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Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier.

Kim H, Bhunia AK - Gut Pathog (2013)

Bottom Line: In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2.ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates.This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Food Science, Molecular Food Microbiology Laboratory, West Lafayette, USA. Bhunia@purdue.edu.

ABSTRACT

Background: Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown.

Methods: Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells.

Results: Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates.

Conclusion: This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

No MeSH data available.


Related in: MedlinePlus

Involvement of SecA2 in adhesion, invasion, and translocation in L. monocytogenes. Analysis of involvement of SecA2 in L. monocytogenes (a) adhesion and invasion, and (b) translocation through paracellular route in Caco-2 cell monolayers in trans-well assay using WT strain F4244, secA2 mutant (ΔsecA2) and secA2 complemented strain (secA2+). Data are average of at least three independent experiments performed in triplicate. Bars marked with different letters (a, b, c) are significantly different at P < 0.05.
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Figure 6: Involvement of SecA2 in adhesion, invasion, and translocation in L. monocytogenes. Analysis of involvement of SecA2 in L. monocytogenes (a) adhesion and invasion, and (b) translocation through paracellular route in Caco-2 cell monolayers in trans-well assay using WT strain F4244, secA2 mutant (ΔsecA2) and secA2 complemented strain (secA2+). Data are average of at least three independent experiments performed in triplicate. Bars marked with different letters (a, b, c) are significantly different at P < 0.05.

Mentions: Earlier, we had shown that the Lap secretion to extracellular milieu is mediated by an auxiliary secretion protein, SecA2 and deletion of secA2 gene in F4244 affected bacterial adhesion and invasion (Figure 6a). Here, we showed that paracellular translocation was also severely impaired in a Δsec2 mutant but restored in the complemented (secA2+) strain (Figure 6b). We also observed similar trend in another strain of L. monocytogenes, 10403S (serovar 1/2a) and the paracellular translocation was significantly (P < 0.05) lower for ΔsecA2 deletion mutant (data not shown). Furthermore, the Δsec2 strain also exhibited reduced dextran permeability in Caco-2 cell trans-well insert model (data not shown). This study further confirms that Lap secretion is SecA2-dependent and impaired SecA2 affects Lap-mediated bacterial paracellular translocation.


Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier.

Kim H, Bhunia AK - Gut Pathog (2013)

Involvement of SecA2 in adhesion, invasion, and translocation in L. monocytogenes. Analysis of involvement of SecA2 in L. monocytogenes (a) adhesion and invasion, and (b) translocation through paracellular route in Caco-2 cell monolayers in trans-well assay using WT strain F4244, secA2 mutant (ΔsecA2) and secA2 complemented strain (secA2+). Data are average of at least three independent experiments performed in triplicate. Bars marked with different letters (a, b, c) are significantly different at P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716925&req=5

Figure 6: Involvement of SecA2 in adhesion, invasion, and translocation in L. monocytogenes. Analysis of involvement of SecA2 in L. monocytogenes (a) adhesion and invasion, and (b) translocation through paracellular route in Caco-2 cell monolayers in trans-well assay using WT strain F4244, secA2 mutant (ΔsecA2) and secA2 complemented strain (secA2+). Data are average of at least three independent experiments performed in triplicate. Bars marked with different letters (a, b, c) are significantly different at P < 0.05.
Mentions: Earlier, we had shown that the Lap secretion to extracellular milieu is mediated by an auxiliary secretion protein, SecA2 and deletion of secA2 gene in F4244 affected bacterial adhesion and invasion (Figure 6a). Here, we showed that paracellular translocation was also severely impaired in a Δsec2 mutant but restored in the complemented (secA2+) strain (Figure 6b). We also observed similar trend in another strain of L. monocytogenes, 10403S (serovar 1/2a) and the paracellular translocation was significantly (P < 0.05) lower for ΔsecA2 deletion mutant (data not shown). Furthermore, the Δsec2 strain also exhibited reduced dextran permeability in Caco-2 cell trans-well insert model (data not shown). This study further confirms that Lap secretion is SecA2-dependent and impaired SecA2 affects Lap-mediated bacterial paracellular translocation.

Bottom Line: In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2.ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates.This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Food Science, Molecular Food Microbiology Laboratory, West Lafayette, USA. Bhunia@purdue.edu.

ABSTRACT

Background: Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown.

Methods: Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells.

Results: Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates.

Conclusion: This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

No MeSH data available.


Related in: MedlinePlus