ABSTRACT
Listeria monocytogenes interaction with the intestinal epithelium is a key step in the infection process. We demonstrated that Listeria adhesion protein (LAP) promotes adhesion to intestinal epithelial cells and facilitates extraintestinal dissemination in vivo. The LAP receptor is a stress response protein, Hsp60, but the precise role for the LAP-Hsp60 interaction during Listeria infection is unknown. Here we investigated the influence of physiological stressors and Listeria infection on host Hsp60 expression and LAP-mediated bacterial adhesion, invasion, and transepithelial translocation in an enterocyte-like Caco-2 cell model. Stressors such as heat (41°C), tumor necrosis factor alpha (TNF-α) (100 U), and L. monocytogenes infection (104 to 106 CFU/ml) significantly (P < 0.05) increased plasma membrane and intracellular Hsp60 levels in Caco-2 cells and consequently enhanced LAP-mediated L. monocytogenes adhesion but not invasion of Caco-2 cells. In transepithelial translocation experiments, the wild type (WT) exhibited 2.7-fold more translocation through Caco-2 monolayers than a lap mutant, suggesting that LAP is involved in transepithelial translocation, potentially via a paracellular route. Short hairpin RNA (shRNA) suppression of Hsp60 in Caco-2 cells reduced WT adhesion and translocation 4.5- and 3-fold, respectively, while adhesion remained unchanged for the lap mutant. Conversely, overexpression of Hsp60 in Caco-2 cells enhanced WT adhesion and transepithelial translocation, but not those of the lap mutant. Furthermore, initial infection with a low dosage (106 CFU/ml) of L. monocytogenes increased plasma membrane and intracellular expression of Hsp60 significantly, which rendered Caco-2 cells more susceptible to subsequent LAP-mediated adhesion and translocation. These data provide insight into the role of LAP as a virulence factor during intestinal epithelial infection and pose new questions regarding the dynamics between the host stress response and pathogen infection.
Listeria monocytogenes is a food-borne pathogen which causes severe opportunistic illness in humans by crossing the intestinal epithelial barrier to gain access to deeper tissues (21, 56). Physiologically stressed individuals, including pregnant women and those who are immunocompromised, are at greatest risk for listeriosis. In these hosts, Listeria is able to cross the blood-brain barrier to affect the central nervous system and the feto-placental barrier to infect the fetus in pregnant women, which may cause spontaneous abortion or stillbirth.
Since it is a food-borne pathogen, the initial interaction of L. monocytogenes with the intestinal epithelium is crucial for establishing infection and promoting bacterial spread to extraintestinal sites. Adhesion is mediated by bacterial factors, including fibronectin binding protein (FbpA), ActA, Ami, CtaP, and LapB. FbpA binds fibronectin in the intestinal epithelium and on hepatocytes (17). ActA, a protein required for actin-based motility during intracellular infection, also promotes adhesion, via host cell proteoglycans (1). Ami, an autolysin amidase, contributes to adhesion via interaction with an unknown host receptor (41). CtaP, a cysteine transport-associated protein, is also involved in adhesion to host cells (61), and LapB, a newly identified PrfA-regulated virulence protein, is involved in both adhesion to and invasion of host cells (49).
Members of the internalin (Inl) family of proteins mediate adhesion to and invasion of a variety of host cell types. Following oral infection, InlB, InlC, and InlJ mediate binding to the human intestinal mucin Muc2 (38), and InlJ also adheres to some cell types, including intestinal epithelial cells (51). InlA drives invasion of intestinal epithelial cells via interaction with the host receptor E-cadherin, a major component of adherens junctions (40), while InlB promotes deeper infection by binding to the receptor c-Met on cells of the endothelium and on hepatocytes (14). InlA-facilitated invasion is associated with systemic spread of L. monocytogenes, as InlA deletion mutants exhibit reduced translocation to extraintestinal sites in guinea pigs (36) and in transgenic mice expressing human E-cadherin (37). Despite the clear role of InlA in mediating epithelial invasion, in vivo studies demonstrate animal mortality following oral infection with InlA mutants or in animals lacking an InlA-specific E-cadherin molecule (3, 8, 16, 30). Such reports have identified additional virulence factors which promote intestinal pathogenesis of L. monocytogenes, independent of InlA. For example, the peptidoglycan hydrolase Auto aids in epithelial cell invasion (7), and virulence invasion protein (Vip) mediates invasion of intestinal epithelial cells by binding to the host receptor Gp96 (8). In transgenic mice expressing human E-cadherin, a Δvip strain exhibited reduced translocation to the mesenteric lymph nodes, liver, and spleen, to levels comparable to those of an ΔinlA mutant. These studies indicate that transit of Listeria across the tight intestinal barrier depends on the concerted action of multiple virulence factors, by mechanisms which are yet unclear.
Our lab has identified Listeria adhesion protein (LAP), a 104-kDa alcohol acetaldehyde dehydrogenase (lmo1634), as a putative adhesion factor which promotes binding to cell lines of intestinal origin (29, 31, 32, 46) and promotes translocation to the liver and spleen following oral infection of mice (6, 31). LAP is present on the bacterial cell wall and is secreted by the SecA2 system (6). The secreted form of LAP, in conjunction with the cell wall-localized form, promotes full LAP-mediated interaction with host cells, possibly by reassociating with the bacterial cell wall (6, 29).
We previously identified human heat shock protein 60 (Hsp60) as the epithelial receptor for LAP, and we demonstrated reduced LAP-mediated adhesion in Caco-2 cells following treatment of the Caco-2 cell surface with an Hsp60-specific antibody (58). Despite our previous indication that LAP is important for pathogenesis during the intestinal stage of Listeria infection (31), the exact contributions of LAP and Hsp60 to intestinal pathogenesis are unclear.
Although Hsp60 performs chaperone functions primarily within the cell cytoplasm and mitochondrial matrix (28), the presence of it and other heat shock proteins has been found on the cytoplasmic membranes of various mammalian cells, and surface localization of these chaperones is now generally accepted (5, 8, 55). Others have also identified heat shock proteins as pathogen ligands: Staphylococcus aureus FbpA associates with Hsp60 to mediate cell invasion (19), the hepatitis B virus HBx protein forms complexes with host Hsp60 and Hsp70 during infection (63), and Brucella abortus exploits host Hsp70 for invasion of placental trophoblasts and induction of abortion in pregnant mice (59). The use of a host Hsp as a pathogen receptor is an intriguing phenomenon, because Hsp expression is elevated in response to physiological stressors, which include changes in temperature as well as bacterial and viral infections (4, 39, 45). Despite mounting evidence that certain pathogens use heat shock proteins as receptors, little information exists on the potential relationship between infection, the heat shock protein response, and subsequent implications for host-pathogen interaction.
