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Infection and Immunity logoLink to Infection and Immunity
. 2014 Sep;82(9):3802–3810. doi: 10.1128/IAI.02012-14

Streptococcus pneumoniae ClpL Modulates Adherence to A549 Human Lung Cells through Rap1/Rac1 Activation

Cuong Thach Nguyen a, Nhat-Tu Le a,*, Thao Dang-Hien Tran a, Eun-Hye Kim a, Sang-Sang Park a, Truc Thanh Luong a, Kyung-Tae Chung b, Suhkneung Pyo a, Dong-Kwon Rhee a,✉
Editor: A Camilli
PMCID: PMC4187815  PMID: 24980975

Abstract

Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens.

INTRODUCTION

Heat shock proteins (HSPs) are highly conserved and abundant proteins that are produced by cells to protect from various insults, such as oxidative stress, infection, and inflammation. During infection, both host cells and pathogens are confronted by dramatic alterations in their surroundings, and HSP induction is important for pathogen survival. Generally, HSPs are molecular chaperones which refold stress-induced denatured proteins and protect the host from stress (1). Some members of the HSP family play an important role in the immune system via binding to antigens and presenting them to immune cells (2). Bacterial HSP60 (GroEL) binds to eukaryotic cells (3) or to Toll-like receptor 4 (4, 5) and partially influences bacterial adherence to host cells. HSP70 (DnaK) stimulates cytokine secretion via the CD14-dependent pathway (6). HSP70 and HSP90 act as bacterial antigens, and immunity to these proteins contributes to protection of humans from infection (7, 8). However, the role of HSP100 proteins in host cells during bacterial infection has not yet been characterized.

Bacterial ligand binding to host cell receptors during invasion can trigger signaling events that subsequently affect gene transcription, cell shape, adhesion, and endocytosis (9, 10). Some bacterial pathogens, such as Salmonella, Shigella, and Listeria, subvert the host cell through modulating the actin cytoskeleton and migrate into the cytoplasm by inducing unidirectional actin polymerization (11). Actin cytoskeletal structures include cortical actin, stress fibers, lamellipodia, and microspikes. The actin polymerization process is essential for the actin-generated motility of bacterial pathogens, which enables them to move into the infected cell (12). Moreover, remodeling of preexisting actin filaments is mainly controlled by the ubiquitous small Rho GTPases (13). In particular, RhoA, Rac1, and Cdc42 are small Rho GTPases that have been shown to be targets of bacterial effector molecules (14) that trigger remodeling of the cellular actin cytoskeleton. Moreover, small GTPases, including RhoA, Rac1, Cdc42, Rab9A, and Rab23, play important roles during Streptococcus infection (15–18). Although Streptococcus invasion has been reported to be mediated by RhoA, Rac1, and Cdc42 (16), invasion by the major adhesin PspC/CbpA involves Cdc42 and not Rac1 or RhoA (17). Thus, the role of small GTPases in host responses to Streptococcus infection is not well understood and needs to be further resolved.

Actin filament reorganization is a dynamic process which is modulated by actin binding proteins (19). Among these proteins, Arp2/3 complex or formins promote the nucleation of actin, whereas cofilin severs actin filaments (19). Thus, cofilin enhances site-directed actin polymerization in vivo (19). Cofilin is activated by dephosphorylation via the slingshot (SSH) family of protein phosphatases and chronophin, whereas it is inactivated by phosphorylation via Rac1-regulated LIM-kinases (LIMKs) and testicular protein kinases (TESKs) (20). Activation of cofilin increases the number and length of filopodia by stimulating englongation of lamelipodial F-actin filaments underlying the plasma membrane (21).

Streptococcus pneumoniae (pneumococcus) is the major causative agent of bacterial meningitis, bacteremia, otitis media, and community-acquired pneumonia (22). During pneumococcal infection, the host cells respond to bacteria by producing antibodies or activating immune cells to restrict proliferation of the pathogen. However, S. pneumonia utilizes virulence factors such as its capsule, pneumolysin, PspA, and LytA to inhibit the host immune system and evade the host defense system. Therefore, studies on host-bacteria interactions are required for efficient intervention to limit bacterial infection. Previously, the ClpL protein, a member of the HSP100/Clp (caseinolytic protease) family, was found mainly in Gram-positive organisms. In addition, we showed that while ClpL was localized in membrane and cytosol fractions at 30°C, after heat shock ClpL was found in cell wall, membrane, and cytosol fractions (23), suggesting that ClpL can translocate to the cell wall and, possibly, cell surface. ClpL is not secreted, based on Western blotting data from our lab (data not shown). ClpL was also demonstrated to have chaperone functions and modulate virulence gene expression (24). Moreover, the ClpL chaperone represses pneumococcal adherence to host cells and induces secretion of tumor necrosis factor alpha through a mechanism dependent on actin polymerization (25). However, much is still unknown, including how S. pneumonia infection modulates gene expression and leads to stimulation of the actin cytoskeleton and the mechanism for how ClpL inhibits adherence.

