Katia Maalouf

Characterisation of Pga1, a putative Candida albicans cell wall protein necessary for proper adhesion and biofilm formation.

The fungal pathogen Candida albicans is a leading causative agent of death in immunocompromised individuals. Many factors have been implicated in virulence including filamentation‐inducing transcription factors, adhesins, lipases and proteases. Many of these factors are glycosylphosphatidylinositol‐anchored cell surface antigenic determinant proteins. Pga1 is one such protein shown to be upregulated during cell wall regeneration. The purpose of this study was to characterise the role Pga1 plays by creating a homozygous pga1 null strain and comparing the phenotype with the parental strain. It was observed that the mutant strain showed less oxidative stress tolerance and an increased susceptibility to calcofluor white, a cell surface disrupting agent that inhibits chitin fibre assembly which translated as a 40% decrease in cell wall chitin content. Furthermore, the mutant exhibited a 50% reduction in adhesion and a 33% reduction in biofilm formation compared with the parental strain, which was reflected as a slight reduction in virulence. Our data suggest that Pga1 plays an important role in cell wall rigidity and stability. It was also observed that the pga1 null was over filamentous on both liquid and solid media and exhibited increased resistance to SDS suggesting upregulation of filamentation‐inducing genes and cell surface components to partially compensate for the deletion.

The Antiproliferative Effect of Kefir Cell-Free Fraction on HuT-102 Malignant T Lymphocytes

Kefir is produced by adding kefir grains (a mass of proteins, polysaccharides, bacteria, and yeast) to pasteurized milk; it has been shown to control several cellular types of cancer, such as Sarcoma 180 in mice, Lewis lung carcinoma, and human mammary cancer. Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia, which is a fatal disease with no effective treatment. The current study aims at investigating the effect of a cell-free fraction of kefir on HuT-102 cells, which are HTLV-1-positive malignant T-lymphocytes. Cells were incubated with different kefir concentrations: the cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir cell-free fraction on the proliferation of HuT-102 cells was then assessed. The levels of transforming growth factor (TGF)-alpha mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction. Finally, the growth inhibitory effects of kefir on cell cycle progression and/or apoptosis were assessed by flow cytometry. The maximum cytotoxicity recorded at 80 microg/microL for 48 hours was only 43%. The percent reduction in proliferation was very significant, dose and time dependent, and reached 98% upon 60-microg/microL treatment for 24 hours. Kefir cell-free fraction caused the downregulation of TGF-alpha, which is a cytokine that induces the proliferation and replication of cells. Finally, a marked increase in cell cycle distribution was noted in the pre-G1 phase. In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1-positive malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended.

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes

Background: Adult lymphoblastic leukemia (ALL) is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer. Purpose: The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1)-negative malignant T-lymphocytes. Methods: Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α), transforming growth factor- beta1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA) and flow cytometry. Results: The maximum cytotoxicity recorded after 48-hours treatment with 80 μg/μL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF-β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG1 phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was further confirmed by Cell Death Detection ELISA. However, kefir did not affect the mRNA expression of metalloproteinases needed for the invasion of leukemic cell lines. Conclusion: In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1-negative malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended.

A modified lipid composition in Fabry disease leads to an intracellular block of the detergent-resistant membrane-associated dipeptidyl peptidase IV

Fabry disease is an X-linked lysosomal storage disorder that leads to abnormal accumulation of glycosphingolipids due to a deficiency of alpha-galactosidase A (AGAL). The consequences of these alterations on the targeting of membrane proteins are poorly understood. Glycosphingolipids are enriched in Triton-X-100- resistant lipid rafts [detergent-resistant membranes (DRMs)] and play an important role in the transport of several membrane-associated proteins. Here, we show that In fibroblasts of patients suffering from Fabry disease, the colocalization of AGAL with the lysosomal marker LAMP2 is decreased compared with wild-type fibroblasts concomitant with a reduced transport of AGAL to lysosomes. Furthermore, overall composition of membrane lipids in the patients’ fibroblasts as well as in DRMs reveals a substantial increase in the concentration of glycolipids and a slight reduction of phosphatidylethanolamine (PE). The altered glycolipid composition in Fabry fibroblasts is associated with an intracellular accumulation and impaired trafficking of the Triton-X-100 DRM-associated membrane glycoprotein dipeptidyl peptidase IV (DPPIV) in transfected Fabry cells, whereas no effect could be observed on the targeting of aminopeptidase N (ApN) that is not associated with this type of DRM. We propose that changes in the lipid composition of cell membranes in Fabry disease disturb the ordered Triton X-100 DRMs and have implications on the trafficking and sorting of DRM-associated proteins and the overall protein–lipid interaction at the cell membrane. Possible consequences could be altered signalling at the cell surface triggered by DRM-associated proteins, with implications on gene regulation and subsequent protein expression.

Case study on the pathophysiology of Fabry Disease: Abnormalities of cellular membranes can be reversed by substrate reduction in vitro

It is still not entirely clear how α-galactosidase A (GAA) deficiency translates into clinical symptoms of Fabry disease (FD). The present communication investigates the effects of the mutation N215S in FD on the trafficking and processing of lysosomal GAA and their potential association with alterations in the membrane lipid composition. Abnormalities in lipid rafts (LRs) were observed in fibroblasts isolated from a male patient with FD bearing the mutation N215S. Interestingly, LR analysis revealed that the distribution of cholesterol and flotillin-2 are distinctly altered in the Fabry fibroblasts when compared with that of the wild-type cells. Furthermore, increased levels of glycolipid globotriaosylceramide 3 (Gb3) and sphingomyelin (SM) were observed in non-raft membrane fractions of Fabry cells. Substrate reduction with N-butyldeoxynojirimycin (NB-DNJ) in vitro was capable of reversing these abnormalities in this patient. These data led to the hypothesis that alterations of LRs may contribute to the pathophysiology of Morbus Fabry. Furthermore, it may be suggested that substrate reduction therapy with NB-DNJ might be a promising approach for the treatment of GAA deficiency at least for the selected patients.