A Lesson in Survival, by Giardia lamblia

In the relationships between host and parasites, there is a cross-talk that involves diverse mechanisms developed by two different genetic systems during years of evolution. On the one hand, immunocompetent hosts have developed effective innate and acquired immune responses that are used to restrict or avoid parasitism. On the other hand, parasites evade the immune response, expressing different antigens on their surface or by using other specific mechanisms, such as nutrient depletion. In this review, we analyze the survival mechanisms used by the protozoan parasite Giardia lamblia during infection. In particular, we examine the multiple roles played by the enzyme arginine deiminase during colonization of the gut, also involving the parasite's mechanism of antigenic variation. Potential drug targets for the treatment of giardiasis are also discussed

Vacuolar protein sorting receptor in Giardia lamblia.

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells

Vacuolar Protein Sorting Receptor in Giardia lamblia

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells

The YQII motif of GlVps contributes to receptor stabilization.

<p>(A) GlVps_-YQII–HA (green) is observed in the cytosol (cyt) of trophozoites probable in small vesicles (white arrows in insert) by IFA and confocal microscopy. pm: plasma membrane. (B) GlVps_-YQII–HA (green) do not colocalizes with BiP (red) in the ER (Merge). Inset (a) magnifies a region of the cell and shows that the green and red fluorescence are well separated. Differential interference contrast microscopy (b) is shown as insert. Scatter plot (panel on the left) correspond to the colocalization analysis. (C) Partial colocalization of GlVps_-YQII–HA (green) and μ2 (red) is observed in the PV region (Merge). Inset (a) magnifies a region of the cell where the green and red fluorescence partially overlap in the PVs. Differential interference contrast microscopy (b) is shown as insert. Bars, 10 μm. Scatter plot of the two labels shows the colocalization (left panel). Pearson's coefficient (PC). Manders' Overlap coefficient (M). (D) GlVps-HA and GlVps_-YQII–HA are detected by immunoblotting using anti-HA mAb in <i>GlVps-ha</i>, <i>GlVps_-YQII–ha</i> trophozoites, respectively. No detection of these receptors was observed in wild-type cells. Proteolytic processing is observed for GlVps_-YQII–HA in comparison with GlVps–HA. Relative molecular weights of protein standards (kDa) are indicated on the left.</p

GlVps and AcPh colocalized throughout the lysosomal pathway.

<p>(A) Direct IFA and confocal microscopy show the colocalization (Merge) of GlVps (green) and AcPh-V5H₆ (red) using directed labeled anti-HA and anti-V5, respectively. Inset magnifies a region of the cell and shows colocalization of the green and red fluorescence in yellow (a). Differential interference contrast microscopy (b) is shown as insert. Scatter plot of the two labels confirms the colocalization (right panel). Bar, 10 μm. (B) AcPh/GlVps and AcPh/GlVps_-YQII interaction was detected by the ability of yeast cells (AH109) to grow on selective plates TDO. No interaction was observed in the high-stringency QDO medium. Controls of the methodology include testing of pESCP-AD/pµ1-BD (protein-protein interaction) and pGlVps-AD/pGBKT7 (autoactivation).</p

GlVps and the medium subunit of AP1 interact via the YQII motif.

<p>(A) Densitometric assessment of one representative RT–PCR experiment shown on bottom. The amount of 1000 nt antisense RNA from the vector is only observed in −µ1 trophozoites. Reduction of endogenous μ1 mRNA levels is observed in −µ1, but not in +µ1 or wild-type cells (wt). Similar expression of <i>glvps</i> mRNA in wild-type, +µ1 and −µ1 cells was observed. (*p<0,0001). (B) GlVps-HA is observed in the cytoplasm in µ1-depleted cells. In cell expressing µ1 (+µ1), GlVps-HA possesses a reticular-perinuclear distribution. Merge panels of green fluorescence and differential interference contrast microscopy for +µ1 and −µ1 trophozoites are shown. Bar, 10 μm. (C) GlVps-HA is detected by immunoblotting using anti-HA mAb in +µ1 (a) and −µ1 (b) trophozoites. The proteolytic processing of GlVps-HA observed in −µ1 trophozoites, differs from the processing of GlVPS_-YQII-HA in cells expressing µ1 (c). Relative molecular weights of protein standards (kDa) are indicated on the left. (D) The yeast two-hybrid assay demonstrates that GlVps (GlVps-AD) but not GlVps_-YQII (GlVps-AD lacking the lysosomal motif) interacts with μ1 (μ1-BD) (left panel). GlVps (GlVps-AD) does not interact with the μ2 subunit of AP2 (μ2-BD) (right panel). Interaction is noticed by the growth of yeast colonies in plates lacking tryptophan, leucine and histidine [TDO (triple-dropout medium) plates] and in the high-stringency medium that also lacked adenine (QDO). Controls of the methodology include testing of pESCP-AD/pµ1-BD or pGlLRP-AD/pµ2-BD (protein-protein interaction) and pGlVps-AD/pGBKT7 (autoactivation).</p

AcPh localization and activity.

<p>(A) Schematic representation of the <i>acph</i> gene containing the GGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTA. CCGGT and CATCATCATCATCATCAT, coding to the V5 epitope and six histidine residues, respectively. A 3D reconstruction of the gene product tagged with V5-H6 using the hidden Markov models (HMMs) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043712#pone.0043712-Soding1" target="_blank">[84]</a> and MODELLER <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043712#pone.0043712-Sali1" target="_blank">[85]</a> is also represented. (B) IFA and confocal microscopy show AcPh-V5 predominantly in the ER but also in the nuclear envelope and PVs (arrowheads). DIC: Differential interference contrast microscopy. (C) Acid phosphatase activity on the PVs and bare zone is observed by using the specific substrate ELF97 at pH 5.5. Alkaline phosphatase activity was not detected in trophozoites at pH ≥7.0. Nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bar, 10 μm.</p