Our objectives in this study were to determine how LAP and Hsp60 mediate interaction of Listeria with intestinal epithelial cells and to evaluate the influence of Listeria infection on host Hsp60 expression. Here we demonstrate that the interaction of LAP and host Hsp60 promotes adherence to and translocation across intestinal epithelial monolayers. We also provide evidence that low levels of L. monocytogenes infection increase expression of host Hsp60, which may in turn lead to greater LAP-mediated association of Listeria with intestinal epithelial cells. This study reveals a novel mechanism by which Listeria may interact with the intestinal epithelial barrier and provides early evidence of how infection-induced expression of host heat shock proteins may promote host-pathogen interaction.
MATERIALS AND METHODS
Bacterial strains and growth conditions. L. monocytogenes F4244 (wild type [WT]; serovar 4b), the isogenic lap-deficient insertion mutant KB208 (lap strain), and lap-complemented CKB208 (lap+) were used as described previously (6, 32) (Table 1). An inlA deletion mutant (ΔinlA; AKB301) and its complement (inlA+; AKB302) were generated for this study. All L. monocytogenes strains were grown in brain heart infusion (BHI) broth (Becton Dickinson) at 37°C, unless indicated otherwise. The lap strain was grown with erythromycin (5 μg/ml) at 42°C, the lap+ strain was grown with erythromycin (5 μg/ml) and chloramphenicol (5 μg/ml) at 37°C, and the inlA+ strain was grown with chloramphenicol (5 μg/ml) at 37°C.
Bacterial strains, cell lines, plasmids, and primers used in this study
Bacterial mutagenesis and complementation.A modification of splicing by overlap extension (SOE) was used to generate a 5′-3′ in-frame deletion of the inlA open reading frame (ORF) (ΔinlA) (10). Oligonucleotide primers InlAUSF and InlAUSR (Table 1) generated a 403-bp product from the 5′ end of the inlA locus. A 400-bp 3′ product was amplified using primers InlADSF and InlADSR (Table 1). The 5′ and 3′ products were combined in equimolar concentrations in a ligation reaction mixture to yield an 803-bp 5′-3′ SOE fragment. This product was digested with BamHI and SacI, cloned into pGEM-T Easy (Promega, Madison, WI), and subcloned into the temperature-sensitive pAUL-A shuttle vector (11). Electrocompetent L. monocytogenes F4244 WT cells were transformed with the construct and subjected to temperature-dependent allelic exchange (10, 11). In-frame deletion of inlA was confirmed by PCR, using primers InlAUSF and InlADSR. Immunoblotting confirmed the absence of InlA but the presence of LAP in the L. monocytogenes ΔinlA strain.
To complement the ΔinlA mutant with the inlA gene (inlA+), oligonucleotide primers InlABamF and InlASphR (Table 1) were used to amplify the entire inlA ORF from L. monocytogenes F4244 genomic DNA. Amplified inlA was cloned via BamHI and SphI sites into pGEM-T Easy and subcloned into pMGS101 (22). Electrocompetent L. monocytogenes ΔinlA cells were transformed with pMGS101-inlA, and positive transformants were selected by plating in the presence of chloramphenicol.
Cell culture.Secondary human enterocyte-like Caco-2 cells (HTB37; American Type Culture Collection) were grown in Dulbecco's modified Eagle medium (DMEM; Invitrogen) containing 10% fetal bovine serum (D10F) (Atlanta Biologicals) at 37°C under 7% CO2 in a humidified incubator. Cells (passages 25 to 35) were seeded at approximately 5 × 104 to 10 × 104 cells/well into 12- or 24-well plates (Corning), and cell monolayers were used between 10 and 14 days later. Confluence was typically achieved at 4 to 5 days, and by 10 days, Caco-2 monolayers were polarized (24).
Antibodies.An anti-Hsp60 antibody was generated via immunization of a New Zealand White rabbit with purified human Hsp60 (Assay Design, Ann Arbor, MI). Antisera were collected at the Purdue University Small Animal Care Facilities, and anti-Hsp60 antibody was purified using a protein G affinity column. An anti-InlA antibody was generated via immunization of a New Zealand White rabbit with purified truncated InlA (amino acid residues 36 to 496) (54). Antisera were collected, and anti-InlA antibody was purified by use of a protein A column. Other antibodies included an anti-β-actin (43 kDa) monoclonal antibody (MAb) (Abcam), an anti-ZO1 polyclonal antibody (PAb) (Invitrogen), anti-LAP MAb H7, and IgG control MAb C11E9, from our lab.
RNA interference and overexpression of Hsp60.Short hairpin RNA (shRNA) constructs targeting human hsp60 mRNA (SureSilencing shRNA-HSPD1) and a nontargeting control shRNA vector (SABiosciences, Frederick, MD) were used to generate a Caco-2 cell line with stable suppression of Hsp60 expression (Table 1). To create a Caco-2 cell line exhibiting overexpression of Hsp60, full-length human hsp60 cDNA was subcloned from pOTB7-hsp60 (Open Biosystems) into the expression vector pcDNA3.1 (Invitrogen) (Table 1). The integrity of hsp60 within the expression construct (pCDNA3.1-hsp60) was verified by sequencing. A pCDNA3.1 plasmid without an insert was used as a vector control. Caco-2 cell transfections were performed using Lipofectamine LTX Plus reagent according to the manufacturer's protocol (Invitrogen). Stable transformants were selected by growth in D10F containing 800 μg/ml Geneticin sulfate (G418) (Sigma). Hsp60 expression levels were monitored by reverse transcription-PCR (RT-PCR) and Western blotting.
