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Infection and Immunity, June 2007, p. 2996-3005, Vol. 75, No. 6
0019-9567/07/$08.00+0     doi:10.1128/IAI.01716-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Modulation of Adherence, Invasion, and Tumor Necrosis Factor Alpha Secretion during the Early Stages of Infection by Streptococcus pneumoniae ClpL

Le Nhat Tu,^1, Hye-Yoon Jeong,^1, Hyog-Young Kwon,¹ Abiodun D. Ogunniyi,² James C. Paton,² Suhk-Neung Pyo,¹ and Dong-Kwon Rhee^1*

College of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea,¹ School of Molecular and Biomedical Science, The University of Adelaide, Adelaide 5005, Australia²

Received 26 October 2006/ Returned for modification 19 December 2006/ Accepted 21 March 2007

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Heat shock proteins (HSPs) play a pivotal role as chaperones in the folding of native and denatured proteins and can help pathogens penetrate host defenses. However, the underlying mechanism(s) of modulation of virulence by HSPs has not been fully determined. In this study, the role of the chaperone ClpL in the pathogenicity of Streptococcus pneumoniae was assessed. A clpL mutant adhered to and invaded nasopharyngeal or lung cells much more efficiently than the wild type adhered to and invaded these cells in vitro, as well as in vivo, although it produced the same amount of capsular polysaccharide. However, the level of secretion of tumor necrosis factor alpha (TNF-) from macrophages infected with the clpL mutant was significantly lower than the level of secretion elicited by the wild type during the early stages of infection. Interestingly, treatment of the human lung epithelial carcinoma A549 and murine macrophage RAW 264.7 cell lines with cytochalasin D, an inhibitor of actin polymerization, increased adherence of the mutant to the host cells. In contrast, cytochalasin D treatment of RAW 264.7 cells decreased TNF- secretion after infection with either the wild type or the mutant. However, pretreatment of cell lines with the actin polymerization activator jasplakinolide reversed these phenotypes. These findings indicate, for the first time, that the ClpL chaperone represses adherence of S. pneumoniae to host cells and induces secretion of TNF- via a mechanism dependent upon actin polymerization during the initial infection stage.

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Heat shock proteins (HSPs) are extremely well conserved in prokaryotes, as well as in eukaryotes, and are induced by various stress factors, such as starvation, exposure to free radicals, and heat shock (19, 35). HSPs can be classified into the HSP100, HSP70, HSP60, and small HSP families depending on their molecular weights and are ubiquitous in prokaryotes and eukaryotes (19). HSPs play a pivotal role in the folding of native and denatured proteins and thus promote cell protection and survival (19, 35). Bacterial HSPs are induced during infection (29, 51), are required for adherence and invasion (7, 33, 37), and can help pathogens struggling to penetrate host defenses. Although attenuation of virulence by mutation of HSP genes has been documented, the underlying mechanism(s) by which HSPs modulate virulence has not been fully elucidated.

Streptococcus pneumoniae (pneumococcus), a gram-positive and naturally transformable organism, causes a variety of potentially life-threatening infections, such as pneumonia, bacteremia, and meningitis (52). It is carried asymptomatically in the nasopharynx of healthy individuals, and this serves as a major reservoir for pneumococcal infections. Pneumococci experience heat shock in the host during penetration from the nasopharyngeal niche (30 to 34°C) into the bloodstream (37°C), and this change can trigger a rapid and transient increase in the levels of HSPs (27). This also provokes dramatic morphological changes, as well as changes in gene expression; pneumococci in the nasopharynx have predominantly the transparent colony phenotype and have been reported to express less capsule and more choline binding protein A (CbpA), whereas pneumococci in the bloodstream have predominantly the opaque colony morphology and tend to produce more capsule and less CbpA (22, 48). These physiological changes and environmental stresses could provoke induction of HSP genes and may lead to modulation of expression of virulence genes controlled by HSPs. Heat shock may also assist in the survival of fevers that develop as a result of the infection.

Recently, we demonstrated that ClpL, a member of the HSP100/Clp (caseinolytic protease) family found mainly in gram-positive organisms, has a chaperone function and also modulates virulence gene expression (27). However, the specific mechanism(s) by which this occurs remains largely unknown. In this study, we investigated the effect of clpL mutation on pneumococcal pathogenesis. Interestingly, a clpL mutant showed massively greater adherence to and invasion of epithelial cells, but it elicited lower tumor necrosis factor alpha (TNF-) secretion by macrophages during the early stages of infection. These effects were dependent on actin polymerization. However, the clpL mutant was more susceptible to macrophage killing and exhibited a level of virulence similar to that of the wild type in both systemic and intranasal mouse challenge studies. Here we show, for the first time, that the ClpL chaperone represses adherence of S. pneumoniae to host cells and induces secretion of TNF- during the initial stages of infection, possibly via a mechanism dependent upon actin polymerization.

