ActA prevents association of ubiquitinated proteins and p62/SQSTM1 with <i>L</i>. <i>monocytogenes</i> in a strain-independent manner.

<p>(A) Confocal images of RAW 264.7 macrophages infected for 1 h with wild-type 10403S <i>L</i>. <i>monocytogenes</i>, stained for Ub. (B) Quantification of Ub colocalization with intracellular wild-type 10403S or 10403S Δ<i>actA</i> bacteria over time. (C) Quantification of Ub colocalization with intracellular wild-type EGD-e or EGD-e Δ<i>actA</i> bacteria over time. (D) Confocal images of RAW 264.7 macrophages infected for 1 h with wild-type EGD-e <i>L</i>. <i>monocytogenes</i>, stained for p62/SQSTM1. (E) Quantification of p62/SQSTM1 colocalization with intracellular wild-type 10403S or 10403S Δ<i>actA</i> bacteria over time. (F) Quantification of p62/SQSTMI colocalization with intracellular wild-type EGD-e or EGD-e Δ<i>actA</i> bacteria over time. Scale bar: 5 μm.</p

The Δ<i>actA</i> mutant of <i>L</i>. <i>monocytogenes</i> evades autophagy in a protein synthesis-dependent manner.

<p>(A) Quantification of the percentage of <i>L</i>. <i>monocytogenes</i> that are LC3⁺ in RAW 264.7 macrophages infected with either 10403S, 10403S Δ<i>actA</i>, EGD-e, EGD-e Δ<i>actA</i> bacteria for: 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). *** P value is < 0.0001 (two-way ANOVA with Bonferroni correction) (B) Quantification of the percentage of <i>L</i>. <i>monocytogenes</i> that are Ub⁺ in RAW 264.7 macrophages infected with either 10403S, 10403S Δ<i>actA</i>, EGD-e, EGD-e Δ<i>actA</i> bacteria for: 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). (C) Quantification of the percentage of <i>L</i>. <i>monocytogenes</i> that are p62/SQSTMI⁺ in RAW 264.7 macrophages infected with either 10403S, 10403S Δ<i>actA</i>, EGD-e, EGD-e Δ<i>actA</i> bacteria for: 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM).</p

LC3 recruitment to <i>L</i>. <i>monocytogenes</i> strains 10403S and EGD-e follows distinct kinetics.

<p>(A) Confocal images of RAW 264.7 macrophages transfected with GFP-LC3 and infected for 1 h with wild-type 10403S <i>L</i>. <i>monocytogenes</i>. Scale bar: 5 μm. (B) Quantification of LC3 colocalization with intracellular wild-type 10403S or isogenic Δ<i>actA</i> mutant bacteria over time in RAW 264.7 macrophages. (C) Quantification of LC3 colocalization with intracellular wild-type EGD-e or isogenic Δ<i>actA</i> mutant bacteria over time in RAW 264.7 macrophages.</p

InlK does not prevent association of ubiquitinated proteins with <i>L</i>. <i>monocytogenes</i> strain 10403S.

<p>(A) Quantification of the percentage of <i>L</i>. <i>monocytogenes</i> that are LC3⁺ in RAW 264.7 macrophages infected with <i>L</i>. <i>monocytogenes</i> with the 10403S background: wildtype (10403S), LLO deficient (Δ<i>hly</i>), ActA deficient (Δ<i>actA</i>), InlK deficient (Δ<i>inlK</i>) and InlK and ActA deficient (Δ<i>inlK</i>Δ<i>actA</i>): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). **<i>P</i> value is < 0.001 (two-way ANOVA with Bonferroni correction). (B) Quantification of the percentage of <i>L</i>. <i>monocytogenes</i> that are LC3⁺ in RAW 264.7 macrophages infected with <i>L</i>. <i>monocytogenes</i> with the 10403S background: wildtype (10403S), LLO deficient (Δ<i>hly</i>), ActA deficient (Δ<i>actA</i>), InlK deficient (Δ<i>inlK</i>) and InlK and ActA deficient (Δ<i>inlK</i>Δ<i>actA</i>): 8 h (-CM) or 3 h, followed by 5 h CM treatment (+CM). ***<i>P</i> value is < 0.001 (two-way ANOVA with Bonferroni correction).</p

Lipidation of LC3 is required for its recruitment to the Δ<i>actA</i> mutant of <i>L</i>. <i>monocytogenes</i> strain EGD-e.