Analysis of Hsp60 expression in Caco-2 cell fractions by SDS-PAGE and immunoblotting.Caco-2 cell monolayers were washed twice with cold phosphate-buffered saline (PBS) (0.1 M; pH 7.0), and membrane and intracellular proteins were isolated using a Mem-Per eukaryotic protein extraction kit (Pierce) (64). Protein preparations were desalted (Zeba desalting spin columns; Pierce), precipitated using acetone, and resuspended in sample solvent (4.6% SDS, 0.5% β-mercaptoethanol, PBS, pH 7.0). The protein concentration was determined by a reducing agent-compatible bicinchoninic acid (BCA) assay (Pierce). A lactate dehydrogenase (LDH) assay was performed with acetone-precipitated protein fractions dissolved in DMEM to rule out contamination of membrane fractions with intracellular proteins (data not shown). Equal protein concentrations (15 μg/lane) from Caco-2 membrane and cytosolic fractions were separated by SDS-PAGE (10% acrylamide). Proteins were transferred to Immobilon-P membranes (Millipore) and immunoprobed with anti-Hsp60 PAb (1.4 mg/ml; 1:1,000 dilution) and β-actin-specific MAb (1.8 mg/ml; 1:1,000 dilution). Bands were detected using horseradish peroxidase-coupled anti-mouse or anti-rabbit antibodies (Jackson Immuno Research, West Grove, PA) with enhanced chemiluminescence substrate (Pierce) and were developed on X-ray films. To compare reaction intensities, average band densities were determined with Quantity One software (Bio-Rad).
Bacterial adhesion and invasion assays.Fresh bacterial cultures were washed and resuspended in D10F and then added to Caco-2 monolayers at a multiplicity of infection (MOI) of ∼10. To measure bacterial adhesion, monolayers were washed after 1 h of infection, and adherent bacteria were enumerated by being plated on BHI agar as previously described (32). For bacterial invasion, monolayers were washed after 1 h of infection and incubated with D10F containing gentamicin (50 μg/ml) for 1 h (47). Caco-2 cells were lysed with 0.1% Triton X, and internalized bacteria were enumerated by plate counting as described before (47).
Transepithelial bacterial translocation assay.Caco-2 monolayers were grown to confluence on Transwell filter inserts (4-μm pore size; Corning) and then placed in 12-well tissue culture plates. Transepithelial electrical resistance (TEER) of polarized monolayers was measured (Voltometer; Millipore), and those with a minimum TEER of about 200 Ω/cm2 (range, 190 to 209 Ω/cm2) were used for translocation experiments (15, 27). Furthermore, to ensure monolayer integrity on Transwell inserts, Caco-2 cells from representative experiments were immunostained using antibodies specific to the tight junction proteins ZO-1, occludin, and claudin and Cy5- and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies and were examined by confocal microscopy as described in “Fluorescence microscopy.” Bacteria (MOI, ∼10) were added to the apical well of the Transwell system, and after 2 h at 37°C in 7% CO2, liquid was collected from the basal well and translocated bacteria were enumerated by plate counting (15). Initial experiments were conducted to determine the appropriate length of time for monitoring bacterial translocation through Caco-2 monolayers, and 2 h was chosen because at this time point bacteria in the basal well most closely represented the number of translocated microorganisms, with minimal posttranslocation replication (data not shown).
Effect of Hsp60 expression on Listeria interaction with Caco-2 cells.To evaluate the role of Hsp60 expression in LAP-mediated infection, Caco-2 cells were subjected to heat stress (41°C) or tumor necrosis factor alpha (TNF-α) (Sigma) exposure at 37°C (100 U/ml D10F) (20) for 1 h, followed by a 3-h recovery period at 37°C with 7% CO2. Flow cytometric analyses were performed to determine that 3 h was the optimum recovery period required for surface Hsp60 expression in Caco-2 cells following heat shock (data not shown). Hsp60 expression in Caco-2 cells was monitored by immunoblotting. Bacterial adhesion and translocation were measured as described above.
Influence of direct or indirect exposure of Caco-2 cells to Listeria on Hsp60 expression.Caco-2 cells were exposed to various doses (104, 106, and 108 CFU/ml, corresponding to MOIs of 0.1, 10, and 100, respectively) of the L. monocytogenes WT or lap strain or of Listeria innocua for 1 h, followed by a 3-h recovery in D10F containing gentamicin (50 μg/ml). Initial flow cytometric analyses determined that 3 h was the optimal recovery period for surface Hsp60 expression following L. monocytogenes infection (data not shown). To monitor Hsp60 expression following direct bacterial exposure, bacteria were added to the apical side of Caco-2 monolayers grown in a 12-well plate. For indirect exposure, bacteria were separated from monolayers via a Transwell filter insert (0.4-μm pore size; Corning). Hsp60 expression in Caco-2 cells was monitored by immunoblotting as described above.
Influence of initial (primary) Listeria infection on susceptibility to subsequent (secondary) LAP-mediated adhesion and transepithelial translocation.A series of experiments was conducted to determine whether Listeria-induced Hsp60 expression affects host cell susceptibility to subsequent (secondary) LAP-mediated infection.
The first experiment was conducted to determine whether initial exposure to WT L. monocytogenes or L. innocua would affect susceptibility to subsequent L. monocytogenes adhesion. L. monocytogenes and L. innocua were chosen for the initial infection because they differed in the ability to induce Caco-2 cell Hsp60 expression. Untransfected Caco-2 cells were exposed to 106 CFU/ml (MOI, ∼10) of the L. monocytogenes WT or lap strain or of L. innocua for 1 h (primary infection) or were left untreated. Noninternalized bacteria were killed by gentamicin (50 μg/ml) treatment, and after a 3-h recovery period in gentamicin-containing D10F, cells were thoroughly washed and exposed again to WT L. monocytogenes (106 CFU/ml) as a secondary infection. Adherent bacteria from the secondary infection were enumerated at 1 h postinfection as described above. To account for the number of bacteria that were internalized during the primary infection and had undergone intracellular replication, control wells were included which had been subjected only to the 1-h primary infection and then maintained in D10F with gentamicin instead of being exposed to the secondary infection. Intracellular bacteria were enumerated from these control wells, and the average value was subtracted from the number of adherent bacteria after the secondary infection.
A second set of experiments was conducted to determine the roles of Hsp60 and LAP in mediating susceptibility to secondary infection. In these experiments, control shRNA (control-sh; mock-transfected)- or shRNA-Hsp60-transfected Caco-2 cells were exposed to L. monocytogenes in a primary infection or were left uninfected. Caco-2 cells were exposed to L. monocytogenes in a primary infection or were left uninfected. Cells were recovered for 3 h as in the first experiment, as described above, and then were exposed to the WT, lap, lap+, ΔinlA, or inlA+ strain in a secondary infection. Secondary bacterial adhesion and translocation were measured in separate experiments. As in the first experiment (above), control wells were included to account for intracellular bacteria resulting from the primary L. monocytogenes infection.
