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CD44 facilitates adherence of Streptococcus equi subsp. zooepidemicus to LA-4 cells
Microbial Pathogenesis 128 (2019) 250–253
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
CD44 facilitates adherence of Streptococcus equi subsp. zooepidemicus to LA-4 cells
T
Qiang Fua,b, Wenwen Lic, Shun Lib, Xianjie Zhaob, Honglin Xieb, Xi Zhangb, Kangjian Lib, Chunquan Mab, Xiaohong Liua,∗ a
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, China School of Life Science, Foshan University, Guangdong, 528225, China c Laboratory Animal Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Streptococcus equi subsp. zooepidemicus CD44 Adherence
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) causes a wide variety of infections in many species. CD44 is a transmembrane adhesion molecule, expressed by various cell types, which has been implicated in several infection processes. The aim of this study was to examine the role of CD44 in S. zooepidemicus adherence to LA-4 cells (mouse lung adenoma). Dose-dependent adhesion with LA-4 may be effectively studied by flow cytometry. Adherence of S. zooepidemicus is reduced after treatment of cells with anti-CD44 antibody. Treatment of S. zooepidemicus with recombinant CD44 significantly reduced bacteria adherence. In addition, CD44 can directly bind to wild-type S. zooepidemicus, while the binding was decreased in the capsule deletion isogenic mutant. These data suggest that CD44 facilitates adherence of S. zooepidemicus to LA-4 cells.
1. Introduction
2. Materials and methods
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) caused variety of infections in many species, including horses, dogs, and pigs [1–4]. These infections have led to significant welfare problems and economic costs, most notably in the training of racehorses. We recently reported that S. zooepidemicus infection led to a significant increase in CD44 mRNA level, suggesting that CD44 plays an important role in S. zooepidemicus infection [5]. CD44 is a transmembrane adhesion molecule, which is expressed on various cell types, including epithelial cells [6,7]. The effect of CD44 on host response is different, depending on the pathogen species and the primary site of infection [8–10]. Several studies have indicated CD44 is involved in the process of host defense against pathogenic microorganisms, including the binding and processing of pathogens [11,12]. In addition, recent studies have shown that S. zooepidemicus capsule contributes to the attachment and invasion of host cells [13,14]. However, the contribution of CD44 to S. zooepidemicus attachment is not well defined. Here, we assessed the role of CD44 in S. zooepidemicus adherence to LA-4 cells (mouse lung adenoma), focusing on the interference with S. zooepidemicus capsule and transmembrane CD44, in addition to specific antibodies and recombinant CD44 protein treatment.
2.1. Bacterial strain and growth conditions
∗
S. zooepidemicus wild-type strain C55138 (China Institute of Veterinary Drug Control) and isogenic mutant ΔhasB were maintained in our laboratory. Bacteria were cultured in aerobic conditions at 37 °C on tryptone soya broth (TSB; Oxoid) or tryptone soya agar (TSA; Oxoid) supplemented with 5% newborn calf serum. 2.2. Cell culture Maintenance of LA-4 cells (ATCC CCL-196), a murine bronchial epithelial cell line, required Ham's Fe12K medium (Gibco); additional supplements of 2 mM L-glutamine, and 15% heat-inactivated fetal calf serum. Cell lines were grown at 37 °C in a humidified atmosphere with 5% CO2 and passaged with trypsin-EDTA (ethylenediaminetetraacetic acid) when confluent. 2.3. Adhesion of bacteria to LA-4 cells Fluorescent staining of S. zooepidemicus was performed according the method used in the previous study, with some modification [15].
Corresponding author. State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-sen University, Guangzhou, 510006, China. E-mail address: [email protected] (X. Liu).
https://doi.org/10.1016/j.micpath.2019.01.016 Received 18 March 2018; Received in revised form 8 January 2019; Accepted 8 January 2019 Available online 09 January 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Increased induction of CD44 on LA-4 cells after S. zooepidemicus exposure. (A) CD44 mRNA levels were determined by real-time RT-PCR. (B) Expression of CD44 on LA-4 cells was determined by flow cytometry. The data are showed as the means ± SD analyzed by Student's t-tests using GraphPad Prism version 5.0 (*, P < 0.05; **, P < 0.01).
Fig. 3. Time course studies of the adherence of FAMSE-labeled S. zooepidemicus to LA-4 cells. Cells were incubated with S. zooepidemicus C55138 at MOI of 100:1 for 30, 60, 90, 120, 180 min at 37 °C, and adherence was analyzed by flow cytometry. The results are presented as the percentages of LA-4 cells with attached S. zooepidemicus (black bars) and the FL-1 fluorescence emitted from the attached S. zooepidemicus as indicated by MFI ± standard deviation (white bars).
