Antimicrobial Peptides Human Beta-Defensin-2 and -3 Protect the Gut During Candida albicans Infections Enhancing the Intestinal Barrier Integrity: In Vitro Study

Abstract

The intestinal mucosa is composed of a monolayer of epithelial cells, which is highly polarized and firmly united to each other thanks to the presence of proteins complexes, called Tight junctions (TJs). Alteration of the mucus layer and TJs causes an increase in intestinal permeability, which can lead to a microbial translocation and systemic disorders. Candida albicans, in addition to its role of commensal, is an opportunistic pathogen responsible for disseminated candidiasis, especially in immunocompromised subjects where the dysbiosis leads to damage of the intestinal mucosal barrier . In this work, we used a line of intestinal epithelial cells able to stably express the genes that encodes human beta defensin-2 (HBD-2) and -3 (HBD-3) to monitor the invasion of C. albicans in vitro. Defensins are a group of antimicrobial peptides (AMPs) found in different living organisms, and are involved in the first line of defense in the innate immune response against pathogens. The results obtained show that the presence of antimicrobial peptides improves the expression of TJs and increases the Trans Epithelial Electrical Resistence value. In addition, the invasive ability of C. albicans in transfected cells is significantly reduced, as well as the expression levels of genes involved in the apoptotic pathway. Through the study of interaction between antimicrobial peptides and microbiota we will be able in the future to better understand the mechanisms by which they exert the host defense function against intestinal pathogens.

The Intestinal Biofilm of Pseudomonas aeruginosa and Staphylococcus aureus Is Inhibited by Antimicrobial Peptides HBD-2 and HBD-3

“…Caco-2 cells (Human Caucasian colon adenocarcinoma cells, ATCC ® HTB-37™) were routinely cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, Waltham, MA, USA), supplemented with 1% Penstrep, 1% glutamine, and 10% foetal calf serum (Gibco, USA) at 37 • C at 5% CO 2 . After transfection, the cells were grown in the presence of 250 µg/mL blasticidin (InvivoGen, San Diego, CA, USA) in a sterile 25 cm 2 flask at a concentration of 3 × 10 5 to confluence for 21 days to reach full differentiation and polarisation [3,10]. The culture medium was changed every two days.…”

“…For the evaluation of the expression levels of genes associated with biofilm formation in P. aeruginosa and S. aureus, the mRNA of viable sessile cells, obtained following biofilm formation as previously described, was extracted using the Tri-Reagent as per manufacturer's instructions. Two hundred nanograms of mRNA were reverse-transcribed (Expand Reverse Transcriptase, Roche Dignostic, Italy) into complementary DNA (cDNA) using random hexamer primers (Random hexamers, Roche Diagnostic, Italy) at 42 • C for 45 min, according to the manufacturer's instructions [3,10].…”

“…All PCR reactions were run in triplicate. The specificity of the amplification products was verified by electrophoresis on a 2% agarose gel and visualisation by ethidium bromide staining [3,10].…”

“…In addition to microbiota, the cells of the intestinal epithelium represent the second barrier in the line of defense between the host and the environment. This occurs through the presence of tight junctions (TJs), which physically strengthen the barrier [3], and by the production of molecules, such as cytokines, chemokines and antimicrobial peptides (AMPs), capable of activating the mechanisms of the innate immune system [4][5][6]. AMPs are highly conserved peptides expressed constitutively, or induced by microorganisms through mechanisms involving the activation of Pattern Recognition Receptors (PRRs) in intestinal epithelial cells [7,8].…”

“…We recently [10] created a Caco-2 cell line stably transfected with the genes encoding the HBD-2 and HBD-3 antimicrobial peptides and evaluated their role during acute infections with Salmonella enterica subsp. enterica serovar typhimurium [10] and C. albicans [3].…”