Fluorescence microscopy.To evaluate surface localization of Hsp60 by immunofluorescence microscopy, Caco-2 cells were grown to confluence in D10F on LabTek chamber slides (Nunc, ThermoFisher Scientific). Cells were washed with Hanks balanced salt solution (HBSS) (Cellgro) and then incubated with anti-Hsp60 PAb (diluted 1:250 in HBSS) for 1 h at 37°C. After being washed with HBSS containing 1% bovine serum albumin (BSA), cells were incubated with FITC-conjugated monovalent secondary Fab fragment (diluted 1:250 in HBSS) for 1 h at 37°C. Propidium iodide (1 μg/ml in HBSS) was added for 10 min to stain dead cells, and then monolayers were washed with HBSS and fixed with 4% paraformaldehyde diluted 1:1 in HBSS at room temperature for 10 min. Cells were mounted with ProLong Gold antifade reagent (Molecular Probes) and examined under an epifluorescence microscope (Leica, Wetzlar, Germany) equipped with Spot software (Sterling Heights, MI).
To evaluate intracellular Hsp60 levels, confluent Caco-2 cells were fixed with paraformaldehyde as described above and then permeabilized with 0.01% Triton X in HBSS. Monolayers were immunoprobed at room temperature with anti-Hsp60 PAb (1:250) and FITC-conjugated monovalent secondary Fab fragment (1:250), as well as with anti-ZO1 PAb (1:250) (Molecular Probes) and Cy5-conjugated monovalent Fab fragment (1:250), as described above. In some cases, cells were treated with Hoechst dye (0.5 μg/ml in HBSS) for nuclear (blue) staining. Images were visualized at the Purdue Life Science Fluorescence Imaging Facility, using a Zeiss LSM 710 confocal fluorescence microscope (Carl Zeiss, Jena, Germany) with a 63×/1.4 water immersion objective. Images were acquired and processed (colocalization) using Zeiss LSM Image Browser software (Carl Zeiss, Jena, Germany).
Statistical analysis.The SAS program (Cary, NC) was employed for statistical analyses. Differences between pairs were assessed by Wilcoxon rank sum analysis, and the Proc GLM test was used for comparison of data from more than three groups (SAS, Cary, NC). P values of <0.05 were considered significant.
RESULTS
LAP-Hsp60 association promotes L. monocytogenes adhesion to Caco-2 cells.We previously demonstrated that a lap-deficient mutant exhibited reduced adhesion to Caco-2 cells (32, 58). In this study, to confirm the role of the Hsp60-LAP interaction in promoting adhesion, we evaluated LAP-mediated adhesion when levels of cellular Hsp60 were altered. Induction of Caco-2 Hsp60 expression via heat stress (41°C) or exposure to TNF-α (100 U/ml) (20) led to increased levels of Hsp60 in both intracellular (44% increase for heat shock and 50% increase for TNF-α exposure) and plasma membrane (69% increase for heat shock and 80% increase for TNF-α exposure) protein fractions (Fig. 1A). Adhesion of the WT, but not the lap strain, to heat-stressed and TNF-α-treated Caco-2 cells increased 2.2-fold (P = 0.01) and 1.9-fold (P = 0.03), respectively, compared to that with unstressed Caco-2 cells (Fig. 1B). The contribution of Hsp60 to this interaction was further confirmed by pretreating stressed cells with an Hsp60-specific antibody prior to infection, which reduced adhesion of the WT to levels similar to those with unstressed Caco-2 cells (P = 0.01) (Fig. 1B).
Influence of Caco-2 Hsp60 expression on LAP-mediated adhesion of L. monocytogenes. (A) Immunoblot showing the level of Hsp60 expression in Caco-2 plasma membrane (Mem-Hsp60) and intracellular (Int-Hsp60) fractions after heat stress (41°C, 1 h) and TNF-α exposure (100 U/ml, 1 h). β-Actin (43 kDa) was used as an internal control. Proteins were loaded at 15 μg/well. (B) Adhesion of WT, lap (mutant), and lap+ (lap-complemented) L. monocytogenes strains to Caco-2 monolayers following Caco-2 heat stress (41°C) and TNF-α (100 U/ml) exposure and after treatment with anti-Hsp60 PAb (1 μg/ml). Adhesion data are averages for at least three independent experiments performed in quadruplicate (n ≥ 12). Error bars represent standard errors of the means (SEM). Lowercase letters (a and b) represent significant differences (P < 0.05) in adhesion of bacterial strains.
To further examine the effect of Hsp60 on Listeria adhesion, we reduced the levels of endogenous Hsp60 by transfecting Hsp60-specific shRNA into Caco-2 cells, which resulted in reductions of Hsp60 protein expression of approximately 70% in intracellular fractions and 90% in plasma membrane fractions (Fig. 2 B). Hsp60 suppression in Caco-2 cells was also confirmed by RT-PCR (Fig. 2A). Partial suppression of Hsp60 by RNA interference was reported by others (9, 13); complete Hsp60 suppression may not be possible because it has important antiapoptotic functions in cultured cell lines (25). Adhesion of the WT decreased >4.5-fold in Hsp60-suppressed cells, whereas adhesion of the lap strain was unchanged (Fig. 2C). Overexpression of Hsp60 via pcDNA3.1-hsp60 resulted in a 60% Hsp60 increase in intracellular protein fractions and a 75% increase in plasma membrane protein fractions (Fig. 2B). Adhesion of the WT increased 2.8-fold (P = 0.006) for cells overexpressing Hsp60, while adhesion of the lap strain was unaffected (Fig. 2C).
Analysis of L. monocytogenes adhesion to Caco-2 cells following Hsp60 knockdown by shRNA (shRNA Hsp60−) or Hsp60 overexpression (Hsp60+) or adhesion to control-sh-transfected cells (Caco-2 cells transfected with noncoding shRNA). (A) RT-PCR analysis of hsp60 expression (23 cycles) in Caco-2 cells following shRNA-Hsp60 knockdown. 18S RNA was used as an internal control. (B) Immunoblot showing Hsp60 suppression (shRNA Hsp60−) and overexpression (Hsp60+) in Caco-2 plasma membrane (Mem-Hsp60) and intracellular (Int-Hsp60) fractions. β-Actin (43 kDa) was used as an internal control. Proteins were loaded at 15 μg/well. (C) Adhesion of L. monocytogenes WT, lap (mutant), and lap+ (lap-complemented) strains to control-sh, shRNA-Hsp60, and Hsp60+ Caco-2 monolayers. Adhesion data are averages for at least three independent experiments performed in quadruplicate (n ≥ 12) and are presented with SEM. Lowercase letters (a, b, and c) indicate significant differences (P < 0.05) in adhesion of individual bacterial strains to Caco-2 monolayers expressing varied levels of Hsp60.
