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Role of Syndecan-4 in the cellular invasion of Orientia tsutsugamushi
Microbial Pathogenesis 36 (2004) 219–225 www.elsevier.com/locate/micpath
Role of Syndecan-4 in the cellular invasion of Orientia tsutsugamushi Hang-Rae Kim, Myung-Sik Choi, Ik-Sang Kim* Department of Microbiology and Immunology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea Received 1 September 2003; received in revised form 6 November 2003; accepted 15 December 2003
Abstract Cell surface heparan sulfate proteoglycans (HSPGs) play a critical role in the cellular invasion of intracellular bacteria and are presumed to have a role in the infection of host cells by Orientia tsutsugamushi. Previously, we showed that O. tsutsugamushi infection decreased markedly after treating host cells with heparinase, which suggests that HSPGs play an important role in oriential infection. We tested oriential infection in REF-Syn4 cells over-expressing syndecan-4, and in REF-Syn4AS cells in which the expression of syndecan-4 was down regulated by transfecting with anti-sense syndecan-4 cDNA. Oriential infection was found to be dependent on the expression level of syndecan-4 on the cell surface. Furthermore, the infectivity of O. tsutsugamushi was specifically reduced by treating O. tsutsugamushi with the purified recombinant core protein of syndecan-4 (Syn4E). These results suggest that the core protein of syndecan-4 and the heparin/heparan sulfate chain of syndecan play an important role in oriential infection by O. tsutsugamushi. q 2004 Elsevier Ltd. All rights reserved. Keywords: Orientia tsutsugamushi; Syndecan-4; Rat embryo fibroblast; Invasion
1. Introduction Orientia tsutsugamushi is an obligate intracellular bacterium, and the causative agent of scrub typhus [1,2]. The disease is characterized by fever, rash, eschar, pneumonitis, meningitis, and disseminated intravascular coagulation (DIC), often leading to multiorgan failure [3,4]. O. tsutsugamushi usually infects a variety of cells in vitro and in vivo. These include endothelial cells [5 – 7], macrophages [8], and polymorphonuclear leukocytes [9] in humans and in experimental animals. After attachment to the host cell plasma membrane, O. tsutsugamushi induces its own uptake through a process called induced phagocytosis [10,11]. After being taken up into a phagosome, O. tsutsugamushi escapes and enters the cytoplasm. Cytosolic O. tsutsugamushi bacteria propel themselves from the cell periphery to the microtubule-organizing center (MTOC) using microtubules and dyneins. This is followed by replication in the perinuclear area [12]. Attachment to host cells and tissues is an essential step in the infectious process of intracellular bacteria, and O. tsutsugamushi probably attaches to host cells in a glycosaminoglycan (GAG)-mediated manner [13,14]. * Corresponding author. Tel.: þ 82-2-740-8304; fax: þ82-2-743-0881. E-mail address: [email protected] (I.-S. Kim). 0882-4010/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2003.12.005
The treatment of host L929 cells with heparan sulfate (HS) was found to inhibit O. tsutsugamushi infection in a dose-dependent manner, whereas treatments with other GAGs, such as chondroitin sulfate had no effect [14]. Since O. tsutsugamushi can invade a wide range of host cell types [13,15], it has been postulated that the attachment of O. tsutsugamushi might require the recognition of a ubiquitous common surface feature, such as heparan sulfate proteoglycans (HSPGs). HSPGs serves as receptors for cell attachment for a number of viruses, such as herpes simplex virus, cytomegalovirus, human immunodeficiency virus, pseudorabies virus, varicella zoster virus, foot-and-mouth disease virus, and respiratory syncytial virus, and for a number of bacteria, like Neisseria gonorrhoeae, Neisseria meningitidis, Borrelia burgdorferi, Chlamydia trachomatis, Helicobacter pylori, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus mutans [16 –18]. Proteoglycans consist of a protein core and GAG chains, which are covalently linked [19]. Syndecan, which is one of the proteoglycans present in mammals, is one of the principal HSPGs on the mammalian cell surface [20,21]. Moreover, syndecan-4 is the only syndecan component found on the majority of mammalian cell surfaces [22].
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Syndecans are known to play an important role in the attachment and uptake processes of several microbial pathogens [23 –25]. Although these studies were performed in vitro, the results indicate that syndecans may be one of the targeted surface cellular receptors present on susceptible host cells. However, no specific relation between HSPGs and oriential infection has been shown. Thus, we investigated whether the infection of host cells depends on the level of the syndecan-4 and its core protein.
2. Results 2.1. The importance of the membrane-bound HS chain To investigate the involvement of membrane bound heparin/HS in oriential infection, host cells were treated with heparinase III, which specifically hydrolyzes the glycosidic linkage present in HS [26]. Treatment with heparinase III greatly reduced the infection of various cell lines by O. tsutsugamushi. However, the infectivity of O. tsutsugamushi was not recovered by exogenously added heparin (Fig. 1). The addition of heparin and/or heparinase III was ineffective at blocking oriential infection completely, which implies the possible involvement of molecules containing membrane-bound heparin in oriential infection. In particular, these results suggest that certain HSPGs present ubiquitously in most eukaryotic cells may mediate oriential infection. 2.2. Syndecan-4 mediates oriential infection Since syndecan-4 is the only known ubiquitous component of HSPGs [22,27], we investigated its role in the infection of host cells by O. tsutsugamushi. Parental rat embryo fibroblast (REF), REF-Syn4, and REF-Syn4AS
Fig. 1. Effect of exogenous heparin on oriential infection after heparinase III treatment. Cells were treated with 25 mU heparinase III (Hept’nase) for 3 h at 37 8C, and then infected with O. tsutsugamushi in the presence and in the absence of 100 ug/ml heparin for 1 h at 4 8C on a rocking platform.
