Listeriolysin O Affects the Permeability of Caco-2 Monolayer in a Pore-Dependent and Ca2+-Independent Manner

Cajnko, Miša Mojca; Marušič, Maja; Kisovec, Matic; Rojko, Nejc; Benčina, Mojca; Caserman, Simon; Anderluh, Gregor

doi:10.1371/journal.pone.0130471

Cited by 21 publications

(20 citation statements)

References 86 publications

(91 reference statements)

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“…Finally, we checked the possibility of protein activation in a cellular setup. We had previously used Caco-2 intestinal epithelial cells for monitoring pore-forming activity of LLO 36 . The cells were exposed to the mutant at pH 7.4 and after brief incubation washed with media at two different pH-values.…”

Section: Resultsmentioning

confidence: 99%

Engineering a pH responsive pore forming protein

Kisovec

Rezelj

Knap

et al. 2017

Sci Rep

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Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

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Section: Resultsmentioning

confidence: 99%

Engineering a pH responsive pore forming protein

Kisovec

Rezelj

Knap

et al. 2017

Sci Rep

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“…44,45 Consistent with our results, virulence factors of Listeria monocytogenes, such as the pore-forming toxin listeriolysin O, induced a significant drop in TEER in a dose-dependent manner in the Caco-2 monolayer. 46 The integrity of the intestinal epithelial cell layer is crucial for morphological function of the intestinal tissue, and cell polarity, microvilli, and actin cytoskeleton play an essential role on cell activity. 47 The present study used an in vitro model to investigate how T. gondii explores Caco-2 enterocytes.…”

Section: Discussionmentioning

confidence: 99%

Toxoplasma gondiiInfection Promotes Epithelial Barrier Dysfunction of Caco-2 Cells

Briceño

Nascimento

Nogueira

et al. 2016

J Histochem Cytochem.

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SummaryAfter oral infection, Toxoplasma gondii invades intestinal cells, induces breakdown of intestinal physiology and barrier functions, and causes intestinal pathology in some animal species. Although parasites' invasion into host cells is a known phenomenon, the effects of T. gondii infection in the intestinal barrier are still not well established. To evaluate morphological and physiological modifications on the colorectal adenocarcinoma-derived Caco-2 cell line during T. gondii infection, microvilli, tight junction integrity, and transepithelial electrical resistance (TEER) were investigated under infection. It was observed that the dextran uptake (endocytosis) and distribution were smaller in infected than in noninfected Caco-2 cells. The infection leads to the partial loss of microvilli at the cell surface. Claudin-1, zonula occludens-1 (ZO-1), and occludin expressions were colocalized by immunofluorescence and presented discontinuous net patterns in infected cells. Immunoblotting analysis at 24 hr postinfection revealed decreasing expression of occludin and ZO-1 proteins, whereas claudin-1 presented similar expression level compared with noninfected cells. T. gondii decreased TEER in Caco-2 cells 24 hr after infection. Our results suggest that T. gondii infection may lead to the loss of integrity of intestinal mucosa, resulting in impaired barrier function. (J Histochem Cytochem 64:459-469, 2016)

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“…After exposed to AGEs (100 µg/ml) or AGEs (100 µg/ml) with SalA (0.3 µM or 3 µM) for 24 h, HRGECs trans-endothelial electrical resistance (TEER) was measured using Millipore Millicell ERS (Merck Millipore, USA) with probe MERSSTX01 and ERS-2 Epithelial volt-ohm meter as previously described [28]. Cell permeability was evaluated by passage of FITC-dextran (Sigma, USA).…”

Section: Methodsmentioning

confidence: 99%

Salvianolic Acid A Protects Against Diabetic Nephropathy through Ameliorating Glomerular Endothelial Dysfunction via Inhibiting AGE-RAGE Signaling

Hou

Qiang

Zhao

et al. 2017

Cell Physiol Biochem

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Background/Aims: Glomerular endothelium dysfunction leads to the progression of renal architectonic and functional abnormalities in early-stage diabetic nephropathy (DN). Advanced glycation end products (AGEs) and receptor for AGEs (RAGE) are proved to play important roles in diabetic nephropathy. This study investigated the role of Salvianolic acid A (SalA) on early-stage DN and its possible underlying mechanism. Methods: In vitro AGEs formation and breaking rate were measured to illustrate the effect of SalA on AGEs. Type 2 diabetic nephropathy rats were induced by high-fat diet and low-dose streptozocin (STZ). After eight-week treatment with SalA 1 mg/kg/day, 24h-urine protein, creatinine clearance was tested and renal structural injury was assessed by PAS and PASM staining. Primary glomerular endothelial cell permeability was evaluated after exposed to AGEs. AGEs-induced RhoA/ROCK and subsequently activated disarrange of cytoskeleton were assessed by western blot and immunofluorescence. Results: Biochemical assay and histological examination demonstrated that SalA markedly reduced endothelium loss and glomerular hyperfiltration, suppressed glomerular hypertrophy and mesangial matrix expansion, eventually reduced urinary albumin and ameliorated renal function. Further investigation suggested that SalA exerted its renoprotective effects through inhibiting AGE-RAGE signaling. It not only inhibited formation of AGEs and increased its breaking in vitro, but also reduced AGEs accumulation in vivo and downregulated RAGE expression. SalA restored glomerular endothelial permeability through suppressing AGEs-induced rearrangement of actin cytoskeleton via AGE-RAGE-RhoA/ ROCK pathway. Moreover, SalA attenuated oxidative stress induced by AGEs, subsequently alleviated inflammation and restored the disturbed autophagy in glomerular endothelial cell and diabetic rats via AGE-RAGE-Nox4 axis. Conclusion: Our study indicated that SalA restored glomerular endothelial function and alleviated renal structural deterioration through inhibiting AGE-RAGE, thus effectively ameliorated early-stage diabetic nephropathy. SalA might be a promising therapeutic agent for the treatment of diabetic nephropathy.

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Listeriolysin O Affects the Permeability of Caco-2 Monolayer in a Pore-Dependent and Ca2+-Independent Manner

Cited by 21 publications

References 86 publications

Engineering a pH responsive pore forming protein

Engineering a pH responsive pore forming protein

Toxoplasma gondiiInfection Promotes Epithelial Barrier Dysfunction of Caco-2 Cells

Salvianolic Acid A Protects Against Diabetic Nephropathy through Ameliorating Glomerular Endothelial Dysfunction via Inhibiting AGE-RAGE Signaling

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