Bacterial strains, plasmids, and growth conditions

Bacterial strains and plasmids used in this study are listed in Supplementary Table 1. All Listeria species were grown in tryptic soy broth containing 0.6% yeast extract (TSBYE; Becton Dickinson) or in Luria-Bertani broth (LB, 0.5% NaCl, 1% tryptone peptone, and 0.5% yeast extract) at 37 °C with shaking for 16–18 h. The L. monocytogenes (Lm) F4244 (WT, serovar 4b) and L. innocua (Lin) F4248 were grown in TSBYE. The isogenic lap-deficient insertional mutant strain (lap^─, KB208) was grown in TSBYE with erythromycin (5 μg/mL) at 42 °C. The lap^─ stain complemented with the Lm lap (lap^─+lap^Lm, CKB208) was grown in TSBYE containing erythromycin (5 μg/ml) and chloramphenicol (Cm; 5 μg/ml) at 37 °C. The Lm F4244 (WT) strain expressing the green fluorescent protein (GFP) were grown in LB containing erythromycin (5 μg/ml) at 37 °C. S. enterica serovar Typhimurium ver. Copenhagen was grown in TSBYE at 37 °C.

To express the Lin LAP in the isogenic lap-deficient insertional mutant strain (lap^─, KB208), the Lin lap gene was cloned in the Listeria expression vector pMGS101⁶⁸ and electrotransformed into the lap-deficient insertional mutant strain (lap^─, KB208) and the resulting strain was designated lap^─+lap^Lin and was grown in TSBYE containing erythromycin (5 μg/ml) and chloramphenicol (Cm; 5 μg/ml) at 37 °C.

The Lactobacillus casei ATCC334 wild-type (LbcWT) strain was used as a host to express LAP from Lin and Lm were cultured in deMan Rogosa Sharpe broth (MRS, Becton Dickinson) at 37 °C for 18–20 h under anaerobic conditions. To recover this strain from fecal and intestinal samples during the animal study, a vancomycin-resistant strain of L. casei was selected by sequentially culturing the bacterium in increasing concentrations of vancomycin (300 µg/ml). To generate the bioengineered lactobacilli expressing LAP from Lin and Lm, the entire lap gene (2.6 kb) from Lm F4244 (WT) was amplified by PCR and inserted into pLP401T⁶⁹ containing the pAmy promoter and electrotransformed³⁸ into the selected vancomycin-resistant L. casei strain.

Briefly, the genomic DNA of Lm F4244 and Lin F4248 was extracted and the entire lap gene (2.6 kb) from of Lm (lap^Lm) and Lin (lap^Lin) amplified with PCR using the primers: LAPN-F 5′- GACCATGGATGGCAATTAAAGAAAATG-3′ and LAPX-R-5′-GACTCGAGTCAAACACCTTTGTAAG-3′ (Integrated DNA Technologies, Supplementary Table 1)³⁸. The amplified DNA products were cloned into pGEM-T Easy Vector (Promega) and designated pGEM-LAP^Lm and pGEM-LAP^Lin, respectively. The Lactobacilli expression vector, pLP401T containing the pAmy promoter was used⁶⁹. The plasmids were digested with NcoI and XhoI, inserted into expression vector pLP401T, and designated pLP401T-LAP^Lm and pLP401T-LAP^Lin. To remove the terminator, which stabilizes the plasmid in E. coli, pLP401T-LAP^Lm/Lin was digested with NotI, and pLP401T-LAP^Lm and pLP401T-LAP^Lin were obtained via self-ligation. Self-ligated pLP401T-LAP^Lm and pLP401T-LAP^Lin were used for electroporation into competent vancomycin-resistant L. casei cells. Competent vancomycin-resistant L. casei cells were prepared by incubation of 2% culture in fresh MRS broth containing 0.5% sucrose and 0.5% glycine at 37 °C until OD600 reached to 0.5–0.8. The cells were harvested (3900×g for 5 min at 4 °C), washed twice with washing buffer (0.5 M sucrose, 10% glycerol), and collected. The cells were resuspended in the same washing buffer and stored at −80 °C. For electroporation, 50 µl of competent cells mixed with 0.5 µg of purified plasmid DNA in an ice-cold cuvette with a 2-mm electrode gap. The electric pulse was delivered by the Gene Pulser XcellTM electroporation system (Bio-Rad) using the following parameter settings: 1.5 kV, 200 Ω, and 25 mF. After electroporation, competent cells were recovered in 1 ml of MRS containing 0.5 M sucrose, 20 mM MgCl₂, 2 mM CaCl₂ at 37 °C for 2 h in a water bath. Electroporated L. casei cells were then incubated at 37 °C for 3 h. Transformants harboring pLP401T-LAP^Lm and pLP401T-LAP^Lin were subsequently selected on MRS agar containing 2 µg/ml erythromycin that were incubated at 37 °C overnight for 72 h. The resulting bioengineered lactobacilli probiotic (BLP) expressing LAP from Lm and Lin were designated LbcLAP^Lm (AKB906) and LbcLAP^Lin (AKB907), respectively. Confirmation of the identity of the lap gene in the LbcLAP^Lm and LbcLAP^Lin strain was done using PCR and sequencing. The selected vancomycin-resistant L. casei strain carrying the pLP401T empty vector (Lbc^Vec) was used as a control. The bioengineered strains and the Lbc^Vec were maintained in MRS broth containing erythromycin (2 µg/ml) under anaerobic conditions at 37 °C.