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Bacterial strains and cell culture conditions. Nonencapsulated S. pneumoniae strain CP1200 (10) and its isogenic clpL derivative HYK104 (27) and encapsulated type 2 strain D39 (= NCTC7466) (1) and its isogenic clpL derivative HYK304 (27) were cultured in Casitone-tryptone-based medium, brain heart infusion broth, or Todd-Hewitt broth as described previously (10, 27). Human nasopharyngeal carcinoma epithelial cell line Detroit 562, the human lung epithelial carcinoma A549 cell line, and the murine macrophage RAW 264.7 cell line were obtained from the American Type Culture Collection. Detroit 562 cells were cultured at 37°C in the presence of 95% air-5% CO₂ in minimal essential medium (Gibco-BRL) supplemented with 10% (vol/vol) fetal bovine serum and 1 mM sodium pyruvate. A549 and RAW 264.7 cells were cultured at 37°C in the presence of 95% air-5% CO₂ in Dulbecco modified Eagle medium (DMEM) with 4.5 g/liter glucose, 10% fetal bovine serum (Gibco BRL), 100 U/ml penicillin G, and 100 µg/ml streptomycin as the basic medium, and fetal bovine serum was added at a concentration of 10%. In DMEM, no difference between growth of the wild type and growth of the clpL mutant was observed over a 5-h incubation period.

Complementation test. For the complementation test, clpL was cloned into the S. pneumoniae shuttle plasmid pMV158 as follows. The clpL gene was amplified with primers 5'-C CTG CAG ATG AAC AAC AAT TTT AAT AA-3' and 5'-C AAG CTT TTA GAC TTT CTC ACG AAT AAC CAA-3', which incorporate PstI and HindIII restriction enzyme sites, respectively, from D39 genomic DNA as described previously (41). The forward and reverse primers bind 320 bp upstream and 486 bp downstream, respectively, of clpL. The resultant 2.1-kb fragment was digested with PstI and HindIII and cloned into the corresponding restriction sites of pMV158 to generate pLNT001. For transformation of S. pneumoniae clpL mutant HYK304 with pLNT001, the method of Blue and Mitchell (5) was used. Briefly, precompetent pneumococci were grown to an A₅₅₀ of 0.1 in brain heart infusion broth supplemented with 1 mM CaCl₂, after which 100 ng/ml of competence-stimulating peptide 1 was added and the culture was incubated for a further 15 min at 37°C. Plasmid pLNT001 was then added to the competent cells and incubated at 37°C for another 3 h. Subsequently, tetracycline-resistant (1 µg/ml) transformants were screened by colony PCR using the primers indicated above to select recombinant clones harboring pLNT001. The presence of the clpL gene was further confirmed by Western immunoblot analysis (data not shown).

Tissue culture assays. Adherence and invasion assays were performed as described previously (28). Briefly, A549, RAW 264.7, or Detroit 562 cells were grown to confluence in 12-well tissue culture plates and washed three times with phosphate-buffered saline (PBS) (pH 7.2), after which 1 ml of culture medium (without antibiotics) was added per well. Exponential-phase cultures of D39 and isogenic clpL mutant derivatives of this strain were harvested by centrifugation, washed with PBS, and resuspended in DMEM or minimal essential medium. Monolayers were infected with 2 x 10⁷ bacteria (bacterium/cell ratio or multiplicity of infection [MOI], 100:1), followed by 1, 2, and 3 h of incubation at 37°C. Fresh medium containing 10 µg/ml penicillin and 200 µg/ml gentamicin was added to each well to kill extracellular bacteria. After an additional 1 h of incubation, the monolayers were washed with PBS, and the cells were detached from the plates by treatment with 0.25% trypsin-0.02% EDTA and then lysed by addition of Triton X-100 (0.025% in PBS). Appropriate dilutions were plated on Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) agar to determine the numbers of viable bacteria.

To determine the total numbers of adherent and intracellular bacteria, infected monolayers were washed as described above and then trypsinized, lysed, and plated quantitatively without antibiotic treatment. All samples were assayed in triplicate, and each assay was repeated at least three times.

Survival of pneumococci in RAW 264.7 cells was examined as described previously (28). Cell monolayers were infected with 2 x 10⁷ CFU of pneumococci (bacterium/cell ratio, 100:1) in DMEM without antibiotics and then incubated for 2 h at 37°C. The cells were washed three times with PBS, and fresh medium containing 10 µg/ml of penicillin and 200 µg/ml of gentamicin was added to kill extracellular bacteria (time zero of the assay). To enumerate intracellular pneumococci at different times after infection, supernatants were removed, and the cells were washed with PBS and then lysed with Triton X-100 as described above. Serial dilutions of the lysate from each well were plated on THY agar. The number of CFU was determined after 24 h of incubation at 37°C. Three independent assays were carried out (in triplicate) for each bacterial strain.

Labeling of the pneumococci with FITC. Pneumococci were labeled with fluorescein isothiocyanate (FITC) as described previously (3). Briefly, bacteria (10⁸ CFU/ml) were mixed with FITC (1 mg/ml; Sigma) dissolved in a buffer containing 0.05 M Na₂CO₃ and 0.1 M NaCl at 4°C for 1 h, washed five times with PBS, and resuspended in DMEM to a final concentration of 10⁸ CFU/ml. For the adherence assay, FITC-labeled pneumococci (10⁸ CFU/ml) were added to the A549 cell monolayer (MOI, 100), incubated at 37°C for 2 h, and subsequently washed five times to remove nonadherent bacteria. Adherent bacteria were directly counted using a fluorescent microscope (Axiovert 200 M time-lapse microscope; Carl Zeiss). Adherence was expressed as the number of attached bacteria per field of view.

Intranasal challenge. Intranasal challenge was carried out essentially as described previously (28). Before challenge, bacteria were cultured at 37°C overnight on blood agar (supplemented with erythromycin when appropriate) and then grown in THY medium for approximately 4 h at 37°C to obtain ca. 2 x 10⁸ CFU/ml (A₅₅₀, 0.3). The concentration of each bacterial culture in THY medium was then adjusted to ca. 10⁹ CFU/ml. Groups of five CD1 mice (5 weeks old) were infected intranasally with 10 µl of either D39 or HYK304 (ca. 2 x 10⁷ CFU/mouse). The survival of mice was monitored four times daily for the first 5 days, twice daily for the next 5 days, and then daily until 21 days after challenge.

