Cargando…

Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells

Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanni...

Descripción completa

Detalles Bibliográficos
Autores principales: Ramirez-Sánchez, Diana Aurora, Navarro-Lleó, Noemi, Bäuerl, Christine, Campista-León, Samuel, Coll-Marqués, José María, Pérez-Martínez, Gaspar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9230732/
https://www.ncbi.nlm.nih.gov/pubmed/35744660
http://dx.doi.org/10.3390/microorganisms10061142
_version_ 1784735139663708160
author Ramirez-Sánchez, Diana Aurora
Navarro-Lleó, Noemi
Bäuerl, Christine
Campista-León, Samuel
Coll-Marqués, José María
Pérez-Martínez, Gaspar
author_facet Ramirez-Sánchez, Diana Aurora
Navarro-Lleó, Noemi
Bäuerl, Christine
Campista-León, Samuel
Coll-Marqués, José María
Pérez-Martínez, Gaspar
author_sort Ramirez-Sánchez, Diana Aurora
collection PubMed
description Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains—Lacticaseibacillus paracasei BL23 and Lacticaseibacillus rhamnosus GG—in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.
format Online
Article
Text
id pubmed-9230732
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-92307322022-06-25 Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells Ramirez-Sánchez, Diana Aurora Navarro-Lleó, Noemi Bäuerl, Christine Campista-León, Samuel Coll-Marqués, José María Pérez-Martínez, Gaspar Microorganisms Article Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains—Lacticaseibacillus paracasei BL23 and Lacticaseibacillus rhamnosus GG—in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration. MDPI 2022-05-31 /pmc/articles/PMC9230732/ /pubmed/35744660 http://dx.doi.org/10.3390/microorganisms10061142 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ramirez-Sánchez, Diana Aurora
Navarro-Lleó, Noemi
Bäuerl, Christine
Campista-León, Samuel
Coll-Marqués, José María
Pérez-Martínez, Gaspar
Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells
title Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells
title_full Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells
title_fullStr Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells
title_full_unstemmed Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells
title_short Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells
title_sort factors affecting spontaneous endocytosis and survival of probiotic lactobacilli in human intestinal epithelial cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9230732/
https://www.ncbi.nlm.nih.gov/pubmed/35744660
http://dx.doi.org/10.3390/microorganisms10061142
work_keys_str_mv AT ramirezsanchezdianaaurora factorsaffectingspontaneousendocytosisandsurvivalofprobioticlactobacilliinhumanintestinalepithelialcells
AT navarrolleonoemi factorsaffectingspontaneousendocytosisandsurvivalofprobioticlactobacilliinhumanintestinalepithelialcells
AT bauerlchristine factorsaffectingspontaneousendocytosisandsurvivalofprobioticlactobacilliinhumanintestinalepithelialcells
AT campistaleonsamuel factorsaffectingspontaneousendocytosisandsurvivalofprobioticlactobacilliinhumanintestinalepithelialcells
AT collmarquesjosemaria factorsaffectingspontaneousendocytosisandsurvivalofprobioticlactobacilliinhumanintestinalepithelialcells
AT perezmartinezgaspar factorsaffectingspontaneousendocytosisandsurvivalofprobioticlactobacilliinhumanintestinalepithelialcells