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

Cajnko MM, Marušić M, Kisovec M, Rojko N, Benčina M, Caserman S, Anderluh G - PLoS ONE (2015)

Bottom Line: The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed.Both toxins exhibit similar effects on epithelium morphology and physiology.Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia.

ABSTRACT
Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.

No MeSH data available.


Related in: MedlinePlus

Pore formation with LLO or EqtII in Caco-2 cells.A Hemolytic activity of LLOA318C-L334C in the reduced state (filled symbols) or the oxidized state (open symbols). Average ± S.D, n = 5. B Binding of LLO and LLOA318C-L334C to multilamellar vesicles. M, molecular weight markers (the bottom band is 50 kDa and the upper is 60 kDa). t, total applied protein; P, protein associated with the pelleted fraction; S, protein remaining in the supernatant after centrifugation. C Time course of the relative drop in TEER (left) after apical application of oxidized (white) or reduced (black) LLOA318C-L334C or EqtIIV8C-K69C and a relative drop in TEER (right) 3 minutes after apical application of oxidized (dark gray) or reduced (white) LLOA318C-L334C or EqtIIV8C-K69C. Data represent means of percent of initial values. Error bars are the standard error of the mean calculated for 2 to 3 independent, experiments. D Time course of SYTOX Green staining after application of 125 nM (black), 62.5 nM (dark gray), 31.3 nM (gray) and 15.6 nM (light gray) LLO (left) or 100 nM (black), 50 nM (dark gray) and 25 nM (gray) EqtII (rigt) and control (white squares). E Confocal microscopy of SYTOX Green staining after application of 125 nM LLO or 100 nM EqtII. Bar in panel C represents 30 μM.
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pone.0130471.g004: Pore formation with LLO or EqtII in Caco-2 cells.A Hemolytic activity of LLOA318C-L334C in the reduced state (filled symbols) or the oxidized state (open symbols). Average ± S.D, n = 5. B Binding of LLO and LLOA318C-L334C to multilamellar vesicles. M, molecular weight markers (the bottom band is 50 kDa and the upper is 60 kDa). t, total applied protein; P, protein associated with the pelleted fraction; S, protein remaining in the supernatant after centrifugation. C Time course of the relative drop in TEER (left) after apical application of oxidized (white) or reduced (black) LLOA318C-L334C or EqtIIV8C-K69C and a relative drop in TEER (right) 3 minutes after apical application of oxidized (dark gray) or reduced (white) LLOA318C-L334C or EqtIIV8C-K69C. Data represent means of percent of initial values. Error bars are the standard error of the mean calculated for 2 to 3 independent, experiments. D Time course of SYTOX Green staining after application of 125 nM (black), 62.5 nM (dark gray), 31.3 nM (gray) and 15.6 nM (light gray) LLO (left) or 100 nM (black), 50 nM (dark gray) and 25 nM (gray) EqtII (rigt) and control (white squares). E Confocal microscopy of SYTOX Green staining after application of 125 nM LLO or 100 nM EqtII. Bar in panel C represents 30 μM.

Mentions: To show that formation of pores at plasma membranes results in a drop in TEER we applied double cysteine mutants, LLOA318C-L334C and EqtIIV8C-K69C. Pore formation by proteins is commonly associated with significant conformational rearrangements that allow insertion of the protein in the membrane and formation of the final pore. These rearrangements can be blocked by introducing two cysteine residues at particular positions, so that upon formation of the disulfide bond the protein is locked in a certain position and pore formation cannot be complete. In order to block permeabilizing activity of LLO we introduced cysteine residues to two helices of the transmembrane helix 2 region, A318C and L334C, which during pore formation rearranges and forms one of the transmembrane β-hairpins [66]. This double mutant did not exhibit hemolytic activity in the oxidized state, but was hemolytic when protein was reduced prior to our measurements (Fig 4A). We have also verified that the LLOA318C-L334C bound to the lipid membranes to the same extent in reduced or oxidized state (Fig 4B). When 1 μM LLOA318C-L334C was applied under oxidative conditions, the drop in TEER was about 20% of the initial value. However, when 125 nM LLOA318C-L334C was applied under reducing conditions the drop in TEER was over 80% (Fig 4C). We have previously characterized EqtIIV8C-K69C mutant, which prevents dislocation of the N-terminal region of EqtII, which is crucial for pore formation. This mutant can bind to membranes in a similar manner as the wild-type protein, but it cannot form pores [53,67]. Under reducing conditions, 100 nM EqtIIV8C-K69C caused over 70% drop in TEER, but the mutant did not affect TEER in oxidative conditions, even if it was applied at four times higher concentration (Fig 4C). Results on mutant proteins indicate that the drop in TEER is in large a consequence of pore formation and not toxin binding to the cell membrane, since TEER values drop significantly only when a functional pore is formed.


