mRNA expression of cell cycle regulatory proteins (BTG3, GADD45A) in HCT-8 cells upon parasite interaction.

<p>Expression levels are expressed in arbitrary units.</p

The role of arginine in intestinal infection.

<p>An overview of the intestinal epithelial lining is given in B, with a villus and a crypt being depicted. Proliferating cells in the crypts migrate towards the tip of the villus, undergoing differentiation, and are shed at the tip of the villus after cell death. <i>Giardia</i> trophozoites sit all along the epithelial surface (with exception of the Paneth cells that are most down in the crypts) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045325#pone.0045325-Aley1" target="_blank">[47]</a>. A trophozoite attached to an IEC is shown in A. Dotted lines indicate molelcules released by the trophozoite. Arginine is actively taken up by the parasite and converted into CO₂, NH₃ and ornithine along the dihydrolase pathway, indicated by the enzymes ADI (arginine deiminase), OCT (ornithine carbamoyl transferase) and CK (carbamate kinase). Ornithine is released via an arginine-ornithine antiporter, blocks the cationic amino acid transporters (CAT) of IECs and thereby uptake of arginine. In addition, arginine is degraded extracellular by ADI and OCT released from <i>Giardia</i> upon interaction. All this leads to reduced arginine availability within the host IEC. As shown within this study, the consequences are reduced polyamine levels in IECs that result in cell cycle arrest via upregulation of cell cycle block genes (GADD45A, BTG3). This reduced cell proliferation in the crypts could lower intestinal cell turnover and reduce cell differentiation along the villus-axis, allowing the parasite to live in a more stable environment. The reduced arginine availability in IECs also leads to decreased production of the antimicrobial agent nitric oxide (NO) via nitric oxide synthases (NOS) in all cells of the intestinal epithelial lining.</p

Amino acid analysis of the medium of the interaction between IECs (Caco2 clone TC7) and <i>Giardia</i> trophozoites (isolate WB).

<p>Arbitrary units are used.</p

Effects of ADI on IEC numbers.

<p>Triplicates of IEC numbers were monitored by MTT assay. In A, IECs (Caco2 and HCT-8 cells), were treated with medium only (medium, white bars), with WB trophozoites (WB, black bars) or with WB trophozoites overexpressing ADI (WB-ADI, grey bars) and measured after 4 d. In B, two silver stained SDS-PAGE gels are shown visualizing the purified ADI and OCT at the expected Mw of 65 kDA and 40 kDA respectively (arrows). In C and D, TC7 and HCT-8 cells were treated with 27.5 U/L ADI from <i>Giardia</i> and measured after 4 d (TC7 cells) and 40 h (HCT-8 cells). ADI induced effects were abolished by addition of arginine to 0.4 mM (ADI +Arg). Corresponding controls are given (buffer; ADIb, which is the boiled protein control; Arg that shows that arginine addition alone does not affect the cells). Cell numbers are expressed as relative to buffer ctrl, P-values are displayed. Note the reduction in cell number upon addition of ADI.</p

Growth curves of Caco2 cells in arginine-free medium compared to arginine- and citrulline-supplemented medium.

<p>Graphs are given for cells growing in complete medium (+Arg, rectangles), in medium without arginine (-Arg, circles) and arginine-free medium complemented with citrulline (+Citr, triangles). In A, cell numbers for proliferating Caco2 cells were measured by MTT assay; in B differentiated Caco2 cells were used. Samples were set up in triplicates, cell numbers expressed as relative to +Arg (d1). P-values calculated in comparison to complete medium values are shown for –Arg and for +Citr. After 3 d, growth was significantly reduced without arginine and citrulline could partially replace for the omitted arginine. In C, argininosuccinate synthase (ASS) mRNA expression in proliferating Caco2 cells at day 2 is displayed for the 3 different growth conditions, as assessed by qPCR in quadruplicates. Note the increase in ASS expression in the presence of citrulline.</p

DNA content profiles of HCT-8 cells upon <i>Giardia</i>-interaction.

<p>The DNA of HCT-8 cells was stained with propidium iodide and analyzed by flow cytometry. The solid lined histograms represent control cells, whereas the dotted lines show profiles of cells after 24 h (A, B and C) and 36 h (D, E and F) of interaction with WB trophozoites. A and D display controls with only water added; in B and E, arginine was added to 0.4 mM; in C and F, citrulline was added to 0.4 mM. Note the histogram shifts upon parasite addition that could be completely reverted by the addition of citrulline.</p

Proposed classification of SpdS inhibitors.

<p>Proposed classification of SpdS inhibitors.</p

Properties of <i>Pf</i>SpdS inhibitors.

<p>Properties of <i>Pf</i>SpdS inhibitors.</p

Ligand binding to <i>Pf</i>SpdS.

<p>(A) Pathways for substrates, products, and inhibitors. The enzyme is represented schematically by a black outline indicating its binding sites and their conformational changes upon ligand binding: left, MTA cavity (triangle; fully formed in the free enzyme, unchanged with bound ligands); middle, central aminopropyl cavity (distorted circle in the free enzyme; becomes circular with bound ligands); right, putrescine site plus distal aminopropyl cavity (distorted oval in the free enzyme; becomes rectangular with bound ligands, some of which do not fill the distal cavity completely). Coloring of schematically represented compounds follows the colored boxes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.g002" target="_blank">Fig 2</a>: aminopropyl donor substrate and its product, beige; polyamine substrates and products, green; inhibitors, blue. The double arrows show binding equilibria between forms; the single arrows indicate enzymatic reaction. Letters and numbers under each shape correspond to the classes defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.t003" target="_blank">Table 3</a> and depicted in panel B. (B) Classification of ligands. Upper left, the schematic representation of the active site introduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.g001" target="_blank">Fig 1</a> and used here to identify ligand-binding locations. Lower left box, the indicated substrates and products are shown as stick cartoons placed in the appropriate active-site locations; right box, representation as in the left box showing the active-site positions of the compounds listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.t003" target="_blank">Table 3</a>. Numbers and letters below each entry correspond to classes defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.t003" target="_blank">Table 3</a> and represented in panel A. Placements in the active-site locations are based on the respective crystal structures except for S_2a* and S_2b*, which are artificially positioned in the structure with dcAdoMet only based on structures P_2a and P_2b with MTA, and I_2b*, which is artificially positioned in the structure with dcAdoMet based on structure I_3a of NACD with MTA.</p

Overall structure and active site of <i>Pf</i>SpdS.

<p>(A) Monomer architecture. The N-terminal beta-sheet domain is light blue; the C-terminal Rossmann fold domain is dark blue. The active site is indicated in a cleft between the two domains, marked by a stick model of bound MTA and putrescine (green, based on PDB ID: 4BP1). The gatekeeper loop spanning the active site is shown in orange; when ligands are bound it adopts a loop-3₁₀ helix-loop structure that is approximated in the representation shown. (B) Active site. Labeled yellow shaded or outlined oval shapes of different sizes represent the indicated parts of the active site referred to in the text. The larger dcAdoMet site (black-outlined oval at right) is conceptually divided into an MTA cavity (large upper shaded oval) and a central aminopropyl cavity (small lower shaded oval). The putrescine site (central black-outlined oval) is adjacent to a distal aminopropyl cavity (shaded oval at upper left). The substrates dcAdoMet and putrescine are represented as stick cartoons with green carbon atoms and other atoms in atomic colors (blue nitrogen, red oxygen, and yellow sulfur); these substrates do not occur together in any existing crystal structure because the enzyme reaction would occur. The cartoon is a composite based on separate structures with dcAdoMet and with MTA and putrescine.</p