FEMS Microbiology Letters, 362, 2015, fnv022 doi: 10.1093/femsle/fnv022 Advance Access Publication Date: 11 February 2015 Research Letter
R E S E A R C H L E T T E R – Pathogens & Pathogenicity
Streptococcus suis serotype 2 strains can induce the formation of neutrophil extracellular traps and evade trapping Jianqing Zhao1,2 , Shan Pan2 , Lan Lin2 , Lei Fu2 , Chao Yang2 , Zhongmin Xu2 , YanMin Wei2 , Meilin Jin2 and Anding Zhang1,2,∗ 1
Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, Hubei 430070, P.R. China and 2 College of Veterinary Medicine, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, Hubei, 430070, P.R. China ∗ Corresponding author: Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, Hubei 430070, P.R. China. Tel: (+86) 27 87282608; Fax: +86-27-87282608; E-mail: [email protected]; One sentence summary: Streptococcus suis serotype 2 strains can induce the formation of neutrophil extracellular traps and evade trapping by the CPS structure. Editor: Ezio Ricca
ABSTRACT Streptococcus suis (S. suis) ranks among the five most important porcine pathogens worldwide and occasionally threatens human health, especially in people that come into close contact with pigs or pork products. Streptococcus suis serotype 2 (SS2) is considered to be the most pathogenic and prevalent capsular type. As a first line of immune defense against SS2 infection, neutrophils can eliminate the invader not only by phagocytosis but also by neutrophil extracellular traps (NETs)-mediated killing. SS2 can resist phagocytosis through polysaccharide capsule (CPS), but how this strain evades the effects of NETs remains to be determined. The present study demonstrated that the epidemic strain 05ZY, the highly pathogenic strain P1/7 and the intermediately pathogenic strain A7 could induce the formation of NETs. Furthermore, SS2 strains could successfully resist NETs-mediated killing, and the CPS structure contributed to this resistance by escaping the trapping. Therefore, the CPS structure not only contributed to the SS2 strains’ resistance to phagocytosis-mediated killing but also played an essential role in evading NETs trapping and further killing in vitro. This study strengthens our understanding of how S. suis can evade innate immune surveillance and elimination. Key words: Streptococcus suis (S. suis); neutrophil extracellular traps (NETs); polysaccharide capsule (CPS)
INTRODUCTION Streptococcus suis (S. suis) is a major swine pathogen that is responsible for severe economic losses in the porcine industry and also represents a significant threat to human health (Segura 2009; Wertheim et al. 2009; Gottschalk 2012). Streptococcus suis serotype 2 (SS2) is considered to be the most pathogenic and prevalent capsular type among the 33 serotypes (types 1 to 31, 33
and 1/2), especially in European and Asian countries (Gottschalk et al. 2013). Two recent large-scale outbreaks of human SS2 epidemics in China have increased awareness of the public health threat, particularly because the cases presented with septic shock (streptococcal toxic shock-like syndrome, STSLS), indicating that new, highly virulent bacterial variants were emerging in Asia (Segura 2009; Ye et al. 2009; Zheng et al. 2011). However,
Received: 23 January 2015; Accepted: 7 February 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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comparative genome research indicated that the highly virulent Chinese strain had not received any toxic genes (Chen et al. 2007; Ye et al. 2009) and suggested that the known genomic islands may regulate gene expression to escape host immune surveillance and elimination (Li et al. 2008). Neutrophils, as a first line of immune defense against bacterial infection, play an important role in controlling S. suis infection (Chabot-Roy et al. 2006). Neutrophils can kill microbes through receptor-mediated capture and phagocytosis, which is the most widely accepted mechanism through which neutrophils destroy pathogens. In addition, neutrophils can also attack pathogens using a novel antimicrobial mechanism called neutrophil extracellular traps (NETs)-mediated bacterial killing (Brinkmann et al. 2004). This mechanism is initiated when, upon stimulation with interleukin-8, phorbol myristate acetate (PMA), or lipopolysaccharide, the lobular shape of the neutrophilic nucleus is lost. Next, the nuclear envelope disintegrates, resulting in the mixing of the nucleus, cytoplasmic and granular components. The cell membrane then ruptures and results in the release of decondensed chromatin into the extracellular space and the subsequent formation of 15–25-nm chromatin fibers (Brinkmann et al. 2004; Fuchs et al. 2007). The fibers interact with high concentrations of histones and antimicrobial molecules, providing these NETs with the ability to restrict pathogens at the site of infection and to ultimately destroy any bacteria they come into contact with (Jaillon et al. 2007). Pathogenic strains have evolved various means of evading or subverting NETs, such as the synthesis of products that inhibit the release of NETs (Riyapa et al. 2012), modifications of their surface structures to escape trapping (Wartha et al. 2007) and the secretion of deoxyribonuclease to disintegrate the NETs (Sumby et al. 2005; Beiter et al. 2006). A recent study indicated that SS2 pathogenic strain 10 could degrade the NETs with S. suis-secreted nuclease A, thus evading the NETs’ antimicrobial activity (de Buhr et al. 2014). The goal of this study was to determine whether this evasion strategy is employed by different pathogenic SS2 strains, especially the highly virulent Chinese strains. The present study employed the epidemic strain 05ZY, the highly pathogenic strain P1/7 and the intermediately pathogenic strain A7 to demonstrate that SS2 strains with different levels of virulence could induce the formation of NETs but could also evade their antimicrobial activity in vitro. Our study also indicated that the polysaccharide capsule (CPS) structure not only contributed to the SS2 strains’ resistance to phagocytosismediated killing but also played a major role in the evasion of trapping and further killing by the NETs.
MATERIALS AND METHODS Bacterial strains and growth conditions The epidemic SS2 strain 05ZY was isolated from the brain of a diseased piglet during the SS2 outbreak in China in 2005 (Zhang et al. 2012). The isogenic mutant cpsEF (cpsEF) were constructed in strain 05ZY according to the method described as the construction of the isogenic hp0197 mutant (Zhang et al. 2012). The S. suis strain A7 was isolated from the brain of a diseased pig in China in 2007 and shows intermediate pathogenicity in pigs and mice (Hu et al. 2011). The virulent SS2 strain P1/7 was originally isolated from a pig dying of meningitis and shows a high degree of pathogenicity in pigs and mice (Holden et al. 2009). The S. suis strains were grown in tryptone soy broth or tryptone soy broth agar (Becton Dickinson, Sparks, MD, USA) at 37◦ C under aerobic conditions.
Ethics statement C57BL/6 mice were purchased from the Laboratory Animal Center of Hubei Province (Permit Number: 42000500001445). The mice were euthanized using CO2 to limit their suffering before isolating the neutrophils. The study was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals Monitoring Committee of Hubei Province, China, and the protocol was approved by the Committee on the Ethics of Animal Experiments at the College of Veterinary Medicine, Huazhong Agricultural University.
Isolation and purification of mouse bone marrow neutrophils Mouse bone marrow neutrophils were obtained from 10- to 15week-old specific pathogen-free mice and purified according to the method described before (Su et al. 2008). The bone marrow from the femurs and tibias was flushed with HBSS-Prep [Ca-Mg-free HBSS supplemented with 20 mM NA-HEPES (pH 7.4) and 0.5% FCS] with a 25-gauge needle. The whole bone marrow was centrifuged, and the RBC was hypotonically lysed with 0.2% NaCl. This solution was restored to isotonicity with 1.2% NaCl and then filtered over a 70-μm nylon cell strainer. The solution was centrifuged and resuspended in HBSS-Prep and then applied over a 62% Percoll gradient. The Percoll solution was then centrifuged at 1000 g for 30 min. At the end of the gradient centrifugation, there will be a sharp interface atop the 62% Percoll (these are immature cells and non-granulocytic lineages) and a more cloudy pellet (the neutrophils). Carefully remove and discard the cells at the interface, the HBSS-Prep and the upper part of the 62% Percoll. Transfer the pellet to another tube, wash twice with 1640 RPMI, resuspend in medium and count. Greater than 90% neutrophil purity was confirmed with incubation with Hoechst 33258 stain for 10 min and visualization with the laser scanning microscopy system using the 40× object, zoom = 4.0 (Olympus fv1000 mp).
Induction of NETs by SS2 strains To compare the induction of NETs by the SS2 strains, SS2 strains were inoculated into the purified neutrophils, and NETs formation was visualized by staining with the extracellular nucleic acid dye SYTOX Green (100 nM) and examined under the fluorescence microscopy (20× object, OLYMPUS IX70) at 60, 120, 180 and 240 min post-inoculation. The extracellular nucleic acid was quantitated according to the following procedure with the QubitTM dsDNA HS Assay Kits (Invitrogen). The cells together with supernatant were collected gently and then centrifuged at 1000 rpm for 5 min to discard cells. 10 μL supernatant (the volume was defined as ‘x’) was added to 190 μL prepared working solution and vortexed for 2–3 s. After incubation for two minutes, the fluorescence values R of the mixtures was read by Qubit 2.0 Fluorometer (the fluorescence values was defined as ‘QF value’), and the concentration of all the samples was calculated using the following equation: concentration of the sample = QF value × ( 200 ). x
Detection of NETs-mediated killing and phagocytic killing by neutrophils The neutrophil killing assays were performed according to the method described by Wartha et al. (2007) with minor
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modifications. For the phagocytic killing assay, purified neutrophils (N+ ) or 1640 RPMI medium (N− ) were incubated with 10% mouse serum, and the SS2 strains were added at an MOI of 1 and incubated for 90 min. The total number of bacteria was quantified by calculating the colony-forming units (cfu) on the plates after serial dilutions. The percentage of bacteria killed by the neutrophils was calculated according to the following formula: phagocytic killing% = [(cfuN − – cfuN + ) / (cfuN − )] × 100. The NETs killing assay was performed according to the method described by Wartha et al. (2007). The purified neutrophils (N+ ) or RPMI 1640 medium (N− ) was activated with 200 nM PMA for 4 h and then incubated with cytochalasin B (10 μg mL–1 , Aladdin) for 15 min to inhibit phagocytosis. Subsequently, the SS2 strains were added at an MOI of 0.01. After 30 min, the viable bacteria were quantified by serial dilutions of the thoroughly scraped well contents. The percentage of non-phagocytic killing of bacteria by PMA-induced NETs was calculated according to the following formula: non-phagocytic killing% = [(cfuN − – cfuN + ) / (cfuN − )] × 100.
Visualization of phagocytosis To visualize phagocytosis, the purified murine neutrophils were seeded on cell culture dishes with glass bottoms, and the adhered neutrophils were infected with the SS2 strains labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA SE), a green fluorescent dye at an MOI of 10 in the presence of 5% mouse serum. After a brief centrifugation (1000 rpm, 1 min) followed by an incubation period of 90 min, the samples were washed and then incubated with 2 mg mL–1 of trypan blue for 15 min to quenched adherent bacteria (Rest and Speert 1994). After washing, the samples were fixed with 4% paraformaldehyde. Then, the nuclear dye Hoechst 33258 was added and incubated for 10 min. Finally, all of the dishes were washed with PBS for three times and observed by the laser scanning microscopy system using the 40× object, zoom = 4.0 (Olympus fv1000mp).
NETs trapping assay To detect NETs trapping, the seeded purified neutrophils were activated with 200 nM PMA for 4 h and then infected with the SS2 strains labeled with the green fluorescent dye CFDA SE at an MOI of 10. After a brief centrifugation followed by an incubation period of 5 min, the samples were washed five times and then fixed with 4% paraformaldehyde. Subsequently, the NETs’ structure was labeled with the extracellular nuclear acid dye SYTOX orange (Invitrogen). Finally, the samples were then observed with the laser scanning microscopy system using the 40× object, zoom = 1.0. To quantify the trapping activity of the NETs toward the different strains, the average numbers of bacteria trapped by NET structure in 20 randomly selected microscopy fields were used to compare the NETs’ ability to capture specific SS2 strains.
Statistical analysis Unless otherwise specified, the data were analyzed using two-tailed, unpaired t test with Welch’s correction, and all of the assays were repeated at least three times. The data were expressed as the means ± standard error of the mean.
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RESULTS AND DISCUSSIONS Streptococcus suis induced the formation of NETs Mouse bone marrow neutrophils were isolated and purified, and nearly all of the isolated cells showed a lobulated nucleus, a characteristic of neutrophils (Fig. S1, Supporting Information). SS2 virulent strains can induce the formation of NETs (de Buhr et al. 2014). However, it is unknown whether this is a universal phenomenon for strains with different degrees of virulence. To address this question, the present study employed the three following virulent SS2 strains: the epidemic strain 05ZY that was isolated in China during an SS2 outbreak in 2005, contained 89K pathogenicity island and could cause STSLS; the reference SS2 strain P1/7 that showed a high degree of pathogenicity to pigs and mice; and strain A7, which showed intermediate pathogenicity to mice and pigs. To compare the induction of NETs, these strains were used to infect the purified neutrophils (MOI = 100), and NETs formation was examined using the extracellular nucleic acid dye SYTOX Green at 60, 120, 180 and 240 min post-infection (p.i.). NETs formation could be observed easily based on loss of the lobular shape of the neutrophilic nucleus and the formation of long DNA fibers. All of the strains tested could induce NETs formation at an early time point (120 min p.i.) (Fig. 1A). The quantity assay for detecting extracellular nucleic acid indicated that all these SS2 strains could result in the nucleic acid release (Fig. 1B) at 60 min post-incubation. Therefore, the selected SS2 strains with different level of virulence could induce NETs formation.
SS2 strains can resist NETs-mediated killing The SS2 strains could not inhibit and even induced the formation of NETs. This finding prompted us to evaluate whether these strains could efficiently resist NETs killing. To address this question, 05ZY, P1/7 and A7 were inoculated into PMA-induced NETs at an MOI of 0.01, and the resistance of these strains to NETs-mediated killing was determined by indirect quantification of the killed strains as described by Wartha et al. (2007). As expected, no detectable killing of 05ZY or P1/7 was observed in this assay (N.D.), and very few A7 strain (3.1 ± 3.1%) were killed by the non-phagocytic killing (Fig. 2A). This result indicated that NETs structure could not help to efficiently kill these pathogens, and it also meant that these SS2 strains could efficiently resist NETs-mediated killing at least in vitro. The result was also consistent with the expectation that these strains could resist neutrophil phagocytosis. Very low levels of 05ZY and P1/7 could be phagocytized by neutrophils, while A7 could be phagocytized by neutrophils at relatively higher levels (15.5 ± 0.9%) (Fig 2B and C). It is well known that the CPS structure of SS2 strains is responsible for their resistance to phagocytosis, and this understanding was also supported by our study. The isogenic cpsEF mutant (cpsEF), which originated from strain 05ZY and doesn’t produce a CPS structure (Fig. S2, Supporting Information), could be engulfed by neutrophils efficiently (37.3 ± 2.9%) (Fig. 2B and C). Furthermore, in a comparison with the encapsulated strains, the unencapsulated isogenic cpsEF mutant (cpsEF) could also easily be killed by nonphagocytic killing (17.2 ± 2.2%) (Fig. 2A). This result indicated that CPS structure not only played an essential role in the resistance to phagocytosis but also contributed to the ability of SS2 strains to escape non-phagocytic killing which mainly indicated NETs killing.
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Figure 1. Induction of NETs by SS2 strains. (A) Purified murine neutrophils were incubated with SS2 strains at MOI = 100 or PMA (200 nM) in the presence of the extracellular DNA dye SYTOX Green. NETs formation was visualized under fluorescence microscopy using the 20× object (OLYMPUS IX70) at 60, 120, 180 and 240 min post-incubation. The neutrophils in the absence of SS2 strains or PMA were served as a negative control (NC). The scale bars in the figure represent 50 μm. (B) Purified murine neutrophils were incubated with SS2 strains at MOI = 100, and the extracellular nucleic acid was quantitated at 60 min post-incubation. The ‘∗ ’ indicated that there was a significant difference (P < 0.01) between the infected cells (A7, P1/7, 05ZY and PMA) and the NC.
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Figure 2. SS2 strains resist NETs-mediated killing and phagocytosis-mediated killing. (A) For the NETs-mediated killing assay, SS2 strains with different degrees of virulence (05ZY, P1/7 and A7), and the isogenic cpsEF mutant (cpsEF) derived from 05ZY were used to determine the degree of non-phagocytic killing of these strains by PMA-induced NETs. Therefore, the non-phagocytic killing mainly indicated NETs killing. The ‘∗ ’ indicated that there was a significant difference (P < 0.05) between the groups. (B) For the phagocytosis-mediated killing assay, SS2 strains with different degrees of virulence (05ZY, P1/7 and A7) and the isogenic cpsEF mutant (cpsEF) were used to determine degree of phagocytosis-mediated killing in each strain. The ‘N.D.’ indicated that no detectable bacteria were recovered. The ‘∗ ’ indicated that a significant difference (P < 0.01) was existed in the two groups. (C) To visualize phagocytosis, adhered neutrophils were infected with SS2 strains labeled with the green fluorescent dye CFDA SE (CFDA, green fluorescent dye) at an MOI of 10. After incubation for 90 min, the dye of extracellular bacteria was bleached with trypan blue. Finally, the cells were further stained with Hoechst 33258 (Hoechst, blue fluorescent dye) and visualized with laser scanning microscopy system using the 40× object, zoom = 4.0 (Olympus fv1000 mp). The scale bars in the figure represent 15 μm.
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Figure 3. CPS structure enables SS2 strains to escape NETs trapping. (A) For the visualization assay, SS2 strains with different degrees of virulence (05ZY, P1/7 and A7), the isogenic cpsEF mutant (cpsEF), which originated from 05ZY were inoculated into PMA-induced NETs at an MOI of 10. All of the SS2 strains were pre-stained with green CFDA SE fluorescent dye (CFDA, green fluorescent dye). After a 5 min incubation, all of the samples were washed five times, and the extracellular nucleic acid was stained with SYTOX Orange Nucleic Acid Stain (SYTOX Orange, red fluorescent dye). The bacteria in the DNA fibers were determined as trapped by NETs structure. The scale bars in the figure represent 50 μm. (B) To quantify the trapping activity of the NETs against the different strains, the average numbers of bacteria trapped by NETs in 20 randomly selected microscopy fields were used to compare the ability of the NETs to trapping specific SS2 strains. The ‘∗ ’ meant there was a significant difference (P < 0.01) between the two groups.
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The CPS structure enables SS2 strains to escape NETs trapping The DNA fibers that comprise the NETs interact with histones and antimicrobial molecules; these interactions provide the NETs the ability to destroy any bacteria that they contact (Jaillon et al. 2007). SS2 strains are susceptible to antimicrobial molecules such as lysozyme in vitro (Fittipaldi et al. 2008). Furthermore, all three of the strains examined in this study are susceptible to the host antibacterial peptide LL-37 in vitro, indicating that none of these strains should resist killing by host antimicrobial molecules. Together, these findings prompted us to consider whether the resistance of SS2 strains to NETs-mediated killing was due to successfully escaping from trapping. To test this hypothesis, all of the strains were inoculated into PMA-induced NETs at an MOI of 10 to detect capture by the NETs. As shown by a fluorescence-based assay, no 05ZY, P1/7 or A7 cells were trapped efficiently (Fig. 3A and B). This result suggested that these SS2 strains could successfully escape trapping by the NETs, and this further resulted in increased resistance to killing. It is well known that the CPS structure of Pneumococcus plays an essential role in resistance to NETs trapping (Wartha et al. 2007). CPS is recognized as an essential virulence factor in S. suis due to its antiphagocytosis effects. This study demonstrated that the CPS of the SS2 strains could resist NETs-mediated killing (Fig. 2A). We observed that the unencapsulated isogenic mutant cpsEF could be trapped by NETs (Fig. 3A). Therefore, the CPS structure enabled the SS2 strains to escape trapping and killing by the NETs. To date, many virulence-associated factors have been identified due to isogenic mutants that showed significantly decreased virulence. Comparative genome studies indicated that several genes contribute to virulence by regulating the expression of genes responsible for CPS synthesis, such as CcpA (Willenborg et al. 2011) and HP0197 (Zhang et al. 2012). In addition to its antiphagocytosis effect, the present study demonstrated the essential role of CPS in resisting NETs-mediated killing, which may also contribute to the immune evasion of SS2. This result would further strengthen our understanding of how certain genes could contribute to virulence by regulating CPS structure. In summary, this present study demonstrated that the epidemic strain 05ZY, the highly pathogenic strain P1/7 and the intermediately pathogenic strain A7 could induce the formation of NETs. Furthermore, these SS2 strains could efficiently resist non-phagocytic killing, which suggested that these SS2 strains could efficiently resist NETs-mediated killing. Because the SS2 strains were susceptible to the antimicrobial molecules that are the major bactericidal components of NETs, we tested whether the resistance was the result of evading capture. Our hypothesis was confirmed by observations that all of the tested SS2 strains but not the unencapsulated isogenic cpsEF mutant could escape trapping efficiently. Therefore, our study demonstrated that the SS2 strains could induce NETs formation and that the CPS structure enabled these strains to evade trapping and further killing.
SUPPLEMENTARY DATA Supplementary data is available at FEMSLE online.
ACKNOWLEDGEMENTS We thank American Journal Experts (AJE) for English language editing. The authors have declared that no competing interests exist.
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FUNDINGS This work was supported by the National Natural Science Foundation of China (31172328, 31272544), the 973 Program (2012CB518805), Program for New Century Excellent Talents and the Fundamental Research Funds for the Central University (2011PY006). Conflict of interest statement. None declared.
REFERENCE Beiter K, Wartha F, Albiger B, et al. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 2006;16:401–7. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532–5. Chabot-Roy G, Willson P, Segura M, et al. Phagocytosis and killing of Streptococcus suis by porcine neutrophils. Microb Pathogenesis 2006;41:21–32. Chen C, Tang J, Dong W, et al. A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 Chinese isolates. PLoS One 2007;2:e315. de Buhr N, Neumann A, Jerjomiceva N, et al. The Streptococcus suis DNAse SsnA contributes to degradation of NETs and evasion of NET-mediated antimicrobial activity. Microbiology 2014;160: 385—95 Fittipaldi N, Sekizaki T, Takamatsu D, et al. Significant contribution of the pgdA gene to the virulence of Streptococcus suis. Mol Microbiol 2008;70:1120–35. Fuchs TA, Abed U, Goosmann C, et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007;176:231–41. Gottschalk M. Streptococcocis. In: Zimmerman JJ, Karriker LA, Ramirez A, et al., (eds). Diseases of Swine, 10th edn. Ames, IA,USA: Blackwell Publishing, 2012,841–55. Gottschalk M, Lacouture S, Bonifait L, et al. Characterization of Streptococcus suis isolates recovered between 2008 and 2011 from diseased pigs in Quebec, Canada. Vet Microbiol 2013;162:819–25. Holden MT, Hauser H, Sanders M, et al. Rapid evolution of virulence and drug resistance in the emerging zoonotic pathogen Streptococcus suis. PLoS One 2009;4:e6072. Hu P, Yang M, Zhang A, et al. Comparative genomics study of multi-drug-resistance mechanisms in the antibioticresistant Streptococcus suis R61 strain. PLoS One 2011;6: e24988. Jaillon S, Peri G, Delneste Y, et al. The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps. J Exp Med 2007;204: 793–804. Li M, Wang C, Feng Y, et al. SalK/SalR, a two-component signal transduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2. PLoS One 2008;3:e2080. Rest RF, Speert DP. Measurement of nonopsonic phagocytic killing by human and mouse phagocytes. Method Enzymol 1994;236:91–108. Riyapa D, Buddhisa S, Korbsrisate S, et al. Neutrophil extracellular traps exhibit antibacterial activity against burkholderia pseudomallei and are influenced by bacterial and host factors. Infect Immun 2012;80:3921–9. Segura M. Streptococcus suis: an emerging human threat. J Infect Dis 2009;199:4–6.
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Su X, Johansen M, Looney MR, et al. CD47 deficiency protects mice from lipopolysaccharide-induced acute lung injury and Escherichia coli pneumonia. J Immunol 2008;180: 6947–53. Sumby P, Barbian KD, Gardner DJ, et al. Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. P Natl Acad Sci USA 2005;102:1679–84. Wartha F, Beiter K, Albiger B, et al. Capsule and D-alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps. Cell Microbiol 2007;9: 1162–71. Wertheim HF, Nghia HD, Taylor W, et al. Streptococcus suis: an emerging human pathogen. Clin Infect Dis 2009;48:617–25.
Willenborg J, Fulde M, de Greeff A, et al. Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis. Microbiology 2011;157:1823–33. Ye C, Zheng H, Zhang J, et al. Clinical, experimental, and genomic differences between intermediately pathogenic, highly pathogenic, and epidemic Streptococcus suis. J Infect Dis 2009;199:97–107. Zhang A, Chen B, Yuan Z, et al. HP0197 contributes to CPS synthesis and the virulence of Streptococcus suis via CcpA. PLoS One 2012;7:e50987. Zheng X, Zheng H, Lan R, et al. Identification of genes and genomic islands correlated with high pathogenicity in Streptococcus suis using whole genome tilling microarrays. PLoS One 2011;6:e17987.
R E S E A R C H L E T T E R – Pathogens & Pathogenicity
Streptococcus suis serotype 2 strains can induce the formation of neutrophil extracellular traps and evade trapping Jianqing Zhao1,2 , Shan Pan2 , Lan Lin2 , Lei Fu2 , Chao Yang2 , Zhongmin Xu2 , YanMin Wei2 , Meilin Jin2 and Anding Zhang1,2,∗ 1
Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, Hubei 430070, P.R. China and 2 College of Veterinary Medicine, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, Hubei, 430070, P.R. China ∗ Corresponding author: Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, Hubei 430070, P.R. China. Tel: (+86) 27 87282608; Fax: +86-27-87282608; E-mail: [email protected]; One sentence summary: Streptococcus suis serotype 2 strains can induce the formation of neutrophil extracellular traps and evade trapping by the CPS structure. Editor: Ezio Ricca
ABSTRACT Streptococcus suis (S. suis) ranks among the five most important porcine pathogens worldwide and occasionally threatens human health, especially in people that come into close contact with pigs or pork products. Streptococcus suis serotype 2 (SS2) is considered to be the most pathogenic and prevalent capsular type. As a first line of immune defense against SS2 infection, neutrophils can eliminate the invader not only by phagocytosis but also by neutrophil extracellular traps (NETs)-mediated killing. SS2 can resist phagocytosis through polysaccharide capsule (CPS), but how this strain evades the effects of NETs remains to be determined. The present study demonstrated that the epidemic strain 05ZY, the highly pathogenic strain P1/7 and the intermediately pathogenic strain A7 could induce the formation of NETs. Furthermore, SS2 strains could successfully resist NETs-mediated killing, and the CPS structure contributed to this resistance by escaping the trapping. Therefore, the CPS structure not only contributed to the SS2 strains’ resistance to phagocytosis-mediated killing but also played an essential role in evading NETs trapping and further killing in vitro. This study strengthens our understanding of how S. suis can evade innate immune surveillance and elimination. Key words: Streptococcus suis (S. suis); neutrophil extracellular traps (NETs); polysaccharide capsule (CPS)
INTRODUCTION Streptococcus suis (S. suis) is a major swine pathogen that is responsible for severe economic losses in the porcine industry and also represents a significant threat to human health (Segura 2009; Wertheim et al. 2009; Gottschalk 2012). Streptococcus suis serotype 2 (SS2) is considered to be the most pathogenic and prevalent capsular type among the 33 serotypes (types 1 to 31, 33
and 1/2), especially in European and Asian countries (Gottschalk et al. 2013). Two recent large-scale outbreaks of human SS2 epidemics in China have increased awareness of the public health threat, particularly because the cases presented with septic shock (streptococcal toxic shock-like syndrome, STSLS), indicating that new, highly virulent bacterial variants were emerging in Asia (Segura 2009; Ye et al. 2009; Zheng et al. 2011). However,
Received: 23 January 2015; Accepted: 7 February 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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comparative genome research indicated that the highly virulent Chinese strain had not received any toxic genes (Chen et al. 2007; Ye et al. 2009) and suggested that the known genomic islands may regulate gene expression to escape host immune surveillance and elimination (Li et al. 2008). Neutrophils, as a first line of immune defense against bacterial infection, play an important role in controlling S. suis infection (Chabot-Roy et al. 2006). Neutrophils can kill microbes through receptor-mediated capture and phagocytosis, which is the most widely accepted mechanism through which neutrophils destroy pathogens. In addition, neutrophils can also attack pathogens using a novel antimicrobial mechanism called neutrophil extracellular traps (NETs)-mediated bacterial killing (Brinkmann et al. 2004). This mechanism is initiated when, upon stimulation with interleukin-8, phorbol myristate acetate (PMA), or lipopolysaccharide, the lobular shape of the neutrophilic nucleus is lost. Next, the nuclear envelope disintegrates, resulting in the mixing of the nucleus, cytoplasmic and granular components. The cell membrane then ruptures and results in the release of decondensed chromatin into the extracellular space and the subsequent formation of 15–25-nm chromatin fibers (Brinkmann et al. 2004; Fuchs et al. 2007). The fibers interact with high concentrations of histones and antimicrobial molecules, providing these NETs with the ability to restrict pathogens at the site of infection and to ultimately destroy any bacteria they come into contact with (Jaillon et al. 2007). Pathogenic strains have evolved various means of evading or subverting NETs, such as the synthesis of products that inhibit the release of NETs (Riyapa et al. 2012), modifications of their surface structures to escape trapping (Wartha et al. 2007) and the secretion of deoxyribonuclease to disintegrate the NETs (Sumby et al. 2005; Beiter et al. 2006). A recent study indicated that SS2 pathogenic strain 10 could degrade the NETs with S. suis-secreted nuclease A, thus evading the NETs’ antimicrobial activity (de Buhr et al. 2014). The goal of this study was to determine whether this evasion strategy is employed by different pathogenic SS2 strains, especially the highly virulent Chinese strains. The present study employed the epidemic strain 05ZY, the highly pathogenic strain P1/7 and the intermediately pathogenic strain A7 to demonstrate that SS2 strains with different levels of virulence could induce the formation of NETs but could also evade their antimicrobial activity in vitro. Our study also indicated that the polysaccharide capsule (CPS) structure not only contributed to the SS2 strains’ resistance to phagocytosismediated killing but also played a major role in the evasion of trapping and further killing by the NETs.
MATERIALS AND METHODS Bacterial strains and growth conditions The epidemic SS2 strain 05ZY was isolated from the brain of a diseased piglet during the SS2 outbreak in China in 2005 (Zhang et al. 2012). The isogenic mutant cpsEF (cpsEF) were constructed in strain 05ZY according to the method described as the construction of the isogenic hp0197 mutant (Zhang et al. 2012). The S. suis strain A7 was isolated from the brain of a diseased pig in China in 2007 and shows intermediate pathogenicity in pigs and mice (Hu et al. 2011). The virulent SS2 strain P1/7 was originally isolated from a pig dying of meningitis and shows a high degree of pathogenicity in pigs and mice (Holden et al. 2009). The S. suis strains were grown in tryptone soy broth or tryptone soy broth agar (Becton Dickinson, Sparks, MD, USA) at 37◦ C under aerobic conditions.
Ethics statement C57BL/6 mice were purchased from the Laboratory Animal Center of Hubei Province (Permit Number: 42000500001445). The mice were euthanized using CO2 to limit their suffering before isolating the neutrophils. The study was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals Monitoring Committee of Hubei Province, China, and the protocol was approved by the Committee on the Ethics of Animal Experiments at the College of Veterinary Medicine, Huazhong Agricultural University.
Isolation and purification of mouse bone marrow neutrophils Mouse bone marrow neutrophils were obtained from 10- to 15week-old specific pathogen-free mice and purified according to the method described before (Su et al. 2008). The bone marrow from the femurs and tibias was flushed with HBSS-Prep [Ca-Mg-free HBSS supplemented with 20 mM NA-HEPES (pH 7.4) and 0.5% FCS] with a 25-gauge needle. The whole bone marrow was centrifuged, and the RBC was hypotonically lysed with 0.2% NaCl. This solution was restored to isotonicity with 1.2% NaCl and then filtered over a 70-μm nylon cell strainer. The solution was centrifuged and resuspended in HBSS-Prep and then applied over a 62% Percoll gradient. The Percoll solution was then centrifuged at 1000 g for 30 min. At the end of the gradient centrifugation, there will be a sharp interface atop the 62% Percoll (these are immature cells and non-granulocytic lineages) and a more cloudy pellet (the neutrophils). Carefully remove and discard the cells at the interface, the HBSS-Prep and the upper part of the 62% Percoll. Transfer the pellet to another tube, wash twice with 1640 RPMI, resuspend in medium and count. Greater than 90% neutrophil purity was confirmed with incubation with Hoechst 33258 stain for 10 min and visualization with the laser scanning microscopy system using the 40× object, zoom = 4.0 (Olympus fv1000 mp).
Induction of NETs by SS2 strains To compare the induction of NETs by the SS2 strains, SS2 strains were inoculated into the purified neutrophils, and NETs formation was visualized by staining with the extracellular nucleic acid dye SYTOX Green (100 nM) and examined under the fluorescence microscopy (20× object, OLYMPUS IX70) at 60, 120, 180 and 240 min post-inoculation. The extracellular nucleic acid was quantitated according to the following procedure with the QubitTM dsDNA HS Assay Kits (Invitrogen). The cells together with supernatant were collected gently and then centrifuged at 1000 rpm for 5 min to discard cells. 10 μL supernatant (the volume was defined as ‘x’) was added to 190 μL prepared working solution and vortexed for 2–3 s. After incubation for two minutes, the fluorescence values R of the mixtures was read by Qubit 2.0 Fluorometer (the fluorescence values was defined as ‘QF value’), and the concentration of all the samples was calculated using the following equation: concentration of the sample = QF value × ( 200 ). x
Detection of NETs-mediated killing and phagocytic killing by neutrophils The neutrophil killing assays were performed according to the method described by Wartha et al. (2007) with minor
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modifications. For the phagocytic killing assay, purified neutrophils (N+ ) or 1640 RPMI medium (N− ) were incubated with 10% mouse serum, and the SS2 strains were added at an MOI of 1 and incubated for 90 min. The total number of bacteria was quantified by calculating the colony-forming units (cfu) on the plates after serial dilutions. The percentage of bacteria killed by the neutrophils was calculated according to the following formula: phagocytic killing% = [(cfuN − – cfuN + ) / (cfuN − )] × 100. The NETs killing assay was performed according to the method described by Wartha et al. (2007). The purified neutrophils (N+ ) or RPMI 1640 medium (N− ) was activated with 200 nM PMA for 4 h and then incubated with cytochalasin B (10 μg mL–1 , Aladdin) for 15 min to inhibit phagocytosis. Subsequently, the SS2 strains were added at an MOI of 0.01. After 30 min, the viable bacteria were quantified by serial dilutions of the thoroughly scraped well contents. The percentage of non-phagocytic killing of bacteria by PMA-induced NETs was calculated according to the following formula: non-phagocytic killing% = [(cfuN − – cfuN + ) / (cfuN − )] × 100.
Visualization of phagocytosis To visualize phagocytosis, the purified murine neutrophils were seeded on cell culture dishes with glass bottoms, and the adhered neutrophils were infected with the SS2 strains labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA SE), a green fluorescent dye at an MOI of 10 in the presence of 5% mouse serum. After a brief centrifugation (1000 rpm, 1 min) followed by an incubation period of 90 min, the samples were washed and then incubated with 2 mg mL–1 of trypan blue for 15 min to quenched adherent bacteria (Rest and Speert 1994). After washing, the samples were fixed with 4% paraformaldehyde. Then, the nuclear dye Hoechst 33258 was added and incubated for 10 min. Finally, all of the dishes were washed with PBS for three times and observed by the laser scanning microscopy system using the 40× object, zoom = 4.0 (Olympus fv1000mp).
NETs trapping assay To detect NETs trapping, the seeded purified neutrophils were activated with 200 nM PMA for 4 h and then infected with the SS2 strains labeled with the green fluorescent dye CFDA SE at an MOI of 10. After a brief centrifugation followed by an incubation period of 5 min, the samples were washed five times and then fixed with 4% paraformaldehyde. Subsequently, the NETs’ structure was labeled with the extracellular nuclear acid dye SYTOX orange (Invitrogen). Finally, the samples were then observed with the laser scanning microscopy system using the 40× object, zoom = 1.0. To quantify the trapping activity of the NETs toward the different strains, the average numbers of bacteria trapped by NET structure in 20 randomly selected microscopy fields were used to compare the NETs’ ability to capture specific SS2 strains.
Statistical analysis Unless otherwise specified, the data were analyzed using two-tailed, unpaired t test with Welch’s correction, and all of the assays were repeated at least three times. The data were expressed as the means ± standard error of the mean.
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RESULTS AND DISCUSSIONS Streptococcus suis induced the formation of NETs Mouse bone marrow neutrophils were isolated and purified, and nearly all of the isolated cells showed a lobulated nucleus, a characteristic of neutrophils (Fig. S1, Supporting Information). SS2 virulent strains can induce the formation of NETs (de Buhr et al. 2014). However, it is unknown whether this is a universal phenomenon for strains with different degrees of virulence. To address this question, the present study employed the three following virulent SS2 strains: the epidemic strain 05ZY that was isolated in China during an SS2 outbreak in 2005, contained 89K pathogenicity island and could cause STSLS; the reference SS2 strain P1/7 that showed a high degree of pathogenicity to pigs and mice; and strain A7, which showed intermediate pathogenicity to mice and pigs. To compare the induction of NETs, these strains were used to infect the purified neutrophils (MOI = 100), and NETs formation was examined using the extracellular nucleic acid dye SYTOX Green at 60, 120, 180 and 240 min post-infection (p.i.). NETs formation could be observed easily based on loss of the lobular shape of the neutrophilic nucleus and the formation of long DNA fibers. All of the strains tested could induce NETs formation at an early time point (120 min p.i.) (Fig. 1A). The quantity assay for detecting extracellular nucleic acid indicated that all these SS2 strains could result in the nucleic acid release (Fig. 1B) at 60 min post-incubation. Therefore, the selected SS2 strains with different level of virulence could induce NETs formation.
SS2 strains can resist NETs-mediated killing The SS2 strains could not inhibit and even induced the formation of NETs. This finding prompted us to evaluate whether these strains could efficiently resist NETs killing. To address this question, 05ZY, P1/7 and A7 were inoculated into PMA-induced NETs at an MOI of 0.01, and the resistance of these strains to NETs-mediated killing was determined by indirect quantification of the killed strains as described by Wartha et al. (2007). As expected, no detectable killing of 05ZY or P1/7 was observed in this assay (N.D.), and very few A7 strain (3.1 ± 3.1%) were killed by the non-phagocytic killing (Fig. 2A). This result indicated that NETs structure could not help to efficiently kill these pathogens, and it also meant that these SS2 strains could efficiently resist NETs-mediated killing at least in vitro. The result was also consistent with the expectation that these strains could resist neutrophil phagocytosis. Very low levels of 05ZY and P1/7 could be phagocytized by neutrophils, while A7 could be phagocytized by neutrophils at relatively higher levels (15.5 ± 0.9%) (Fig 2B and C). It is well known that the CPS structure of SS2 strains is responsible for their resistance to phagocytosis, and this understanding was also supported by our study. The isogenic cpsEF mutant (cpsEF), which originated from strain 05ZY and doesn’t produce a CPS structure (Fig. S2, Supporting Information), could be engulfed by neutrophils efficiently (37.3 ± 2.9%) (Fig. 2B and C). Furthermore, in a comparison with the encapsulated strains, the unencapsulated isogenic cpsEF mutant (cpsEF) could also easily be killed by nonphagocytic killing (17.2 ± 2.2%) (Fig. 2A). This result indicated that CPS structure not only played an essential role in the resistance to phagocytosis but also contributed to the ability of SS2 strains to escape non-phagocytic killing which mainly indicated NETs killing.
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Figure 1. Induction of NETs by SS2 strains. (A) Purified murine neutrophils were incubated with SS2 strains at MOI = 100 or PMA (200 nM) in the presence of the extracellular DNA dye SYTOX Green. NETs formation was visualized under fluorescence microscopy using the 20× object (OLYMPUS IX70) at 60, 120, 180 and 240 min post-incubation. The neutrophils in the absence of SS2 strains or PMA were served as a negative control (NC). The scale bars in the figure represent 50 μm. (B) Purified murine neutrophils were incubated with SS2 strains at MOI = 100, and the extracellular nucleic acid was quantitated at 60 min post-incubation. The ‘∗ ’ indicated that there was a significant difference (P < 0.01) between the infected cells (A7, P1/7, 05ZY and PMA) and the NC.
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Figure 2. SS2 strains resist NETs-mediated killing and phagocytosis-mediated killing. (A) For the NETs-mediated killing assay, SS2 strains with different degrees of virulence (05ZY, P1/7 and A7), and the isogenic cpsEF mutant (cpsEF) derived from 05ZY were used to determine the degree of non-phagocytic killing of these strains by PMA-induced NETs. Therefore, the non-phagocytic killing mainly indicated NETs killing. The ‘∗ ’ indicated that there was a significant difference (P < 0.05) between the groups. (B) For the phagocytosis-mediated killing assay, SS2 strains with different degrees of virulence (05ZY, P1/7 and A7) and the isogenic cpsEF mutant (cpsEF) were used to determine degree of phagocytosis-mediated killing in each strain. The ‘N.D.’ indicated that no detectable bacteria were recovered. The ‘∗ ’ indicated that a significant difference (P < 0.01) was existed in the two groups. (C) To visualize phagocytosis, adhered neutrophils were infected with SS2 strains labeled with the green fluorescent dye CFDA SE (CFDA, green fluorescent dye) at an MOI of 10. After incubation for 90 min, the dye of extracellular bacteria was bleached with trypan blue. Finally, the cells were further stained with Hoechst 33258 (Hoechst, blue fluorescent dye) and visualized with laser scanning microscopy system using the 40× object, zoom = 4.0 (Olympus fv1000 mp). The scale bars in the figure represent 15 μm.
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Figure 3. CPS structure enables SS2 strains to escape NETs trapping. (A) For the visualization assay, SS2 strains with different degrees of virulence (05ZY, P1/7 and A7), the isogenic cpsEF mutant (cpsEF), which originated from 05ZY were inoculated into PMA-induced NETs at an MOI of 10. All of the SS2 strains were pre-stained with green CFDA SE fluorescent dye (CFDA, green fluorescent dye). After a 5 min incubation, all of the samples were washed five times, and the extracellular nucleic acid was stained with SYTOX Orange Nucleic Acid Stain (SYTOX Orange, red fluorescent dye). The bacteria in the DNA fibers were determined as trapped by NETs structure. The scale bars in the figure represent 50 μm. (B) To quantify the trapping activity of the NETs against the different strains, the average numbers of bacteria trapped by NETs in 20 randomly selected microscopy fields were used to compare the ability of the NETs to trapping specific SS2 strains. The ‘∗ ’ meant there was a significant difference (P < 0.01) between the two groups.
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The CPS structure enables SS2 strains to escape NETs trapping The DNA fibers that comprise the NETs interact with histones and antimicrobial molecules; these interactions provide the NETs the ability to destroy any bacteria that they contact (Jaillon et al. 2007). SS2 strains are susceptible to antimicrobial molecules such as lysozyme in vitro (Fittipaldi et al. 2008). Furthermore, all three of the strains examined in this study are susceptible to the host antibacterial peptide LL-37 in vitro, indicating that none of these strains should resist killing by host antimicrobial molecules. Together, these findings prompted us to consider whether the resistance of SS2 strains to NETs-mediated killing was due to successfully escaping from trapping. To test this hypothesis, all of the strains were inoculated into PMA-induced NETs at an MOI of 10 to detect capture by the NETs. As shown by a fluorescence-based assay, no 05ZY, P1/7 or A7 cells were trapped efficiently (Fig. 3A and B). This result suggested that these SS2 strains could successfully escape trapping by the NETs, and this further resulted in increased resistance to killing. It is well known that the CPS structure of Pneumococcus plays an essential role in resistance to NETs trapping (Wartha et al. 2007). CPS is recognized as an essential virulence factor in S. suis due to its antiphagocytosis effects. This study demonstrated that the CPS of the SS2 strains could resist NETs-mediated killing (Fig. 2A). We observed that the unencapsulated isogenic mutant cpsEF could be trapped by NETs (Fig. 3A). Therefore, the CPS structure enabled the SS2 strains to escape trapping and killing by the NETs. To date, many virulence-associated factors have been identified due to isogenic mutants that showed significantly decreased virulence. Comparative genome studies indicated that several genes contribute to virulence by regulating the expression of genes responsible for CPS synthesis, such as CcpA (Willenborg et al. 2011) and HP0197 (Zhang et al. 2012). In addition to its antiphagocytosis effect, the present study demonstrated the essential role of CPS in resisting NETs-mediated killing, which may also contribute to the immune evasion of SS2. This result would further strengthen our understanding of how certain genes could contribute to virulence by regulating CPS structure. In summary, this present study demonstrated that the epidemic strain 05ZY, the highly pathogenic strain P1/7 and the intermediately pathogenic strain A7 could induce the formation of NETs. Furthermore, these SS2 strains could efficiently resist non-phagocytic killing, which suggested that these SS2 strains could efficiently resist NETs-mediated killing. Because the SS2 strains were susceptible to the antimicrobial molecules that are the major bactericidal components of NETs, we tested whether the resistance was the result of evading capture. Our hypothesis was confirmed by observations that all of the tested SS2 strains but not the unencapsulated isogenic cpsEF mutant could escape trapping efficiently. Therefore, our study demonstrated that the SS2 strains could induce NETs formation and that the CPS structure enabled these strains to evade trapping and further killing.
SUPPLEMENTARY DATA Supplementary data is available at FEMSLE online.
ACKNOWLEDGEMENTS We thank American Journal Experts (AJE) for English language editing. The authors have declared that no competing interests exist.
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FUNDINGS This work was supported by the National Natural Science Foundation of China (31172328, 31272544), the 973 Program (2012CB518805), Program for New Century Excellent Talents and the Fundamental Research Funds for the Central University (2011PY006). Conflict of interest statement. None declared.
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