In this study, we found that the ClpL protein induces Rap1 and Rac1 activation during pneumococcal infection. Wild-type (WT) S. pneumonia infection increased phosphorylation of cofilin (inactivated form) via Rap1 and Rac1 and showed lower filopodium formation than infection with a clpL mutant (ΔclpL), which resulted in lower adherence for WT than ΔclpL to A549 host cells.

MATERIALS AND METHODS

Bacterial strains and cell cultures.

S. pneumoniae encapsulated type 2 strain D39 (NCTC7466) and the clpL mutant (ΔclpL) (24), pspC mutant (26), and type 1 strain ATCC 6301 and the clpL mutant (ATCC 6301-ΔclpL) (23) were cultured in Todd-Hewitt broth as described previously (24). S. pneumoniae nonencapsulated CP1200, a derivative of Rx1, and its isogenic clpL derivative (HYK1) were cultured in Casitone-tryptone-based medium (CAT) as described previously (24). The A549 (ATCC CCL-185) human lung epithelial carcinoma cell line was maintained at 37°C in a humidified incubator at 95% air–5% CO2, grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (Cambrex Bio Science, Walkersville, MD) and 1× penicillin-streptomycin (PAA Laboratories GmbH, Pasching, Austria).

Labeling of pneumococci with FITC.

Pneumococci were labeled with fluorescein isothiocyanate (FITC) as described previously (27). Briefly, bacteria (108 CFU ml−1) were mixed with FITC (1 mg ml−1; Sigma) dissolved in a buffer containing 0.05 M Na2CO3 and 0.1 M NaCl at 4°C for 1 h, washed 5 times with phosphate-buffered saline (PBS), and resuspended in DMEM to a final concentration of 108 CFU ml−1.

Western blot assay.

Cells were infected with pneumococci (multiplicity of infection [MOI], 100) or incubated with 200 ng/ml of purified ClpL protein. After infection or incubation, cells were washed with PBS and then 50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% protease cocktail (RIPA buffer) was added. Cell lysates were collected and subjected to Western blotting as described previously (23).

Rap1 activation assay.

Rap1 activity was assayed by using a Rap1 activation kit (Upstate Biotechnology, Lake Placid, NY). Rap1-GTP from various infected lysates was pulled down for 1 h at 4°C by using a glutathione S-transferase fusion protein that corresponded to the human Rap1 binding domain of RalGDS, precoupled to glutathione-Sepharose beads. Next, beads were washed with ice-cold lysis buffer, resuspended in SDS sample buffer, and subjected to SDS-PAGE followed by Western blotting with anti-Rap1 antibody (Upstate Biotechnology, Lake Placid, NY) to detect GTP-Rap1 (28).

Endogenous Rho GTPase activity assays.

Cells were grown in a 6-well dish to 40% confluence and treated with small interfering RNA (siRNA) prior to infection. RhoA, Rac1, and Cdc42 activation was evaluated by using a RhoA, Rac1, and Cdc42 activation kit (Cytoskeleton, Denver, CO). Cell lysates were incubated with GST-rhotekin polo-box domain (PBD) protein beads (for active RhoA) or GST-PAK PBD protein beads (for active Rac1 and Cdc42) for 1 h at 4°C with gentle rotation. Beads were washed, and pulled-down proteins were subjected to SDS-PAGE followed by Western blotting with anti-RhoA, anti-Rac1, or anti-Cdc42 antibody (Upstate Biotechnology) to detect the presence of the GTP-bound forms (14, 15).

Transfection.

Control siRNA (siCo; sc-37007), Rap1 siRNA (siRap1; sc-36384), and Rac1 siRNA (siRac1; sc-36351) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To knock down Rap1 or Rac1 expression with siRNA, 50 nM siRap1 or 100 nM siRac1 was added to 100 μl of serum-free DMEM without antibiotics. Transfection was carried out per the manufacturer's suggestions.