To induce the expression of LAP, the BLP strains, were grown in modified MRS broth (1% w/v protease peptone, 0.5% w/v yeast extract, 0.2% w/v meat extract, 0.1% v/v Tween 80, 37 mM C₂H₃NaO₂, 0.8 mM MgSO₄, 0.24 mM MnSO₄, 8.8 mM C₆H₁₄N₂O₇ in 0.2 M potassium phosphate (dibasic), pH 7.0) supplemented with mannitol (1% w/v) at 37 °C under anaerobic conditions. Growth curves for all three strains were generated and LAP expression was verified by Western blotting, and immunofluorescence staining using anti-LAP mAb³⁸.

Mammalian cells

The human colon carcinoma Caco-2 cell line (ATCC # HTB37) from 25 to 35 passages and the MDCK cell line (ATCC # CCL34) from 10 to 20 passages were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 4 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (FBS; Atlanta Biologicals). The Caco-2 cells presenting stable suppression of Hsp60 mRNA (Hsp60^─), or presenting a non-targeting control shRNA vector (Hsp60^Vec) or exhibiting a constitutive overexpression of Hsp60 were previously developed (Hsp60⁺)³² and cultured in DMEM supplemented with 4 mM L- glutamine, 1 mM sodium pyruvate, 10% FBS, and 800 μg/ml Geneticin.

Mice

Female mice (A/J: 6–8 weeks of age) that are highly sensitive to oral Lm challenge^31,37,46 were purchased from Jackson Laboratories. Upon arrival, animals were provided ad lib feed (Rodent Diet 5001, LabDiet), sterile deionized water, and acclimatized for 5 days before the experiment. Shepherd’s™ ALPHA-dri^® (alpha-cellulose) was used for bedding. A cycle of 12-h artificial light and 12-h darkness was maintained. Relative humidity was 50–60% and the temperature was 20–25 °C. Mice were randomly assigned to eight different groups. Overnight cultures of each Lactobacillus strains (LbcWT, LbcLAP^Lm, and LbcLAP^Lin) grown in modified MRS broth were collected and centrifuged at 3500×g for 15 min. After three washes in phosphate-buffered saline (PBS, pH 7.4) the pellets were and resuspended in sterile deionized water at 4–8 × 10⁹ CFU/ml and replenished daily with fresh Lactobacillus cultures for 10 days. Naive animals received only water.

On the day of the challenge, food and water were withdrawn 5 h before oral gavage. The 12-h grown Lm F4244 (WT, clinical isolate) resuspended in 100 μl of PBS, (pH 7.4) containing ~5 × 10⁸ CFU were administered orally using a stainless-steel ball-end feeding tube (Popper). The uninfected naive mice received only PBS (pH 7.4)³⁷. The food was returned 1 hpi, and the mice were sacrificed 24 h (day 11) and 48 hpi (day 12, 17, and 22) using CO₂ asphyxiation. Animals were observed for clinical signs, such as ruffled hair, movement and recumbency, and their feeding and drinking habits. Body weights were also recorded during the experiments.

The animal procedure was approved by the Purdue University Animal Care and Use Committee (PACAUC approval No.1201000595) who adheres to the recommendations of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Western blotting

To assess the expression of LAP in the BLP strains or Listeria strains were grown as above. To isolate cell wall-associated proteins washed bacterial pellets from 10 ml of overnight-grown cultures were resuspended in 0.5 ml protein extraction buffer (0.5% SDS, 10 mM Tris at pH 6.9), and incubated at 37 °C for 30 min with agitation³⁷. The samples were centrifuged (14,000×g, 10 min, 4 °C), and the supernatants (containing cell wall-associated proteins) were retained. To isolate secreted proteins, cell-free culture supernatants were concentrated by 10% trichloroacetic acid (w/v) and centrifuged (10,000×g for 20 min at 4 °C)³⁷. The resulting pellet was resuspended and washed with ice-cold acetone, centrifuged, and residual acetone was evaporated. The pellet was resuspended in alkaline rehydration buffer (100 mM Tris-base, 3% SDS, 3 mM DTT, pH 11), and boiled for 10 min. To isolate total bacterial proteins (whole-cell lysates from bacterial pellets) the Bacterial Protein Extraction (B-PER) (Thermo Fisher Scientific) reagent was used.