LAP-Hsp60 interaction promotes Listeria monocytogenes translocation through epithelial monolayers.Although adhesion is a crucial step in L. monocytogenes pathogenesis, binding of bacteria to epithelial cells does not always directly correlate with dissemination to target organs (30). We therefore investigated whether LAP was implicated in Listeria invasion of intestinal epithelial cells, which could contribute to the differences in systemic dissemination observed between LAP-deficient and WT L. monocytogenes strains (6, 31). In gentamicin protection invasion assays, internalization of the WT was greater than that of the lap strain (P = 0.003), but both strains were significantly more invasive than the ΔinlA strain (Fig. 3). However, neither Hsp60 suppression nor overexpression (Fig. 3) influenced invasion of the WT, lap, or ΔinlA strain (Fig. 3), indicating that the LAP-Hsp60 interaction may not be directly responsible for invasion of L. monocytogenes into epithelial cells.
Analysis of L. monocytogenes WT, lap, lap+, ΔinlA, and inlA+ strain invasion of Caco-2 cells following Hsp60 knockdown by shRNA (shRNA Hsp60−) or Hsp60 overexpression (Hsp60+) or of control-sh-transfected cells (Caco-2 cells with scrambled shRNA). Invasion results are averages for at least three independent experiments performed in quadruplicate (n ≥ 12) and are presented with SEM. Lowercase letters (a to c) indicate significant differences (P < 0.05) in invasion among bacterial strains.
We next sought to determine the role of LAP in translocation through Caco-2 monolayers. Caco-2 monolayer integrity was verified by confocal microscopy, with visible tight junction borders and no alteration in cellular distribution of tight junction proteins before and after treatment with L. monocytogenes (Fig. 4). Furthermore, TEER values for intact Caco-2 cell monolayers ranged from 190 ± 2 to 209 ± 6 (Table 2) and were consistent in all our experiments throughout this study, and these values are within the range reported by others (15, 27). As a positive control, treatment of monolayers with cytochalasin D induced a very low TEER (Table 2) (60). Following 2 h of bacterial exposure (MOI, ∼10), translocation of the lap strain through Caco-2 monolayers was 60% less than that of the WT (P = 0.02) (Fig. 5 B); however, there were no apparent differences in TEER values between WT- and lap mutant-treated Caco-2 cells (Table 2). Increasing Caco-2 Hsp60 expression via endogenous overexpression enhanced WT and lap+ strain translocation 2-fold (P = 0.001) and 1.6-fold (P = 0.01), respectively, but did not influence translocation of the lap strain (Fig. 5B). shRNA suppression of Hsp60 decreased translocation of the WT >3-fold (P = 0.004), but transit of the lap strain to the basal compartment (Fig. 5B) was not affected. Similarly, addition of exogenous Hsp60 to the surfaces of Caco-2 cells resulted in greater translocation of the WT (P = 0.001) but not of the lap strain (data not shown). These data indicate a clear role for the LAP-Hsp60 interaction in mediating transepithelial translocation of L. monocytogenes.
Confocal microscopic analysis of tight junction integrity in uninfected (A) and L. monocytogenes-infected (2 h) (B) Caco-2 monolayers grown on Transwell filters. Monolayers were labeled with antibodies specific for the tight junction proteins ZO-1, occludin, and claudin (red) and for Hsp60 (green) and with a nuclear stain (blue). The images reveal visible tight junction borders in both uninfected and infected Caco-2 monolayers, with no alteration in cellular distribution of tight junction proteins or in cellular damage.
Analysis of transepithelial translocation of L. monocytogenes through polarized Caco-2 cell monolayers grown on a Transwell filter insert (see the text for further details). (A) Immunoblot showing LAP and InlA expression in L. monocytogenes WT and ΔinlA strains. Each lane was loaded with 15 μg of total cell protein. (B) Transepithelial translocation of L. monocytogenes WT, lap, lap+, ΔinlA, and inlA+ cells through Caco-2 monolayers following Hsp60 knockdown by shRNA [shRNA Hsp60(−)] or Hsp60 overexpression (Hsp60+) or through control-sh-transfected cells (Caco-2 cells transfected with noncoding shRNA). (C) Translocation of WT, lap, lap+, and ΔinlA cells and of ΔinlA cells pretreated with anti-LAP MAb (1 μg/ml) or an IgG control (MAb C11E9) (1 μg/ml) through control-sh-transfected Caco-2 monolayers. Data are averages for at least three independent translocation experiments performed in quadruplicate (n ≥ 12) and are presented with SEM. Lowercase letters (a to c) indicate significant differences (P < 0.05) in translocation of individual bacterial strains through Caco-2 monolayers expressing various levels of Hsp60.
TEER values for Caco-2 cells before and after treatments
Surprisingly, translocation of the ΔinlA strain through Caco-2 monolayers was nearly 2-fold greater than that of the WT (P = 0.025) and was reversed to WT levels when the strain was complemented with inlA (Fig. 5B and C). Furthermore, ΔinlA strain translocation was influenced by Hsp60 expression, similar to that of the WT, as translocation decreased in shRNA-Hsp60-transfected Caco-2 monolayers (Fig. 5B). To test for LAP-mediated translocation in the ΔinlA strain, which expresses normal levels of LAP (Fig. 5A), the ΔinlA strain was coated with a LAP-specific antibody prior to Caco-2 cell infection. Antibody treatment resulted in a 60% reduction in translocation (P = 0.02) (Fig. 5C), indicating that LAP-mediated translocation occurs in the ΔinlA strain. The observance of increased translocation in the absence of epithelial cell invasion suggests that LAP-mediated translocation of the ΔinlA strain may occur via a noninvasive, or paracellular, mechanism. Such paracellular translocation might necessitate localization of Hsp60 near Caco-2 paracellular borders. Confocal microscopic analysis revealed low levels of Hsp60 colocalization with Caco-2 cell tight junctions (Fig. 6 C).
