Quercetin reduces Streptococcus suis virulence by inhibiting suilysin activity and inflammation
Introduction
Streptococcus suis (S. suis), one of the most important pathogens in the swine industry, is a gram-positive bacterium commonly found worldwide that can cause major economic losses and several fatal infections, including streptococcal toxic shock syndrome (STSS), meningitis and arthritis [[1], [2], [3]]. In the United States alone, this bacterium has resulted in the loss of hundreds of millions of dollars per year [4]. S. suis is considered an emerging and serious threat to public safety, as it is an opportunistic zoonotic pathogen [3]. To date, more than 30 serotypes of S. suis have been identified based on the specificity of the capsule antigens [5], and serotype 2 is the most frequently isolated serotype from clinically infected pigs worldwide [6,7];most meningitis cases are caused by S. suis type 2 (SS2) [5]. This bacterium poses a major hazard to pig producers and pork processors who have close contact with pigs and pork products or consumers who eat undercooked pork [5,8]. Approximately 1650 cases of human S. suis infections were reported worldwide before 2014 [9]. In the southeast Asian countries Vietnam and Thailand, S. suis infection is one of the leading causes of bacterial meningitis in adults [10,11]. In 1998 and 2005, two severe outbreaks of S. suis occurred in China. Hundreds of people were infected, and dozens of deaths occurred in a short period of time, which caused a major public health crisis, incalculable economic losses and serious consequences [12,13]. S. suis infections have caused serious public health issues, and effective treatment methods and strategies are urgently needed.
More than half of Chinese patients infected by SS2 in the aforementioned outbreak in 2005 had an unusual clinical manifestation of STSS, including disseminated intravascular coagulation, which was associated with platelet-bacterium interactions, and these patients showed increased mortality [[13], [14], [15]]. Suilysin (SLY), a secreted toxin of most virulent S. suis strains, was reported as the sole stimulatory factor for platelet activation and aggregation in the infections caused by S. suis. Moreover, the SLY-negative SS2 strain 1330 could not induce platelet aggregation [16], suggesting SLY may play a crucial role in DIC of STSS induced by SS2. SLY is an important exocrine toxin of SS2, a member of the cholesterol-dependent cytolysin (CDC) family, which can create pores in cholesterol-containing membranes and then lead to cell lysis [17,18]. The role and mechanism of SLY in human infections with SS2 are still unclear; however, the worldwide clinical case records show that SLY-positive strains led to more severe symptoms in China and southeast Asia than SLY-negative strains in North America [10,19,20]. In addition, Takeuchi et al. found that S. suis ST1, a strain producing high levels of SLY, caused meningitis more often than strains that produced low levels of SLY or no SLY [21]. In addition, the SS2 strain 05ZYH33 with increased SLY production showed hypervirulence in vivo and in vitro compared to S. suis 1940, a strain with lower levels of SLY production, which had a higher ability to penetrate the epithelium and survive in the bloodstream [22]. Moreover, SLY could also stimulate the release of heparin binding protein, induce inflammatory responses and increase the vascular permeability in mice [23]. Furthermore, SLY played an important role in a mouse model of SS2 meningitis. The mice infected with high levels of the SLY-producing strain had survival rates of less than 10%; however, in another group of mice used as a control, which was infected with a low level of the SLY strain, or a sly knockout strain, only a few deaths were observed, and mortality decreased significantly. Compared with the group infected with the strain producing high levels of SLY, the control group showed significantly reduced colony colonization and inflammation in the brain. These results further demonstrated that SLY was a crucial factor contributing toSS2 meningitis [21]. Thus, SS2 requires a series of virulence factors to successfully infect and cause host disease; SLY plays a crucial role in this process and may be related to STSS and meningitis. Therefore, SLY could be a potential new target for the treatment of SS2 infection.