were examined for the cell surface expressions of syndecan4 and syndecan-2 by flow cytometry. REF-Syn4 overexpressed syndecan-4 [28], whereas REF-Syn4AS, which was created by stable transfection with anti-sense syndecan-4 cDNA, under-expressed syndecan-4. These two cell lines showed a significant decrease in the expressions of surface syndecan-4, whereas syndecan-2 increased in both versus the parent cells (Fig. 2A). Oriential infection in REF-Syn4 cells was increased by one-fourth or one-third versus REF cells (Fig. 2C). And, the decrease in cell surface expression shown by REF-Syn4AS significantly reduced oriential infectivity by one-third or one-half (Fig. 2B and C). Thus, the expression level of syndecan-4 seemed to be correlated with oriential infection, and syndecan-4 appeared to be necessary for infection. However, these findings do not provide sufficient evidence to enable us to suggest that syndecan-4 core protein is directly related to the process of oriential infection. 2.3. Inhibition of oriential infection by the core protein of syndecan-4 (Syn4E) To investigate which component of syndecan-4 is directly involved in this process, purified ectodomain region of syndecan-4 (Syn4E) and syndecan-2 (Syn2E) were obtained by purifying the cell extract of an over-expressing recombinant bacterial clone, which was kindly provided by Dr ES Oh (Center for Cell Signaling Research, Ewha Womans University) [29 –31]. Syn2E and Syn4E, recombinant core proteins of syndecan-2 and syndecan-4, were tested to determine whether they influence the infection of HMEC-1, HeLa, REF, or REF-Syn4AS by O. tsutsugamushi. Syn2E was chosen and used as the control to verify the specificity of syndecan-4, due to its high similarity to Syn4E at the amino acid sequence level [32]. In the presence of Syn4E (100 ug/ml), the infection of HMEC-1, HeLa, REF, or REF-Syn4 cells by O. tsutsugamushi was reduced by one-third or one-half in a dosedependent manner. On the other hand, Syn4E inhibited the infection of L929 by O. tsutsugamushi at a higher concentration (200 ug/ml) (Fig. 3A). The specificity of Syn4E was demonstrated when the addition of Syn2E to host cells was found not to inhibit infection (Fig. 3B). Syn2E only inhibited the infection of HMEC-1 cell line, and then only slightly. The infection of human umbilical vein endothelial cells (HUVECs) by O. tsutsugamushi also was inhibited by adding Syn2E (data not shown). It appears that the core protein of syndecan-2 might be involved in the infection of endothelial cells by O. tsutsugamushi. These results show that the core protein of syndecan-4 specifically inhibits oriential infection in various cell lines. To directly address the involvement core protein of syndecan-4, we used CHO pgsA-745 cells. This cell line is a defective in xylosyltransferase activity, which leads to the absence or to a much reduced level of GAG chains on the HSPG core proteins [33]. We found that the addition of
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Fig. 2. Effect of the expression level of syndecan-4 on oriential infection. (A) REF cells were stably transfected with antis-sense syndecan-4 cDNA and the expression levels of syndecan protein was analyzed by flow cytometry using anti-syndecan-2 or syndecan-4 antibodies. IgG was used as control. (B) Representative immunofluorescence microscopic images of cells infected with O. tsutsugamushi were taken using a digital camera. Cells were fixed in methanol and labeled with KI-37, a Mab against the O. tsutsugamushi 56 kDa protein, and a FITC-conjugated secondary antibody. Green spots indicate Orientia and the red background indicates the cell (magnification, £ 400). (C) Cells were infected with O. tsutsugamushi for 30 min on a rocking platform. Following infection for 90 min at 37 8C, the infected cells were washed three times with PBS and subsequently incubated in fresh medium for 2 h at 37 8C. An asterisk indicates that the levels of infection in REF cells and REF-Syn4 or REF-Syn4AS cells were significantly different (*, P , 0:01; **, P , 0:005; ***, P , 0:001). Abbreviations: REF, rat embryo fibroblast; REF-Syn4, REF over-expressing syndecan-4; REF-Syn4AS, REF transfected with anti-sense syndecan-4 cDNA; Syn-2, anti-syndecan-2 antibody; Syn-4, anti-syndecan-4 antibody; Ig-G, immunoglobulin G.
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Fig. 3. Effect of the core protein of syndecan-4 on oriential infection. (A) For dose-dependency, Syn4E was added with oriential inoculum onto growing cells at the indicated concentrations, and incubated for 1 h at 4 8C on a rocking platform. Oriential infection was initiated by moving the cells to 37 8C. Following infection for 90 min at 37 8C, the infected cells were washed and incubated as specified in Fig. 2. An asterisk indicates that a significant difference between the levels of infection shown by cells treated with Syn4E versus the untreated control cells (*, P , 0:05; **, P , 0:01; ***, P , 0:005). (B) For selectivity, Syn2E or Syn4E was added with oriential inoculum onto growing cells. An asterisk indicates a significant difference between the levels of infection of Syn2E treated cells and Syn4E treated cells (*, P , 0:05; **, P , 0:01).
Syn4E markedly reduced oriential infectivity to one-half of that of Syn2E (Fig. 4).
3. Discussion O. tsutsugamushi can invade a wide range of host cells [13,15], and attaches to the cellular surface in a HSPG-mediated manner [14]. After digesting host cells with heparinase III and then infecting host cells by O. tsutsugamushi, we found that oriential infection was reduced in various cell lines, such as L929, HMEC-1, or HeLa, and that the infectivity of O. tsutsugamushi was not recovered by exogenous heparin treatment (Fig. 1).