Influence of L. monocytogenes infection on Hsp60 expression in Caco-2 cells. (A) Immunoblot analysis of the level of Hsp60 expression in Caco-2 plasma membrane (Mem-Hsp60) and intracellular (Int-Hsp60) fractions after exposure to L. monocytogenes (Lm) at 1 × 104, 1 × 106, or 1 × 108 CFU/ml. β-Actin was used as an internal control. Proteins were loaded at 15 μg/well. Blots are representative of at least three individual experiments. (B) Microscopic analysis of Hsp60 (green) on the surfaces of uninfected or L. monocytogenes-infected (106 CFU/ml [MOI of 10:1] and 108 CFU/ml [MOI of 1,000:1]) Caco-2 cells. Caco-2 cells were labeled with anti-Hsp60 antibody and an FITC-conjugated secondary antibody. Propidium iodide was used to stain dead cells (red). Samples were viewed on a Leica fluorescence microscope with a 40× objective. Bars, 10 μm. (C) Confocal microscopic analysis of intracellular Hsp60 (FITC; green) expression in Caco-2 cells left uninfected (top panels) or infected with 106 CFU/ml L. monocytogenes (bottom panels). The tight junction protein ZO1 was labeled (Cy5; red) for the purpose of visualizing cell borders. Yellow highlighting indicates areas where Hsp60 and ZO-1 are colocalized (right panels). Hsp60 expression was more abundant in L. monocytogenes-infected cells than in uninfected cells.
L. monocytogenes infection at a low dosage influences Hsp60 expression.Recent reports indicate that bacterial and viral infections can induce a host heat shock response (4, 39, 62). We sought to determine whether L. monocytogenes infection might influence Hsp60 expression in intestinal epithelial cells by infecting Caco-2 cells with various doses (104, 106, and 108 cells) of L. monocytogenes and monitoring protein expression. Infection with each dose of L. monocytogenes increased hsp60 transcript levels, as analyzed by RT-PCR (data not shown), as well as levels of Hsp60 protein in plasma membrane and intracellular protein fractions (Fig. 6A). However, the greatest increase in Hsp60 was in the plasma membrane following infection with 104 or 106 cells of L. monocytogenes (Fig. 6A). Fluorescence microscopy confirmed increased levels of surface (Fig. 6B) and intracellular (Fig. 6C) Hsp60 and demonstrated that colocalization of Hsp60 with Caco-2 cell tight junctions increased following L. monocytogenes infection (Fig. 6C).
To determine whether Hsp60 expression occurred specifically in response to L. monocytogenes or in response to general bacterial factors, we exposed Caco-2 cells to 106 CFU/ml of the L. monocytogenes WT or lap strain or nonpathogenic L. innocua and monitored Hsp60 expression by Western blotting (Fig. 7A) and flow cytometry (Fig. 7B). The Hsp60 level increased in both intracellular (62% increase) and membrane (80% increase) fractions following infection with L. monocytogenes but was not dependent upon LAP expression, since the lap mutant had similar levels to those of the WT (Fig. 7A), and did not increase after L. innocua exposure. These data indicate that the Hsp60 response may be specific to L. monocytogenes. Furthermore, no change in Hsp60 expression was observed when bacterial cells were separated from Caco-2 cells by use of a 0.4-μm filter (indirect exposure) (Fig. 7), suggesting that direct bacterial contact with epithelial cells, irrespective of LAP expression, is required and that diffusible microbial factors may not induce Hsp60 expression.
Hsp60 expression in Caco-2 cells following direct versus indirect exposure to Listeria. (A) Immunoblot analysis of Hsp60 levels in Caco-2 plasma membrane (Mem-Hsp60) and intracellular (Int-Hsp60) fractions following direct or indirect (via separation by a 0.4-μm-pore-size filter) exposure to the L. monocytogenes (Lm) WT, the L. monocytogenes lap strain, or L. innocua at 106 CFU/ml (MOI, 10:1). β-Actin was used as an internal control. Blots are representative of three individual experiments. Proteins were loaded at 15 μg/well. Lanes marked with an open box indicate increased Hsp60 expression compared to that of controls. (B) Flow cytometric analysis of Hsp60 expression on the surfaces of Caco-2 cells following direct or indirect exposure to L. monocytogenes WT or lap strain. Data are averages for 3 experiments, with treatments run in quadruplicate (n = 12), and are presented as fold changes in the number of Caco-2 cells expressing surface Hsp60 compared to uninfected cells. Data are presented with SEM. Lowercase letters (a and b) represent significant differences (P < 0.05) in surface Hsp60 expression in Caco-2 cells due to direct versus indirect bacterial exposure.
Caco-2 cells expressing high levels of Hsp60 are more susceptible to subsequent L. monocytogenes infection.Pathogen-induced Hsp60 expression is not unprecedented (4, 39); however, it is intriguing in the case of L. monocytogenes infection of intestinal epithelial cells, since Hsp60 is the receptor for LAP-mediated infection. We therefore designed a series of cell-based studies to test whether L. monocytogenes-induced Hsp60 expression would render host cells more susceptible to further LAP-mediated (secondary) infection. First, control (untransfected) Caco-2 monolayers were exposed to WT L. monocytogenes or to L. innocua in a primary infection; after a 3-h recovery period with gentamicin, cells were then subjected to secondary infection by L. monocytogenes. The number of L. monocytogenes organisms bound to Caco-2 cells resulting from the secondary infection was significantly greater (P = 0.032) following a primary L. monocytogenes exposure than in previously uninfected cells or in monolayers which were first exposed to L. innocua (Fig. 8A). These data suggest that initial infection with L. monocytogenes may render host cells more susceptible to further (secondary) infection by this pathogen, but this alone does not imply involvement of Hsp60 or LAP in susceptibility to secondary infection. Therefore, a second experiment was conducted with control-sh (transfected with shRNA noncoding vector)- and shRNA-Hsp60-transfected Caco-2 cells, where the primary infection microorganism was the WT and the secondary microorganism was the WT, lap, or ΔinlA strain (Fig. 8B). In control-sh-transfected Caco-2 cells, we observed significantly greater adhesion of the WT (3.3-fold increase) (P = 0.01) and the ΔinlA strain (4.4-fold increase) (P = 0.025), but not the lap mutant, following primary L. monocytogenes WT infection than that to cells which were previously uninfected (Fig. 8B). This indicates that primary infection rendered the Caco-2 cells more susceptible to LAP-mediated infection, in a manner which was independent of InlA. There was no apparent effect of primary infection on adhesion of any microorganism during secondary infection in shRNA-Hsp60-transfected Caco-2 cells (Fig. 8C).