To enumerate bacteria in different organs after intranasal challenge, mice were sacrificed at 6, 12, 24, and 36 h postinfection, and the blood, lungs, spleen, brain, and nasopharynx were removed aseptically and then washed three times with PBS (140 mM NaCl, 3 mM KCl, 10 mM NaH₂PO₄, 1.5 mM KH₂PO₄; pH 7.3). Samples were then homogenized in PBS with a tissue homogenizer (model 200, double insulated; PRO Scientific Inc., Oxford, CT) on ice, serially diluted as appropriate in sterile PBS, and plated in duplicate on blood agar containing the appropriate antibiotic(s). Subsequently, the plates were incubated for approximately 16 h at 37°C in an atmosphere containing 95% air and 5% CO₂, after which colonies were counted and averages for replicates were determined.

Measurement of CPS. Capsular polysaccharide (CPS) was prepared as previously described (31). CPS preparations were made by resuspending pneumococci grown on blood agar plates in 150 mM Tris-HCl (pH 7.0)-1 mM MgSO₄ so that the A₅₅₀ was 5. A 1-ml aliquot was pelleted by centrifugation in a microcentrifuge at 15,500 x g. The pellet was resuspended in 0.5 ml of 150 mM Tris-HCl (pH 7.0)-1 mM MgSO₄. Autolysis of the bacteria was induced by addition of 0.1% (wt/vol) deoxycholate (Sigma) and incubation at 37°C for 15 min. The samples were then incubated with 100 U of mutanolysin, 50 µg of DNase I (Roche Applied Science), and 50 µg of RNase A (Roche Applied Science) at 37°C for 18 h. After this, the samples were incubated with 50 µg of proteinase K at 56°C for 4 h prior to storage at –20°C. The total amounts of CPS produced by encapsulated strain D39 and its isogenic clpL mutant (HYK304) were determined by Western blotting using type 2 antiserum.

Cell fractionation studies. Exponentially growing cells were collected by centrifugation, and sucrose-induced protoplast formation was performed as described previously (28). Briefly, cells were converted to protoplasts by incubation at 30°C for 1 h with 1 M sucrose buffer (1 M sucrose, 100 mM Tris-HCl [pH 7.6], 2 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride). Centrifugation at 13,000 x g for 20 min was used to separate the cell wall fraction (supernatant) from the protoplasts (pellet). The protoplasts were subjected to osmotic lysis by dilution with 19 volumes of hypotonic buffer (100 mM Tris-HCl [pH 7.6], 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA). Lysates were centrifuged initially at 5,000 x g for 5 min to remove cell debris and then at 50,000 x g for 30 min to obtain the cytoplasmic fraction (supernatant) and the membrane fraction (pellet).

Determination of cytokine secretion after infection. RAW 264.7 or A549 cells (2 x 10⁵ cells/well) were infected with 2 x 10⁷ CFU of pneumococci (MOI, 100). After infection, supernatant was collected and centrifuged at 5,500 x g for 20 min to remove the bacterial pellet, and the level of secreted TNF- or interleukin-8 (IL-8) was measured using an enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences) as recommended by the supplier. Curves were fitted to sigmoidal dose-response curves and compared using an F test (GraphPad Prism, version 4.0; GraphPad Software).

RNA techniques. RAW 264.7 cells were preincubated with 0.5 µg/ml cytochalasin D, 100 µM cycloheximide, or 10^–12 M jasplakinolide for 2 h prior to infection with either D39 or the clpL mutant. After infection, total RNA was extracted using the TRIZOL reagent (Invitrogen) and further digested with DNase I (Sigma). cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (SuperBio) as instructed by the supplier. The levels of mRNAs for TNF- and β-actin (internal control) were determined by PCR using PCR PreMix (Maxime PCR PreMix kit i-Taq; iNtRON Biotech) according to the instructions provided. The primers used for TNF- and β-actin were primers 5'-AGT GAC AAG CCT GTA GC-3' and 5'-CTC CTG GTA TGA GAT AGC-3' and primers 5'-TGA CAG GAT GCA GAA GGA GA-3' and 5'-GCT GGA AGG TGG ACA GTG AG-3', which generated 239- and 133-bp products, respectively. The primers used for IL-8 were primers 5'-ATG ACT TCC AAG CTG GCC GTG-3' and 5'-TTA TGA ATT CTC AGC CCT CTT CAA AAA CTT CTC-3', which generated a 379-bp product.

Statistical analysis. Statistical differences between the medians of groups were analyzed by the Mann-Whitney U test (two-tailed, unpaired). ELISA data were expressed as averages ± standard errors of the means for triplicate wells. A difference was considered statistically significant if the P value was <0.05.