To extract the proteins from Caco-2 cells, cells were seeded in six-well plates for 14–21 days. The cells were washed, scraped from the bottom of six-well plates, suspended in PBS, and pelleted by centrifugation. The detergent-insoluble (membrane) and the detergent-soluble (cytosolic) proteins were isolated using a Mem-Per Eukaryotic Protein Extraction Kit (Thermo Fisher Scientific). Halt proteases and phosphatase inhibitors (Thermo Fisher Scientific) were used during all protein extraction procedures. The protein concentrations were determined by BCA assay (Thermo Fisher Scientific) and separated on SDS-PAGE gels (10–12.5% polyacrylamide) and electro-transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were then blocked in 5% nonfat dry milk (NFDM) in 0.1 M Tris-buffered saline, pH 7.5 (TBS) containing 0.1% Tween 20 (TBST) for at least 1 h. All primary antibodies were diluted in 5% NFDM in TBST and incubated overnight. Primary antibodies (Supplementary Table 1) against LAP, InlA, NamA were used at (1 µg/ml). Antibodies against Hsp60 (mAb), occludin, and MEK 1/2 were used at 1:1000 dilution, and β-actin at 1:2000 dilution. The HRP-linked secondary antibodies; anti-rabbit IgG or anti-mouse IgG (1:2000 dilution in 5% NFDM in TBST) were incubated at 37 °C for 1 h, and a chemiluminescence method was performed using LumiGLO reagent (Cell Signaling) before visualization in the Chemi-Doc XRS system (Bio-Rad). To immunoprobe, the same membrane with another antibody, the originally bound antibodies were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) according to the manufacturer’s protocol. The average reaction intensities of the bands were determined using Quantity One software (Bio-Rad) and presented as the mean ± SEM after normalization to the loading control and are presented as % change relative to the control (untreated cells set at 100%). Immunoblots data are representative of three independent experiments.

Adhesion, invasion, and translocation profiles of Listeria monocytogenes and Lactobacillus casei strains

To analyze the adhesion or invasion profiles of Lm or L. casei strains (LbcWT, Lbc^vec, LbcLAP^Lm, or LbcLAP^Lin) strains, overnight bacterial cultures were washed three times with PBS and resuspended in DMEM containing 10% fetal calf serum (D10F). Bacterial cultures were then added to polarized Caco-2 monolayers (cultured for up to 14–21 days) at a multiplicity of infection (MOI) of ~10 or ~50 for Lm strains or a multiplicity of exposure (MOE) of ~10 for L. casei strains³².

To measure bacterial adhesion, monolayers were rinsed in DMEM after 1 h of incubation at 37 °C in 5% CO₂ and were lysed with 0.1% Triton X-100 in PBS to release the adherent bacteria. The adherent bacteria (CFU) were enumerated by plating serial dilutions of the lysates on Listeria selective modified oxford (MOX) agar plates for Lm counts and on Lactobacillus selective MRS agar plates for L. casei counts.

For bacterial invasion, Caco-2 monolayers were rinsed in DMEM after 1 h of bacterial exposure as described above and incubated with D10F supplemented with gentamicin (50 µg/ml) for an additional 1 h at 37 °C in 5% CO₂ to kill the extracellular bacteria. Caco-2 cells were then rinsed in DMEM, lysed with 0.1% Triton X-100 in PBS and the internalized bacteria were enumerated by plating the serial dilution of the lysates on agar plates as above.

To analyze the in vitro translocation efficiencies of Lm or L. casei strains, Caco-2 cells were grown as monolayers on Transwell inserts with 3.0-μm pores (Corning-Costar) for up to 14–21 days³². The integrity of monolayers was monitored by measuring the TEER (Millicells Voltmeter, Millipore) and at least 200 Ω/cm² (±10) was used as the standard. Overnight bacterial cultures were washed with PBS, resuspended in D10F, and were added to the apical side of the Transwell at an MOI of ~50 for Lm strains or MOE of ~10 for L. casei strains. After 2 h incubation at 37 °C in 5% CO₂, the liquid from the basal well was serially diluted and the translocated bacteria were enumerated by plating. The percent of bacteria adhered, invaded to or translocated across the Caco-2 monolayers was calculated: (the number of viable bacteria recovered at the basal side/The number of viable bacteria added to the apical side) × 100.

Immunofluorescence staining of live bacterial cells

The expression of LAP in the BLP and Listeria strains was assessed by immunofluorescence staining. Briefly, the stationary phase-grown (at 37 °C) cultures of L. casei in mMRS or Listeria in TSBYE (0.6%), were harvested by centrifugation, washed twice with cold PBS, and incubated with the mouse anti-LAP mAb-H7 (1:50 in 5% BSA in PBS) at 37 °C for 1 h and washed in cold PBS⁷⁰. Bacterial cells were then incubated with Alexa-488-conjugated goat anti-mouse secondary antibody (Cell Signaling, 1:50 in 5% BSA in PBS) at 37 °C for 1 h, washed at least four times with 0.5% Tween 20 in PBS and viewed in a Nikon A1R confocal microscope (equipped with 405-nm/Argon/561-nm lasers) using a Plan APO VC 60X/1.40 NA oil immersion objective with the Nikon Elements software (Nikon Instruments Inc.).