Fig. 2. Adherence of FAMSE-labeled S. zooepidemicus to LA-4 cells. Cells were incubated with S. zooepidemicus C55138 at MOI of 1:1, 10:1, 100:1, 500:1, 1000:1 for 60 min at 37 °C, and adherence was analyzed by flow cytometry. (A) Dot plot display mode of FL-1 versus side scatter (SSC) after the incubation of the cells with labeled bacteria. (B) The results are presented as the percentages of LA-4 cells with attached S. zooepidemicus (black bars) and the FL-1 fluorescence emitted from the attached S. zooepidemicus as indicated by MFI ± standard deviation (white bars).
Fig. 4. Pretreatment of LA-4 cells with anti-CD44 antibody reduced S. zooepidemicus adherence to cells. LA-4 cells were immersed in medium alone, the isotype control, or anti-CD44 for 30 min at 37 °C, and adherence was analyzed by flow cytometry (*, P < 0.05; **, P < 0.01 versus the cells treated with isotype control).
statically co-incubated with LA-4 cells at bacterial cell ratio (BCR) of 1:1, 10:1, 100:1, 500:1, and 1000:1 for 60 min at 37 °C. Time course study of the adherence was performed at a BCR of 100:1 for 30, 60, 90, 120, 180 min. Following incubation, the LA-4 cells were fixed with 2% formaldehyde and analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the FL-1 channel to measure fluorescence intensity.
Briefly, 6-carboxyfluorescein succinimidyl ester (FAMSE; Molecular Probes, 34 μg) was added to 10 ml S. zooepidemicus in phosphate-buffered saline (PBS) and the bacterial cells were incubated with gentle rotation for 20 min at 37 °C. Following staining, the bacterial cells were harvested by centrifugation at 12,000×g, washed once in a large volume of PBS, and suspended in PBS. Labeled bacterial cells were 251
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S. zooepidemicus was incubated with 1 ng of mouse CD44 for 30 min at 37 °C. After incubation, the bacteria were centrifuged at 12,000×g, and the excess CD44 was removed by washing the bacteria twice in PBS. The adherence assay was performed as described above at a BCR of 100:1. 2.5. Binding of CD44 to S. zooepidemicus Binding of CD44 to S. zooepidemicus was performed according the method of previously study [16]. Wild-type S. zooepidemicus or capsule deletion mutant ΔhasB were incubated with 1 ng of mouse CD44 for 30 min at 37 °C. Then, the bacteria were centrifuged for 15 min at 12,000×g and incubated with FITC-conjugated CD44 antibody (BioLegend) for 30 min on ice. After incubation, bacterial cells were centrifuged, washed, and fixed with paraformaldehyde. The binding was determined on a FACSCalibur flow cytometer (Becton Dickinson). Forward and side scatter properties were used to exclude debris and aggregates, and 30,000 gated events were recorded. M1 marker set to include > 98% unlabeled bacteria. The percentage of fluorescent bacteria (brighter than M1 fluorescence intensity units on the FL1 axis) was calculated for each sample.
Fig. 5. Pretreatment of S. zooepidemicus with recombinant CD44 protein inhibit S. zooepidemicus adherence to LA-4 cells. Labeled S. zooepidemicus were incubated with mouse CD44 for 30 min at 37 °C, and adherence was analyzed by flow cytometry (*, P < 0.05; **, P < 0.01 versus the cells incubated with BSA-treated S. zooepidemicus).
3. Results 3.1. S. zooepidemicus exposure increased induction of CD44 on LA-4 cells To investigate whether S. zooepidemicus influences CD44 expression on LA-4 cells, relative quantification of gene transcript was examined by real-time PCR. The real-time PCR revealed that CD44 mRNA level was increased from S. zooepidemicus exposure (Fig. 1A). In addition, the expression of CD44 on LA-4 cells after S. zooepidemicus exposure was determined by flow cytometry. Treatment of LA-4 cells with S. zooepidemicus increased CD44 expression (Fig. 1B). The results confirmed that expression of CD44 on LA-4 cells was significantly up-regulated after S. zooepidemicus exposure. 3.2. Flow cytometric analysis of S. zooepidemicus adherence to LA-4 cells
Fig. 6. Binding of CD44 to wild-type S. zooepidemicus and capsule deletion mutantΔhasB. (A) Binding of CD44 to S. zooepidemicus was detected by flow cytometry using FITC-conjugated CD44 antibody. (B) Examples of flow cytometry histograms for binding of CD44 to wild-type S. zooepidemicus and capsule deletion mutant ΔhasB are shown, BSA treated cells are represented in shaded histograms and CD44 treated cells open histograms.