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

Cajnko MM, Marušić M, Kisovec M, Rojko N, Benčina M, Caserman S, Anderluh G - PLoS ONE (2015)

Pore formation with LLO or EqtII in Caco-2 cells.A Hemolytic activity of LLOA318C-L334C in the reduced state (filled symbols) or the oxidized state (open symbols). Average ± S.D, n = 5. B Binding of LLO and LLOA318C-L334C to multilamellar vesicles. M, molecular weight markers (the bottom band is 50 kDa and the upper is 60 kDa). t, total applied protein; P, protein associated with the pelleted fraction; S, protein remaining in the supernatant after centrifugation. C Time course of the relative drop in TEER (left) after apical application of oxidized (white) or reduced (black) LLOA318C-L334C or EqtIIV8C-K69C and a relative drop in TEER (right) 3 minutes after apical application of oxidized (dark gray) or reduced (white) LLOA318C-L334C or EqtIIV8C-K69C. Data represent means of percent of initial values. Error bars are the standard error of the mean calculated for 2 to 3 independent, experiments. D Time course of SYTOX Green staining after application of 125 nM (black), 62.5 nM (dark gray), 31.3 nM (gray) and 15.6 nM (light gray) LLO (left) or 100 nM (black), 50 nM (dark gray) and 25 nM (gray) EqtII (rigt) and control (white squares). E Confocal microscopy of SYTOX Green staining after application of 125 nM LLO or 100 nM EqtII. Bar in panel C represents 30 μM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0130471.g004: Pore formation with LLO or EqtII in Caco-2 cells.A Hemolytic activity of LLOA318C-L334C in the reduced state (filled symbols) or the oxidized state (open symbols). Average ± S.D, n = 5. B Binding of LLO and LLOA318C-L334C to multilamellar vesicles. M, molecular weight markers (the bottom band is 50 kDa and the upper is 60 kDa). t, total applied protein; P, protein associated with the pelleted fraction; S, protein remaining in the supernatant after centrifugation. C Time course of the relative drop in TEER (left) after apical application of oxidized (white) or reduced (black) LLOA318C-L334C or EqtIIV8C-K69C and a relative drop in TEER (right) 3 minutes after apical application of oxidized (dark gray) or reduced (white) LLOA318C-L334C or EqtIIV8C-K69C. Data represent means of percent of initial values. Error bars are the standard error of the mean calculated for 2 to 3 independent, experiments. D Time course of SYTOX Green staining after application of 125 nM (black), 62.5 nM (dark gray), 31.3 nM (gray) and 15.6 nM (light gray) LLO (left) or 100 nM (black), 50 nM (dark gray) and 25 nM (gray) EqtII (rigt) and control (white squares). E Confocal microscopy of SYTOX Green staining after application of 125 nM LLO or 100 nM EqtII. Bar in panel C represents 30 μM.
Mentions: To show that formation of pores at plasma membranes results in a drop in TEER we applied double cysteine mutants, LLOA318C-L334C and EqtIIV8C-K69C. Pore formation by proteins is commonly associated with significant conformational rearrangements that allow insertion of the protein in the membrane and formation of the final pore. These rearrangements can be blocked by introducing two cysteine residues at particular positions, so that upon formation of the disulfide bond the protein is locked in a certain position and pore formation cannot be complete. In order to block permeabilizing activity of LLO we introduced cysteine residues to two helices of the transmembrane helix 2 region, A318C and L334C, which during pore formation rearranges and forms one of the transmembrane β-hairpins [66]. This double mutant did not exhibit hemolytic activity in the oxidized state, but was hemolytic when protein was reduced prior to our measurements (Fig 4A). We have also verified that the LLOA318C-L334C bound to the lipid membranes to the same extent in reduced or oxidized state (Fig 4B). When 1 μM LLOA318C-L334C was applied under oxidative conditions, the drop in TEER was about 20% of the initial value. However, when 125 nM LLOA318C-L334C was applied under reducing conditions the drop in TEER was over 80% (Fig 4C). We have previously characterized EqtIIV8C-K69C mutant, which prevents dislocation of the N-terminal region of EqtII, which is crucial for pore formation. This mutant can bind to membranes in a similar manner as the wild-type protein, but it cannot form pores [53,67]. Under reducing conditions, 100 nM EqtIIV8C-K69C caused over 70% drop in TEER, but the mutant did not affect TEER in oxidative conditions, even if it was applied at four times higher concentration (Fig 4C). Results on mutant proteins indicate that the drop in TEER is in large a consequence of pore formation and not toxin binding to the cell membrane, since TEER values drop significantly only when a functional pore is formed.

Bottom Line: The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed.Both toxins exhibit similar effects on epithelium morphology and physiology.Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia.

ABSTRACT
Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.

No MeSH data available.


Related in: MedlinePlus