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Cryptosporidium parvum is a protozoan parasite that causes intestinal malabsorptive syndrome and malnutrition. Considering the importance of di-tripeptide absorption for nutritional status, we previously investigated the regulation of PepT1 transporter in the suckling rat model of acute cryptosporidiosis and showed that PepT1 protein expression and activity were not modified in the parasitized intestine. Here we used confocal microscopy performed on intestinal villi to determine the subcellular localization of PepT1 together with f-actin and parasites. For this purpose, confocal microscopy using vibratome thick sections was developed on the distal small intestine, the preferential site of parasite implantation. Results showed major heterogeneity of apical PepT1 expression among enterocytes, which did not correlate with actin staining or parasite implantation. These results underscore the importance of considering the effect of C. parvum at the cellular scale and not only in the entire epithelium.

Cryptosporidium parvum is a parasitic protozoa increasingly appreciated as a cause of intestinal malabsorptive syndrome leading to malnutrition and/or growth failure. Because a major mechanism for apical peptide absorption by small intestine is via the proton-coupled transporter PepT1, we investigated the expression and functionality of this transporter in our model of acute cryptosporidiosis. Four-day-old Sprague-Dawley rats were inoculated by gavage with 5 x 10(5) oocysts of C. parvum and killed at Day 12 (peak of the infection) or Day 21 (spontaneous clearance of the parasite). PepT1 expression and functionality were quantified in the distal small intestine, preferential site of C. parvum implantation, and in the proximal small intestine, free of parasite, using Western blot and Ussing chambers, respectively. No difference in total PepT1 protein expression or in glycyl-sarcosine fluxes was observed in C. parvum-infected rats compared with controls either on Day 12 or on Day 21, both in the proximal and in the distal small intestine. However, a significant decrease of apical membrane protein expression of PepT1 was observed in C. parvum-infected enterocytes compared with controls. This maintained dipeptide transport observed despite villous atrophy and decreased expression of the protein at the brush-border membrane strongly suggest a transient upregulation of PepT1 activity, probably related to gamma-interferon regulation.

This study aimed to explore the metabolic consequences of cryptosporidiosis in an acute experimental model both at the peak of infection and after parasite clearance. Four-day-old suckling rats were infected with 10(6) oocysts of Cryptosporidium parvum. At the peak of infection (day 8 PI), C. parvum resulted in a dramatic reduction both in nutrient intake (-50%) and body weight (16.3+/-5.2 vs 27.3+/-1.0 g, P<0.01) with a decrease in both lean body mass and adipose tissue. Muscular fractional and absolute synthesis rate were reduced (-15 and -55%, respectively). After parasite clearance (day 17 PI), body weight remained reduced in formerly infected animals (37.8+/-8.0 vs 47.8+/-4.2 g, P<0.01) whereas nutrient intake normalized and fractional synthesis rate slightly increased (+22%) compared to controls. Overall, our results show that the impact and consequences of cryptosporidiosis are far greater than generally appreciated, leading to major malnutrition in suckling rats.

In order to assess the impact of Cryptosporidium parvum on host intestinal physiology, we investigated absorption of the two principal amino acids in dam's milk (leucine, glutamate), using Ussing chambers and RT-PCR analyses. Experiments were performed in both heavily (ileum) and mildly (duodenum) infected segments of the small intestine at the peak of infection [day 8 post-infection (PI)] and after spontaneous clearance of the parasite (day 17 PI). At day 8 PI, amino acid fluxes across the mucosa were decreased throughout the small intestine (P<0.01) and EAAT3 mRNA expression was reduced ( from -49% to -28%). At day 17 PI, leucine and glutamate fluxes were normalized but the decrease in EAAT3 mRNA levels persisted (from -31% to -46%). Our results demonstrate that cryptosporidiosis induces major amino acid malabsorption involving the entire small intestine which is not counterbalanced by any up-regulation, even after spontaneous clearance of the parasite.

Cryptosporidium parvuminfection induces amino acid malnutrition leading to growth retardation in children. Owing to the nutritional efficiency of peptides compared to free amino acids and the resistance of the di-tripeptide transporter PepT1 to mucosal injury, we analyzed the intestinal expression of PepT1 during experimental acute cryptosporidiosis in suckling rats from day 4 to day 50. PepT1 mRNA levels were increased at the peak of infection (day 10) all along the small intestine and normalized after spontaneous clearance of the parasite (day 21). Immunolocalization of PepT1 showed that its expression was maintained in the brush border membrane of enterocytes in infected rats from day 4 to day 50 all along the small intestine. Our results suggest a transcriptional up-regulation during acute cryptosporidiosis in response to both C. parvum-induced malnutrition and parasite implantation. As no treatment is available, a semi-elemental diet should be considered part of the treatment of cryptosporidiosis.

In the present study. we explored the nutritional consequences of cryptosporidiosis. In order to ascertain the direct responsibility of C. parvum for impairment of staturoponderal development observed during the infection in neonatal animals, we investigated the absorption of two major components of the total amino acids in dam's milk (leucine and glutamate) across the ileal mucosa. The infection resulted in significant (47% and 34%, respectively) reductions in leucine and glutamate fluxes (P<0.01). Moreover, the leucine aminopeptidase and alkaline phosphatase activities were reduced in the infected ileal mucosa. Interestingly, the reduction in weight gain, which began at day 6 post-infection (PI), persisted until day 20 PI, although no cryptosporidia were detected in the ileal mucosa after day 12 PI. We thus provide evidence that the malabsorption of amino acids during cryptosporidiosis contributes to impairing the development of neonatal animals, with consequences that persist beyond eradication of the parasite.

Cryptosporidiosis is an important cause of diarrhea associated with growth retardation in children and severe malnutrition in immunocompromised patients. The pathophysiology is poorly understood. In the suckling rat model, we show that C. parvum infection impairs net electrogenic transport across the ileal mucosa without involvement of prostaglandins, as well as trans- and paracellular permeability and leucine and glutamate absorption. These results provide evidence for the development of an intestinal malabsorptive syndrome during cryptosporidiosis. Unspecific process such as villous atrophy and inflammatory cytokines secretion should be regarded as possible mediators of this syndrome. However, specific mechanisms have to be considered since C. parvum induces a rearrangement of the host enterocyte cytoskeleton which might impaired intracellular trafficking thus reducing the membrane expression of nutrient transporters. Infection and malnutrition are known to be tightly associated, making each other worse. As no specific efficient therapy exists, cryptosporidiosis-induced malnutrition must be taken into account when establishing therapeutic scheme.