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Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells.

Cemma M, Lam GY, Stöckli M, Higgins DE, Brumell JH - PLoS ONE (2015)

Bottom Line: The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface.Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells.Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.

ABSTRACT
Listeria monocytogenes is an intracellular bacterial pathogen that can replicate in the cytosol of host cells. These bacteria undergo actin-based motility in the cytosol via expression of ActA, which recruits host actin-regulatory proteins to the bacterial surface. L. monocytogenes is thought to evade killing by autophagy using ActA-dependent mechanisms. ActA-independent mechanisms of autophagy evasion have also been proposed, but remain poorly understood. Here we examined autophagy of non-motile (ΔactA) mutants of L. monocytogenes strains 10403S and EGD-e, two commonly studied strains of this pathogen. The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface. However, only strain EGD-e ΔactA displayed colocalization with the autophagy marker LC3 at 8 hours post infection. A bacteriostatic agent (chloramphenicol) was required for LC3 recruitment to 10403S ΔactA, suggesting that these bacteria produce a factor for autophagy evasion. Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells. However, deletion of inlK in either the wild-type or ΔactA background of strain 10403S had no impact on autophagy evasion by bacteria, indicating it does not play an essential role in evading autophagy. Replication of ΔactA mutants of strain EGD-e and 10403S was comparable to their parent wild-type strain in macrophages. Thus, ΔactA mutants of L. monocytogenes can block killing by autophagy at a step downstream of protein ubiquitination and, in the case of strain EGD-e, downstream of LC3 recruitment to bacteria. Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.

No MeSH data available.


Related in: MedlinePlus

InlK does not prevent association of ubiquitinated proteins with L. monocytogenes strain 10403S.(A) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). **P value is < 0.001 (two-way ANOVA with Bonferroni correction). (B) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). ***P value is < 0.001 (two-way ANOVA with Bonferroni correction).
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pone.0125856.g005: InlK does not prevent association of ubiquitinated proteins with L. monocytogenes strain 10403S.(A) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). **P value is < 0.001 (two-way ANOVA with Bonferroni correction). (B) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). ***P value is < 0.001 (two-way ANOVA with Bonferroni correction).

Mentions: Cossart and colleagues recently suggested that the bacterial virulence factor InlK may contribute to the evasion of autophagy by L. monocytogenes EGD-e [18]. Thus, we tested a possible role for InlK in autophagy evasion by strain 10403S. 10403S mutants deficient in either InlK (ΔinlK) or InlK and ActA (ΔinlKΔactA) did not exhibit any differences in LC3 colocalization compared to wild-type bacteria (Fig 5A). We conclude that InlK does not play an essential role in autophagy evasion by L. monocytogenes strain 10403S in the presence or absence of ActA.


Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells.

Cemma M, Lam GY, Stöckli M, Higgins DE, Brumell JH - PLoS ONE (2015)

InlK does not prevent association of ubiquitinated proteins with L. monocytogenes strain 10403S.(A) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). **P value is < 0.001 (two-way ANOVA with Bonferroni correction). (B) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). ***P value is < 0.001 (two-way ANOVA with Bonferroni correction).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4430529&req=5

pone.0125856.g005: InlK does not prevent association of ubiquitinated proteins with L. monocytogenes strain 10403S.(A) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). **P value is < 0.001 (two-way ANOVA with Bonferroni correction). (B) Quantification of the percentage of L. monocytogenes that are LC3+ in RAW 264.7 macrophages infected with L. monocytogenes with the 10403S background: wildtype (10403S), LLO deficient (Δhly), ActA deficient (ΔactA), InlK deficient (ΔinlK) and InlK and ActA deficient (ΔinlKΔactA): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). ***P value is < 0.001 (two-way ANOVA with Bonferroni correction).
Mentions: Cossart and colleagues recently suggested that the bacterial virulence factor InlK may contribute to the evasion of autophagy by L. monocytogenes EGD-e [18]. Thus, we tested a possible role for InlK in autophagy evasion by strain 10403S. 10403S mutants deficient in either InlK (ΔinlK) or InlK and ActA (ΔinlKΔactA) did not exhibit any differences in LC3 colocalization compared to wild-type bacteria (Fig 5A). We conclude that InlK does not play an essential role in autophagy evasion by L. monocytogenes strain 10403S in the presence or absence of ActA.

Bottom Line: The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface.Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells.Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.

ABSTRACT
Listeria monocytogenes is an intracellular bacterial pathogen that can replicate in the cytosol of host cells. These bacteria undergo actin-based motility in the cytosol via expression of ActA, which recruits host actin-regulatory proteins to the bacterial surface. L. monocytogenes is thought to evade killing by autophagy using ActA-dependent mechanisms. ActA-independent mechanisms of autophagy evasion have also been proposed, but remain poorly understood. Here we examined autophagy of non-motile (ΔactA) mutants of L. monocytogenes strains 10403S and EGD-e, two commonly studied strains of this pathogen. The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface. However, only strain EGD-e ΔactA displayed colocalization with the autophagy marker LC3 at 8 hours post infection. A bacteriostatic agent (chloramphenicol) was required for LC3 recruitment to 10403S ΔactA, suggesting that these bacteria produce a factor for autophagy evasion. Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells. However, deletion of inlK in either the wild-type or ΔactA background of strain 10403S had no impact on autophagy evasion by bacteria, indicating it does not play an essential role in evading autophagy. Replication of ΔactA mutants of strain EGD-e and 10403S was comparable to their parent wild-type strain in macrophages. Thus, ΔactA mutants of L. monocytogenes can block killing by autophagy at a step downstream of protein ubiquitination and, in the case of strain EGD-e, downstream of LC3 recruitment to bacteria. Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.

No MeSH data available.


Related in: MedlinePlus