<p>(A, B) Confocal images of RAW 264.7 macrophages transfected with GFP-LC3 (A) or GFP-LC3^G120A (B) and infected for 4 h with EGD-e Δ<i>actA L</i>. <i>monocytogenes</i>. Scale bar: 5 μm. (C,D) Quantification of LC3 or GFP-LC3^G120A colocalization with intracellular wild-type EGD-e (C) or EGD-e Δ<i>actA</i> (D) bacteria at 4 h p.i. in RAW 264.7 macrophages. (E) Quantification of LC3 or GFP-LC3^G120A colocalization with intracellular 10403S Δ<i>actA</i> bacteria infected for 3 h, followed by 5 h CM treatment (+CM) in RAW 264.7 macrophages. (F) Quantification of LAMP1 colocalization with intracellular wild-type 10403S or isogenic Δ<i>actA</i> mutant bacteria from 1 to 8 h p.i. in RAW 264.7 macrophages. (G) Quantification of LAMP1 colocalization with intracellular wild-type EGD-e or isogenic Δ<i>actA</i> mutant bacteria from 1 to 8 h p.i. in RAW 264.7 macrophages.</p

CSLM analysis of <i>L. monocytogenes</i> biofilm production.

<p>Results presented are the means ±SD from two independent experiments performed in triplicate.</p><p>Student's <i>t-test</i> indicated a statistically significant difference between biofilm thickness formed by <i>L. monocytogenes</i> 10403S compared to mutant bacterial strains (p ≤ 0.05).</p><p>CSLM analysis of <i>L. monocytogenes</i> biofilm production.</p

Transmission and scanning electron microscopy analysis of <i>L. monocytogenes</i> EPS production.

<p><i>L. monocytogenes</i> 10403S bacteria in biofilms formed on dialysis tubing membranes (regenerated cellulose) (A) (bar = 100 nm) or planktonic bacteria grown in broth culture (B) (bar = 500 nm) were examined by TEM at 72 hours post-inoculation. (C) SEM of a <i>L. monocytogenes</i> biofilm developed on regenerated cellulose at 24 hours post-inoculation (bar = 10 µm). Arrows indicate EPS. For TEM, samples were fixed with 25% glutaraldehyde, rinsed with cacodylate buffer and stained with ruthenium red to visualize EPS material. For SEM, samples were rinsed with multiple dilutions of ethanol prior to visualization.</p

Biofilm formation by Δ<i>phoPR</i> and Δ<i>dltABCD L. monocytogenes</i>.

<p>Bacterial strains were inoculated into TSBYE media in 96-well plates and grown at 35°C for 24 hours. Cultures were then diluted 1:10 into fresh HTM media with 3% glucose and 0.1 mg/mL each cysteine and methionine in new 96-well PVC microtiter plates. Plates were incubated at 35°C for 96 hours, rinsed with dH₂O using a semi-automated cell washer, stained with crystal violet, rinsed with acetic acid and the OD₅₉₅ ±SD determined using a spectrophotometer. The data presented are representative of three independent experiments. *, p <0.05 (One-way ANOVA test).</p

Identified <i>L. monocytogenes</i> biofilm-formation genes.

<p><i>Putative functions were obtained from <a href="http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html</a></i>.</p><p><i>Based on DNA homologies with the L. monocytogenes 10403S genome database; lmrg refers to genetic loci within strain 10403S</i>.</p><p><i>% Compared to wild-type L. monocytogenes 10403S biofilm formation in two independent experiments</i>.</p><p>Identified <i>L. monocytogenes</i> biofilm-formation genes.</p

Scanning electron microscopy of a bean sprout inoculated with <i>L. monocytogenes</i>.

<p>Sterile bean sprouts were placed in HTM agar media with 3% glucose and inoculated with 10 µl of a 1:10 dilution of a 24-hour culture of 10403S. Following a 24 hour incubation, bean sprouts were processed for scanning electron microscopy (A) Bean sprout (bar = 1 mm) (B) magnified view of the white square from (A) (bar = 100 µm). (C) Bean sprout vegetative tissue colonized with <i>L. monocytogenes</i> (bar = 10 µm) (D) magnification of (C) (bar = 10 µm).</p