TGF-β production and expression of mesenchymal markers in A549 cells.

<p>(<b>A</b>) Quantification of TGF-β in supernatants from cells cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar). (<b>B</b>) Western blot of cell lysates loads analyzing expression levels of N-cad, (first lane) and Vimentin (second lane) in cells cultured in NG (white bar), HG (black bar) or OG (gray bar) conditions with or without 2 ng/mL TGF-β. Signal intensities were normalized, with GAPDH as loading control, and relative intensities of N-cad (<b>C</b>) and Vimentin (<b>D</b>) are shown. The results are representative of 3 independent experiments. Quantitative analyses are shown as mean ± standard deviation. P values were calculated using the Student's t test. *<i>P≤0.01</i>; **P<i>≤0.005</i>.</p

Analysis of cell morphology and motility.

<p>Cell morphology (<b>A</b>); cell motility (<b>B</b>) and cell circularity (<b>C</b>) of A549 cells treated in NG, HG or OG conditions with (right panel) or without TGF-β (left panel). Representative photos are presented. Tracks of 50 random individual cells on gold solution (<b>D</b>) were measured using the Scion Image program represented as squared pixels, and are shown as mean ± SD. NG (white bar), HG (black bar) or OG (gray bar). *P≤0,005.</p

Effect of hyperglycemia onfFN biosynthesis.

<p>Western blot of A549 cell lysates cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar) medium with (+) or without (−) TGF-β, showing expression of total FN (first lane) and onfFN (second lane) (<b>A</b>). Signal intensities were normalized, with GAPDH as loading control, and relative intensities of total FN (<b>B</b>) and onfFN (<b>C</b>) are shown. (<b>D</b>) Western blot of A549 total FN (first lane) and onfFN (second lane) immunoprecipitated from cell lysates by FDC-6 mAbs, submitted (+) or not (−) to the remotion of <i>O</i>-glycosylation. Human plasma FN (pFN, 0.5 µg) was used as control. qRT-PCR analysis of gene that codifies IIICS domain of onfFN (<b>E</b>) and GalNacT6 (<b>F</b>) respectively. Graph shows one of three independent experiments as mean ± SD. * <i>P≤0.005</i>. Effect of anti-TGF-β blocking antibody in the expression of total FN (first line) and onfFN (second line) (<b>G</b>).</p

Human evolutionary loss of epithelial Neu5Gc expression and species-specific susceptibility to cholera

<div><p>While infectious agents have typical host preferences, the noninvasive enteric bacterium <i>Vibrio cholerae</i> is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key <i>V</i>. <i>cholerae</i> virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB₅ cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid <i>N</i>-glycolylneuraminic acid (Neu5Gc) and the consequent excess of <i>N</i>-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient <i>Cmah</i>^<i>-/-</i> mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of <i>Cmah</i>^<i>-/-</i> mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, <i>V</i>. <i>cholerae</i> evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.</p></div

Schematic representation of VcN-mediated remodeling of mucosal complex gangliosides to GM1 and possible VcN-independent mechanisms that lead to increased susceptibility to Ctx intoxication in the absence of Neu5Gc.

<p>VcN cleaves α2–8 and α2-3-linked sialic acids from di-, tri-, and tetra-sialylated gangliosides to generate the monosialylated ganglioside, GM1. Importantly, VcN is not able to cleave the α2-3-linked sialic acid in the GM1 and thus it the optimal target for Ctx is retained. Di-, tri-, tetra-sialylated gangliosides from WT mice (containing α2-8-linked Neu5Gc) are resistant to VcN cleavage and thus much less GM1 is generated (a—left panels). In contrast, VcN more efficiently cleaves di-, tri-, and tetra-sialylated gangliosides from <i>Cmah</i>^<i>-/-</i> mice, resulting in increased binding of Ctx (a—right panels). In keeping with this rationale, the remodeling of human gangliosides to GM1 that is catalyzed by VcN (b—right panels) occurs at much higher levels than in non-human mammalian cells (b—left panels). We show here that there is also an unknown VcN-independent mechanism by which the small intestine of <i>Cmah</i>^<i>-/-</i> mice is more susceptible to Ctx intoxication. We speculate that the increased levels of ion secretion across the small intestine of <i>Cmah</i>^<i>-/-</i> mice produced by Ctx also rely on changes in the hydrophobic/hydrophilic properties of the cell surface glycocalyx (c). Epithelial cells surfaces may contain up to 10⁸ residues of sialic acids. Thus, the abundance of hydroxyl groups in Neu5Gc-positive cells increases the hydrophilicity of their glycocalyx. On the other hand, the glycocalyx of human cells should be less hydrophilic since it only contains glycans with Neu5Ac. Although not studied here, it is also possible that CFTR glycosylation differs in the absence of Neu5Gc, perhaps modulating its trafficking to the cell suface in response to Ctx (c).</p

Human cells expressing Neu5Gc are resistant to VcN activity in generating Ctx binding and cell intoxication.

<p>(a-b) DMB-HPLC analysis for sialic acid incorporation in the cell surface glycans of T84 cells fed with 5mM of Neu5Gc (right histogram) or 5mM of Neu5Ac (left histogram) as a control. Arrow indicates the Neu5Gc-corresponding peak in Neu5Gc fed cells that is absent in Neu5Ac fed cells. NT = Not treated. (c) Whole cell ELISA (6 wells for each condition) for Ctx binding to T84 cells with or without VcN treatment. (d) Quantification of cAMP production induced by CtxAB5 with or without VcN treatment calculated for three different wells (biological triplicates) for each condition. Graphs represent one of three independent experiments. Black bars—Neu5Gc fed cells. Gray bars Neu5Ac expressing cells. (*p<0.05 and **p<0.005).</p

The presence of Neu5Gc compromises VcN activity and generation of epitopes for Ctx binding, compared to Neu5Ac.

<p>(a) Glass slides were printed with the indicated synthetic structures and incubated with increasing amounts of VcN. The resulting slides were probed with the bacterial Hsa-BR adhesin to detect exposed α2-3-linked mono sialic acid. Monosialogangliosides (b) and di-tritetrasialogangliosides (c) isolated from mice small intestine mucosa or from the brain (d and e) were treated with increasing amounts of VcN and incubated with 10ug/mL of biotin-conjugated B subunit of Ctx. Samples of gangliosides isolated from the brain of mice genetically engineered to express Neu5Gc in the brain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007133#ppat.1007133.ref025" target="_blank">25</a>] are referred as NestinCre⁺-<i>Cmah</i>^<i>tg</i> in figure (e). For all samples, Ctx binding was measured after incubation of the wells with HRP-Streptavidin and OPD substrate. The graphs are representative of three independent technical replicates using the batch of gangliosides isolated as described in the methods (*p<0.05).</p

Increased susceptibility of <i>Cmah</i>^<i>-/-</i> small intestine to Ctx-induced fluid secretion.

<p>(a) Representative pictures of Intestinal loops of WT and <i>Cmah</i>^<i>-/-</i> treated with 100uL of PBS containing 10μg/mL of Ctx during 4 h. (b) Quantification of fluid accumulation in intestinal loops treated with 100uL of PBS containing 10μg/mL of Ctx plus and minus 10mU of VcN during 4 h, based on the loop weight/length ratio. (c) Ussing chamber measurement of ion secretion from small intestine fragments (black bars for WT and gray bars for <i>Cmah</i>^<i>-/-</i>) exposed to 10ug/mL of active CtxAB5 for 4 h (n = 4) or Forskolin exposure (*p<0.05). (d) Measurement of FITC recovery in the serum of mice treated with FITC-dextran by oral gavage.</p