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Adherence of and invasion by the clpL mutant in vitro and in vivo. Previously, we demonstrated that a clpL mutant had almost the same level of virulence as the wild type in a mouse intraperitoneal challenge study (27). However, when the pneumococcus is injected intraperitoneally, it may experience less environmental change than it would experience if it were administered via the intranasal route. Since expression of ClpL could be induced by environmental stresses, such as a shift from the nasopharynx temperature to the blood temperature, it is conceivable that the clpL mutant would be less able to survive in the host. To check this possibility, we investigated the effects of clpL mutation on virulence after intranasal challenge of mice. However, there was no significant difference in survival in mice between the wild type (D39) and the clpL mutant (data not shown). Nevertheless, this does not exclude the possibility of subtle modulation of virulence traits during the initial stage of infection, for example, binding of the pneumococci to the host cells and subsequent triggering of innate host defenses. Therefore, we assessed the adherence and invasion capacities of the clpL mutant using human nasopharyngeal carcinoma epithelial cells (Detroit 562), human lung alveolar carcinoma (type II pneumocyte) cells (A549), and murine macrophage RAW 264.7 cells. Unexpectedly, the adherence of the clpL mutant to Detroit 562 cells after 1, 2, and 3 h of infection was 29-, 16-, and 26-fold greater, respectively, than the adherence of the wild-type strain (Fig. 1A). The adherence of the mutant to A549 cells after 2 and 3 h of infection was 75- and 56-fold greater, respectively, than the adherence of the wild type (Fig. 1B). Furthermore, the adherence of the clpL mutant to RAW 264.7 cells after 1, 2, and 3 h of infection was 8-, 20-, and 21-fold greater, respectively, than the adherence of the wild type (Fig. 1C). The invasion of both Detroit 562 and A549 cells by the clpL mutant was up to 5.3-fold greater than the invasion by the wild type (Fig. 1D and E), and the invasion of RAW 264.7 cells by the clpL mutant was 2.7-fold greater (Fig. 1F). These results clearly indicate that the clpL mutant could adhere and invade more efficiently than its otherwise isogenic wild-type parent in vitro.

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FIG. 1. Adherence to and invasion of epithelial cells by S. pneumoniae D39 and its isogenic clpL mutant in vitro. (A and D) Adherence to and invasion of Detroit 562 cells. (B and E) Adherence to and invasion of A549 cells. (C and F) Adherence to and invasion of RAW 264.7 cells. Cells were infected with 2 x 107 CFU of D39 or HYK304 (MOI, 100). For adherence, the monolayer was washed after infection, and the total number of bacteria in each well was determined by viable cell counting. For invasion, extracellular bacteria were removed by treatment with penicillin and gentamicin after infection. The monolayer was then washed extensively, and the number of intracellular bacteria was determined by viable cell counting. One asterisk, P < 0.05 for a comparison with the wild-type strain-infected group; two asterisks, P < 0.01 for a comparison with the wild-type strain-infected group; three asterisks, P < 0.001 for a comparison with the wild-type strain-infected group. The error bars indicate the standard deviations for three independent experiments.

To further confirm the preferential adherence of the clpL mutant, live wild-type and clpL mutant pneumococci were labeled with FITC and used in adherence assays. Again, the clpL mutant showed preferential adherence compared with the wild type (Fig. 2A). To ascertain that this phenotype was due to the clpL mutation, a complementation test was carried out with a recombinant clpL mutant clone expressing functional ClpL. The level of adherence of the complemented clpL mutant was similar to that of wild-type bacteria (Fig. 2B), demonstrating that the preferential adherence was due to the clpL mutation. When purified ClpL (10 to 80 µg/ml) was added in the clpL mutant infection assay, the adherence of the mutant decreased significantly in a concentration-dependent manner, down to 43% of the adherence of the untreated group (data not shown). This suggests that ClpL can inhibit adherence of pneumococci to the cell lines.

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FIG. 2. Preferential adherence of S. pneumoniae clpL mutant to epithelial cells in vitro. (A) Adherence to A549 cells. FITC-labeled pneumococci (108 CFU ml–1) were added to an A549 cells monolayer (MOI, 100) and incubated at 37°C for 2 h, and subsequently the monolayer was washed extensively and the adherent bacteria were counted with a fluorescent microscope. Adherence was expressed as the number of attached bacteria per appropriate area viewed with the microscope. (B) Complementation test. To verify the preferential adherence of the clpL mutant, the clpL gene was cloned in the clpL mutant. The levels of adherence of the various strains were determined as described in the legend to Fig. 1. Two asterisks, P < 0.01 for a comparison with the wild-type strain-infected group; three asterisks, P < 0.001 for a comparison with the wild-type strain-infected group. The bars and error bars indicate the means and standard deviations, respectively, for three independent experiments.

To corroborate the in vitro findings, the adherence to and invasion of the mouse nasopharynx and lungs by the mutant were compared to the adherence to and invasion of the mouse nasopharynx and lungs by the wild type after intranasal challenge of mice. The numbers of the clpL mutant cells in the nasopharynges of the mice were 3.3-, 1.7-, 4.6-, and 40,000-fold higher than the numbers of the wild-type cells after 6, 12, 24, and 36 h of infection, respectively (Fig. 3A). Bacteria were not detected in the lungs and spleens after 6 h of infection for both wild-type strain- and clpL mutant-infected mice. However, after 12, 24, and 36 h of infection, the viable counts of the clpL mutant bacteria in the lungs were 39-, 109-, and 9,000-fold higher than the viable counts for the wild type in infected mice (Fig. 3C). In addition, the numbers of clpL mutant bacteria in the blood, spleen, and brain were up to 73,487-fold higher than the numbers of the wild-type bacteria (Fig. 3B, D, and E). This clearly demonstrates that ClpL is involved in modulation of adherence and invasion in vivo, as well as in vitro.