Inhibitor treatment.

C3 exoenzyme (Cytoskeleton Inc.), NSC23766 (Calbiochem), mevastatin, and geranylgeranyltransferase I inhibitor GGTI-298 were purchased from Sigma-Aldrich. For each inhibitor, the following concentrations were used: C3 exoenzyme at a concentration of 0.5 μg/ml for 4 h (29); NSC23766 (with a 50% inhibitory concentration [IC50] of 50 μM, which was shown to not affect the activity of Cdc42 or RhoA) at a concentration of 100 μM for 2 h; mevastatin at a concentration of 10 μM for 18 h (30); GGTI-298 at 5 μM for 4 h (IC50, 3 μM) (31).

Purification of non-histidine-tagged ClpL.

To clone non-histidine-tagged ClpL in Escherichia coli, the full-length clpL open reading frame (ORF), encoding ClpL amino acids 1 to 701, was amplified with two primers from CP1200 DNA: HYG5 (5′-GGCCCATATGAATAACAACTTTAATAATTTTA-3′, with an NdeI site inserted) and HYG4 (5′-GGCCGAGCTCTTAGACTTTCTCACGAATAAC-3′, with KpnI and SacI sites inserted). The amplified ORF was digested with NdeI and SacI and ligated into NdeI- and SacI-digested pET30(a), to generate the plasmid pKHY003. PCR-amplified clones were verified by nucleotide sequencing. To clone recombinant ClpL, E. coli BL21(DE3) was transformed with pKHY003 and grown at 37°C to an optical density at 550 nm of 0.6. Expression of the recombinant protein was induced by addition of isopropyl-β-d-thiogalactopyranoside at a final concentration of 0.1 mM. Subsequently, the culture was harvested by centrifugation, and the pellet was resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 10% glycerol, 1 mM dithiothreitol, 1 mM PMSF). The resuspended cells were lysed by freezing and thawing followed by sonication. After centrifugation, the cell lysate supernatant was incubated with 0.04% polyethyleneimine and then centrifuged at 15,000 × g for 20 min. Non-histidine-tagged ClpL protein in the precipitate was further purified with a DEAE-Sepharose column.

Rhodamine-phalloidin staining.

A549 cells were infected with pneumococci (MOI, 500) or treated with 200 ng/ml of purified ClpL protein for the indicated times to assay changes in F-actin staining as described previously (32). Cells were briefly washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. The cells were washed with PBS 3 times (5 min/wash). Cells were permeabilized with 1% Triton X-100 in PBS for 5 min. Cells were washed, and nonspecific binding was blocked by incubating the samples in 1% bovine serum albumin (BSA) in PBS overnight at 4°C. F-actin staining was performed using rhodamine-phalloidin (1:1,000; Molecular Probes) in BSA/PBS to reduce background. After washing 3 times (5 min/wash), coverslips were mounted (Prolong antifade kit; Molecular Probes) and examined by confocal microscopy (LSM 510 Meta DuoScan; Carl Ziess Micro Imaging GmbH, Germany).

Adherence assays.

Adherence assays were performed as described previously (24). Briefly, A549 cells were infected with pneumococci (MOI, 100). To determine the total numbers of adherent bacteria, infected monolayers were washed with PBS and cells were detached from the plates by treatment with 0.25% trypsin–0.02% EDTA and then lysed by the addition of 0.025% Triton X-100 in H2O. Appropriate dilutions were plated on Todd-Hewitt medium supplemented with yeast extract (THY) agar to determine the number of viable bacteria. All samples were assayed in triplicate, and each assay was repeated three times.

For sequential competitive adherence assays, A549 cells were infected with either D39 WT or the clpL mutant (MOI, 100) for 30 or 60 min. The adhered bacteria were washed away with PBS and killed by the antibiotics penicillin (10 μg/ml) and gentamicin (200 μg/ml). Next, the pretreated cells were infected with D39 S. pneumoniae for 2 h and total numbers of adherent bacteria were determined by plating on THY agar. The additional 2-h time point reflects events before invasion.

Statistical analysis.

Statistical differences between groups were analyzed via a one-way analysis of variance (ANOVA; Tukey's test). All results are representative of three independent experiments. Statistically significant differences (P values) were determined at levels of <0.05, <0.01, and <0.001.