Inhibition of Listeria monocytogenes adhesion, invasion, and translocation by L. casei strains

To investigate the ability of L. casei strains to inhibit the adhesion, invasion, and translocation, Caco-2 (Caco-2, Hsp60^Vec, Hsp60^─ or Hsp60⁺) were used³⁸. MDCK cells were cultured for 4–5 days. Briefly, overnight cultures of L. casei strains (LbcWT, Lbc^vec, LbcLAP^Lm, or LbcLAP^Lin) were washed three times with PBS, resuspended in D10F and were added to each well of cultured Caco-2 or MDCK cells (MOE ~10) and incubated for 24 h at 37 °C in 5% CO₂. Unbound bacteria were removed by rinsing monolayers with DMEM. Overnight-grown Lm-WT (F4244) cultures were washed with PBS, resuspended in D10F was added to Caco-2 or MDCK cells (MOI ~50) and incubated for 1 h at 37 °C with 5% CO₂. The cell monolayers were then rinsed three times with DMEM and lysed with 0.1% Triton X-100 in PBS. The adhered bacterial counts were determined as above.

To investigate the ability of L. casei strains to inhibit the invasion of Lm, L. casei strains were added to Caco-2 or MDCK cells (MOE~10, Caco-2, Hsp60^Vec, Hsp60^─, or Hsp60⁺) for 24 h and then infected with Lm (MOI ~50) as above. To determine the intracellular Lm counts, the cell monolayers were incubated in D10F supplemented with gentamycin (50 μg/mL) to kill the extracellular bacteria for an additional 1 h at 37 °C with 5% CO₂. Epithelial cells were lysed, and the invaded bacteria were enumerated.

To investigate the ability of L. casei strains to inhibit the translocation of Lm, the Caco-2 (Caco-2, Hsp60^Vec, Hsp60^─, or Hsp60⁺) or MDCK monolayers grown on Transwell filter inserts as above. L. casei cultures were first added to the apical wells (MOE, 10) and incubated at 37 °C with 5% CO₂ for 24 h. Unbound bacteria were removed by rinsing cells with DMEM. Overnight-grown Lm culture resuspended in D10F were added to epithelial monolayers (MOI ~50) and incubated at 37 °C for 2 h with 5% CO₂. The liquid from the basal well was removed, serially diluted, and plated on MOX agar plates to enumerate Lm. The percent of Lm adhered, invaded to or translocated across cell monolayers was calculated as above.

Antimicrobial activity

To assess the antilisterial activity of L. casei cultures, Petri plates were poured with sterile brain heart infusion (BHI; Neogen) agar (1.5% agarose) and overlaid with sterile BHI soft agar (0.8% agarose) seeded with 20 µl of freshly (12 h) grown Lm F4244. Wells of 7.0-mm diameter was dug aseptically with a cork borer and 100 µl of L. casei cultures (LbcWT, Lbc^vec, LbcLAP^Lm, or LbcLAP^Lin) grown to a stationary phase in mMRS broth at 37 °C were loaded per well. As a positive control, 100 µl of Pediococcus acidilactici strain H (pediocin producer) grown to a stationary phase in MRS broth at 37 °C and 100 µl of vancomycin (100 mg/ml) were loaded per well. Plates were kept at 4 °C for 15 min for absorption and incubated at 37 °C for 24 h to observe the zones of inhibition.

Growth characteristics of BLP strains in growth media and artificial gastrointestinal fluids

For growth curve experiments, the L. casei strains (LbcWT, Lbc^vec, LbcLAP^Lm, and LbcLAP^Lin) were inoculated (1%) in MRS or mMRS containing the appropriate antibiotics and incubated at 37 °C and absorbance at 600 nm was determined at 2-h intervals for a period of 12 h and at 24 h by using a microplate reader (Bio-Rad). The assay was repeated three times with duplicate samples.

The survival of L. casei strains exposed sequentially to the simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF-I and SIF-II, to simulate gastric phase, enteric phase 1 and enteric phase 2, respectively), over 6 h (2 h for each step) period was monitored by plating. SGF contained pepsin (3 g/L) and lipase (0.9 mg/L) (Sigma-Aldrich), pH 1.2–1.5 (adjusted using 1 N HCl). Both SIF-I and SIF-II contained bile (bovine bile; 10 g/L, Sigma-Aldrich) and porcine pancreatin (1 g/L; Sigma-Aldrich), but SIF-I pH was 4.3–5.2 and SIF-II pH was 6.7–7.5 (adjusted using alkaline solution; 150 ml of 1 N NaOH, 14 g of PO₄H₂Na.2H₂0 and deionized water up to 1 L).