S. suis infection can trigger a series of severe inflammatory reactions, especially arthritis and meningitis [5]. An excessive inflammatory response is a manifestation of STSS induced by SS2 [24]. Several studies have suggested that the increasing permeability through the blood-brain barrier during SS2 infection was stimulated by inflammatory reactions or mediators that occurred during adhesion and infection [25]. In addition, SS2 could stimulate monocytes/macrophages and produce pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and monocyte chemotactic protein-1 (MCP-1) [26,27]. In addition, SS2 infection results in recognition of toll-like receptor 2 (TLR 2) and activation of members of the mitogen-activated protein kinase (MAPK) pathway, including c-Jun N-terminal kinase (JNK) and p38MAPK extracellular-signal-regulated kinase (ERK), which are closely related to the inflammatory response and can also regulate cell differentiation, proliferation and death [28,29]. A series of inflammatory reactions induced by SS2 are also an important part of the pathogenic process. Therefore, attenuating the inflammatory response of SS2 may be an important strategy for the treatment of SS2 infection.
Antimicrobial therapy is currently the main method for clinical treatment of SS2 infection [30], but bacterial resistance is becoming increasingly serious, especially given that the rate of development of new antibiotics is much lower than the emergence of bacterial resistance [31]. To effectively improve or eliminate the current unfavorable situation in the fight against S. suis infection, new anti-infective strategies and agents are urgently needed. In recent years, anti-virulence strategies have been recognized as promising and effective approaches to fight the rapid development of drug resistance [32]. Targeting virulence factors does not affect the normal growth of bacteria [33] and does not put a strong survival selection pressure on bacteria, which may delay the emergence of drug resistance. However, treatment with anti-virulence agents could significantly reduce the pathogenicity of pathogens [32,33]. At present, anti-virulence strategies using specific antibodies against α-hemolysin can significantly inhibit the virulence of S. aureus in vitro and in vivo [34].
Quercetin is a natural flavonoid that can be extracted from a variety of plants and fruits and has various pharmacological and biological effects, including antiviral, antioxidation, anticancer and anti-inflammatory effects [[35], [36], [37], [38]]. Recent studies have found that quercetin could inhibit obesity and obesity-induced inflammation in cells and mice, which could also inhibit angiogenesis in nasopharyngeal carcinoma [39,40]. In addition, quercetin induced the differentiation of bone marrow mesenchymal stem cells [41]. However, the effect of quercetin in infections caused by SS2 and the interaction with SLY have not been reported thus far. In this study, we demonstrate that the natural bioflavonoid compound quercetin could inhibit the activity of SLY by preventing SLY oligomerization and significantly reduce the inflammatory response in SS2 infected cells and the mortality in infected mice. Thus, our results suggested quercetin may be a promising therapeutic candidate to treat SS2 infections.
Section snippets
Bacterial strain and regents
The strain used in this study was the highly virulent SS2 ZY05719, which was isolated from pigs and kindly provided by Professor Hongjie Fan from Nanjing Agricultural University (Nanjing, China). Cell culture medium (Dulbecco's modified Eagle's medium, DMEM) was purchased from Invitrogen. Fetal bovine serum (FBS) was purchased from Biological Industries (BI). The bacterial medium Todd-Hewitt broth (THB) was obtained from Qingdao Hope Bio-Technology (Qingdao, China). Dimethyl sulfoxide (DMSO)
Quercetin inhibits SLY pore-forming activity without interfering with SS2 viability
The MIC of the natural flavonoid quercetin (Fig. 1A) on SS2 was more than 1024 μg/mL; additionally, no significant difference was observed for the growth of SS2 in the presence of various concentrations of quercetin (Fig. 1B). These data suggested that quercetin had no anti-SS2 activity and did not affect the viability of SS2. After SS2 was co-cultured with quercetin, the hemolytic activity of SS2 culture supernatants was evaluated by hemolysis assays. As shown in Fig. 1C, the bacterial culture
Discussion
Similarly, anti-virulence strategies can also be used for SS2infections. Among the various virulence factors, SLY plays an important role in SS2 pathogenicity, as deletion of SLY can significantly reduce the pathogenicity of SS2 in mice [21]. In addition, SLY can increase the vascular permeability of mice, which is related to STSS [23]. Strains producing high-level SLY, but not strains with weak SLY secretion, are more likely to cause meningitis in humans [21]. In addition, SLY could facilitate
Conflicts of interest
None of the authors have a financial interest in any of the products, devices, or Materials mentioned in this manuscript. The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the National Key Technology R&D Program (No. 2016YFD05013), the National Natural Science Foundation of China (grant 31602109) and the Jilin Scientific and Technological Development Program under Grant 20190103147JH.
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These authors contributed equally to this article.