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FIG. 3. Bacterial recovery from the nasopharynges (A), blood (B), lungs (C), spleens (D), and brains (E) of CD1 mice after intranasal challenge. Two groups of CD1 mice (20 mice/group) were challenged intranasally with either D39 or HYK304 using 2 x 107 CFU/mouse. At 6, 12, 24, and 36 h postinfection, five mice from each group were sacrificed, and the number of recovered bacteria was determined by plating on blood agar in the presence of the appropriate antibiotics. One asterisk, P < 0.05 for a comparison with the wild-type strain-infected group; two asterisks, P < 0.01 for a comparison with the wild-type strain-infected group; three asterisks, P < 0.001 for a comparison with the wild-type strain-infected group. The bars and error bars indicate the means and standard errors of the means (n = 5), respectively, for each time.

CPS content of the clpL mutant. Adherence of pneumococci could be affected by the thickness of the capsule layer, and nonencapsulated strains are known to adhere more efficiently than encapsulated strains adhere (45). It has been shown previously that the clpL mutation does not affect the level of expression of pneumococcal surface proteins, such as CbpA (a known adhesin), pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), and LytA, as determined by Western blot analysis (27). However, the amount of CPS in the clpL mutant was not determined. To verify that the clpL mutant produces less capsule, its total CPS content was determined by Western blotting, as described previously (31). Interestingly the total amount of CPS produced by the clpL mutant was not different from the total amount of CPS produced by the wild type (data not shown), demonstrating that mutation of clpL does not have any effect on capsule expression.

Viability of the clpL mutant at later stages of infection in vivo. Although the clpL mutant showed greater adherence and invasion, there was no significant difference in overall survival in mice between the wild type and the mutant after intranasal challenge, suggesting that the number of mutant cells might decrease to levels similar to the levels of the wild-type cells at the later stages of infection. As expected, after 48 and 96 h of infection, the viable counts of the mutant in the nasopharynx were 5.3- and 790-fold lower than the viable counts of the wild type, respectively, and the viable counts of the mutant in the lungs were 3.3- and 180-fold lower, respectively (Fig. 4). These results indicate that the clpL mutant is significantly more susceptible to host clearance mechanisms during the later stages of infection after intranasal challenge.

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FIG. 4. Viability of S. pneumoniae clpL mutant at a later stage of infection. Two groups of mice (15 mice/group) were challenged intranasally with either D39 or HYK304 (2 x 107 CFU/mouse). Five mice from each group were sacrificed at 24, 48, and 96 h postinfection, and the number of recovered bacteria was determined by plating on blood agar. One asterisk, P < 0.05 for a comparison with the wild-type strain-infected group; two asterisks, P < 0.01 for a comparison with the wild-type strain-infected group. (A) Nasopharynx. (B) Lungs. The bars and error bars indicate the means and standard errors of the means (n = 5), respectively, for each time.

Susceptibility of the clpL mutant to macrophages and autolysis in vitro. To investigate the underlying mechanism for the reduced fitness of the clpL mutant at the later stages of infection, autolysis under stress conditions and survival in macrophages were determined. In the presence of a low dose of penicillin (0.06 µg/ml), the viability of the clpL mutant decreased much more rapidly than the viability of the wild type decreased, and the levels of viable mutant cells were only 3.7, 1.5, and 12.1% of the levels of viable wild-type bacteria after 2, 4, and 6 h incubation, respectively (Fig. 5A). This clearly demonstrates that the clpL mutant is highly susceptible to penicillin-induced lysis.

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FIG. 5. Susceptibility of S. pneumoniae clpL mutant to macrophage and penicillin killing. (A) Bacteria were suspended in THY broth supplemented with penicillin (0.06 µg ml–1), which was followed by incubation for 2, 4, and 6 h and plating on THY agar to determine the number of viable bacteria. , D39; , D39 plus penicillin; , HYK304; •, HYK304 plus penicillin. (B) Cells were infected with 2 x 107 CFU of pneumococci (MOI, 100). At 2 h postinfection, extracellular bacteria were removed by treatment with antibiotics. After this, the monolayer was incubated further. Thereafter, the number of internalized bacteria was determined by viable cell counting. One asterisk, P < 0.05 for a comparison with the wild-type strain-infected group; two asterisks, P < 0.01 for a comparison with the wild-type strain-infected group. The symbols and error bars indicate the means and standard deviations, respectively, for three independent experiments.

The susceptibility of the clpL mutant to macrophage killing was investigated by determining its survival in RAW 264.7 murine macrophages. The cells were infected with wild-type or mutant bacteria for 2 h, and antibiotics were added to kill extracellular bacteria. Subsequently, the viability of the bacteria was determined by counting the number of intracellular bacteria. Two hours after antibiotic treatment, the viability of the mutant in RAW 264.7 cells was 55-fold lower than the viability of the wild type (Fig. 5B). Three hours after antibiotic treatment, no clpL mutant bacteria were detected, whereas the number of viable wild-type bacteria remained approximately 2.8 x 10² CFU/cell. These results suggest that the clpL mutant is highly susceptible to stress conditions, such as the presence of hydrolytic enzymes and oxidative chemicals.

Modulation of adherence and invasion by ClpL is mediated via actin polymerization. Since phagocytosis involves cytoskeletal rearrangement, membrane trafficking, and recruitment of proteins from the cytoplasm to the cell surface (16, 43), the effects of inhibitors of actin polymerization and protein synthesis on increased adherence of the the clpL mutant were investigated. When A549 cells were pretreated with cytochalasin D, an inhibitor of actin polymerization, both the adherence of and invasion by the clpL mutant were significantly increased 4.0- and 3.9-fold, respectively, whereas the invasion of A549 cells by wild-type bacteria was decreased, demonstrating that the adherence of and invasion by the clpL mutant could be modulated by actin polymerization (Fig. 6A and C). Pretreatment of RAW 264.7 cells with latrunculin A, an inhibitor of actin polymerization, resulted in adherence of and invasion by the clpL mutant similar to the adherence and invasion seen with cells treated with cytochalasin D (data not shown). In contrast, when A549 and RAW 264.7 cells were pretreated with jasplakinolide, an activator of actin polymerization, the adherence of and invasion by the wild type increased significantly (up to 2.7-fold), whereas the adherence of and invasion by the clpL mutant decreased (Fig. 6E, F, G, and H), corroborating our suggestion that adherence is mediated by actin polymerization via ClpL. In addition, adherence of the mutant to the host cells increased and decreased in a concentration-dependent manner when the host cells were pretreated with cytochalasin D and jasplakinolide, respectively (data not shown). Furthermore, when A549 cells were pretreated with cycloheximide, a eukaryotic translation inhibitor, the adherence to and invasion by the wild type decreased, whereas the adherence of and invasion by the clpL mutant increased, indicating that new protein synthesis is required for adherence to and invasion of the host cells by the wild-type bacteria but not by the clpL mutant (Fig. 6B and D). Since the increase in adherence to and invasion of host cells by the clpL mutant after cytochalasin D treatment was greater than the increase observed after cycloheximide treatment, actin polymerization seems to play a more significant role than new protein synthesis in the process.

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FIG. 6. Mediation of adherence to and invasion of A549 and RAW 264.7 cells by D39 and HYK304 in vitro via actin polymerization. For adherence, A549 and RAW 264.7 cells were pretreated for 2 h with either 0.5 µg ml–1 of cytochalasin D (A), 100 µM cycloheximide (B), or 10–12 M jasplakinolide (A549 cells [E] and RAW 264.7 cells [F]) and then infected with 2 x 107 CFU of D39 or HYK304 (MOI, 100). After infection, each monolayer was washed, and the total number of bacteria in each well was determined by viable cell counting. For invasion, A549 and RAW 264.7 cells were pretreated for 2 h with either 0.5 µg ml–1 of cytochalasin D (C), 100 µM cycloheximide (D), or 10–12 M jasplakinolide (A549 cells [G] and RAW 264.7 cells [H]) and then infected with pneumococci as described above. After infection, extracellular bacteria were removed by treatment with penicillin and gentamicin. After removal of the antibiotics, the monolayer was washed, and intracellular bacteria were enumerated by viable cell counting. One asterisk, P < 0.05 for a comparison with the control group; two asterisks, P < 0.01 for a comparison with the control group; three asterisks, P < 0.001 for a comparison with the control group. The error bars indicate the standard deviations for three independent experiments.

Requirement for ClpL and actin polymerization in TNF- secretion. Since TNF- is a key cytokine produced during the early stages of pneumococcal infection (4), it is conceivable that increased adherence to and invasion by the clpL mutant of the host cells might stimulate cytokine secretion. To examine this possibility, the level of secreted TNF- after infection of RAW 264.7 cells with the clpL mutant was compared to the level of secreted TNF- after infection of RAW 264.7 cells with the wild type. Surprisingly, after 1 h of infection, the level of TNF- secreted by cells infected with the mutant was only 39.6% of the level secreted by cells infected with the wild type. However, the level of TNF- secreted by cells infected with the mutant increased thereafter and reached almost the same level as the level secreted by the cells infected with the wild type 3 h postinfection (Fig. 7A). Thereafter, TNF- secretion by the clpL mutant-infected cells decreased more rapidly, indicating that the mutation modulates secretion of TNF- in RAW 264.7 cells (data not shown). To further corroborate the reduced TNF- secretion at the mRNA level, the TNF- mRNA level was determined by reverse transcriptase PCR (RT-PCR). Correspondingly, the TNF- mRNA level did not increase significantly in the clpL mutant-infected cells compared to the level in uninfected cells, whereas it increased significantly (2.5- and 3.1-fold, respectively) after 30 and 60 min of infection in cells infected with wild-type bacteria compared to the level in the uninfected cells (Fig. 7C). This indicates that ClpL mediates induction of TNF- expression in the RAW 264.7 cells. When A549 cells were infected with the mutant, the IL-8 mRNA level (as determined by RT-PCR) was lower than the level found in cells infected with the wild type (Fig. 7E). The observation that the mutant infection induced lower TNF- (Fig. 7C) and IL-8 (Fig. 7E) mRNA levels in both RAW 264.7 and A549 cells than the wild-type infection induced suggests that the mutant is less potent for induction of cytokine production.

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FIG. 7. Modulation of TNF- and IL-8 secretion from RAW 264.7 and A549 cells via actin polymerization. (A and B) Cells were pretreated for 2 h with either cytochalasin D (A) or jasplakinolide (Jas) (B) and then infected with 2 x 107 CFU of D39 or HYK304 (MOI, 100). After infection, RAW 264.7 cell supernatant was collected, and the level of secreted TNF- was determined by ELISA. One asterisk, P < 0.05 for a comparison with the either the wild-type strain-infected or control group; two asterisks, P < 0.01 for a comparison with either the wild-type strain-infected or control group. The bars and error bars indicate the means and standard deviations, respectively, for three independent experiments. (C, D, and E). Agarose gel analyses of TNF- and IL-8 mRNA. Cells were pretreated for 2 h with either cytochalasin D (C and E) or jasplakinolide (D) and then infected with 2 x 107 CFU of D39 or HYK304 (MOI, 100). After infection, RAW 264.7 (C and D) or A549 (E) cells were collected, and the TNF- (C and D) and IL-8 (E) mRNA levels were determined by RT-PCR, using β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control.