Overnight cultures of L. casei strains were washed and resuspended in SGF (100 ml) and incubated at 37 °C, with agitation (150 rpm for 2 h) (gastric phase), and bacterial counts were monitored every 30 min for 2 h. The cells from SGF were pelleted and transferred sequentially into SIF-I, and SIF-II, incubated each at 37 °C for 2 h to simulate the initial and final phases of intestinal digestion. L. casei counts were enumerated on MRS agar plates and the assay was repeated three times with duplicate samples. The level of LAP expression in BLP cultures following exposure to SGF and SIF (I and II) was also monitored by western blotting using anti-LAP mAb. The survival of BLP strains in water is also ensured during animal treatment in a 24-h cycle.

Enumeration of L. monocytogenes and L. casei in mouse organs and samples

Mice were euthanized by CO₂ asphyxiation at 10 days following probiotic treatment or 24 (day 11) and 48 (day 12, 17, 22) hpi, and the intestine (duodenum, jejunum, ileum, cecum, and colon), blood, MLN, spleen, liver, kidney were aseptically collected.

To assess the L. casei loads in the lumen, the entire intestinal contents were removed and homogenized using a tissue homogenizer (Bio Spec) in 5 ml of PBS containing 0.1% Tween 20 (PBS-T). To assess the mucosal-associated bacteria (Lm or L. casei), the entire length of the intestine (duodenum–colon, for Lm) or the segments of the intestine (ileum, cecum, and colon, for L. casei) was flushed with ice-cold sterile PBS that removed the luminal contents and loosely adherent bacteria. The whole intestine was homogenized in 9 ml of buffered Listeria enrichment broth (BLEB) containing 0.1% Tween 20 and selective antimicrobial agents (Neogen) to enumerate Lm. The intestinal segments were homogenized in 4.5 ml PBS-T to enumerate L. casei. To assess invaded bacterial counts (Lm or L. casei), intestinal segments (ileum, cecum, and colon) were treated with gentamicin (100 µg/ml) in DMEM for 2 h at room temperature to kill extracellular bacteria. After five washes in DMEM, the intestinal segments were homogenized in 1 ml of PBS-T. Fecal pellets were weighed, and then suspended in PBS (150 mg/ml) and homogenized using sterile toothpicks.

Aseptically harvested extraintestinal organs/tissues were homogenized using a tissue homogenizer in 4.5 ml (spleen, kidney, and MLN) or 9 ml (liver) of BLEB containing 0.1% Tween 20 and selective antimicrobial agents (Neogen) for Lm counts and in PBS-T for L. casei counts. To enumerate Listeria, LbcWT and BLP strains (LbcLAP^Lm and LbcLAP^Lin), the tissue and fecal homogenates were serially diluted in PBS and plated on MOX agar plates containing selective antibiotics (Neogen), MRS agar containing vancomycin (300 µg/ml), and MRS agar containing vancomycin (300 µg/ml) and erythromycin (2 µg/ml), respectively. No colonies were detected on MRS + vancomycin plates from mock-treated (naive) animals or animals that received Lm only.

Blood was collected using a 1 ml syringe with a 21-G needle by cardiac puncture. To enumerate the burdens of Listeria in the blood, 50 μl of blood was diluted with 450 μl of BLEB immediately following collection and samples were serially diluted and plated as above. In specific experiments, a section of the ileum (~2 cm) or colon (~2 cm) was fixed overnight in 10% formalin for histopathology or immunostaining.

Clinical sign score

After infection, mice were monitored and scored³ blindly by a veterinarian for disease severity by two parameters: weight loss (>95% of initial weight = 0, 95–90% initial weight = 1, 90–80% initial weight = 2, and <80% initial weight = 3) and morbidity (score of 0–5 for each symptom; labored breathing, response to external stimuli, movement, recumbency, and ruffled fur).

Immunohistochemistry, Alcian blue staining, and histopathology

Mouse tissues were fixed in 10% neutral buffered formalin for 24–48 h, placed in a Sakura Tissue-Tek VIP6 tissue processor for dehydration through graded ethanol (70%, 80%, 95%, and 100%), cleaned in xylene, and embedded in Leica Paraplast Plus paraffin. Tissue sections (4 µm) were made using a Thermo HM355S microtome. Sections were mounted on charged slides and dried for 30–60 min in a 60 °C oven. All slides were deparaffinized through three changes of xylene (5 min each) and rehydrated through graded ethanol as above in a Leica Autostainer XL. Slides are stained in Gill’s II hematoxylin blue and counterstained in an eosin/phloxine B mixture using the Leica Autostainer XL. Finally, slides were dehydrated, cleared in xylene, and mounted with coverslips in a toluene-based mounting media (Leica MM24).