Fig. 4. Effect of the syndecan-4 core protein on oriential infection in a glycosaminoglycan-deficient cell line (CHO pgsA-745). Syn2E or Syn4E was added with oriential inoculum onto growing cells, and incubated for 1 h at 4 8C on a rocking platform. Oriential infection was initiated by moving the cells to 37 8C, as specified in Fig. 3. An asterisk indicates a significant difference between that the levels of inhibition to oriential infection shown by Syn4E of Syn2E treated cells versus the untreated control (**, P , 0:005).
These results suggested that some common surface feature of many different kinds of host cells may be required for cellular invasion by O. tsutsugamushi. Although no relation between HSPGs and the mediation of oriential infection bas been revealed, it seemed reasonable to investigate the possibility that O. tsutsugamushi interacts with the ubiquitously surface-expressed HSPG, syndecan-4. Syndecans can bind to a diverse group of ligands, for example, matrix components, growth factors, lipolytic enzymes, protease inhibitors, and transcriptional regulators. And, they modulate the activities of these ligands through via their HS chains [34]. Syndecans are known to serve as adhesion and internalization receptors during the cellular invasion of intracellular bacteria [16 –18]. Since a certain specific component of HSPG was suggested to be involved in the uptake of N. gonorrhoeae [25], Fresissler et al. suspected that the over-expressions of syndecan-1 and/or syndecan-4 were a cause of an increase in invasion of epithelial cells by N. gonorrhoeae. However, transfection with syndecan-1 and syndecan-4 mutant constructs lacking the intracellular domain, resulted in the inhibition of bacterial invasion [25]. Saphire et al. recently reported that HIV-1 attaches three- to five-fold better to cells expressing syndecan-1 than cells expressing glypican1 [23]. Furthermore, they showed that the introduction of syndecan-1 into non-permissive cells rendered them more susceptible, by promoting HIV-1 attachment [23]. Moreover, when syndecan was expressed in non-permissive cells, HIV-1 exhibited a higher capacity for the cellular surface via the HS chains of syndecan exclusively [24]. These results show that syndecans, acting as receptor molecules, mediate the attachment of microorganisms to host cells. Moreover, HSPGs, including syndecan and perlecan, can directly mediate an endocytic pathway for
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the internalization of atherogenic lipoproteins [35 – 37]. Since syndecan-4 provides a mechanical association between extracellular ligands and the actin cytoskeleton [38], the clustering of syndecan-4 stimulates the rearrangement of the actin cytoskeleton into ordered stress fibers. However, the recruitment of syndecans into focal complexes is dependent on the actin cytoskeleton, and may be disrupted by cytochalasin D [39]. To clarify the role of syndecan-4, a widely expressed transmembrane HSPG, we investigated whether oriential infection is affected by the cell surface expression of syndecan-4. Although the expression of syndecan-4 was not completely depleted in REF-Syn4AS cells, oriential infection was markedly decreased by . 50%. Moreover, the over-expression of syndecan-4 consistently led to the enhancement of oriential infection in REF-Syn4 cells by . 30%. These results demonstrate that the expression level of syndecan-4 is closely correlated with oriential infection. Next, we investigated whether the core protein of syndecan-4 (Syn4E) affects oriential infection of the host cells by O. tsutsugamushi. The addition of Syn4E inhibited the infection of various host cells by O. tsutsugamushi in a dose-dependent manner (Figs. 3 and 4). However, Syn2E did not significantly affect oriential infection, through O. tsutsugamushi infectivity was reduced slightly in HMEC-1 cells (Fig. 3). Further investigations are needed to determine how Syn2E inhibits oriential infection in HMEC-1 cells. However, it could be suggested that the core protein of syndecan-4 is involved in oriential infection, and that syndecan-4 is a candidate receptor molecule of oriential infection, along with heparin/HS. Although our results show the importance of syndecan-4 in the infection of host cells by O. tsutsugamushi, further investigation is needed, particularly into the role of syndecan-4 in the mechanism of cellular invasion of O. tsutsugamushi.
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(1 ug/ml; Sigma Chemical Co., St Louis, MO), epidermal growth factor (10 ng/ml; Life Technologies), penicillin (100 U/ml), and streptomycin (100 ug/ml). The CHO pgsA-745 (CRL 2242), HeLa (CCL 2), and L929 (CCL 1) cell-lines were purchased from the American Type Culture Collection, Manassas, VA, and cultured in Ham’s F12 nutrient mixture medium (Life Technologies) or in Dulbecco’s modified Eagle’s medium (DMEM; Life technologies) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 ug/ml) [12,14]. 4.2. Preparation of O. tsutsugamushi inoculum O. tsutsugamushi was propagated in monolayers of L929 cells as described previously [41]. When more than 90% of the cells were infected, as determined using an indirect immunofluorescent-antibody technique [42], the cells were collected, homogenized with a glass Dounce homogenizer (Wheaton Inc., Millville, NJ), and centrifuged at 500g for 5 min. The supernatant was then centrifuged at 10,000g for 10 min, the oriential pellet resuspended in DMEM containing 10% FBS, and stored in liquid nitrogen until required. The titer of infectivity of the inoculum was determined as described previously [12]. Briefly, two-fold serially diluted oriential samples were inoculated onto L929 cell layers in a 24-well tissue culture plate containing 12 mm diameter glass cover slips. The plate was centrifuged at 1450g for 10 min at RT. After an 8-h incubation in a humidified 5% CO2 atmosphere at 37 8C, the culture medium was removed. The cells were then washed with phosphate buffered saline (PBS), fixed in 100% methanol, and stained, as described in immunofluorescent assay below. The ratio of infected cells to the counted number of cells was determined microscopically and the number of infected cell-counting units (ICU) of the sample was calculated [8]. A total of 0.5– 2 £ 107 ICU of O. tsutsugamushi was used to infect cultured cells in the 24-well tissue culture plate.