Adherence to LA-4 cells of S. zooepidemicus was analyzed by flow cytometry using the FL-1 channel to determine the mean fluorescence intensity (MFI). The MFI marker M was set to include less than 2% of the negative control (uninfected) cells (Fig. 2A). The percentage of LA4 cells with S. zooepidemicus attached was identified by gating on fluorescence. There was an increase in MFI in LA-4 cells incubated with increasing numbers of S. zooepidemicus (Fig. 2B). Furthermore, the percentage of LA-4 cells with S. zooepidemicus attached increased with a corresponding increase in the BCR (Fig. 2B). As the time of exposure to bacteria increased, the MFI in LA-4 cells and the percentage of LA4 cells with S. zooepidemicus attached increased accordingly (Fig. 3).
2.4. Effect of CD44 antibodies and recombinant CD44 protein on bacterial adherence
3.3. Adherence of S. zooepidemicus is reduced after treatment of epithelial cells with CD44 antibodies
Inhibition of S. zooepidemicus adherence by CD44 antibodies was analyzed. LA-4 cells were washed with PBS and treated with CD44 antibodies (BD Pharmingen, 20 μg/ml) or control antibodies (BD Pharmingen, 20 μg/ml) for 30 min at 37 °C. After washing to remove excess antibodies, cells were incubated with labeled S. zooepidemicus for 30 min at a BCR of 100:1. LA-4 cells were washed and fixed with 2% formaldehyde prior to flow cytometry. The complete CDS of Mus musculus CD44 mRNA (cDNA clone MGC: 58,984 IMAGE: 4910789) was synthesized by Sangon Biotech Shanghai Co Ltd. The amino acid sequence of CD44 was expressed in E. coli strains BL21 as described in our previous study [16]. The ability of recombinant CD44 protein to inhibit labeled bacteria adherence to LA-4 cells was analyzed. Labeled
To investigate whether CD44 antibodies influence binding of S. zooepidemicus to LA-4 cells, the cells were pretreated with CD44 antibodies and isotypes. Treatment of LA-4 cells with anti-CD44 antibodies significantly reduced bacterial adherence to LA-4 cells (Fig. 4). 3.4. Recombinant CD44 protein inhibit S. zooepidemicus adherence to LA4 cells The contribution of the CD44 activity to the binding of S. zooepidemicus to LA-4 cells was investigated by pretreatment of S. zooepidemicus with CD44. Both the MFI value and the percentage of LA-4 cells with S. zooepidemicus attached to cells incubated with CD44-treated S. zooepidemicus was significantly smaller than the cells incubated with 252
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Research of Emerging Animal (KLPREAD201801-03).
BSA-treated S. zooepidemicus (Fig. 5). 3.5. Binding of CD44 to wild-type S. zooepidemicus and capsule deletion mutant ΔhasB
Diseases
in Foshan
University
References
The contribution of the CD44 activity to the binding of S. zooepidemicus to LA-4 cells was investigated by wild-type S. zooepidemicus and capsule deletion mutant ΔhasB. The MFI value of LA-4 cells incubated with S. zooepidemicus ΔhasB was significantly smaller than that of LA4 cells incubated with wild-type S. zooepidemicus (Fig. 6).
[1] N. Decaro, V. Mari, V. Larocca, M. Losurdo, G. Lanave, M.S. Lucente, et al., Molecular surveillance of traditional and emerging pathogens associated with canine infectious respiratory disease, Vet. Microbiol. 192 (2016) 21–25. [2] K. Gruszynski, A. Young, S.J. Levine, J.P. Garvin, S. Brown, L. Turner, et al., Streptococcus equi subsp. zooepidemicus infections associated with Guinea pigs, Emerg. Infect. Dis. 21 (2015) 156–158. [3] S. Velineni, R. DeNegri, S.C. Artiushin, J.F. Timoney, Comparison of specificities of serum antibody responses of horses to clinical infections caused by Streptococcus equi or zooepidemicus, Vet. Microbiol. 180 (2015) 253–259. [4] M.R. Petersen, B. Skive, M. Christoffersen, K. Lu, J.M. Nielsen, M.H. Troedsson, et al., Activation of persistent Streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis, Vet. Microbiol. 