For immunohistochemistry, after deparaffinization, antigen retrieval was done in the appropriate buffer using a BioCare decloaking chamber at 95 °C for 20 min. Slides were cooled at room temperature for 20 min dipped into TBST. The rest of the staining was carried out at room temperature using a BioCare Intellipath stainer. Slides were incubated with 3% hydrogen peroxide in water for 5 min, or Bloxall block for 10 min for antibody labeling. Slides were rinsed with TBST and incubated in 2.5% normal goat or horse serum for 20 min. Excess reagents were removed, and a primary antibody or antibody cocktail was applied at the appropriate dilution for 30 min. Primary antibodies (Supplementary Table 1) include antibodies to Listeria (1:100 dilution), ZO-1 (1:100 dilution), LAP (1:1000 dilution), Muc2 (1:500 dilution), Ki67 (1:100 dilution), cleaved caspase-3 (1:200 dilution), IL-10 (1:100 dilution), TGFβ (1:250 dilution), CD3 (1:200 dilution), CD4 (1:100 dilution), CD8α (1:1000 dilution), F4/80 (1:200 dilution), FoxP3 (1:200 dilution), CD11c (1:500 dilution), and NKp46 (1:100 dilution). Negative control slides were stained with their respective isotype controls (Supplementary Table 1) at 1–2 µg/mL for 30 min. After TBST rinse (twice) the secondary antibody was applied for 30 min, rinsed (twice) in TBST before reaction with Vector ImmPACT DAB (Vector Labs) for 5 min. Slides that were probed with two antibodies were counterstained with ImmPACT Vector Red (Vector Labs). Slides were rinsed in water and counterstained with hematoxylin. Tissue sections were also stained with Alcian blue for goblet cell counts.

For histopathology, a microscopic examination was performed by a board-certified veterinary pathologist who was blinded to the treatment groups and the interpretation was based on standard histopathological morphology. The extent of mouse ileal lesions was determined by using a semi-quantitative method that included the amount of inflammatory infiltrate and the percentage of goblet cells comprising the villous epithelium. The mouse ileal tissues were scored on a scale of 0–3 for the aforementioned two parameters yielding a maximum score of 6. A histomorphological scale for assessing inflammation in the lamina propria of the mucosa is provided as follows: 3, marked amounts (sheets of granulocytes expanding the width of the villous tip); 2, moderate amounts (sheets of granulocytes at the base of the villous); 1, mild amounts (multifocal scattering); and 0, none observed. To estimate percentage of goblet cells loss, following scale was used: 3, 50% or greater; 2, 25–50%; 1, 11–25%; and 0, <10%.

For H&E-stained and immunoperoxidase-stained tissues were imaged using a DMLB microscope (Leica) with ×40/0.25 NA HC FL PLAN or a ×100/1.40 NA HC FL PLAN oil immersion objective and a DFC310 FX (Leica) camera controlled by Leica Application Suite. Post-acquisition processing, including the stitching of tiled images, was performed using Leica Application Suite (Leica). Immunoperoxidase-stained positive cells such as immune cell infiltrate were counted manually on tiled images in a blinded manner. For each experiment, immunoperoxidase-stained positive cells from 25 villi in the tissue sections of three to four individual animals per treatment were recorded. Each point represents an average of 25 villi from a single mouse.

Survival study

For the survival study, a single LD₅₀ dose (estimated by preliminary experiments) of ~2.5 × 10⁹ CFU/mouse (100 µl volume) was administered per oral route using a stainless-steel ball-end feeding tube (Popper) and observed for 10 days and mortality was recorded. Animal body weight was also recorded, and mice were sacrificed and considered deceased, if weight loss was greater than 25% for two consecutive measurements, as mandated by PACUC regulation.

Competitive exclusion of L. monocytogenes by L. casei strains

Bacterial cultures were prepared as above and were suspended in D10F to a final concentration of 1 × 10⁷ CFU/ml. For competitive adhesion, Lm was co-inoculated with each of the L. casei strains (LbcWT, Lbc^vec, LbcLAP^Lm, or LbcLAP^Lin, 1:1 ratio) to Caco-2 cell monolayer to achieve an MOI/E of 50 and incubated at 37 °C for 1 h with 5% CO₂. The cell monolayers were rinsed three times with D10F and lysed with 0.1% Triton X-100 in PBS. Adherent bacteria were enumerated by plating the serial dilution of resulting lysates on Lactobacillus selective MRS agar and Listeria selective MOX agar plates. The percent of bacteria adhered to cells was calculated as described above.