RESULTS

S. pneumoniae ClpL inhibits pneumococcal adherence.

Adherence of encapsulated ΔclpL to Detroit, A549, and RAW 264.7 cells was previously shown to be higher than that of encapsulated D39 WT (25). In addition, when adherence of nonencapsulated CP1200 and its isogenic clpL mutant (HYK1) was examined, adherence of HYK1 was also higher than the wild type (Fig. 1A). Thus, it is likely that ClpL inhibits S. pneumoniae adherence to host cells. To test this idea, purified ClpL (10 to 80 ng/ml) was included in the encapsulated ΔclpL adherence assay mixtures. Adherence of ΔclpL to A549 cells decreased significantly in a ClpL protein concentration-dependent manner, down to 25% of the adherence of the untreated group (Fig. 1B), whereas DnaJ protein did not produce any effect on pneumococcal adherence (Fig. 1C), demonstrating that ClpL can inhibit adherence of ΔclpL to host lung cells. Subsequent adherence assays were performed to confirm that ClpL inhibits the adherence of pneumococci to host lung cells. When A549 cells were pretreated with either WT or ΔclpL (followed by washing and killing the remaining bacteria with antibiotics) and the adherence of WT pneumococcus was determined, pretreatment of the cells with WT pneumococci decreased adherence compared to pretreatment with mutant pneumococci (Fig. 1D). Thus, S. pneumoniae ClpL inhibits pneumococcal adherence.

FIG 1.

FIG 1

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S. pneumoniae ClpL inhibits pneumococcal adherence. (A) A549 cell monolayers were infected with pneumococcus CP1200 or its isogenic ClpL derivative (HYK1) for 1, 2, and 3 h. Nonadherent pneumococci were washed off, and the number of adherent pneumococci was determined by plating on THY agar. (B and C) Nontagged ClpL (B) or DnaJ (C) protein was added to A549 cells and incubated for 3 h prior to infection with the clpL mutant. After 2 h of infection, the numbers of adherent pneumococci were determined as described in Materials and Methods. (D) A549 cells were infected with WT and ΔclpL for 30 and 60 min, followed by washing and killing extracellular bacteria. The cells were then infected with WT for 2 h, and the numbers of adherent pneumococci were determined as described in Materials and Methods. The data are representative of three independent experiments. The statistical differences were analyzed via an ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Small GTPase-mediated actin polymerization is required for pneumococcal adherence to A549 cells.

Microarray analysis showed that pneumococcal infection increased expression of Rap1 and certain genes involved with the actin cytoskeleton (data not shown). Also, Rap1 and small GTPases such as Rho and Rac-1 are known to modulate actin polymerization (33–35). To confirm the role of actin polymerization in pneumococcal adherence, A549 cells were treated with inhibitors of Rap1 (GGTI-298), Rho (C3 exoenzyme), and Rac1 (NSC23766), as well as a pan-Rho-GTPase inhibitor (mevastatin), prior to infection, and pneumococcal adherence to A549 cells was determined. Rap1 inhibitor treatment significantly decreased adherence of both WT and ΔclpL pneumococci to the host A549 cells (Fig. 2A). Consistently, treatment with Rac1 and Rho inhibitors also significantly decreased adherence of both WT and ΔclpL pneumococci to the host A549 cells in dose-dependent manner (Fig. 2B to D). Consistently, the inhibitors inhibited serotype 1 pneumococcal adherence to A549 cells (Fig. 2E to H). These results suggest that small GTPase-mediated actin polymerization plays a role in pneumococcal adherence.

FIG 2.

FIG 2

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Small GTPase-mediated actin polymerization is required for pneumococcal adherence to A549 cells. A549 cell monolayers were pretreated with inhibitors for the indicated times as described in Materials and Methods. The cells were then infected with type 2 (A to D) or type 1 ATCC 6301 (E to H) pneumococci for 2 h, nonadherent pneumococci were washed off, and adherent pneumococci numbers were determined by plating on THY agar. The data are representative of results from three independent experiments. The statistical differences were analyzed via an ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the untreated cells).

ClpL is required for Rap1, Rac1 induction, and cofilin/p-cofilin expression during infection.

Next, when Rap1 levels in D39 WT- and ΔclpL-infected cells were compared, total Rap1 levels were significantly induced in D39 infected cells and slightly induced in ΔclpL-infected cells (Fig. 3A). Moreover, active GTP-bound Rap1 levels were much higher in D39-infected cells at 2 and 4 h postinfection than in ΔclpL-infected cells (Fig. 3A). These results demonstrated that pneumococcal infection induces Rap1 but that D39 WT infection induces Rap1 to a greater extent than ΔclpL infection.

FIG 3.

FIG 3

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ClpL is required for Rap1 and Rac1 induction and cofilin/p-cofilin expression during infection. (A and B) A549 cells were infected with pneumococci for 1, 2, or 4 h. After infection, cells were washed with PBS and lysed. The lysates were then subjected to Western blot analysis using the indicated antibodies. GTP-bound active Rap1 and Rho GTPases in the lysates were pulled down with a binding domain to the active forms. The pull-down assay mixtures were washed with lysis buffer and subjected to SDS-PAGE followed by Western blotting with the indicated antibodies. The data are representative of results from three independent experiments. (C) Cell lysates from the experiment shown in panel A were subjected to Western blotting for cofilin/p-cofilin detection. The band density of cofilin/p-cofilin was measured by using ImageJ, and differences were analyzed via an ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the uninfected cells).

RhoA is activated by pneumolysin (15), whereas Rac1 and Cdc42 are activated by pneumococcus serotype 2 and serotype 35A infections, respectively (17, 36). To confirm the involvement of small RhoGTPases in WT and ΔclpL pneumococcal infections, the expression of small RhoGTPases was determined in GTPase activity assays. We observed that D39 WT infection induced a significant increase in total Rac1 expression at 1, 2, and 4 h postinfection, whereas ΔclpL infection induced a marginal increase in total Rac1 levels (Fig. 3A). Consistently, D39 infection significantly increased active Rac1 levels at 2 h and 4 h postinfection, whereas ΔclpL infection caused a significant decrease in active Rac1. However, neither D39 WT nor ΔclpL infection induced an increase in total Cdc42 levels or active Cdc42. Also, neither WT nor ΔclpL infection induced total or active RhoA expression (Fig. 3B). These results indicate that ClpL influences the activation of Rap1 and Rac1 during serotype 2 pneumococcal infection.

Recent studies indicated that Rap1 and Rac1 regulate the phosphorylation of cofilin, which binds to actin to mediate actin turnover, subsequently modulating cytoskeleton dynamics and inducing actin rearrangement (34, 37). Therefore, we examined cofilin and p-cofilin levels after serotype 2 pneumococcal infections. ΔclpL infection induced cofilin expression at a slightly higher level than the D39 infection. In contrast, ΔclpL infection induced p-cofilin, the inactive form, at levels significantly lower than the D39 infection (Fig. 3C and D). Therefore, ClpL appears to decrease cofilin activity during infection, thus affecting the actin cytoskeleton and adherence. We also compared cofilin and p-cofilin levels after D39 and complemented strain infection. As expected, cofilin/p-cofilin expression in the complemented strain was the same as WT (see Fig. S1 in the supplemental material). Taken together, ClpL modulates activation of cofilin during pneumococcal infection.

Purified ClpL protein induces Rap1 and Rac1 and cofilin/p-cofilin expression in A549 cells.

Our results showed that D39 WT infection induced higher levels of Rap1, Rac1, and p-cofilin than ΔclpL infection, indicating that ClpL likely plays a role in modulation of Rap1, Rac1, and cofilin/p-cofilin expression during infection. To confirm this, purified untagged rClpL was incubated with A549 cells, and total and active Rap1 or Rac1 and cofilin levels were determined. Purified rClpL induced Rap1 expression in a time-dependent manner (Fig. 4A). Active Rap1 expression was induced significantly after 15 and 30 min, which decreased after 60 min of incubation (Fig. 4A). Consistently, total and active Rac1 expression was also induced after 5 min of incubation (Fig. 4B).

FIG 4.

FIG 4

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Purified ClpL protein induces Rap1 and Rac1 and cofilin/p-cofilin expression in A549 cells. (A and B) A549 cells were seeded on 6-well plates and incubated with 200 ng/ml of purified ClpL protein for the indicated times. Cell lysates were subjected to pull-down assays followed by Western blotting to detect Rap1 (A) or Rac1 (B). (C) Alternatively, cell lysates were directly used for Western blot analysis to detect cofilin/p-cofilin. The data are representative of results from four independent experiments. The band density was measured by using ImageJ, and differences were analyzed via an ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

In addition, cofilin was induced and the levels reached a plateau after 30 min of incubation with rClpL. However, further incubation repressed cofilin levels (Fig. 4C). p-cofilin expression was also dramatically induced after 15 min of incubation, but it was repressed after 60 min (Fig. 4C). To exclude the possibility that the observed effects might be due to lipopolysaccharide (LPS) contamination of the purified rClpL protein, the proteins were heat inactivated by boiling for 30 min, since LPS is not inactivated by heat (38), and cofilin/p-cofilin levels were determined. Only rClpL induced cofilin and p-cofilin after 30 min of incubation, while other pneumococcal proteins, such as purified VncR, PspA, and heat-inactivated rClpL, did not (see Fig. S2 in the supplemental material). Taken together, these results suggest that ClpL induces Rap1, Rac1, and cofilin/p-cofilin expression during pneumococcal infection.

Rap1 and Rac1 activation is required for cofilin phosphorylation.

To further verify that Rap1 and Rac1 regulate cofilin/p-cofilin expression during pneumococcal infection, Rap1 or Rac1 expression was knocked down by using siRNA, and cofilin/p-cofilin expression was determined by Western blotting. siRap1 transfection decreased cofilin and p-cofilin expression (Fig. 5A and B), although both D39 and ΔclpL infections increased cofilin and p-cofilin levels significantly at 1, 2, and 4 h postinfection. This suggests that Rap1 is required for cofilin phosphorylation in both D39 WT and ΔclpL infections. However, siRap1 inhibited p-cofilin levels significantly in ΔclpL infection (Fig. 5B).

FIG 5.

FIG 5

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Rap1 is required for cofilin/p-cofilin expression. (A and B) Cells were transfected with siRap1 (50 nM) for 24 h and then infected with pneumococci for 0, 1, 2, and 4 h. Subsequently, cells were lysed, and cell lysates were used for Western blot analysis. (C and D) Quantification of results from the experiments shown in panels A and B. The data are representative of results from four independent experiments. Ctrl and siCo represent noninfection controls and nonspecific siRNA-treated infection controls, respectively. The statistical differences were analyzed via an ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the uninfected cells and all the other groups, as indicated).

Similarly, siRac1 transfection decreased both cofilin and p-cofilin expression significantly in both WT- and ΔclpL-infected cells (Fig. 6). Interestingly, following siRac1 transfection, cofilin expression was inhibited time dependently in D39-infected cells (Fig. 6A), whereas cofilin expression was induced time dependently in ΔclpL-infected cells and reached maximum levels 4 h postinfection (Fig. 6B). However, p-cofilin levels were significantly inhibited by siRac1 transfection in both WT- and ΔclpL-infected cells (Fig. 6A and B), suggesting that Rac1 is required for cofilin phosphorylation.

FIG 6.

FIG 6

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Rac1 is required for cofilin/p-cofilin expression. (A and B) Cells were transfected with siRap1 (100 nM) for 24 h and then infected with pneumococci for 0, 1, 2, and 4 h. Subsequently, cells were lysed, and cell lysates were used for Western blot analysis. (C and D) Quantification of results from the experiments shown in panels A and B. The data are representative of results from four independent experiments. Ctrl and siCo represent noninfection controls and nonspecific siRNA-treated infection controls, respectively. The statistical differences were analyzed via an ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the uninfected cells and all the other groups as indicated).

Taken together, our data provide additional evidence that Rap1 and Rac1 are sufficient for cofilin phosphorylation during D39 and ΔclpL infections.

Rap1/Rac1-mediated actin filaments are required for pneumococcal adherence.

Since p-cofilin levels in ΔclpL-infected cells were significantly lower than in D39-infected cells (Fig. 3C), ClpL appears to inactivate cofilin during infection. Cofilin plays a role in increasing the number and length of filopodia (21). Thus, ΔclpL infection might stimulate formation of filopodia more efficiently than D39 infection. To examine this possibility, cells were infected with pneumocci and F-actin was stained with rhodamine-phalloidin to visualize filopodia by confocal microscopy. We observed that filopodium formation was initiated at 1 h postinfection and increased significantly at 2 and 4 h postinfection in both D39 WT- and ΔclpL-infected cells (data not shown). However, the number and length of filopodia in ΔclpL-infected cells were increased compared to the D39-infected cells (Fig. 7A).

FIG 7.

FIG 7

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Rap1/Rac1 are required for filopodia formation. (A) A549 cells on glass coverslips were pretreated with siRap1 or siRac1 prior to infection with FITC-labeled pneumococci for 24 h or 48 h, respectively. Filopodia were visualized by rhodamine-phallodin staining followed by confocal microscopy. Arrows indicate filopodia. (B and C) The number of cells with filopodia was determined by counting several view fields per coverslip. The data are representative of results from three independent experiments. Ctrl and siCo represent noninfection controls and nonspecific siRNA-treated infection controls, respectively. The statistical differences were analyzed via an ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the uninfected cells and all the other groups as indicated).

To confirm that Rap1 and Rac1 activation is required for filopodium formation, Rap1 and Rac1 expression was knocked down via specific siRNA transfection and filopodium formation was observed. siRap1 transfection inhibited filopodium formation significantly during pneumococcus infection (Fig. 7A and B). Similarly, siRac1 transfection reduced filopodium formation significantly in both D39 WT- and ΔclpL-infected cells (Fig. 7A and C). Taken together, our data indicate that Rap1 and Rac1 play a role in filopodium formation. The filopodia may provide a scaffold for bacteria to bind to the host cell, thus allowing more efficient adherence of ΔclpL to the host cells.

DISCUSSION

HSPs are rapidly induced by heat shock and other stimuli in both Gram-negative and -positive bacteria (39). Once the temperature returns to normal, HSPs decline rapidly to steady-state levels in E. coli (39). In contrast, HSP levels in pneumococci are sustained even 1 h after return to normal temperatures (40), suggesting that pneumococcal HSPs may have different roles than those of E. coli. Bacterial HSP60 (GroEL) and HSP 70 (DnaK) are localized at the cell wall or surface and facilitate cell wall synthesis or help the refolding of cell wall proteins (41–45). Moreover, treatment of Staphylococcus aureus with cefoxitin, a cephalosporin antibiotic, induces GroES, DnaK, and Clp ATPase subunits, such as ClpL (46). In pneumococci, ClpL is present at the cell wall and interacts with the cell wall penicillin binding protein (PBP2x). Thus, the ClpL-deficient mutant is more susceptible to cell wall-active antibiotics, such as penicillin, and has thinner cell walls than the wild type (23). However, it is unknown how ClpL interacts with host cells.

A total of 131 ClpL homologues are found in Gram-positive bacteria (http://www.ncbi.nlm.nih.gov/gene). ClpL homologues are found in Streptococcus (S. pyogenes, S. parauberis, S. suis, S. oralis, S. uberis, S. pneumoniae, S. equi, S. agalactiae, S. parasanguinis, S. dysgalactiae, S. mitis, S. gallolyticus, S. sanguinis, S. pasteurianus, S. thermophiles, S. intermedius, S. salivarius, and S. mutans), Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and lactic acid bacteria comprising Lactococcus sp. (L. lactis), Lactobacillus spp. (L. sakei, L. plantarum, L. casei, L. rhamnosus, L. reuteri, L. delbrueckii, L. salivarius, L. brevis, L. gasseri, L. buchneri), and Leuconostoc spp. (L. gasicomitatum, L. citreum, and L. gelidum). This prevalence of ClpL in Gram-positive bacteria suggests that ClpL may play an important role in these pathogens as well as in lactic acid bacteria for survival during confrontation with harsh environments such as thermal stress during niche change and entrapment by phagocytes followed by exposure to respiratory oxygen species.

Our previous study indicated that the D39 has a longer chain length than the clpL mutant (23). D39 is more resistant to deoxycholate (DOC)-, Triton X-100-, and penicillin-triggered lysis and has a thicker cell wall with mucoid colony morphology as well as a longer generation time compared to the clpL mutant (23). However, CPS levels and surface proteins such as PspC and PspA were not significantly different between D39 and ΔclpL (data not shown). Increased chain length was shown to promote pneumococcal adherence and colonization during infection (47). However, D39 adheres less efficiently than the clpL mutant despite its longer chain length (25), providing evidence that pneumococcal adherence depends not only on chain length but also on the host response to infection.

Host-pathogen interactions often induce major alterations in the actin cytoskeleton and in actin polymerization, which are required for bacterial internalization (11). Rap1 was found to regulate actin filament formation via interaction between profilin and actin, thereby inducing phagocytosis of serum (C3bi)-opsonized zymosans in macrophages (48, 49). Thus, Rap1 is essential for endocytosis and phagocytosis (48, 49). Rac1 is also a key regulator of cell migration and stimulates membrane ruffling (50), and engulfment and internalization of apoptotic cells by airway epithelial cells are Rac1 dependent (51). Rac1 is also necessary for efficient uptake of Yersinia pseudotuberculosis by nonphagocytic cells (52). Here, we showed that GTPase-mediated actin polymerization is required for serotype 2 pneumococcal adherence. Furthermore, S. pneumoniae induces host Rap1 and Rac1 proteins, indicating that Rap1-Rac1 may play an important role in pneumococcal adherence to human lung cells.

Serotype 35A (NCTC10319) pneumococcal adherence by the major adhesin PspC/CbpA involves Cdc42 but not Rac1 (17). Therefore, we also compared Rac1 expression in serotype 2 D39 and the pspC mutant. We found that serotype 2 pspC mutant infection increased total and active Rac1 levels compared to WT infection, indicating that PspC inhibits Rac1 expression and activation during serotype 2 pneumococcal infection (see Fig. S3 in the supplemental material). In addition, our previous results showed that PspC levels in ΔclpL were not significantly different from the D39 WT (24), suggesting that PspC is not involved in ClpL-mediated adherence. Thus, ClpL inhibits pneumococcal adherence, and this adherence inhibition appears to be due to ClpL and not PspC. A previous study reported that an actin polymerization inhibitor did not affect pneumococcal adherence but did affect invasion (17), indicating that actin polymerization is not involved in pneumococcal adherence. In contrast, when we infected A549 human lung cells with S. pneumoniae serotype 2 (NCTC7466), small GTPase inhibitors impaired pneumococcal adherence dose dependently (Fig. 2), suggesting that small GTPase-mediated actin polymerization is involved in pneumococcal adherence. Since serotype difference can generate strain-specific effects on host cells that affect adherence and internalization (53), these inconsistent results could have been due to differences in pneumococcal serotype and host cell type. We note that the previous study used dog kidney epithelium-derived MDCK (Madin-Darby canine kidney) cells, which are not the normal habitat of S. pneumoniae, or Calu3 human lung epithelial cells that expressed endogenous human pIgR (17).

A previous study showed that invasion of Detroit 562, A549, and RAW 264.7 cells by the clpL mutant was greater than that of the wild type, indicating that the clpL mutant invaded more efficiently than the wild-type parent in vitro (25). In this study, our results demonstrated that small GTPase-mediated actin polymerization is required for pneumococcal adherence (Fig. 2). Thus, small GTPase-mediated actin polymerization might be important for pneumococcal invasion during infection, since adherence is the first step for invasion. Therefore, ClpL protein seems to modulate pneumococcal invasion via modulation of small GTPase-mediated actin polymerization during infection.

Rap1-dependent activation of cofilin is critical for early cytoskeletal changes (34). Cofilin depolymerizes actin, increasing the rate of actin cytoskeletal reorganization (54, 55). Thus, the activation of cofilin is important for modulating actin structure and cytoskeleton remodeling. Here, our experimental results using siRap1 and siRac1 demonstrated a significant decrease in cofilin and p-cofilin levels, indicating induction and activation of cofilin by Rap1 and Rac1 in pneumococcal infection.

In this study, ClpL was found to be responsible for the induction and activation of Rap1 and Rac1 during infection. We found that (i) ClpL protein can induce and activate Rap1 and Rac1, increasing cofilin phosphorylation. (ii) ΔclpL infection shows slightly higher levels of cofilin than WT infection. (iii) Rap1 and Rac1 are required for phosphorylation of cofilin. Thus, ΔclpL infection induces higher levels of filopodia than the D39 WT infection. Since filopodia are necessary for bacterial attachment to the host cell surface (56), the adherence of ΔclpL to A549 cells was higher than D39 WT. However, the mechanism by which ClpL transmits signals to the host cell remains unknown. We hypothesize that ClpL interacts with the host cell directly or indirectly. It is possible that ClpL binds directly to unknown receptors of the host cells, or ClpL may modulate certain proteins that bind to the cellular receptors and transmit signals. Since ClpL is a pneumococcal chaperone, it is likely a chaperone for a class of pneumococcal proteins, and the role of one or more of these proteins may be to modify actin structure in the host cell. Further studies are necessary to provide further insights into how pneumococci respond upon initial contact with host cells.

Supplementary Material

Supplemental material
supp_82_9_3802__index.html (1.1KB, html)

ACKNOWLEDGMENTS

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2011-0024794) and by the Korea Science & Engineering Foundation (WCU R33-10045).

Footnotes

Published ahead of print 30 June 2014

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02012-14.

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