (ET, 10 µg) were used for each sample.

View larger version (52K): [in this window] [in a new window]   Fig. 6. During encystation, ADI is translocated to the nuclei and inhibits CWP expression. (A) Results of western blotting that was performed after cytoplasm- and nuclear-fractionation assays using anti-ADI pAb at 24 and 48 hours post-encystation induction in wild-type (ADI) cells. L, cell lysate previous fractionation; C, cytoplasmic fraction; N, nuclear fraction (upper panel). Middle panel: anti-VSP1267 mAb is used to detect cytoplasm- or nuclear-fraction contamination. Lower panel: a representative time course of IFA shows ADI (red) distribution during encystation (ESVs in green). (B) Confocal microscopy and IFA using wild-type cells show that ADI (red) is translocated to the nuclei when the encysting cell is filled with ESVs (arrowheads). CWP1 is stained in green. (C) Top panels: representative differential interference contrast (DIC) and DAPI-staining images show a stable ADI-transgenic culture (Merge). Middle panels: direct IFA shows that trophozoites highly overexpressing ADI-HA (green) in the nuclei do not express CWP2 (red) during encystation (arrowheads in Merge). Bottom panels: closer analysis of three ADI-transgenic cells demonstrated that those expressing ADI-HA (green) in the nuclei (arrowheads in Merge) do not reveal CWP2 (red) expression after 24 hours of encystation, whereas those with low ADI-HA expression do (arrow in Merge). Scale bars: 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Schematic representation of the functions of ADI during growth and encystation. (A) During growth, the trophozoites acquire free arginine from the extracellular medium. Inside the cell, cytoplasmic ADI converts arginine into citrulline, with ATP production occurring at the final enzymatic step of the ADH pathway. (B) ADI is released to the extracellular space when the trophozoites are in contact with human colon epithelial cells and compete with the host NOS for the free arginine, thereby reducing the production of NO. (C) Under low exposure to specific antibodies, ADI acts as a PAD on the cytoplasmic tail of VSP, inducing VSP switching. (D) During the last step of encystation, ADI is translocated from the cytoplasm to the nuclei, turning encystation-specific genes off and ending the process.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter