Microbial Pathogenesis xxx (2014) 1e6
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The impact of mgrA on progression of Staphylococcus aureus sepsis Cai-lin Liu, Zhong-ju Chen, Feng Wang, Hong-yan Hou, Yue Wang, Xu-hui Zhu, Cui Jian, Lei Tian, Shao-zhen Yan, Ling-qing Xu, Zi-yong Sun* Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2014 Received in revised form 30 March 2014 Accepted 31 March 2014 Available online xxx
Sepsis induced by Staphylococcus aureus has worse outcome with the appearance of methicillin-resistant Staphylococcus aureus (MRSA) because of multi-resistance to a large group of antibiotics, which may lead to death from septic shock. Pathogenesis of S. aureus infections are involved in the production of a wide variety of virulence factors. MgrA, a noval global regulator, is a member of the MarR (multiple antibiotic resistance regulator)/SarA (staphylococcal accessory regulator A) family proteins, which plays a key role in regulating the expression of major virulence factors in S. aureus. In the present study, by using a murine model of sepsis, we investigated the role of mgrA in onset and progression of S. aureus induced sepsis. We found that mice inoculated with wild-type strain Newman had significantly higher mortality (p ¼ 0.029), more weight lost, more bacterial load in blood, spleen and kidney, more intense inflammation response, and worse histopathology than mice inoculated with mgrA knockout strain. Our results has provided evidence that mgrA is a global regulator in S. aureus, and play an important role in S. aureus sepsis, could increase mortality and accelerate the onset and development of sepsis. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: mgrA Staphylococcus aureus Sepsis Virulence
1. Introduction Staphylococcus aureus is a major human pathogen that causes a wide variety of diseases, ranging from skin and soft tissue infections to life-threatening illnesses, such as pneumonia, toxic shock syndrome, bacteremia and endocarditis [1,2]. Despite antimicrobial therapy, the morbidity and mortality associated with S. aureus infections remain high, due, in part, to the organism’s ability to develop resistance to virtually all antibiotics, including vancomycin [1,3]. The capacity to cause infection is probably attributable to the organism’s capacity to colonize and survive in host during infection process. Sepsis is a complex and severe infection resulting in multiorgan failure with a fatal outcome, Which is one of the main causes of death in Intensive Care Units and accounts for about 120,491 deaths per year in the US [4]. The pathogenicity of S. aureus is a complex process involving the expression of an array of different virulence factors, including adhesins [e.g. Fibronectinbinding proteins (FnBPs) and protein A] that mediate binding to host cells, enzymes (e.g. proteases and lipases), toxins [e.g.a-hemolysin and PantoneValentine leukocidin (PVL)], phenolsoluble modulins and capsular polysaccharides [5e
* Corresponding author. Tel./fax: þ86 02783663639. E-mail address: [email protected] (Z.-y. Sun).
7]. The expression of virulence factors is coordinately regulated by multiple two component systems and global transcription factors. mgr, a global regulator in S. aureus, first be reported by Luong TT et al., in 2003 [8], which is a member of the MarR (multiple antibiotic resistance regulator)/SarA (staphylococcal accessory regulator A) family proteins and controls expression of w350 genes. It plays a key role in the regulation of the expression of major virulence factors in S. aureus (e.g., a-hemolysin, coagulase, protein A, capsular polysaccharide, protease, and nuclease). Furthermore, mgrA also modulates the expression of efflux pump and autolysins and affect resistance of S. aureus to fluoroquinolone and glycopeptide drugs [9,10]. To elucidate the role of mgrA in onset and progression of S. aureus induced sepsis, a murine model of sepsis has been development in the current study. 2. Results 2.1. mgrA expression regulate the progression of S. aureus sepsis Mice were inoculated with wild-type strain Newman expressing the mgrA gene and mgrA knockout strain lacking mgrA expression with a septic dose of 6.0 106 CFU, respectively (n ¼ 10/group). During the course of experiment (up to 14 days), seven of ten mice in the wild-type Newman group died (mortality rate 70%), and only
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one of ten mice in the mgrA knockout group died (mortality rate 10%). Mice inoculated with the mgrA knockout survived significantly better (log rank ¼ 4.739, p ¼ 0.029) than mice inoculated with wild-type strain Newman (Fig. 1). To investigate weight change, all of mice were inoculated with bacteria with a dose of 3.0 106 CFU (n ¼ 10/group). Our results showed that mice inoculated with the wild-type strain lost significantly more weight during the course of experiment as compared to mgrA knockout strain inoculated mice(up to 14 days) (Fig. 2). 2.2. Assays of CD4þ/CD8þ T cells and serum TNF-a, IL-6 Previous studies have shown that CD4þ/CD8þ T cells significantly changes in the inflammatory response, adaptive CD4þ T cell responses are thought to be regulated by cytokines produced by two distinct CD4þ T cell subsets, Th1 and Th2 cells. Th1 cells produce IL-2 and IFN-g, whereas Th2 cells synthesize IL-4 and IL-10, which promotes the inflammatory response. Cytotoxic T cell (CD8þ T cell) releases IL-4, IL-5, IL-6, and IL-13 [11,12]. As shown in Fig. 3A, CD4þ/CD8þ T cells in blood peaked at about 24 h after induction of sepsis. However, CD4þ/CD8þ T cells in blood was significantly lower in mgrA knockout group than in wild-type strain group at 24 h (p ¼ 0.0011), 48 h (p ¼ 0.0025), and 72 h (p ¼ 0.0243) (Fig. 3A). To investigate how mgrA expression affects the cytokine production, we analyzed differences in inflammatory cytokines in the serum during sepsis. As shown in Fig. 3B, serum levels of IL-6 reached a maximum level at 12 h after the injection of S. aureus, and gradually fell thereafter in both groups, however, significantly higher IL-6 levels were seen in wild-type Newman group than in mgrA knockout group at 4 h (p < 0.0001), 12 h (p < 0.0001), 24 h (p < 0.0001), 48 h (p ¼ 0.0003) and 72 h (p ¼ 0.0012) (Fig. 3B). As shown in Fig. 3C, serum TNF-a levels peaked at about 4 h after injection of bacteria, TNF-a levels were comparable in wild-type Newman group and mgrA knockout group at 1 h, 3 h, 48 h, 72 h, 96 h, and 120 h. However, 4 h after induction of sepsis, mgrA knockout group significantly lower levels of TNF-a than wild-type Newman group at 4 h (p ¼ 0.0032), 12 h (p < 0.0001), 24 h (p < 0.0001) (Fig. 3C). 2.3. Analysis of bacterial load of blood and tissues Blood and tissue bacterial load after animal death or sacrifice (up to 14 days) were shown in Fig. 4. Bacterial load in blood after
Fig. 1. Survival curves of BALB/c mice (n ¼ 10/group) inoculated with 6 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. Mice inoculated with mgrA knockout strain showed significantly improved survival than mice inoculated with the wild-type Newman. (p ¼ 0.029, KaplaneMeier, log-rank test).
Fig. 2. Weight changes of BALB/c mice (n ¼ 10/group) inoculated with 3 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. Comparisons were made using the ManneeWhitney U-test.
challenge by wild-type strain was higher than those of mgrA knockout strain (Fig. 4A). Tissue bacterial load was significantly higher in the kidney and liver of mice in wild-type Newman group compared with mgrA knockout group (Log10 bacteria of kidney: 7.53 0.23 vs 6.02 þ 0.33, p < 0.001; Log10 bacteria of liver:5.82 0.26 vs 4.84 0.29, p ¼ 0.024). However, the bacterial load in spleen had no difference between the two groups (Log10 bacteria of spleen:4.67 0.19 vs 4.51 0.54, p ¼ 0.404) (Fig. 4B). 2.4. Assessment of histopathology Our results indicated that both of the two groups caused slight to moderate histopathology in most tissues at day 1 and day 2 and showed severe histopathology at day 4. As compared with mice inoculated with mgrA knockout strain, the wild-type inoculated mice had significantly more severe histopathology (Fig. 5). 3. Discussion MgrA is a 147-residue protein that belongs to a member of regulatory proteins called SarA protein family by virtue of their homology to SarA of S. aureus [13], which is a pleiotropic regulator that controls autolysis, virulence, biofilm formation, and efflux pump activity in S. aureus [14e16]. In a transcriptional profiling study of the mgrA of S. aureus strain Newman, Luong et al. found that MgrA upregulates 175 genes while downregulating 180 others [8]. In the present study we used a murine sepsis model induced by S. aureus to investigate the impact of mgrA gene expression on the onset and progression of sepsis. Our result showed that mice inoculated with the wild-type strain displayed significantly higher mortality, more weigh lost, and more severe histopathology than mice inoculated with mgrA mutation strain lacking mgrA gene expression. S. aureus can infect almost every tissue in the human host, its pathogenicity is mostly due to the production of various virulence factors, such as hemolysins, leukocidins, immune Modulators, cell wall components, and capsular polysaccharide (CP). Any alteration in regulatory genes will affect the expression of these virulence factors [3,17]. MgrA in S. aureus, a global transcriptional regulator, was identified previously as a key virulence determinant, which in particular plays a key role in the regulation of the expression of major virulence factors in S. aureus (e.g., capsular polysaccharide, protease, a-toxin, nuclease, and protein A) [8,13]. A previous study have shown that MgrA is critical for S. aureus pathogenesis in vivo, and an mgrA mutant strain exhibits 1000e10,000 fold virulence
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Fig. 3. Levels of CD4þ/CD8þ T cells, serum IL-6, and TNF-a, after induction of sepsis by inoculated with 6 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. Blood samples were taken at the indicated time points. A. CD4þ/CD8þ ratio. B. Serum IL-6 levels. C. Serum IL-6 levels. The means SD of ten mice are shown. * ¼ p < 0.05, p-value was obtained by 2-tailed t-test.
reduction in a mouse model of infection [18]. Jonsson IM et al. have demonstrated that mice that were inoculated with the wildtype Newman strain displayed significantly higher rate of mortality, showed a less favorable development of weight and displayed higher frequency and severity of arthritis than mice inoculated with the S. aureus mutant lacking mgrA gene expression in a murine model of hematogenously spread S. aureus arthritis [19]. Our results showed that significantly faster and more severe progression of sepsis in mice inoculated with the
wild-type strain Newman than mice inoculated with mgrA knockout strain. Cytokines are a group of proteins that are expressed by several cell types, act as immune mediators and regulators [20]. Levels of several important cytokines can easily be measured in the peripheral circulation, and therefore, cytokines have long been proposed as potential markers. The most commonly cytokines are tumor necrosis factor (TNF)-a and interleukin (IL)-6. Enhanced TNF-a and IL-6 levels evidently correlate positively with the
Fig. 4. Comparative bacteria loads in blood, liver, spleen, and kidney after mice death or sacrifice with infection inoculated with 3 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. A: blood bacterial load. B: organs bacterial load. The means SD of ten mice are shown. ns ¼ not significant. p-value was obtained by 2-tailed t-test.
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Fig. 5. Representative tissue sections of BALB/c mice at day 2: A: Liver of a mice inoculated with wild-type Newman showed moderate sinusoidal engorgement and focal lobular necrosis. B: Liver of a mice inoculated with mgrA knockout showed slight sinusoidal engorgement and focal lobular necrosis. C: Spleen of a mice inoculated with wild-type Newman showed Mast cell and moderate hemorrhagic necrosis. D: Spleen of a mice inoculated with mgrA knockout showed slight hemorrhagic necrosis. E: Kidney of a mice inoculated with wild-type Newman showed slight renal tubular epithelial cell swelling. F: Kidney of a mice inoculated with mgrA knockout showed no significant histopathology. Original magnification: 400. Hematoxylin-eosin staining was used.
incidence of sepsis [21]. Our results revealed that wild-type strain inoculated mice released significantly higher serum levels of TNF-a and IL-6, along with more severe sepsis progression, than mice inoculated with mgrA knockout strain. T cells secretes IL-2, IFNgand TNF-b, IL-4, IL-9, IL-10 and IL-13 et al. After antigen stimulation, CD4þ T cells are traditionally thought to differentiate into either Th1 or Th2 subsets. Each subset is characterized by its distinct cytokine production profile, mediation of cellular (Th1) or humoral (Th2) immunity. Previous studies have shown that CD4þ/ CD8þ T cells significantly changes in the inflammatory response
[11,12]. In this study, CD4þ/CD8þ T cells ratio was significantly lower in mgrA knockout strain inoculated mice than that wild-type strain inoculated mice at 24 h, 48 h, and 72 h. In vivo survival of S. aureus is also dependent on the ability to evade killing by host macrophages. Most S. aureus clinical isolates express a thin microcapsular layer that is composed of serotype 5, serotype 8 or serotype 336 capsular polysaccharide (CP). Expression of type 5 and type 8 capsule is associated with increased virulence in animal infection models. In vitro phagocytosis assays revealed that the presence of the capsule reduced the uptake of
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cells by neutrophils in the presence of normal serum opsonins, indicating that capsule is anti-opsonic [22,23]. MgrA can upregulate capsular polysaccharide encoding genes, and a lack of the mgrA gene can lead to diminished expression of CPs and thereby to decreased bacterial resistance to phagocytosis and intracellular killing [19]. Indeed, Our results showed that mice inoculated with wild-type strain had significantly more bacterial load in blood and tissue than that mgrA knockout strain inoculated mice. We concluded that mgrA knockout strain had a reduced expression of CP8 due to lack of mgrA gene which lead to decreased bacterial resistance to phagocytosis. In addition, we demonstrated that wildtype strain inoculated mice had faster and more severe histopathology than that mgrA knockout inoculated mice, suggesting that mgrA regulates virulence factors which impact on tissue damage. Once S. aureus invades the tissue, it produces a large number of cell surface-associated components and secreted products that include adhesins, enzymes, toxins, capsular polysaccharides, and other gene products that facilitate tissue colonization, tissue destruction, or immune evasion [24,25]. Our results demonstrated that mice inoculated with mgrA knockout presented significantly lower bacteria load in kidney and liver than that mice inoculated with wild-type strain. Jonsson IM et al. [22] have also reported significantly lower mgrA mutant bacteria load in kidneys both at early and late stages of infection. Previous study have showed [26,27] sortase A was more expressed in the wild-type than in the mgrA mutant strain, and an upregulation of sortase A in vitro results in increased expression of proteins like protein A, clumping factor, and collagen adhesin and thereby efficient adherence to host tissues by wild-type staphylococci. Mutants that lack the mgrA will thus have difficulties to colonize the host, suggesting that MgrA also regulates virulence factors that are of importance for colonization in tissue. In summary, the current study verified previous findings on the role of mgrA as a global regulator in S. aureus, this study showed that mgrA increased mortality and accelerate the onset and development of sepsis, the BALB/c mice inoculated with wild-type strain Newman showed more intense inflammatory response, more blood and tissue bacteria load, and more severe histopathological changes than the mice inoculated with mgrA knockout strain. 4. Material and methods 4.1. Mice Male BALB/c mice (6e8 week old, weight 18e22 g) were maintained separately at the Animal Facility of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, under controlled conditions (specific pathogen free, 22 C, 55% humidity, and 12 h light/dark cycle). All experimental procedures on animals used in this study were performed under a protocol approved by the Huazhong University of Science and Technology Animal Care and Use Committee (Permit Number:SYXK2009-0049). 4.2. Bacterial strains and construction of mgrA knockout strain S. aureus wild-type strain Newman (Biovector Science Lab, Inc, China) and its isogenic mgrA knockout strain were used in this study. To construct mgrA mutant, we amplified by PCR two fragments that flanked the left and right sides of mgrA gene sequence targeted for deletion. The two resulting PCR fragments have a 16base complementary region to facilitate annealing followed by a second round of PCR amplification with outside primers to obtain a single fragment. The fusion product was purified, digested with SmaI, and ligated into shuttle vector pMAD. The resulting
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recombinant pMAD plasmids were transformed first into S. aureus RN4220 and then into Newman strain by electroporation [28,29]. Construction and selection of the putative mgrA mutant was performed as described previously [30]. The plausible allelic replacement mutants were confirmed by PCR and Southern blots with the appropriate probes. The bacterial strains were cultured in Brain-Heart Infusion (BHI) broth at 37 C in air with shaking to log phase. Bacteria were centrifuged at 5000 g for 10 min and washed twice with sterile phosphate buffered saline (PBS). Then, the bacterial suspension was resuspended to an OD600 of 0.4. To verify the correct dilution, an aliquot was serially diluted on Tryptic Soy Agar (TSA) plates and colonies were counted after a 24 h incubation at 37 C. 4.3. Experimental infection of mice Four separate experiments were performed in this study. In all experiments, the mice were inoculated i.v. into tail vein with 0.2 ml suspension of S. aureus, and weighed regularly during the course of experiments. In the first experiment we assessed the impact of mgrA mutation on mortality, ten mice per group were inoculated with a dose of 6.0 106 CFU of S. aureus, a bacteria dose known to induce sepsis (up to 14 days). In the second experiment, mice were inoculated with a dose of 6.0 106 CFU of S. aureus, blood were analyzed for CD4þ/CD8þ T cells, and serum was analyzed IL-6, and TNF-a levels (up to 120 h). In the third experiment, mice were inoculated with a dose of 3.0 106 CFU of S. aureus for assessment of weight change (up to 14 days). In the fourth experiment, mice were inoculated with a dose of 3.0 106 CFU of S aureus for examination of blood and tissue bacteria load and histological analyses (up to 14 days). 4.4. Bacterial counts in blood and tissues The blood added to broth was serially diluted 1:10 in 0.9% NaCl. A 0.2 ml aliquot of each dilution was plated onto Blood Agar. The number of viable cells was estimated after counting on plates and multiplying by the appropriate dilution factor. The number of viable bacteria was expressed as Log10CFU/mL. Tissue segments of spleen, liver and kidney were weighted and homogenized; a 0.1 ml aliquot was then serially diluted 1:10 in sterile sodium chloride. Another aliquot of 0.1 ml of each dilution was plated onto Blood Agar. Plates were incubated at 35 C and the number of viable colonies were counted at each dilution and multiplied by the appropriate dilution factor. The number of viable bacteria was expressed as Log10CFU/g. 4.5. Flow cytometric analysis Blood was collected into heparin containing tubes. Erythrocytes were removed by cell lysis in BD FACS lysing solution (BD Biosciences, Heidelberg, Germany). After washing two times with FACS buffer (phosphate-buffered saline, 0.5% bovine serum albumin, 0.1% sodium azide. BD Biosciences), FITC-anti mouse CD4 and FITC-anti mouse CD8a (Biolegend, California, USA) were added to cell suspensions. All of these cell suspensions were incubated for 30 min at 4 C. After two washes with FACS buffer, the pellets were resuspended in 300 ml FACS buffer followed by analysis with FACScan flow cytometer (Becton Dickinson). 4.6. Enzyme-linked immunosorbent assay (ELISA) for IL-6 and TNFa At the indicated time points after induction of sepsis, approximately 0.5 ml blood were collected by eye enucleated, placed on ice
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and allowed to clot before centrifugation at 3000 g for 10 min. The serum levels of cytokines were measured by a standard sandwich cytokine ELISA procedure, according to the instructions of mouse IL-6 and TNF-a kits (R&D Systems, Minneapolis, MN, USA). 4.7. Histological analyses For evaluation of the extent of tissue damage and cellular response, histological analyses were performed. For the two experimental groups, three mice per group for every time point were submitted to histopathology examination after 1, 2, 4, 7, 14day after inoculation with 3 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. The following organs were collected from each animal: liver, spleen and kidneys. Tissues were fixed with 4% paraformaldehyde, then stained with hematoxylineeosin. 4.8. Statistical analysis Statistical analysis of survival curves was estimated by Kaplane Meier analysis, log-rank test was used. To compare weight change, ManneWhitney U-test was used. Cytokine levels, blood and tissues bacterial counts were expressed as the mean SD, data sets were analyzed using two-tailed Student’s t test. Any p value
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
The impact of mgrA on progression of Staphylococcus aureus sepsis Cai-lin Liu, Zhong-ju Chen, Feng Wang, Hong-yan Hou, Yue Wang, Xu-hui Zhu, Cui Jian, Lei Tian, Shao-zhen Yan, Ling-qing Xu, Zi-yong Sun* Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2014 Received in revised form 30 March 2014 Accepted 31 March 2014 Available online xxx
Sepsis induced by Staphylococcus aureus has worse outcome with the appearance of methicillin-resistant Staphylococcus aureus (MRSA) because of multi-resistance to a large group of antibiotics, which may lead to death from septic shock. Pathogenesis of S. aureus infections are involved in the production of a wide variety of virulence factors. MgrA, a noval global regulator, is a member of the MarR (multiple antibiotic resistance regulator)/SarA (staphylococcal accessory regulator A) family proteins, which plays a key role in regulating the expression of major virulence factors in S. aureus. In the present study, by using a murine model of sepsis, we investigated the role of mgrA in onset and progression of S. aureus induced sepsis. We found that mice inoculated with wild-type strain Newman had significantly higher mortality (p ¼ 0.029), more weight lost, more bacterial load in blood, spleen and kidney, more intense inflammation response, and worse histopathology than mice inoculated with mgrA knockout strain. Our results has provided evidence that mgrA is a global regulator in S. aureus, and play an important role in S. aureus sepsis, could increase mortality and accelerate the onset and development of sepsis. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: mgrA Staphylococcus aureus Sepsis Virulence
1. Introduction Staphylococcus aureus is a major human pathogen that causes a wide variety of diseases, ranging from skin and soft tissue infections to life-threatening illnesses, such as pneumonia, toxic shock syndrome, bacteremia and endocarditis [1,2]. Despite antimicrobial therapy, the morbidity and mortality associated with S. aureus infections remain high, due, in part, to the organism’s ability to develop resistance to virtually all antibiotics, including vancomycin [1,3]. The capacity to cause infection is probably attributable to the organism’s capacity to colonize and survive in host during infection process. Sepsis is a complex and severe infection resulting in multiorgan failure with a fatal outcome, Which is one of the main causes of death in Intensive Care Units and accounts for about 120,491 deaths per year in the US [4]. The pathogenicity of S. aureus is a complex process involving the expression of an array of different virulence factors, including adhesins [e.g. Fibronectinbinding proteins (FnBPs) and protein A] that mediate binding to host cells, enzymes (e.g. proteases and lipases), toxins [e.g.a-hemolysin and PantoneValentine leukocidin (PVL)], phenolsoluble modulins and capsular polysaccharides [5e
* Corresponding author. Tel./fax: þ86 02783663639. E-mail address: [email protected] (Z.-y. Sun).
7]. The expression of virulence factors is coordinately regulated by multiple two component systems and global transcription factors. mgr, a global regulator in S. aureus, first be reported by Luong TT et al., in 2003 [8], which is a member of the MarR (multiple antibiotic resistance regulator)/SarA (staphylococcal accessory regulator A) family proteins and controls expression of w350 genes. It plays a key role in the regulation of the expression of major virulence factors in S. aureus (e.g., a-hemolysin, coagulase, protein A, capsular polysaccharide, protease, and nuclease). Furthermore, mgrA also modulates the expression of efflux pump and autolysins and affect resistance of S. aureus to fluoroquinolone and glycopeptide drugs [9,10]. To elucidate the role of mgrA in onset and progression of S. aureus induced sepsis, a murine model of sepsis has been development in the current study. 2. Results 2.1. mgrA expression regulate the progression of S. aureus sepsis Mice were inoculated with wild-type strain Newman expressing the mgrA gene and mgrA knockout strain lacking mgrA expression with a septic dose of 6.0 106 CFU, respectively (n ¼ 10/group). During the course of experiment (up to 14 days), seven of ten mice in the wild-type Newman group died (mortality rate 70%), and only