179 (2015) 119–125. [5] Q. Fu, P. Xiao, Y. Chen, Z. Wei, X. Liu, CD44 deficiency enhanced Streptococcus equi ssp. zooepidemicus dissemination and inflammation response in a mouse model, Res. Vet. Sci. 115 (2017) 96–101. [6] O. Babasola, K.J. Rees-Milton, S. Bebe, J. Wang, T.P. Anastassiades, Chemically modified N-acylated hyaluronan fragments modulate proinflammatory cytokine production by stimulated human macrophages, J. Biol. Chem. 289 (2014) 24779–24791. [7] C. Wu, T. Thalhamer, R.F. Franca, S. Xiao, C. Wang, C. Hotta, et al., Galectin-9CD44 interaction enhances stability and function of adaptive regulatory T cells, Immunity 41 (2014) 270–282. [8] X. He, X. Shi, S. Puthiyakunnon, L. Zhang, Q. Zeng, Y. Li, et al., CD44-mediated monocyte transmigration across Cryptococcus neoformans-infected brain microvascular endothelial cells is enhanced by HIV-1 gp41-I90 ectodomain, J. Biomed. Sci. 23 (2016) 28. [9] J. Garay, M.B. Piazuelo, S. Majumdar, L. Li, J. Trillo-Tinoco, L. Del Valle, et al., The homing receptor CD44 is involved in the progression of precancerous gastric lesions in patients infected with Helicobacter pylori and in development of mucous metaplasia in mice, Cancer Lett. 371 (2016) 90–98. [10] Q. Fu, L. Hou, P. Xiao, C. Guo, Y. Chen, X. Liu, CD44 deficiency leads to decreased proinflammatory cytokine production in lung induced by PCV2 in mice, Res. Vet. Sci. 97 (2014) 498–504. [11] V. Nadella, Z. Wang, T.S. Johnson, M. Griffin, A. Devitt, Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells, Biochim. Biophys. Acta 1853 (2015) 201–212. [12] K. Rox, M. Rohde, G.S. Chhatwal, R. Muller, Disorazoles block group A streptococcal invasion into epithelial cells via interference with the host factor ezrin, Cell Chem. Biol. 24 (2017) 159–170. [13] B. Xu, X. Pei, Y. Su, Z. Ma, H. Fan, Capsule of Streptococcus equi subsp. zooepidemicus hampers the adherence and invasion of epithelial and endothelial cells and is attenuated during internalization, FEMS Microbiol. Lett. 363 (2016). [14] Z. Wei, Q. Fu, Y. Chen, P. Cong, S. Xiao, D. Mo, et al., The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development, Vaccine 30 (2012) 4670–4675. [15] S. Trouillet, J.P. Rasigade, Y. Lhoste, T. Ferry, F. Vandenesch, J. Etienne, et al., A novel flow cytometry-based assay for the quantification of Staphylococcus aureus adhesion to and invasion of eukaryotic cells, J. Microbiol. Methods 86 (2011) 145–149. [16] Q. Fu, Z. Wei, P. Xiao, Y. Chen, X. Liu, CD44 enhances macrophage phagocytosis and plays a protective role in Streptococcus equi subsp. zooepidemicus infection, Vet. Microbiol. 198 (2017) 121–126. [17] M. Izquierdo, F. Navarro-Garcia, R. Nava-Acosta, J.P. Nataro, F. Ruiz-Perez, M.J. Farfan, Identification of cell surface-exposed proteins involved in the fimbriamediated adherence of enteroaggregative Escherichia coli to intestinal cells, Infect. Immun. 82 (2014) 1719–1724. [18] T.M. VanWagoner, J.M. Atack, K.L. Nelson, H.K. Smith, K.L. Fox, M.P. Jennings, et al., The modA10 phasevarion of nontypeable Haemophilus influenzae R2866 regulates multiple virulence-associated traits, Microb. Pathog. 92 (2016) 60–67. [19] C. Chen, M.L. Mangoni, Y.P. Di, In vivo therapeutic efficacy of frog skin-derived peptides against Pseudomonas aeruginosa-induced pulmonary infection, Sci. Rep. 7 (2017) 8548. [20] G.S. Abu-Ali, L.M. Ouellette, S.T. Henderson, D.W. Lacher, J.T. Riordan, T.S. Whittam, et al., Increased adherence and expression of virulence genes in a lineage of Escherichia coli O157:H7 commonly associated with human infections, PLoS One 5 (2010) e10167. [21] G.J. van der Windt, S. Florquin, A.F. de Vos, C. van't Veer, K.C. Queiroz, J. Liang, et al., CD44 deficiency is associated with increased bacterial clearance but enhanced lung inflammation during Gram-negative pneumonia, Am. J. Pathol. 177 (2010) 2483–2494. [22] S. Backert, N. Tegtmeyer, Type IV secretion and signal transduction of Helicobacter pylori CagA through interactions with host cell receptors, Toxins 9 (2017). [23] M.A. Zafar, S. Hamaguchi, T. Zangari, M. Cammer, J.N. Weiser, Capsule type and amount affect shedding and transmission of Streptococcus pneumoniae, mBio 8 (2017). [24] B.L. Spencer, A.T. Shenoy, C.J. Orihuela, M.H. Nahm, The pneumococcal serotype 15C capsule is partially O-acetylated and allows for limited evasion of 23-valent pneumococcal polysaccharide vaccine-elicited anti-serotype 15B antibodies, Clin. Vaccine Immunol. : CVI 24 (2017). [25] P. Barato, E.R. Martins, G.M. Vasquez, M. Ramirez, J. Melo-Cristino, N. Martinez, et al., Capsule impairs efficient adherence of Streptococcus agalactiae to intestinal epithelium in tilapias Oreochromis sp, Microb. Pathog. 100 (2016) 30–36.
4. Discussion Bacterial adherence to epithelial cells is important for many bacteria to break through the first barrier of the host [17–19]. In the present study, the adherence of S. zooepidemicus to cultured epithelial cells was initially studied using a flow cytometric method as previously described [15,20]. The percentage of LA-4 cells with FAMSE-labeled S. zooepidemicus attached, and the relative amount of S. zooepidemicus attached to LA-4 cells (MFI value) were determined. A significant positive relationship was observed between BCR and MFI values. The binding of S. zooepidemicus to LA-4 cells was found to follow a logarithmic relationship with the percentage of LA-4 cells with S. zooepidemicus attached. The results revealed flow cytometry to be well suited to detect S. zooepidemicus attached to LA-4 cells. CD44 is a transmembrane adhesion molecule that is expressed in most cell types. Several studies have indicated that CD44 is involved in the process of host defense against pathogenic microorganisms, including the binding and processing of pathogens [8,21,22]. The present study analyzed whether CD44 antibodies influence the binding of S. zooepidemicus to LA-4 cells. Treatment of LA-4 cells with anti-CD44 antibodies caused a general reduction of bacterial adherence. Treatment of S. zooepidemicus with recombinant CD44 also resulted in reduction of bacterial adherence. In addition, recombinant CD44 could directly bind to S. zooepidemicus. These results suggest that CD44 has a general involvement in S. zooepidemicus adherence, perhaps because of CD44 as a direct receptor. The capsule of Streptococcus plays an important role in pathogenicity [23–25], and it is also involved in the regulation of Streptococcus adhesion activity. Our previous study showed that the capsule plays an important role in the pathogenicity of S. zooepidemicus [14]. Fluorescently labeled wild-type S. zooepidemicus and capsule mutant were allowed to adhere to cells for 30 min, and the fluorescence of the cells was measured. The MFI value of LA-4 cells incubated with S. zooepidemicus ΔhasB was significantly smaller than the value of LA4 cells incubated with wild-type S. zooepidemicus (Fig. 6). These results demonstrate that S. zooepidemicus adherence to LA-4 cells is upregulated in the presence of capsule. The effect of the CD44 binding was significantly reduced in isogenic strain of S. zooepidemicus lacking capsule, suggesting that CD44 is directly involved in S. zooepidemicus adherence to LA-4 cells. In conclusion, we have shown that blockade of CD44 with antibodies, recombinant protein, and S. zooepidemicus mutant ΔhasB inhibits adherence of S. zooepidemicus. Conflicts of interest The authors declare no conflicts of interest in this work. Acknowledgments This work was supported by the National Natural Science Foundation of China (31872443, 31502047), Project of Department of Education of Guangdong Province (2017KTSCX193), China Agriculture Research System (CARS-36), and Key Laboratory for Preventive
253
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
CD44 facilitates adherence of Streptococcus equi subsp. zooepidemicus to LA-4 cells
T
Qiang Fua,b, Wenwen Lic, Shun Lib, Xianjie Zhaob, Honglin Xieb, Xi Zhangb, Kangjian Lib, Chunquan Mab, Xiaohong Liua,∗ a
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, China School of Life Science, Foshan University, Guangdong, 528225, China c Laboratory Animal Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Streptococcus equi subsp. zooepidemicus CD44 Adherence
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) causes a wide variety of infections in many species. CD44 is a transmembrane adhesion molecule, expressed by various cell types, which has been implicated in several infection processes. The aim of this study was to examine the role of CD44 in S. zooepidemicus adherence to LA-4 cells (mouse lung adenoma). Dose-dependent adhesion with LA-4 may be effectively studied by flow cytometry. Adherence of S. zooepidemicus is reduced after treatment of cells with anti-CD44 antibody. Treatment of S. zooepidemicus with recombinant CD44 significantly reduced bacteria adherence. In addition, CD44 can directly bind to wild-type S. zooepidemicus, while the binding was decreased in the capsule deletion isogenic mutant. These data suggest that CD44 facilitates adherence of S. zooepidemicus to LA-4 cells.
1. Introduction
2. Materials and methods
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) caused variety of infections in many species, including horses, dogs, and pigs [1–4]. These infections have led to significant welfare problems and economic costs, most notably in the training of racehorses. We recently reported that S. zooepidemicus infection led to a significant increase in CD44 mRNA level, suggesting that CD44 plays an important role in S. zooepidemicus infection [5]. CD44 is a transmembrane adhesion molecule, which is expressed on various cell types, including epithelial cells [6,7]. The effect of CD44 on host response is different, depending on the pathogen species and the primary site of infection [8–10]. Several studies have indicated CD44 is involved in the process of host defense against pathogenic microorganisms, including the binding and processing of pathogens [11,12]. In addition, recent studies have shown that S. zooepidemicus capsule contributes to the attachment and invasion of host cells [13,14]. However, the contribution of CD44 to S. zooepidemicus attachment is not well defined. Here, we assessed the role of CD44 in S. zooepidemicus adherence to LA-4 cells (mouse lung adenoma), focusing on the interference with S. zooepidemicus capsule and transmembrane CD44, in addition to specific antibodies and recombinant CD44 protein treatment.
2.1. Bacterial strain and growth conditions
∗
S. zooepidemicus wild-type strain C55138 (China Institute of Veterinary Drug Control) and isogenic mutant ΔhasB were maintained in our laboratory. Bacteria were cultured in aerobic conditions at 37 °C on tryptone soya broth (TSB; Oxoid) or tryptone soya agar (TSA; Oxoid) supplemented with 5% newborn calf serum. 2.2. Cell culture Maintenance of LA-4 cells (ATCC CCL-196), a murine bronchial epithelial cell line, required Ham's Fe12K medium (Gibco); additional supplements of 2 mM L-glutamine, and 15% heat-inactivated fetal calf serum. Cell lines were grown at 37 °C in a humidified atmosphere with 5% CO2 and passaged with trypsin-EDTA (ethylenediaminetetraacetic acid) when confluent. 2.3. Adhesion of bacteria to LA-4 cells Fluorescent staining of S. zooepidemicus was performed according the method used in the previous study, with some modification [15].
Corresponding author. State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-sen University, Guangzhou, 510006, China. E-mail address: [email protected] (X. Liu).
https://doi.org/10.1016/j.micpath.2019.01.016 Received 18 March 2018; Received in revised form 8 January 2019; Accepted 8 January 2019 Available online 09 January 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Increased induction of CD44 on LA-4 cells after S. zooepidemicus exposure. (A) CD44 mRNA levels were determined by real-time RT-PCR. (B) Expression of CD44 on LA-4 cells was determined by flow cytometry. The data are showed as the means ± SD analyzed by Student's t-tests using GraphPad Prism version 5.0 (*, P < 0.05; **, P < 0.01).
Fig. 3. Time course studies of the adherence of FAMSE-labeled S. zooepidemicus to LA-4 cells. Cells were incubated with S. zooepidemicus C55138 at MOI of 100:1 for 30, 60, 90, 120, 180 min at 37 °C, and adherence was analyzed by flow cytometry. The results are presented as the percentages of LA-4 cells with attached S. zooepidemicus (black bars) and the FL-1 fluorescence emitted from the attached S. zooepidemicus as indicated by MFI ± standard deviation (white bars).
Fig. 2. Adherence of FAMSE-labeled S. zooepidemicus to LA-4 cells. Cells were incubated with S. zooepidemicus C55138 at MOI of 1:1, 10:1, 100:1, 500:1, 1000:1 for 60 min at 37 °C, and adherence was analyzed by flow cytometry. (A) Dot plot display mode of FL-1 versus side scatter (SSC) after the incubation of the cells with labeled bacteria. (B) The results are presented as the percentages of LA-4 cells with attached S. zooepidemicus (black bars) and the FL-1 fluorescence emitted from the attached S. zooepidemicus as indicated by MFI ± standard deviation (white bars).
Fig. 4. Pretreatment of LA-4 cells with anti-CD44 antibody reduced S. zooepidemicus adherence to cells. LA-4 cells were immersed in medium alone, the isotype control, or anti-CD44 for 30 min at 37 °C, and adherence was analyzed by flow cytometry (*, P < 0.05; **, P < 0.01 versus the cells treated with isotype control).
statically co-incubated with LA-4 cells at bacterial cell ratio (BCR) of 1:1, 10:1, 100:1, 500:1, and 1000:1 for 60 min at 37 °C. Time course study of the adherence was performed at a BCR of 100:1 for 30, 60, 90, 120, 180 min. Following incubation, the LA-4 cells were fixed with 2% formaldehyde and analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the FL-1 channel to measure fluorescence intensity.
Briefly, 6-carboxyfluorescein succinimidyl ester (FAMSE; Molecular Probes, 34 μg) was added to 10 ml S. zooepidemicus in phosphate-buffered saline (PBS) and the bacterial cells were incubated with gentle rotation for 20 min at 37 °C. Following staining, the bacterial cells were harvested by centrifugation at 12,000×g, washed once in a large volume of PBS, and suspended in PBS. Labeled bacterial cells were 251
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S. zooepidemicus was incubated with 1 ng of mouse CD44 for 30 min at 37 °C. After incubation, the bacteria were centrifuged at 12,000×g, and the excess CD44 was removed by washing the bacteria twice in PBS. The adherence assay was performed as described above at a BCR of 100:1. 2.5. Binding of CD44 to S. zooepidemicus Binding of CD44 to S. zooepidemicus was performed according the method of previously study [16]. Wild-type S. zooepidemicus or capsule deletion mutant ΔhasB were incubated with 1 ng of mouse CD44 for 30 min at 37 °C. Then, the bacteria were centrifuged for 15 min at 12,000×g and incubated with FITC-conjugated CD44 antibody (BioLegend) for 30 min on ice. After incubation, bacterial cells were centrifuged, washed, and fixed with paraformaldehyde. The binding was determined on a FACSCalibur flow cytometer (Becton Dickinson). Forward and side scatter properties were used to exclude debris and aggregates, and 30,000 gated events were recorded. M1 marker set to include > 98% unlabeled bacteria. The percentage of fluorescent bacteria (brighter than M1 fluorescence intensity units on the FL1 axis) was calculated for each sample.
Fig. 5. Pretreatment of S. zooepidemicus with recombinant CD44 protein inhibit S. zooepidemicus adherence to LA-4 cells. Labeled S. zooepidemicus were incubated with mouse CD44 for 30 min at 37 °C, and adherence was analyzed by flow cytometry (*, P < 0.05; **, P < 0.01 versus the cells incubated with BSA-treated S. zooepidemicus).
3. Results 3.1. S. zooepidemicus exposure increased induction of CD44 on LA-4 cells To investigate whether S. zooepidemicus influences CD44 expression on LA-4 cells, relative quantification of gene transcript was examined by real-time PCR. The real-time PCR revealed that CD44 mRNA level was increased from S. zooepidemicus exposure (Fig. 1A). In addition, the expression of CD44 on LA-4 cells after S. zooepidemicus exposure was determined by flow cytometry. Treatment of LA-4 cells with S. zooepidemicus increased CD44 expression (Fig. 1B). The results confirmed that expression of CD44 on LA-4 cells was significantly up-regulated after S. zooepidemicus exposure. 3.2. Flow cytometric analysis of S. zooepidemicus adherence to LA-4 cells
Fig. 6. Binding of CD44 to wild-type S. zooepidemicus and capsule deletion mutantΔhasB. (A) Binding of CD44 to S. zooepidemicus was detected by flow cytometry using FITC-conjugated CD44 antibody. (B) Examples of flow cytometry histograms for binding of CD44 to wild-type S. zooepidemicus and capsule deletion mutant ΔhasB are shown, BSA treated cells are represented in shaded histograms and CD44 treated cells open histograms.
Adherence to LA-4 cells of S. zooepidemicus was analyzed by flow cytometry using the FL-1 channel to determine the mean fluorescence intensity (MFI). The MFI marker M was set to include less than 2% of the negative control (uninfected) cells (Fig. 2A). The percentage of LA4 cells with S. zooepidemicus attached was identified by gating on fluorescence. There was an increase in MFI in LA-4 cells incubated with increasing numbers of S. zooepidemicus (Fig. 2B). Furthermore, the percentage of LA-4 cells with S. zooepidemicus attached increased with a corresponding increase in the BCR (Fig. 2B). As the time of exposure to bacteria increased, the MFI in LA-4 cells and the percentage of LA4 cells with S. zooepidemicus attached increased accordingly (Fig. 3).
2.4. Effect of CD44 antibodies and recombinant CD44 protein on bacterial adherence
3.3. Adherence of S. zooepidemicus is reduced after treatment of epithelial cells with CD44 antibodies
Inhibition of S. zooepidemicus adherence by CD44 antibodies was analyzed. LA-4 cells were washed with PBS and treated with CD44 antibodies (BD Pharmingen, 20 μg/ml) or control antibodies (BD Pharmingen, 20 μg/ml) for 30 min at 37 °C. After washing to remove excess antibodies, cells were incubated with labeled S. zooepidemicus for 30 min at a BCR of 100:1. LA-4 cells were washed and fixed with 2% formaldehyde prior to flow cytometry. The complete CDS of Mus musculus CD44 mRNA (cDNA clone MGC: 58,984 IMAGE: 4910789) was synthesized by Sangon Biotech Shanghai Co Ltd. The amino acid sequence of CD44 was expressed in E. coli strains BL21 as described in our previous study [16]. The ability of recombinant CD44 protein to inhibit labeled bacteria adherence to LA-4 cells was analyzed. Labeled
To investigate whether CD44 antibodies influence binding of S. zooepidemicus to LA-4 cells, the cells were pretreated with CD44 antibodies and isotypes. Treatment of LA-4 cells with anti-CD44 antibodies significantly reduced bacterial adherence to LA-4 cells (Fig. 4). 3.4. Recombinant CD44 protein inhibit S. zooepidemicus adherence to LA4 cells The contribution of the CD44 activity to the binding of S. zooepidemicus to LA-4 cells was investigated by pretreatment of S. zooepidemicus with CD44. Both the MFI value and the percentage of LA-4 cells with S. zooepidemicus attached to cells incubated with CD44-treated S. zooepidemicus was significantly smaller than the cells incubated with 252
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Research of Emerging Animal (KLPREAD201801-03).
BSA-treated S. zooepidemicus (Fig. 5). 3.5. Binding of CD44 to wild-type S. zooepidemicus and capsule deletion mutant ΔhasB
Diseases
in Foshan
University
References
The contribution of the CD44 activity to the binding of S. zooepidemicus to LA-4 cells was investigated by wild-type S. zooepidemicus and capsule deletion mutant ΔhasB. The MFI value of LA-4 cells incubated with S. zooepidemicus ΔhasB was significantly smaller than that of LA4 cells incubated with wild-type S. zooepidemicus (Fig. 6).
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4. Discussion Bacterial adherence to epithelial cells is important for many bacteria to break through the first barrier of the host [17–19]. In the present study, the adherence of S. zooepidemicus to cultured epithelial cells was initially studied using a flow cytometric method as previously described [15,20]. The percentage of LA-4 cells with FAMSE-labeled S. zooepidemicus attached, and the relative amount of S. zooepidemicus attached to LA-4 cells (MFI value) were determined. A significant positive relationship was observed between BCR and MFI values. The binding of S. zooepidemicus to LA-4 cells was found to follow a logarithmic relationship with the percentage of LA-4 cells with S. zooepidemicus attached. The results revealed flow cytometry to be well suited to detect S. zooepidemicus attached to LA-4 cells. CD44 is a transmembrane adhesion molecule that is expressed in most cell types. Several studies have indicated that CD44 is involved in the process of host defense against pathogenic microorganisms, including the binding and processing of pathogens [8,21,22]. The present study analyzed whether CD44 antibodies influence the binding of S. zooepidemicus to LA-4 cells. Treatment of LA-4 cells with anti-CD44 antibodies caused a general reduction of bacterial adherence. Treatment of S. zooepidemicus with recombinant CD44 also resulted in reduction of bacterial adherence. In addition, recombinant CD44 could directly bind to S. zooepidemicus. These results suggest that CD44 has a general involvement in S. zooepidemicus adherence, perhaps because of CD44 as a direct receptor. The capsule of Streptococcus plays an important role in pathogenicity [23–25], and it is also involved in the regulation of Streptococcus adhesion activity. Our previous study showed that the capsule plays an important role in the pathogenicity of S. zooepidemicus [14]. Fluorescently labeled wild-type S. zooepidemicus and capsule mutant were allowed to adhere to cells for 30 min, and the fluorescence of the cells was measured. The MFI value of LA-4 cells incubated with S. zooepidemicus ΔhasB was significantly smaller than the value of LA4 cells incubated with wild-type S. zooepidemicus (Fig. 6). These results demonstrate that S. zooepidemicus adherence to LA-4 cells is upregulated in the presence of capsule. The effect of the CD44 binding was significantly reduced in isogenic strain of S. zooepidemicus lacking capsule, suggesting that CD44 is directly involved in S. zooepidemicus adherence to LA-4 cells. In conclusion, we have shown that blockade of CD44 with antibodies, recombinant protein, and S. zooepidemicus mutant ΔhasB inhibits adherence of S. zooepidemicus. Conflicts of interest The authors declare no conflicts of interest in this work. Acknowledgments This work was supported by the National Natural Science Foundation of China (31872443, 31502047), Project of Department of Education of Guangdong Province (2017KTSCX193), China Agriculture Research System (CARS-36), and Key Laboratory for Preventive
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