To evaluate the effect of actin polymerization on cytokine secretion, RAW 264.7 cells were pretreated with cytochalasin D, and then TNF- secretion was determined. After 1 and 2 h of infection, TNF- secretion from the RAW 264.7 cells infected with the wild type decreased significantly (46 and 72%, respectively). However, the observed reduction in TNF- secretion after cytochalasin D treatment from cells infected with the clpL mutant did not reach statistical significance (Fig. 7A). This result suggests that TNF- secretion is differentially affected by actin polymerization in the presence and absence of ClpL. To check whether modulation of TNF- secretion by cytochalasin D occurred at the level of transcription, TNF- mRNA levels were assessed by RT-PCR. Surprisingly, cytochalasin D pretreatment induced TNF- mRNA expression in both wild-type strain-infected cells and clpL mutant-infected cells (Fig. 7C), demonstrating that TNF- induction can occur independent of ClpL in the presence of cytochalasin D. To corroborate this finding, quantitative RT-PCR was carried out, and similar results were obtained (data not shown). Furthermore, these results suggest that TNF- secretion from wild-type strain-infected cells requires actin polymerization since TNF- mRNA was induced 30 min after cytochalasin D treatment but the level of its secretion was similar to that of the control (untreated) group 3 h after cytochalasin D treatment. In contrast, the TNF- secretion from the clpL mutant-infected cells was much less than that observed for the control group even 3 h after cytochalasin D treatment, although TNF- mRNA was induced 30 min after cytochalasin D treatment. This indicates that ClpL can mediate TNF- secretion via actin polymerization. The F-actin-severing compound latrunculin had an effect similar to the effect of cytochalasin D on adherence of the clpL mutant to the A549 cells (data not shown), whereas the actin-polymerizing agent jasplakinolide reversed the effect in both nonprofessional phagocytes (A549) and professional phagocytes (RAW 264.7) (Fig. 6A, C, E, F, G, and H). This clearly demonstrates that adherence is mediated by actin polymerization via ClpL.

When the clpL mutant was used to infect RAW 264.7 cells, the number of intracellular bacteria in the host cells was significantly higher than the number observed for the corresponding wild-type infection (Fig. 5B), indicating that the clpL mutant is less potent for inducing TNF- secretion and that invasion is not associated with TNF- secretion in the mutant.

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ATP-dependent chaperones play a major role in bacterial survival under stress conditions, when proteins tend to unfold and aggregate. HSP60 (GroEL) and HSP70 (DnaK) have been shown to be required for virulence modulation (6, 42), adherence (12, 14, 37), intracellular replication (25), invasion (44), and expression of TNF- and adhesion molecules (15, 30). Modulation of virulence by HSP100 family members, such as ClpB (8), ClpC (20, 33, 40), ClpE (34), ClpX (13), and ClpP (28, 39), has also been documented. Thus, inactivation of heat shock genes has resulted in virulence attenuation in several pathogens (6, 7, 25, 33, 37, 40, 44). In the present study, we demonstrated for the first time that inactivation of ClpL promoted adherence of S. pneumoniae to host cells, but did so without affecting virulence in mice.

Adherence is an initial stage in the invasion of the host cells by pathogens and involves a number of ligands, such as oligosaccharides and protein adhesins. In S. pneumoniae, the clpL mutation was found not to affect expression of the putative adhesin CbpA or other surface proteins, including PspA, LytA, and PsaA (27). However, the translocation of these proteins to the outside of the cell wall could be facilitated by HSPs other than ClpL, affecting the level of surface expression. To verify this possibility, pneumococcal proteins in subcellular components were fractionated to obtain cytosol, membrane, and extracellular fractions by osmotic shock and subjected to Western blot analysis. GroEL protein was employed as a cytosolic protein marker. Under heat shock conditions, the CbpA and PsaA levels in extracellular, cytosol, and membrane fractions of the clpL mutant were essentially the same as those in the fractions of the wild type (data not shown). Interestingly, the PspA and neuraminidase (NanA) levels in the extracellular fraction of the clpL mutant were much more lower than those in the extracellular fraction of the wild type (H.-Y. Kwon, unpublished data), indicating that additional factors might be involved in increased adherence of the clpL mutant. Such additional factors could be surface-associated exoglycosidases, such as NanA, β-galactosidase (BgaA), and β-N-acetylglucosaminidase (StrH). Although NanA deglycosylates lactoferrin, a secretory component, and immunoglobulin A2, thereby enhancing adherence of S. pneumoniae to components of the airway (24, 46, 47), it is probably not involved since its level in the clpL mutant was lower than its level in the wild type. However, BgaA and StrH, which have been shown to remove galactose and N-acetylglucosamine, respectively, thereby facilitating pneumococcal adherence to epithelial cells (23), or sugar moieties, such as lacto-N-neotetraose and asialoganglioside GM1, which contribute to adherence of pneumococci to the host cells (46), might be involved in increased adherence of the clpL mutant. More work to investigate whether one or more of these factors play some role in the clpL-mediated adherence is warranted.

For high-capacity clearance of pathogens, complement binds to pathogen and forms an immune complex, which interacts with complement receptors on phagocytes, mediating opsonization, phagocytosis, and lysis of pathogens (2). To determine whether the mutant is more susceptible to complement-mediated killing, the bactericidal activity of the ClpL antiserum was determined. Interestingly, the mutant was as sensitive to the ClpL antiserum as the wild type (data not shown), ruling out any enhanced complement sensitivity of the mutant.

Upon microbial infection, activated macrophages initiate phagocytosis of microbes (16, 36). This process relies on a profound rearrangement of the actin cytoskeleton and recruitment of internal membranes derived from endoplasmic reticulum and eventually leads to degradation of the pathogen (36, 43). In this study, cytochalasin D treatment resulted in better adherence of the clpL mutant than of the wild type to the host cells, suggesting that adherence of the clpL mutant to the host is facilitated by inhibition of actin polymerization.

TNF-, the most potent proinflammatory cytokine, is secreted by activated macrophages and induces diverse cellular responses ranging from apoptosis to inflammatory responses (16, 36, 38). Recently, the secretory pathway for TNF- from the Golgi apparatus to the cell surface was found to be mediated by vesicle-associated membrane protein 3, which delivers Golgi apparatus-generated TNF- from the recycling endosome to the site of phagocytic cup formation at the cell surface. This indicates that TNF- is released at the phagocytic cup, thus promoting simultaneous proinflammatory cytokine secretion and phagocytosis of pathogens (32). However, how bacteria participate in modulation of actin rearrangement and TNF- secretion remains unknown. In this study, the clpL mutant elicited decreased TNF- secretion, in spite of its efficient adherence to RAW 264.7 cells, suggesting that ClpL is involved in TNF- secretion. In addition, cytochalasin D treatment decreased TNF- secretion, but jasplakinolide reversed this result in both wild-type strain- and mutant-infected cells, demonstrating that TNF- secretion is stimulated by actin polymerization. However, these actin modulators had differential effects on adherence of the wild type and the mutant to the host cells in both nonprofessional (A549) and professional (RAW 264.7) phagocytes, indicating that ClpL reverses the effects of actin polymerization modulators. At the mRNA level, cytochalasin D treatment induced TNF- expression in both wild-type strain- and mutant-infected cells. Bacterial HSP60 and HSP70 have been documented to induce the secretion of proinflammatory cytokines (15, 30, 53) and to increase epithelial cell proliferation (54). However, although TNF- signaling pathways are well characterized (9, 18, 49), the effects of HSP100 chaperones, including ClpL, on cytokine and TNF- secretion have not been investigated previously. To our knowledge, this is the first report that TNF- secretion could be modulated by bacterial HSPs, as well as by modulators of actin polymerization.

The discrepancy between the level of TNF- (i.e., higher level of TNF- mRNA in the D39-infected cells after 1 h of cytochalasin D treatment than in the mutant-infected cells) and the lower level of TNF- secretion could be due to differential multistage regulation in eukaryotes. Regulation of TNF- secretion can occur during transcription in the nucleus, translation in the ribosome, and secretion via trafficking from the Golgi apparatus to the cell membrane. Therefore, it is likely that an increase in mRNA does not result in an increase in TNF- secretion. Further studies could reveal which stage in the regulation of TNF- secretion is critically affected by ClpL.

Greater adherence of the mutant to the host cells might be due to an increase in cortical F-actin. It has been shown that HSP27 decreases cortical F-actin and inhibits chemotaxis and exocytosis (21). Furthermore, chemotaxis is accompanied by actin polymerization (26). In our experiments, ClpL seemed to decrease cortical F-actin and also inhibited phagocytosis and translocation into the cytoplasm. Therefore, in the clpL mutant, cortical F-actin could be increased, thereby promoting phagocytosis. Cytochalasin D treatment inhibits further actin polymerization, leading to an increase in the level adherence and invasion.

Pneumococci can adapt to new host environments by reversible phase variation (22). The transparent phenotype, favored during nasopharyngeal colonization, is associated with reduced expression of CPS, while the opaque phenotype, favored in the lungs and blood, is associated with high levels of CPS expression (11, 50). Full encapsulation interferes sterically with the attachment of pneumococci to epithelial cells (17, 45, 50). Furthermore, pneumococci in intimate contact with cells or invading cells have been shown to be devoid of CPS by electron microscopy (17). However, the level of encapsulation of the clpL mutant is similar to that of the wild type when the organisms are grown in vitro, although we cannot completely eliminate the possibility the ClpL is involved in modulation of capsular expression in vivo. Further studies on the amount of CPS present on the cell surface upon contact with host epithelial cells are needed to verify this possibility.

Thus, ClpL could repress adherence to the host cells and induce TNF- secretion during the early phase of infection, possibly via a mechanism dependent upon actin polymerization. This study provides some insight into one of the diverse microbial strategies employed during early pathogenesis.

 
This work was supported by Korea Research Foundation grant 2004-015-C00512 and Korea Science and Engineering Foundation (KOSEF) grant R01-2006-000-10504-0 funded by the Korea government (MOST) and by National Health and Medical Research Council of Australia program grant 284214.

 
* Corresponding author. Mailing address: College of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea. Phone: 82 31 2907707. Fax: 82 31 2907727. E-mail: dkrhee{at}skku.edu

Published ahead of print on 2 April 2007.

Editor: J. N. Weiser

L.N.T. and H.-Y.J. contributed equally to this work.

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Infection and Immunity, June 2007, p. 2996-3005, Vol. 75, No. 6
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