Influence of primary Listeria infection on Caco-2 cell susceptibility to subsequent (secondary) L. monocytogenes adhesion. (A) Secondary adhesion of L. monocytogenes WT to normal Caco-2 cells after primary infection with L. monocytogenes WT or L. innocua compared to that with previously uninfected cells. Data are averages for at least three independent experiments performed in quadruplicate (n ≥ 12) and are presented with SEM. Lowercase letters (a and b) represent significant differences (P < 0.05) in adhesion of bacterial strains. (B and C) Adhesion of L. monocytogenes WT, lap, lap+, ΔinlA, and inlA+ strains to control-sh-transfected Caco-2 cells (Caco-2 cells transfected with noncoding shRNA) (B) and to Hsp60 knockdown Caco-2 cells (shRNA Hsp60−) (C) following primary infection with L. monocytogenes WT for 1 h. After primary infection, cells were incubated for 3 h in gentamicin (50 μg/ml)-containing cell culture medium (recovery period) prior to secondary infection for 1 h with L. monocytogenes. Adhesion data are averages for at least three independent experiments performed in quadruplicate (n ≥ 12) and are presented with SEM. Bars marked with asterisks indicate significant differences in secondary bacterial adhesion between previously uninfected and infected Caco-2 monolayers (P < 0.05). Bars marked with lowercase letters (a to e) represent significant (P < 0.05) differences in LAP-mediated adhesion between bacterial strains.
Similarly, in transepithelial translocation studies, we observed an increase in LAP-mediated translocation in control-sh-transfected (Fig. 9A) but not shRNA-Hsp60-transfected (Fig. 9B) Caco-2 cell monolayers following primary L. monocytogenes WT infection. Despite differences observed in LAP-mediated translocation in control-sh-transfected Caco-2 cells, the level of translocation during secondary infection in both Caco-2 cell lines was higher for all microorganisms than that observed in earlier primary infection studies. This may be due to reductions in TEER which resulted from primary WT infection (data not shown). The secondary adhesion and transepithelial translocation findings emphasize the contribution of L. monocytogenes-induced host Hsp60 expression to subsequent LAP-mediated infectivity of L. monocytogenes.
Influence of Listeria-induced Hsp60 expression on subsequent LAP-mediated translocation through Caco-2 monolayers. Translocation of L. monocytogenes WT, lap, lap+, ΔinlA, and inlA+ strains is shown for control-sh-transfected cells (Caco-2 cells transfected with noncoding shRNA) (A) and shRNA-Hsp60-transfected Caco-2 cells (B) following primary infection with L. monocytogenes WT. Caco-2 cell preparation and treatment were the same as those described in the legend to Fig. 7, except that the translocation assay during secondary infection was performed for 2 h. Translocation assays were repeated at least three times in quadruplicate wells (n ≥ 12), and data are presented with SEM. Bars marked with asterisks indicate significant differences in secondary bacterial adhesion between previously uninfected and infected Caco-2 monolayers (P < 0.05), while bars marked with lowercase letters (a to c) represent significant (P < 0.05) differences in LAP-mediated adhesion between bacterial strains.
DISCUSSION
Mechanisms mediating interaction of Listeria with the host intestinal epithelium are critical for establishing a successful infection and involve the concerted action of multiple virulence factors (23). Our lab previously identified LAP as a virulence factor which promotes adhesion to intestinal epithelial cell lines (31) and whose expression is required for full virulence in orally infected mice (6, 31). We identified human Hsp60 as the epithelial receptor for LAP (58), but the precise role of the LAP-Hsp60 interaction in mediating bacterium-epithelial cell interaction was unknown, as was its potential for promoting extraintestinal translocation of bacteria. Others have shown that pathogens can target host heat shock proteins to promote adhesion and invasion (19, 59, 63), and it is also known that stressors, including infection, can induce synthesis of host heat shock proteins (4, 39, 45). However, little information exists about the influence of the host stress response on infection by pathogens which recognize heat shock proteins as receptors. Our objectives were to examine how the LAP-Hsp60 interaction influences bacterial adhesion, invasion, and transepithelial translocation and to determine whether Listeria infection influences Hsp60 expression or LAP-mediated infection of cultured intestinal epithelial cells.
We confirmed that the interaction of LAP with Hsp60 is critical for establishing full adhesion, as Caco-2 cells were significantly less susceptible to LAP-mediated adhesion following shRNA-Hsp60 knockdown than parental Caco-2 cell lines. Similarly, adhesion of WT but not lap bacteria was greater when the cellular level of Hsp60 was increased via constitutive overexpression, heat stress, or TNF-α exposure. Invasion of the WT into Caco-2 cells was inherently greater than that of the lap-deficient mutant, which initially suggested that LAP might also serve as an epithelial invasion factor. However, manipulation of Hsp60 expression had no influence on the invasive capacity of L. monocytogenes, which indicates that the sole interaction of LAP with Hsp60 is not likely to drive invasion of L. monocytogenes. Instead, LAP may promote invasion by interacting with an unknown host receptor, or LAP-mediated adhesion may indirectly facilitate invasion by increasing bacterial association with epithelial cells and promoting interaction of invasion factors such as internalins and Vip with their receptors (8, 38, 40, 51).
Using a Transwell cell culture device that enabled measurement of bacterial translocation across polarized cell monolayers (27), we demonstrated that LAP is involved in promoting transepithelial translocation through Caco-2 cells, as translocation of the WT was greater than that of the lap mutant. This was further confirmed in an Hsp60-suppressed Caco-2 cell line in which WT translocation was significantly impaired but the lap strain showed no effect. Similarly, overexpression of Hsp60 led to greater WT but not lap strain translocation.
Our adhesion and translocation findings support our earlier studies, in which we demonstrated LAP-mediated extraintestinal dissemination of Listeria to the liver and spleen following oral infection of mice (6, 31). However, the question remains as to how LAP-Hsp60-mediated transepithelial translocation occurs, since invasion studies provided no clear role for the LAP-Hsp60 interaction in mediating bacterial entry into intestinal epithelial cells. Interestingly, the L. monocytogenes ΔinlA strain exhibited greater translocation through Caco-2 monolayers than the WT. In the absence of inlA, invasion of L. monocytogenes into intestinal epithelial cells is severely restricted (40). If the LAP-Hsp60 interaction mediates bacterial translocation via a noninvasive route, such as through a paracellular rather than a transcellular pathway (15, 27), then in comparison to fully invasive WT bacteria, more ΔinlA bacteria may be available for LAP-mediated paracellular translocation. This proposed mechanism of a noninvasive route of translocation (Fig. 10) could be tested through creation of a double inlA lap mutation in L. monocytogenes and by comparing translocation of the double mutant to that of the WT, ΔinlA, and lap strains; however, several attempts to generate this double mutant were unsuccessful in our laboratory. Therefore, in lieu of having a lap inlA mutant, we treated the ΔinlA strain with anti-LAP antibody prior to infection of Caco-2 cells to block LAP associated with the bacterial surface. Treatment of the ΔinlA strain with anti-LAP, but not with an IgG control antibody, significantly reduced its translocation in comparison to that of the ΔinlA strain alone. While antibody treatment reduced ΔinlA strain translocation, the levels were not as low as those for the L. monocytogenes lap strain, most likely due to LAP secretion by the ΔinlA strain after antibody treatment. Our data provide strong evidence that LAP may mediate translocation via a noninvasive mechanism, such as through a paracellular pathway, whose molecular mechanism is currently under investigation in our lab.