Analysis of L. casei and L. monocytogenes co-aggregation in vitro

Lm F4244, Lin F4248, LbcWT, LbcLAP^Lm, and LbcLAP^Lin were cultured at 37 °C for 16–18 h in TSBYE (Listeria strains) or mMRS (L. casei strains). All cultures were pelleted by centrifugation at 8000 × g for 3 min and washed with sterile PBS. All cultures were serially diluted to obtain a cell concentration of 10⁶ CFU/ml. Lm or Lin cultures were allowed to interact with the individual probiotic strains (LbcWT, LbcLAP^Lm, or LbcLAP^Lin) at a 1:1 ratio in PBS for 1 h at room temperature with constant agitation on Lab Doctor Revolver (Mid Scientific). Anti-Listeria magnetic Dynabeads (Thermo Fischer Scientific) were used to capture and separate Lm from unbound probiotics. Briefly, 20 μl/ml of bead slurry was added to the bacterial mixtures and allowed to interact for 10 min at room temperature with constant agitation. Beads were magnetically separated and washed with sterile PBS-T three times (10 min each wash) with constant agitation. Beads were serially diluted and plated on MOX and MRS agar for enumeration of Listeria and Lactobacillus, respectively. For blocking the surface-expressed LAP on the BLP strains, all L. casei strains were harvested from 1 ml of overnight-grown culture, and the pellets were washed three times in PBS before the addition of 5 µg/ml of mouse-monoclonal anti-LAP mAb or the isotype control mouse IgG (Santa Cruz Biotechnology). The mixture was incubated at 37 °C for 1 h with gentle shaking and then pelleted, washed five times in the PBS, and used in the aforementioned capture assay. Bead-captured bacteria were also examined under confocal fluorescence microscopy. Images were acquired using a Nikon A1R confocal microscope (equipped with 405-nm/Argon/561-nm lasers) using a Plan APO VC ×60/1.40 NA oil immersion objective with the Nikon NIS Elements software (Nikon Instruments Inc.).

Immunofluorescence staining and confocal microscopy

The mouse ileal or colonic tissue sections were fixed with 10% formalin and embedded in paraffin. The tissues were sectioned (5-µm thick), deparaffinized, and rehydrated for antigen retrieval by immersing the slides in boiling sodium citrate buffer (10 mM, pH 6.0) or 0.01 M Tris/EDTA (pH 9.0), for 10 min. The tissue sections were permeabilized and blocked with PBS containing 0.3% Triton X-100 (Sigma-Aldrich) and 3% normal goat serum (Cell signaling) and immunostained with specific primary antibodies or a cocktail of primary antibodies (Supplementary Table 1) by incubating overnight at 4 °C. Primary antibodies included antibodies to MLCK (1:100 dilution), P-MLC (1:200 dilution), claudin-1 (1:200 dilution), occludin (1:150 dilution), E-cadherin (1:200 dilution), p65 (1:800 dilution), and P-p65 (1:100 dilution). Slides were then rinsed with PBS (three cycles, 5 min), and were incubated with respective Alexa Fluor 488/555/647-conjugated secondary antibody (1:500 dilution) for 2 h at room temperature followed by 3× washing with PBS (5 min each). The nuclei were stained with DAPI (500 ng/ml; Cell signaling), and slides were mounted in ProLong antifade reagent (Invitrogen).

For antibody labeling in cells, Caco-2 cells were grown to 40–50% confluence in eight-chambered slides (Millipore). At the end of the treatment, the cells were fixed with 3.7% formaldehyde in PBS for 20 min and permeabilized and blocked with PBS containing 0.3% Triton X-100 and 3% BSA (Sigma-Aldrich) for 1 h at room temperature and then incubated with primary antibodies (Supplementary Table 1) to LAP (1:50 dilution), ZO-1 (1:100 dilution), (Hsp60 pAb, 1:100 dilution) or similar primary antibodies or a cocktail of primary antibodies at dilutions described above overnight at 4 °C. The cells were then washed with PBS (three cycles, 5 min) and incubated with respective Alexa Fluor 488/555/647-conjugated secondary antibodies (1:500 dilution) for 2 h at room temperature. The nuclei were stained with DAPI (500 ng/ml; Cell Signaling) and slides were mounted in ProLong antifade reagent (Invitrogen).

All images were acquired using a Nikon A1R confocal microscope as above. The X-Z and Y-Z cross-sections were produced by orthogonal reconstructions from z-stack scanning at 0.15-µm intervals taken with ×60 objective in a 5-µm-thick paraffin-embedded tissue section or Caco-2 monolayers. Three-dimensional reconstructions were performed using Nikon NIS Elements software (Nikon Instruments Inc.). Post-acquisition processing was done in Adobe Photoshop.

The p65 and P-p65 nuclear-positive cells were counted and expressed as average nuclear-positive cells per 15 villi from four individual mice per treatment. For quantification of MLCK and P-MLC expression or analysis of redistribution of cell–cell junctional proteins, images of Caco-2 cells or mouse ileum from five different fields from three independent experiments (for Caco-2 cells, representing 90–100 cells) or three individual mice (representing 100–150 epithelial cells) per treatment were acquired. The relative expression levels of MLCK and P-MLC were analyzed by using the NIH ImageJ software. For the analysis of redistribution of cell–cell junctional protein or Lm cells adhered, % of the total number of cells containing intracellular cell–cell junctional protein puncta and the number of Lm cells adhered were manually counted in acquired images and calculated, respectively.

Giemsa staining

To assess bacterial adhesion, Caco-2 cells were grown to 40–50% confluence in eight-chambered slides (Millipore). At the end of the treatment, cell monolayers were stained with Giemsa stain followed by microscopic examination to visualize bacterial attachment qualitatively. Images were acquired using a DMLB microscope (Leica) with a ×100/1.40 NA HC FL PLAN oil immersion objective.

Fluorescent in-situ hybridization (FISH)

After deparaffinization, the enzyme digestion and hybridization were carried out on a BioCare Medical IQ Kinetic slide stainer. Slides were incubated with proteinase K (Dako) at 20 µl/ml in TBST at 37 °C for 10 min, washed with 2× saline sodium citrate (SSC) buffer containing 0.1% Tween 20, dehydrated in graded ethanol (70%, 95%, and 100%) and air-dried. Slides were then incubated with Alexa-488-conjugated-Lcas467 L. casei DNA probe (5′/5Alex488N/CCGTCACGCCGACAACAG-3′) at a 1:60 dilution (0.11 ng/µL) (Supplementary Table 1). The oligomer was denatured at 78 °C for 5 min and then hybridized at 45 °C for 16 h. Slides were washed with 2× SSC, stringency wash with 0.1× SSC carried out at 60 °C, and washed again with 2× SSC. Finally, slides were counterstained with DAPI for 5 min at 1 µg/mL and mounted with coverslips with Prolong Gold. Images were acquired using a Nikon A1R confocal microscope as above.

Biofilm assay

The microtiter plate biofilm assay was used to quantify biofilm formation with slight modifications⁷¹. Briefly, the optical density (OD₅₉₅ nm) of overnight cultures of Lm, S. typhimurium, and L. casei (LbcWT, Lbc^vec, LbcLAP^Lm, or LbcLAP^Lin) was adjusted to 1.2. The cultures were diluted 40-fold in a mixture of (1:1 ratio) brain heart infusion and mMRS broths. One hundred and fifty microliters of diluted monocultures or co-cultures (mixed prior in 1:1 ratio) were aliquoted into wells of a 96-well tissue culture-treated microtiter plate (Corning) and incubated at 32 °C for 48 h. Following incubation, the supernatant from each well was aspirated to remove loosely attached cells, and the wells were washed three times with 10 mM PBS. The microtiter plate was air-dried for 15 min and 150 μl of 0.1% crystal violet (CV) solution was added to each well and incubated for 45 min at room temperature to stain the biofilm cells. Each well was washed four times with sterile water to remove residual CV stain and air-dried for 15 min. Two hundred microliter of 95% ethanol was added into each well and incubated for 15 min at room temperature to destain the biofilm. Finally, the ethanol solution from each well was transferred to a fresh flat-bottom microtiter plate and absorbance at 595 nm was measured. Wells were also imaged before the addition of ethanol.

Analysis of epithelial cell–cell junctional integrity

To test the effect of L. casei strains on cell–cell junctional integrity, the TEER of Caco-2 cells before and after treatment was measured using a Millicell ERS system (Millipore). For analysis of epithelial permeability, 5 mg/ml of 4 kDa FITC-Dextran (FD4; Sigma) in D10F was added to the well (apical side) and translocation of FD4 to the basal side was monitored by a spectrophotometer (Spectramax, Molecular Devices).

Intestinal permeability assay

Four to five hours before sacrifice, mice were orally gavaged with 100 μl of FD4 (15 mg/100 μl, Sigma)³¹. Urine voluntarily excreted during euthanasia, was collected from the tray/bag, and blood was collected by cardiac puncture. Sera and urine were appropriately diluted in PBS and assayed for FD4 by measuring in a spectrophotometer (excitation: 485 nm; emission 520 nm; Molecular devices) The FD4 concentration was calculated using a standard curve generated by serially diluting FD4 in PBS.

Cytokine ELISA

The quantification of TNFα, IL-6, and IFNγ protein levels was performed in the ileal tissue lysates homogenized in cell lysis buffer. The protein concentrations in the ileal tissue lysates were determined by BCA assay and equal amounts of protein were assayed using mouse-specific ELISA kits (Ray Biotech) as per the manufacturer’s instruction.

Statistical analysis

Experimental data were analyzed using Microsoft Excel and GraphPad Prism (La Jolla, CA) software. P values and the type of statistical analysis performed are described in the figure legends. For mouse microbial counts, statistical significance was assessed by the Mann–Whitney test. For the mice survival experiment, the Kaplan–Meyer plot was generated, and a log-rank test was performed. In other experiments, comparisons between treatment and control were performed using the one-way or two-way analysis of variance with Tukey’s multiple-comparison test. Unless otherwise indicated, data for all experiments are presented as the mean ± standard error of the mean (SEM).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.