Norwich Medical School

1 Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrateevoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2. 2 Amino (and imino) acid transport via rPAT2 was pH-dependent, Na þ -independent and electrogenic. At extracellular pH 5.5 (in Na þ -free conditions) proline uptake was saturable (Km 172741 mM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter. 3 PAT2 preferred substrates are L-a-amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4-or 5-membered heterocyclic amino and imino acids such as 2-azetidinecarboxylate, proline and cycloserine, where both D-and L-enantiomers are transported. 4 The major restrictions on transport are side chain size (the ethyl group of a-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH 2 groups, as in b-alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O-methyl esters show either reduced transport (alanine-O-methyl ester) or are excluded. 5 The structural characteristics that determine the substrate specificity of rPAT2 have been identified. This information should prove valuable in the design of selective substrates/inhibitors for PAT1 and PAT2. -Omethyl ester; O-Me-b-Ala, b-alanine-O-methyl ester; O-Me-Gly, glycine-O-methyl ester; O-Me-Pro, proline-Omethyl ester; PAT1, proton-coupled amino acid transporter 1; PAT2, proton-coupled amino acid transporter 2; pH i , intracellular pH; rPAT2, rat proton-coupled amino acid transporter 2; SLC36, solute carrier family 36; Slc36a2, solute carrier family 36 member 2; Tramdorin 1, transmembrane domain-rich protein 1

Giardia lamblia is a major cause of parasite-induced diarrhoea, resides in the upper part of the small intestine lumen. Two genetically distinct lineages (or assemblages) cause human disease. Giardiasis is caused when Giardia trophozoites of either assemblage attach to the intestinal epithelium. This can result in malabsorption, disruption of epithelial barrier function and apoptosis of intestinal epithelial cells. By employing an Ussing chamber, we demonstrated that trans-epithelial electrical resistance (TEER) of human colonic adenocarcinoma derived epithelial cell line (CaCo-2) monolayers decreased after 24 hours of co-culture with the parasite. When these cells were co-cultured with Giardia supernatants, inhibition of the CaCo-2 cell short-circuit current (Isc) in response to forskolin and UTP was observed. This inhibition was governed by the availability of Cl- channels as evidenced with Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS) and ...

Giardia is a major cause of parasite-induced diarrhoea. It resides in the upper part of the small intestine lumen; and it is transmitted by the faecal-oral route, either directly or indirectly. This waterborne pathogen is highly prevalent in industrialized countries. Recent studies, using PCR, suggest that prevalence in the United States (USA) maybe as high as 22% with a high incidence of asymptomatic carriage. In Europe, similar studies suggest that prevalence in Romania may be as high as 91%. In the United Kingdom (UK), case reporting suggests that the prevalence of giardiasis is approximately 2% representing some 3% of all reported cases of gastro-intestinal infectious diseases. Two genetically distinct lineages (or assemblages) cause human disease. Giardiasis is caused when Giardia trophozoites of either assemblage attach to the intestinal epithelium. This can result in malabsorption, disruption of epithelial barrier function and apoptosis of intestinal epithelial cells. By empl...

Giardia lamblia is a major cause of parasite-induced diarrhoea, which resides in the upper part of the small intestine lumen. Giardia is transmitted by the faecal-oral route, either directly or indirectly. This waterborne pathogen is highly prevalent in industrialized countries recent studies using PCR suggest that prevalence in the United States (USA) maybe as high as 22% with a high incidence of asymptomatic carriage. In Europe, similar studies suggest that prevalence in Romania may be as high as 91%. In the United Kingdom (UK), case reporting suggests that the prevalence of giardiasis is approximately 1.89% representing some 3% of all reported cases of gastro-intestinal infectious diseases. Two genetically distinct lineages (or assemblages) cause human disease. Giardiasis is caused when Giardia trophozoites of either assemblage attach to the intestinal epithelium. This can result in malabsorption, disruption of epithelial barrier function and apoptosis of intestinal epithelial cells. Giardia is a common cause of outbreaks of waterborne disease worldwide. In the US from 1991 to 2006 there were 13 outbreaks of Giardia linked to drinking water.
By employing an Ussing chamber, we demonstrated that trans-epithelial electrical resistance (TEER) of human colonic adenocarcinoma derived epithelial cell line (CaCo-2) monolayers decreased after 24 hours of co-culture with the parasite. When these cells were co-cultured with Giardia supernatants, inhibition of the CaCo-2 cell short-circuit current (Isc) in response to forskolin and UTP was observed. This inhibition was governed by the availability of Cl- channels as evidenced with Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS) and Glybenclamide (Gly101). The loss of transpethelial flux in response to the different inhibitors as well as the decrease of the CaCo-2 TEER raised the possibility of the presence of a soluble mediator. SDS-PAGE electrophoresis analysis of the Giardia supernatants identified a number of soluble proteins present in the culture supernatants from assemblages A and B. Disruption of endothelial function was then further investigated by fluorescent microscopy.
Our results suggest there is a soluble factor secreted by Giardia whose activity compromises enterocyte function and in particular the transcytosis of electrolytes. This enterocytes’ disruption by a soluble mediator seems to be quite similar to other well known enterotoxins, such as the potent cholera toxin (CT) produced by Vibrio cholera. Interestingly, these two different toxins are both involved in the disruption of chloride secretion. V.cholerae binds to the enterocytes’ plasma membrane and, releases an enzymatically active subunit that causes an increase of the cyclic-adenosine 5’-monophosphate (cAMP) production. cAMP is involved in chloride secretion by the Cystic Fibrosis conductance Regulator (CFRT) channel; the high level of intracellular cAMP causes a massive secretion of electrolytes and water into the intestinal lumen. Little or no structural damage is observed during a V.cholerae infection. Unlike V.cholerae toxin, Giardia soluble factor seems to induce a lot of structural damage such as brush border absorptive surface loss and diffuse shortening of villi. Even though Giardia soluble factor seems to be at the origin of the enterocytes’ structural changes and the increase of chloride secretion, its pathogenic mechanism requires further study.

Giardia is a major cause of parasitic diarrhoea and inhabits the upper small intestine. It is transmitted by faecal-oral route. Trophozoites attach to the intestinal epithelium, inducing giardiasis. Two genetically distinct lineages (assemblages) cause the human disease. Our work suggests that Giardia supernatant influences the short circuit current (Isc) of Colorectal Adenocarcinoma cell line (CaCo-2). The loss of transepithelial flux in response to different inhibitors has raised the possibility of Giardia-secreted mediators. By Differential Interference Contrast microscopy (DIC), we observed a concordant modification of the CaCo-2 cells morphology after 24 hours incubation with Giardia supernatants at different dilutions. The intestinal cell size significantly decreased when incubated with parasite supernatant, with significant effects being mediated even at high dilution. SDS-PAGE analyses of supernatants identified a number of secreted proteins; showing a majority of such proteins conserved between the two assemblages but also a few assemblage A-specific soluble proteins. Proteomic identification of these potential mediators of pathology is now underway.
Our results show that Giardia secretes soluble mediators which may be able to exert effects even at low concentration and at sites such as the colon which are distal to the infection.