Proposed model of LAP-mediated paracellular translocation in Caco-2 intestinal epithelial monolayers, which occurs independently of InlA-mediated invasion of L. monocytogenes. (A) In WT L. monocytogenes, the interaction of InlA with the epithelial receptor E-cadherin promotes invasion of Caco-2 cells, while interaction of LAP with the epithelial receptor Hsp60 mediates paracellular transepithelial translocation. (B) In an L. monocytogenes ΔinlA strain, the absence of InlA-specific E-cadherin interaction facilitates greater interaction of LAP-Hsp60 and promotes increased paracellular bacterial translocation through Caco-2 monolayers.
L. monocytogenes infection is known to reduce the TEER of epithelial monolayers (33), as also observed in this study (Table 2), which can contribute to paracellular bacterial translocation (15, 27). Although the exact mechanism for a Listeria-induced reduction in TEER is unknown, it has been linked to virulence traits such as actin cytoskeleton disruption following invasion (26, 52, 60), as well as extracellular secretion of LLO (50). Although the presence or absence of LAP expression did not induce changes in TEER (Table 2), it is possible that a weakened epithelial barrier facilitates LAP-Hsp60-mediated bacterial translocation.
The finding that LAP-mediated translocation was greater in the ΔinlA strain was unexpected, given previous findings that L. monocytogenes ΔinlA strains are attenuated for systemic infection following oral administration in animals expressing an InlA-specific E-cadherin (35, 37). It is possible that LAP-Hsp60-mediated translocation contributes to differences in systemic infection observed in vivo between WT and lap strains (6, 31). However, the fact that ΔinlA bacteria translocate more but invade less in vitro yet cause less systemic infection following oral administration in vivo (37) draws into question the relative importance of epithelial cell invasion versus transepithelial translocation during in vivo infection by L. monocytogenes. Certainly, the ability of L. monocytogenes to cross the intestinal epithelial barrier is a complex process involving multiple virulence factors. The importance of InlA-mediated intestinal epithelial invasion during in vivo Listeria infection has been demonstrated clearly (35, 37). Although L. monocytogenes has previously been shown to translocate across epithelial monolayers in vitro (12), our findings of LAP-Hsp60-mediated translocation are the first evidence that listerial translocation is driven by a specific bacterium-host interaction. If transepithelial translocation of L. monocytogenes leads to submucosal phagocytosis, as it does for pathogens such as Campylobacter and Shigella (34, 53), then it could potentiate systemic spread of L. monocytogenes due to the pathogen's ability to survive complete destruction within phagocytes (18). However, in light of our findings that the ΔinlA strain translocates more in vitro, although it is deficient for systemic infection in vivo (37), more studies are needed to determine the specific role of LAP-mediated transepithelial translocation during systemic infection by L. monocytogenes.
The use of human Hsp60 as a receptor for LAP is intriguing given recent findings that infection by bacterial and viral pathogens can induce Hsp expression in host cells. Belles et al. (4) demonstrated that L. monocytogenes intravenous infection of mice increased plasma membrane expression of Hsp60 in spleen and liver lymphocytes. Infections by Salmonella enterica serovar Enteritidis (39) and dengue virus (45) also increased expression of host Hsps in cultured cells. Here we showed that infection with moderate doses (104 to 106 CFU/ml) of L. monocytogenes increased intracellular and surface expression of Hsp60 in Caco-2 monolayers. The Hsp60 response required direct exposure to L. monocytogenes and was not induced by nonpathogenic L. innocua (Fig. 8A). The infection-induced Hsp60 response may be dependent upon pathogen contact or invasion, although it is independent of LAP expression. Since Hsp60 serves as a LAP receptor, we conducted studies to test whether infection-induced Hsp60 expression would affect cellular susceptibility to subsequent LAP-mediated infection. We observed an important role for infection-induced Hsp60 expression in promoting Listeria infection, as both LAP-mediated adhesion and translocation were greater in Caco-2 cells already subjected to primary infection than in previously uninfected cells. This phenomenon was observed only in parental (control-sh) Caco-2 cells, not in Hsp60-suppressed Caco-2 cells, which confirmed the role of Listeria-induced Hsp60 expression in mediating greater susceptibility to LAP-mediated infection.
The finding that Listeria-induced Hsp60 expression leads to greater LAP-mediated infectivity provides evidence of a host-pathogen interplay during infection. Expression of heat shock proteins during infection may protect cellular constituents against damage resulting from pathogen virulence mechanisms and host defenses (42, 43). Surface-expressed Hsp60 may also promote the host immune response by serving as a warning signal (48, 57) and by binding pathogen-associated molecular patterns (PAMPs) to modulate PAMP-induced Toll-like receptor (TLR) signaling (44). However, certain pathogens can use host chaperones to promote infection: S. aureus binds to Hsp60 to promote invasion of epithelial cells (19), Brucella abortus uses Hsp70 to mediate invasion of trophoblasts (59), and expression of Hsp60 by macrophages is critical for dengue virus infection and intracellular replication (45). We demonstrated that Listeria infection induces Hsp60 expression in host cells, which facilitates greater LAP-mediated adhesion and translocation. The role of Hsp60 expression in the broader context of in vivo L. monocytogenes infection is still unknown. However, these studies provide evidence that initial infection of intestinal epithelial cells initiates a host stress response, which promotes LAP-mediated L. monocytogenes infection. These findings pose relevant questions as to how the host heat shock response may affect the outcome of infection by other pathogens which target chaperones as receptors.
ACKNOWLEDGMENTS
We sincerely thank Krishna Mishra for assistance with molecular cloning. We are also grateful to Jennie Sturgis and Gregory Richter for aid with confocal microscopy, to Cathy Ragheb and Cheryl Holdman for assistance with flow cytometry, and to R. Vemulapalli, D. Zhou, B. Applegate, and J. P. Robinson for critical evaluations of the study and for helpful discussions.
Part of this research was supported by Purdue Faculty Scholar Funding, a Bilsland Fellowship, and the U.S. Department of Agriculture (project number 1935-42000-035).
FOOTNOTES
- Received 17 May 2010.
- Returned for modification 22 June 2010.
- Accepted 14 September 2010.
- Copyright © 2010 American Society for Microbiology