4. Materials and methods
4.3. Indirect immunofluorescent assay
4.1. Cell culture
Infected monolayer cells were washed three times with PBS, fixed in 100% methanol for 5 min at -20 8C, and stained as described previously [14,43]. Briefly, O. tsutsugamushi was stained with Mab (KI37), against the O. tsutsugamushi 56 kDa outer membrane protein, and with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (Sigma Chemicals Co.). The cells were also treated with 0.003% Evans blue for background staining. The average number of O. tsutsugamushi per cell was determined microscopically and calculated as follows [14,43]: No. of Orientia per cell ¼ (total number of O. tsutsugamushi)/(total number of infected cells) £ (percentage of infected cells)/ 100. The infectivity (percentage of control) of oriential infection was determined by using untreated control cells, and was calculated as follows: Infectivity (% of control) ¼ 100 £ (No. of Orientia per cell in test group)/
REF cells and REF-Syn4 cells (kindly donated by Dr ES Oh), which over-express syndecan-4, were grown in alphaMEM medium. This medium was supplemented with 5% fetal bovine serum (FBS), 100 U of penicillin per milliliter, and 100 ug of streptomycin per milliliter (all from Life Technologies, Grand Island, NY) in a humidified 5% CO2 atmosphere at 37 8C. For maintenance, REF-Syn4 cells were cultured in media supplemented with 200 ug/ml G418 (Life Technologies). HMEC-1 cells, derived from human dermal microvascular endothelial cells [40], were provided by the Center for Disease Control and Prevention (CDC; Atlanta, GA) The cells were propagated in MCDB 131 medium (Life Technologies) containing 10% FBS, hydrocortisone
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(No. of Orientia per cell in the control group). The average number of O. tsutsugamushi per cell and infectivity (% of control) were used to compare cell infectivities by O. tsutsugamushi. 4.4. O. tsutsugamushi infection experiment Cells grown on 12 mm diameter glass cover slips for 24 h were infected with O. tsutsugamushi (0.5 – 2 £ 107 ICU) in medium for 30 min at RT on a rocking platform. Oriential infection was initiated by moving the cells to a humidified 5% CO2 atmosphere at 37 8C for 90 min [12]. Next, the infected cells were washed three times with PBS, incubated in fresh medium for 2 h at 37 8C, and then analyzed for infectivity as described above. In the inhibition assay involving recombinant core proteins, the cells were incubated with an appropriate concentration of purified recombinant core proteins and oriential inoculum for 1 h at 4 8C on a rocking platform. Oriential infection was initiated by moving the cells to 37 8C for 90 min, and then by following by the procedure described above. For enzymatic treatment, cells grown on 12 mm diameter glass cover slips were treated with 25 mU of heparinase III (EC 4.2.2.8; Sigma Chemicals) in Hanks’ Balanced Salt Solution (HBSS; Life Technologies), supplemented with 0.1% bovine serum albumin (BSA), for 3 h at 37 8C [14,26]. Following incubation with heparinase III enzyme, the cells were washed three times with media. They were then infected with O. tsutsugamushi in the presence and in the absence of 100 ug/ml heparin for 1 h at 4 8C on a rocking platform. The cells were then incubated for 30 min at 37 8C before immunofluorescent staining [12]. 4.5. Recombinant core proteins of syndecan-2 (Syn2E) and syndecan-4 (Syn4E) The ectodomains of syndecan-2 and syndecan-4, lacking the entire cytoplasmic domain, and subcloned into glutathione S-transferase (GST) expression vector pGEX-5X-1, were kindly provided by Dr ES Oh [29–31]. The GST-ectodomains of syndecan-2 (Syn2E) and syndecan-4 (Syn4E) were induced with 0.1 mM isopropyl b-D -thiogalactopyranose (IPTG; Sigma Chemicals Inc.) for 6 h at 37 8C as described previously [29–31]. Briefly, harvested cells were sonicated and centrifuged, and the recombinant core proteins were purified with glutathione-sepharose beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ), according to the manufacturer’s instructions. The recombinant core proteins were dialyzed versus PBS (pH 7.4) before being used. 4.6. Stable transfection of anti-sense syndecan-4 cDNA The anti-sense syndecan-4 cDNA construct was kindly donated by Dr ES Oh. For anti-sense syndecan-4 cDNA, the entire cDNA was excised from pcDNA3 vector as a 700 bp HindIII fragment using a site in the polylinker region and a
restriction site at the stop codon. The HindIII fragment was religated into pcDNA3 and fragment orientation confirmed using a unique Bam HI restriction site of the cDNA [22,31]. This construct was transfected into REF cells using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions. Transfected cell populations were selected based on the continued presence of G418 (Life Technologies). To examine syndecan-4 cell surface expression, the cells were analyzed for the expressional levels of syndecan-2 or syndecan-4 by flow cytometry (FACS) for each individual clone, as described previously [22,31]. Briefly, cells were cultured in 10 cm diameter tissue culture dishes, and then washed with PBS and detached using trypsin (wt/vol)/1 mM EDTA. This was followed by sequential washing in culture medium and FACS buffer (1X PBS, 10% FBS). Cells were then incubated on ice for 1 h with anti-syndecan-2 [31,44] or anti-syndecan-4 [31,45] antibody in FACS buffer, as described previously. The cells were then washed three times with PBS containing 0.05% Tween-20 and incubated with FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in FACS buffer for 30 min on ice. 4.7. Statistical analysis All experiments were performed at least three times in duplicate. The results are presented in bar graphs as averages value ^ the standard error (SE). Differences in results between the control and test groups were compared using a paired Student t test. Significance was accepted at P , 0:05:
Acknowledgements We express our gratitude to Dr Eok-Soo Oh (Center for Cell Signaling Research, Ewha Womans University) for providing us with materials necessary to carry out this work, and to all members of his laboratory for invaluable assistance. This study was supported by a grant from the Ministry of Health and Welfare (grant 01-PJ10-PG6-01GM01-0004).
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Role of Syndecan-4 in the cellular invasion of Orientia tsutsugamushi Hang-Rae Kim, Myung-Sik Choi, Ik-Sang Kim* Department of Microbiology and Immunology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea Received 1 September 2003; received in revised form 6 November 2003; accepted 15 December 2003
Abstract Cell surface heparan sulfate proteoglycans (HSPGs) play a critical role in the cellular invasion of intracellular bacteria and are presumed to have a role in the infection of host cells by Orientia tsutsugamushi. Previously, we showed that O. tsutsugamushi infection decreased markedly after treating host cells with heparinase, which suggests that HSPGs play an important role in oriential infection. We tested oriential infection in REF-Syn4 cells over-expressing syndecan-4, and in REF-Syn4AS cells in which the expression of syndecan-4 was down regulated by transfecting with anti-sense syndecan-4 cDNA. Oriential infection was found to be dependent on the expression level of syndecan-4 on the cell surface. Furthermore, the infectivity of O. tsutsugamushi was specifically reduced by treating O. tsutsugamushi with the purified recombinant core protein of syndecan-4 (Syn4E). These results suggest that the core protein of syndecan-4 and the heparin/heparan sulfate chain of syndecan play an important role in oriential infection by O. tsutsugamushi. q 2004 Elsevier Ltd. All rights reserved. Keywords: Orientia tsutsugamushi; Syndecan-4; Rat embryo fibroblast; Invasion
1. Introduction Orientia tsutsugamushi is an obligate intracellular bacterium, and the causative agent of scrub typhus [1,2]. The disease is characterized by fever, rash, eschar, pneumonitis, meningitis, and disseminated intravascular coagulation (DIC), often leading to multiorgan failure [3,4]. O. tsutsugamushi usually infects a variety of cells in vitro and in vivo. These include endothelial cells [5 – 7], macrophages [8], and polymorphonuclear leukocytes [9] in humans and in experimental animals. After attachment to the host cell plasma membrane, O. tsutsugamushi induces its own uptake through a process called induced phagocytosis [10,11]. After being taken up into a phagosome, O. tsutsugamushi escapes and enters the cytoplasm. Cytosolic O. tsutsugamushi bacteria propel themselves from the cell periphery to the microtubule-organizing center (MTOC) using microtubules and dyneins. This is followed by replication in the perinuclear area [12]. Attachment to host cells and tissues is an essential step in the infectious process of intracellular bacteria, and O. tsutsugamushi probably attaches to host cells in a glycosaminoglycan (GAG)-mediated manner [13,14]. * Corresponding author. Tel.: þ 82-2-740-8304; fax: þ82-2-743-0881. E-mail address: [email protected] (I.-S. Kim). 0882-4010/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2003.12.005
The treatment of host L929 cells with heparan sulfate (HS) was found to inhibit O. tsutsugamushi infection in a dose-dependent manner, whereas treatments with other GAGs, such as chondroitin sulfate had no effect [14]. Since O. tsutsugamushi can invade a wide range of host cell types [13,15], it has been postulated that the attachment of O. tsutsugamushi might require the recognition of a ubiquitous common surface feature, such as heparan sulfate proteoglycans (HSPGs). HSPGs serves as receptors for cell attachment for a number of viruses, such as herpes simplex virus, cytomegalovirus, human immunodeficiency virus, pseudorabies virus, varicella zoster virus, foot-and-mouth disease virus, and respiratory syncytial virus, and for a number of bacteria, like Neisseria gonorrhoeae, Neisseria meningitidis, Borrelia burgdorferi, Chlamydia trachomatis, Helicobacter pylori, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus mutans [16 –18]. Proteoglycans consist of a protein core and GAG chains, which are covalently linked [19]. Syndecan, which is one of the proteoglycans present in mammals, is one of the principal HSPGs on the mammalian cell surface [20,21]. Moreover, syndecan-4 is the only syndecan component found on the majority of mammalian cell surfaces [22].
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Syndecans are known to play an important role in the attachment and uptake processes of several microbial pathogens [23 –25]. Although these studies were performed in vitro, the results indicate that syndecans may be one of the targeted surface cellular receptors present on susceptible host cells. However, no specific relation between HSPGs and oriential infection has been shown. Thus, we investigated whether the infection of host cells depends on the level of the syndecan-4 and its core protein.
2. Results 2.1. The importance of the membrane-bound HS chain To investigate the involvement of membrane bound heparin/HS in oriential infection, host cells were treated with heparinase III, which specifically hydrolyzes the glycosidic linkage present in HS [26]. Treatment with heparinase III greatly reduced the infection of various cell lines by O. tsutsugamushi. However, the infectivity of O. tsutsugamushi was not recovered by exogenously added heparin (Fig. 1). The addition of heparin and/or heparinase III was ineffective at blocking oriential infection completely, which implies the possible involvement of molecules containing membrane-bound heparin in oriential infection. In particular, these results suggest that certain HSPGs present ubiquitously in most eukaryotic cells may mediate oriential infection. 2.2. Syndecan-4 mediates oriential infection Since syndecan-4 is the only known ubiquitous component of HSPGs [22,27], we investigated its role in the infection of host cells by O. tsutsugamushi. Parental rat embryo fibroblast (REF), REF-Syn4, and REF-Syn4AS
Fig. 1. Effect of exogenous heparin on oriential infection after heparinase III treatment. Cells were treated with 25 mU heparinase III (Hept’nase) for 3 h at 37 8C, and then infected with O. tsutsugamushi in the presence and in the absence of 100 ug/ml heparin for 1 h at 4 8C on a rocking platform.
were examined for the cell surface expressions of syndecan4 and syndecan-2 by flow cytometry. REF-Syn4 overexpressed syndecan-4 [28], whereas REF-Syn4AS, which was created by stable transfection with anti-sense syndecan-4 cDNA, under-expressed syndecan-4. These two cell lines showed a significant decrease in the expressions of surface syndecan-4, whereas syndecan-2 increased in both versus the parent cells (Fig. 2A). Oriential infection in REF-Syn4 cells was increased by one-fourth or one-third versus REF cells (Fig. 2C). And, the decrease in cell surface expression shown by REF-Syn4AS significantly reduced oriential infectivity by one-third or one-half (Fig. 2B and C). Thus, the expression level of syndecan-4 seemed to be correlated with oriential infection, and syndecan-4 appeared to be necessary for infection. However, these findings do not provide sufficient evidence to enable us to suggest that syndecan-4 core protein is directly related to the process of oriential infection. 2.3. Inhibition of oriential infection by the core protein of syndecan-4 (Syn4E) To investigate which component of syndecan-4 is directly involved in this process, purified ectodomain region of syndecan-4 (Syn4E) and syndecan-2 (Syn2E) were obtained by purifying the cell extract of an over-expressing recombinant bacterial clone, which was kindly provided by Dr ES Oh (Center for Cell Signaling Research, Ewha Womans University) [29 –31]. Syn2E and Syn4E, recombinant core proteins of syndecan-2 and syndecan-4, were tested to determine whether they influence the infection of HMEC-1, HeLa, REF, or REF-Syn4AS by O. tsutsugamushi. Syn2E was chosen and used as the control to verify the specificity of syndecan-4, due to its high similarity to Syn4E at the amino acid sequence level [32]. In the presence of Syn4E (100 ug/ml), the infection of HMEC-1, HeLa, REF, or REF-Syn4 cells by O. tsutsugamushi was reduced by one-third or one-half in a dosedependent manner. On the other hand, Syn4E inhibited the infection of L929 by O. tsutsugamushi at a higher concentration (200 ug/ml) (Fig. 3A). The specificity of Syn4E was demonstrated when the addition of Syn2E to host cells was found not to inhibit infection (Fig. 3B). Syn2E only inhibited the infection of HMEC-1 cell line, and then only slightly. The infection of human umbilical vein endothelial cells (HUVECs) by O. tsutsugamushi also was inhibited by adding Syn2E (data not shown). It appears that the core protein of syndecan-2 might be involved in the infection of endothelial cells by O. tsutsugamushi. These results show that the core protein of syndecan-4 specifically inhibits oriential infection in various cell lines. To directly address the involvement core protein of syndecan-4, we used CHO pgsA-745 cells. This cell line is a defective in xylosyltransferase activity, which leads to the absence or to a much reduced level of GAG chains on the HSPG core proteins [33]. We found that the addition of
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Fig. 2. Effect of the expression level of syndecan-4 on oriential infection. (A) REF cells were stably transfected with antis-sense syndecan-4 cDNA and the expression levels of syndecan protein was analyzed by flow cytometry using anti-syndecan-2 or syndecan-4 antibodies. IgG was used as control. (B) Representative immunofluorescence microscopic images of cells infected with O. tsutsugamushi were taken using a digital camera. Cells were fixed in methanol and labeled with KI-37, a Mab against the O. tsutsugamushi 56 kDa protein, and a FITC-conjugated secondary antibody. Green spots indicate Orientia and the red background indicates the cell (magnification, £ 400). (C) Cells were infected with O. tsutsugamushi for 30 min on a rocking platform. Following infection for 90 min at 37 8C, the infected cells were washed three times with PBS and subsequently incubated in fresh medium for 2 h at 37 8C. An asterisk indicates that the levels of infection in REF cells and REF-Syn4 or REF-Syn4AS cells were significantly different (*, P , 0:01; **, P , 0:005; ***, P , 0:001). Abbreviations: REF, rat embryo fibroblast; REF-Syn4, REF over-expressing syndecan-4; REF-Syn4AS, REF transfected with anti-sense syndecan-4 cDNA; Syn-2, anti-syndecan-2 antibody; Syn-4, anti-syndecan-4 antibody; Ig-G, immunoglobulin G.
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Fig. 3. Effect of the core protein of syndecan-4 on oriential infection. (A) For dose-dependency, Syn4E was added with oriential inoculum onto growing cells at the indicated concentrations, and incubated for 1 h at 4 8C on a rocking platform. Oriential infection was initiated by moving the cells to 37 8C. Following infection for 90 min at 37 8C, the infected cells were washed and incubated as specified in Fig. 2. An asterisk indicates that a significant difference between the levels of infection shown by cells treated with Syn4E versus the untreated control cells (*, P , 0:05; **, P , 0:01; ***, P , 0:005). (B) For selectivity, Syn2E or Syn4E was added with oriential inoculum onto growing cells. An asterisk indicates a significant difference between the levels of infection of Syn2E treated cells and Syn4E treated cells (*, P , 0:05; **, P , 0:01).
Syn4E markedly reduced oriential infectivity to one-half of that of Syn2E (Fig. 4).
3. Discussion O. tsutsugamushi can invade a wide range of host cells [13,15], and attaches to the cellular surface in a HSPG-mediated manner [14]. After digesting host cells with heparinase III and then infecting host cells by O. tsutsugamushi, we found that oriential infection was reduced in various cell lines, such as L929, HMEC-1, or HeLa, and that the infectivity of O. tsutsugamushi was not recovered by exogenous heparin treatment (Fig. 1).
Fig. 4. Effect of the syndecan-4 core protein on oriential infection in a glycosaminoglycan-deficient cell line (CHO pgsA-745). Syn2E or Syn4E was added with oriential inoculum onto growing cells, and incubated for 1 h at 4 8C on a rocking platform. Oriential infection was initiated by moving the cells to 37 8C, as specified in Fig. 3. An asterisk indicates a significant difference between that the levels of inhibition to oriential infection shown by Syn4E of Syn2E treated cells versus the untreated control (**, P , 0:005).
These results suggested that some common surface feature of many different kinds of host cells may be required for cellular invasion by O. tsutsugamushi. Although no relation between HSPGs and the mediation of oriential infection bas been revealed, it seemed reasonable to investigate the possibility that O. tsutsugamushi interacts with the ubiquitously surface-expressed HSPG, syndecan-4. Syndecans can bind to a diverse group of ligands, for example, matrix components, growth factors, lipolytic enzymes, protease inhibitors, and transcriptional regulators. And, they modulate the activities of these ligands through via their HS chains [34]. Syndecans are known to serve as adhesion and internalization receptors during the cellular invasion of intracellular bacteria [16 –18]. Since a certain specific component of HSPG was suggested to be involved in the uptake of N. gonorrhoeae [25], Fresissler et al. suspected that the over-expressions of syndecan-1 and/or syndecan-4 were a cause of an increase in invasion of epithelial cells by N. gonorrhoeae. However, transfection with syndecan-1 and syndecan-4 mutant constructs lacking the intracellular domain, resulted in the inhibition of bacterial invasion [25]. Saphire et al. recently reported that HIV-1 attaches three- to five-fold better to cells expressing syndecan-1 than cells expressing glypican1 [23]. Furthermore, they showed that the introduction of syndecan-1 into non-permissive cells rendered them more susceptible, by promoting HIV-1 attachment [23]. Moreover, when syndecan was expressed in non-permissive cells, HIV-1 exhibited a higher capacity for the cellular surface via the HS chains of syndecan exclusively [24]. These results show that syndecans, acting as receptor molecules, mediate the attachment of microorganisms to host cells. Moreover, HSPGs, including syndecan and perlecan, can directly mediate an endocytic pathway for
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the internalization of atherogenic lipoproteins [35 – 37]. Since syndecan-4 provides a mechanical association between extracellular ligands and the actin cytoskeleton [38], the clustering of syndecan-4 stimulates the rearrangement of the actin cytoskeleton into ordered stress fibers. However, the recruitment of syndecans into focal complexes is dependent on the actin cytoskeleton, and may be disrupted by cytochalasin D [39]. To clarify the role of syndecan-4, a widely expressed transmembrane HSPG, we investigated whether oriential infection is affected by the cell surface expression of syndecan-4. Although the expression of syndecan-4 was not completely depleted in REF-Syn4AS cells, oriential infection was markedly decreased by . 50%. Moreover, the over-expression of syndecan-4 consistently led to the enhancement of oriential infection in REF-Syn4 cells by . 30%. These results demonstrate that the expression level of syndecan-4 is closely correlated with oriential infection. Next, we investigated whether the core protein of syndecan-4 (Syn4E) affects oriential infection of the host cells by O. tsutsugamushi. The addition of Syn4E inhibited the infection of various host cells by O. tsutsugamushi in a dose-dependent manner (Figs. 3 and 4). However, Syn2E did not significantly affect oriential infection, through O. tsutsugamushi infectivity was reduced slightly in HMEC-1 cells (Fig. 3). Further investigations are needed to determine how Syn2E inhibits oriential infection in HMEC-1 cells. However, it could be suggested that the core protein of syndecan-4 is involved in oriential infection, and that syndecan-4 is a candidate receptor molecule of oriential infection, along with heparin/HS. Although our results show the importance of syndecan-4 in the infection of host cells by O. tsutsugamushi, further investigation is needed, particularly into the role of syndecan-4 in the mechanism of cellular invasion of O. tsutsugamushi.
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(1 ug/ml; Sigma Chemical Co., St Louis, MO), epidermal growth factor (10 ng/ml; Life Technologies), penicillin (100 U/ml), and streptomycin (100 ug/ml). The CHO pgsA-745 (CRL 2242), HeLa (CCL 2), and L929 (CCL 1) cell-lines were purchased from the American Type Culture Collection, Manassas, VA, and cultured in Ham’s F12 nutrient mixture medium (Life Technologies) or in Dulbecco’s modified Eagle’s medium (DMEM; Life technologies) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 ug/ml) [12,14]. 4.2. Preparation of O. tsutsugamushi inoculum O. tsutsugamushi was propagated in monolayers of L929 cells as described previously [41]. When more than 90% of the cells were infected, as determined using an indirect immunofluorescent-antibody technique [42], the cells were collected, homogenized with a glass Dounce homogenizer (Wheaton Inc., Millville, NJ), and centrifuged at 500g for 5 min. The supernatant was then centrifuged at 10,000g for 10 min, the oriential pellet resuspended in DMEM containing 10% FBS, and stored in liquid nitrogen until required. The titer of infectivity of the inoculum was determined as described previously [12]. Briefly, two-fold serially diluted oriential samples were inoculated onto L929 cell layers in a 24-well tissue culture plate containing 12 mm diameter glass cover slips. The plate was centrifuged at 1450g for 10 min at RT. After an 8-h incubation in a humidified 5% CO2 atmosphere at 37 8C, the culture medium was removed. The cells were then washed with phosphate buffered saline (PBS), fixed in 100% methanol, and stained, as described in immunofluorescent assay below. The ratio of infected cells to the counted number of cells was determined microscopically and the number of infected cell-counting units (ICU) of the sample was calculated [8]. A total of 0.5– 2 £ 107 ICU of O. tsutsugamushi was used to infect cultured cells in the 24-well tissue culture plate.
4. Materials and methods
4.3. Indirect immunofluorescent assay
4.1. Cell culture
Infected monolayer cells were washed three times with PBS, fixed in 100% methanol for 5 min at -20 8C, and stained as described previously [14,43]. Briefly, O. tsutsugamushi was stained with Mab (KI37), against the O. tsutsugamushi 56 kDa outer membrane protein, and with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (Sigma Chemicals Co.). The cells were also treated with 0.003% Evans blue for background staining. The average number of O. tsutsugamushi per cell was determined microscopically and calculated as follows [14,43]: No. of Orientia per cell ¼ (total number of O. tsutsugamushi)/(total number of infected cells) £ (percentage of infected cells)/ 100. The infectivity (percentage of control) of oriential infection was determined by using untreated control cells, and was calculated as follows: Infectivity (% of control) ¼ 100 £ (No. of Orientia per cell in test group)/
REF cells and REF-Syn4 cells (kindly donated by Dr ES Oh), which over-express syndecan-4, were grown in alphaMEM medium. This medium was supplemented with 5% fetal bovine serum (FBS), 100 U of penicillin per milliliter, and 100 ug of streptomycin per milliliter (all from Life Technologies, Grand Island, NY) in a humidified 5% CO2 atmosphere at 37 8C. For maintenance, REF-Syn4 cells were cultured in media supplemented with 200 ug/ml G418 (Life Technologies). HMEC-1 cells, derived from human dermal microvascular endothelial cells [40], were provided by the Center for Disease Control and Prevention (CDC; Atlanta, GA) The cells were propagated in MCDB 131 medium (Life Technologies) containing 10% FBS, hydrocortisone
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(No. of Orientia per cell in the control group). The average number of O. tsutsugamushi per cell and infectivity (% of control) were used to compare cell infectivities by O. tsutsugamushi. 4.4. O. tsutsugamushi infection experiment Cells grown on 12 mm diameter glass cover slips for 24 h were infected with O. tsutsugamushi (0.5 – 2 £ 107 ICU) in medium for 30 min at RT on a rocking platform. Oriential infection was initiated by moving the cells to a humidified 5% CO2 atmosphere at 37 8C for 90 min [12]. Next, the infected cells were washed three times with PBS, incubated in fresh medium for 2 h at 37 8C, and then analyzed for infectivity as described above. In the inhibition assay involving recombinant core proteins, the cells were incubated with an appropriate concentration of purified recombinant core proteins and oriential inoculum for 1 h at 4 8C on a rocking platform. Oriential infection was initiated by moving the cells to 37 8C for 90 min, and then by following by the procedure described above. For enzymatic treatment, cells grown on 12 mm diameter glass cover slips were treated with 25 mU of heparinase III (EC 4.2.2.8; Sigma Chemicals) in Hanks’ Balanced Salt Solution (HBSS; Life Technologies), supplemented with 0.1% bovine serum albumin (BSA), for 3 h at 37 8C [14,26]. Following incubation with heparinase III enzyme, the cells were washed three times with media. They were then infected with O. tsutsugamushi in the presence and in the absence of 100 ug/ml heparin for 1 h at 4 8C on a rocking platform. The cells were then incubated for 30 min at 37 8C before immunofluorescent staining [12]. 4.5. Recombinant core proteins of syndecan-2 (Syn2E) and syndecan-4 (Syn4E) The ectodomains of syndecan-2 and syndecan-4, lacking the entire cytoplasmic domain, and subcloned into glutathione S-transferase (GST) expression vector pGEX-5X-1, were kindly provided by Dr ES Oh [29–31]. The GST-ectodomains of syndecan-2 (Syn2E) and syndecan-4 (Syn4E) were induced with 0.1 mM isopropyl b-D -thiogalactopyranose (IPTG; Sigma Chemicals Inc.) for 6 h at 37 8C as described previously [29–31]. Briefly, harvested cells were sonicated and centrifuged, and the recombinant core proteins were purified with glutathione-sepharose beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ), according to the manufacturer’s instructions. The recombinant core proteins were dialyzed versus PBS (pH 7.4) before being used. 4.6. Stable transfection of anti-sense syndecan-4 cDNA The anti-sense syndecan-4 cDNA construct was kindly donated by Dr ES Oh. For anti-sense syndecan-4 cDNA, the entire cDNA was excised from pcDNA3 vector as a 700 bp HindIII fragment using a site in the polylinker region and a
restriction site at the stop codon. The HindIII fragment was religated into pcDNA3 and fragment orientation confirmed using a unique Bam HI restriction site of the cDNA [22,31]. This construct was transfected into REF cells using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions. Transfected cell populations were selected based on the continued presence of G418 (Life Technologies). To examine syndecan-4 cell surface expression, the cells were analyzed for the expressional levels of syndecan-2 or syndecan-4 by flow cytometry (FACS) for each individual clone, as described previously [22,31]. Briefly, cells were cultured in 10 cm diameter tissue culture dishes, and then washed with PBS and detached using trypsin (wt/vol)/1 mM EDTA. This was followed by sequential washing in culture medium and FACS buffer (1X PBS, 10% FBS). Cells were then incubated on ice for 1 h with anti-syndecan-2 [31,44] or anti-syndecan-4 [31,45] antibody in FACS buffer, as described previously. The cells were then washed three times with PBS containing 0.05% Tween-20 and incubated with FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in FACS buffer for 30 min on ice. 4.7. Statistical analysis All experiments were performed at least three times in duplicate. The results are presented in bar graphs as averages value ^ the standard error (SE). Differences in results between the control and test groups were compared using a paired Student t test. Significance was accepted at P , 0:05:
Acknowledgements We express our gratitude to Dr Eok-Soo Oh (Center for Cell Signaling Research, Ewha Womans University) for providing us with materials necessary to carry out this work, and to all members of his laboratory for invaluable assistance. This study was supported by a grant from the Ministry of Health and Welfare (grant 01-PJ10-PG6-01GM01-0004).
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