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one of ten mice in the mgrA knockout group died (mortality rate 10%). Mice inoculated with the mgrA knockout survived significantly better (log rank ¼ 4.739, p ¼ 0.029) than mice inoculated with wild-type strain Newman (Fig. 1). To investigate weight change, all of mice were inoculated with bacteria with a dose of 3.0 106 CFU (n ¼ 10/group). Our results showed that mice inoculated with the wild-type strain lost significantly more weight during the course of experiment as compared to mgrA knockout strain inoculated mice(up to 14 days) (Fig. 2). 2.2. Assays of CD4þ/CD8þ T cells and serum TNF-a, IL-6 Previous studies have shown that CD4þ/CD8þ T cells significantly changes in the inflammatory response, adaptive CD4þ T cell responses are thought to be regulated by cytokines produced by two distinct CD4þ T cell subsets, Th1 and Th2 cells. Th1 cells produce IL-2 and IFN-g, whereas Th2 cells synthesize IL-4 and IL-10, which promotes the inflammatory response. Cytotoxic T cell (CD8þ T cell) releases IL-4, IL-5, IL-6, and IL-13 [11,12]. As shown in Fig. 3A, CD4þ/CD8þ T cells in blood peaked at about 24 h after induction of sepsis. However, CD4þ/CD8þ T cells in blood was significantly lower in mgrA knockout group than in wild-type strain group at 24 h (p ¼ 0.0011), 48 h (p ¼ 0.0025), and 72 h (p ¼ 0.0243) (Fig. 3A). To investigate how mgrA expression affects the cytokine production, we analyzed differences in inflammatory cytokines in the serum during sepsis. As shown in Fig. 3B, serum levels of IL-6 reached a maximum level at 12 h after the injection of S. aureus, and gradually fell thereafter in both groups, however, significantly higher IL-6 levels were seen in wild-type Newman group than in mgrA knockout group at 4 h (p < 0.0001), 12 h (p < 0.0001), 24 h (p < 0.0001), 48 h (p ¼ 0.0003) and 72 h (p ¼ 0.0012) (Fig. 3B). As shown in Fig. 3C, serum TNF-a levels peaked at about 4 h after injection of bacteria, TNF-a levels were comparable in wild-type Newman group and mgrA knockout group at 1 h, 3 h, 48 h, 72 h, 96 h, and 120 h. However, 4 h after induction of sepsis, mgrA knockout group significantly lower levels of TNF-a than wild-type Newman group at 4 h (p ¼ 0.0032), 12 h (p < 0.0001), 24 h (p < 0.0001) (Fig. 3C). 2.3. Analysis of bacterial load of blood and tissues Blood and tissue bacterial load after animal death or sacrifice (up to 14 days) were shown in Fig. 4. Bacterial load in blood after
Fig. 1. Survival curves of BALB/c mice (n ¼ 10/group) inoculated with 6 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. Mice inoculated with mgrA knockout strain showed significantly improved survival than mice inoculated with the wild-type Newman. (p ¼ 0.029, KaplaneMeier, log-rank test).
Fig. 2. Weight changes of BALB/c mice (n ¼ 10/group) inoculated with 3 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. Comparisons were made using the ManneeWhitney U-test.
challenge by wild-type strain was higher than those of mgrA knockout strain (Fig. 4A). Tissue bacterial load was significantly higher in the kidney and liver of mice in wild-type Newman group compared with mgrA knockout group (Log10 bacteria of kidney: 7.53 0.23 vs 6.02 þ 0.33, p < 0.001; Log10 bacteria of liver:5.82 0.26 vs 4.84 0.29, p ¼ 0.024). However, the bacterial load in spleen had no difference between the two groups (Log10 bacteria of spleen:4.67 0.19 vs 4.51 0.54, p ¼ 0.404) (Fig. 4B). 2.4. Assessment of histopathology Our results indicated that both of the two groups caused slight to moderate histopathology in most tissues at day 1 and day 2 and showed severe histopathology at day 4. As compared with mice inoculated with mgrA knockout strain, the wild-type inoculated mice had significantly more severe histopathology (Fig. 5). 3. Discussion MgrA is a 147-residue protein that belongs to a member of regulatory proteins called SarA protein family by virtue of their homology to SarA of S. aureus [13], which is a pleiotropic regulator that controls autolysis, virulence, biofilm formation, and efflux pump activity in S. aureus [14e16]. In a transcriptional profiling study of the mgrA of S. aureus strain Newman, Luong et al. found that MgrA upregulates 175 genes while downregulating 180 others [8]. In the present study we used a murine sepsis model induced by S. aureus to investigate the impact of mgrA gene expression on the onset and progression of sepsis. Our result showed that mice inoculated with the wild-type strain displayed significantly higher mortality, more weigh lost, and more severe histopathology than mice inoculated with mgrA mutation strain lacking mgrA gene expression. S. aureus can infect almost every tissue in the human host, its pathogenicity is mostly due to the production of various virulence factors, such as hemolysins, leukocidins, immune Modulators, cell wall components, and capsular polysaccharide (CP). Any alteration in regulatory genes will affect the expression of these virulence factors [3,17]. MgrA in S. aureus, a global transcriptional regulator, was identified previously as a key virulence determinant, which in particular plays a key role in the regulation of the expression of major virulence factors in S. aureus (e.g., capsular polysaccharide, protease, a-toxin, nuclease, and protein A) [8,13]. A previous study have shown that MgrA is critical for S. aureus pathogenesis in vivo, and an mgrA mutant strain exhibits 1000e10,000 fold virulence
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Fig. 3. Levels of CD4þ/CD8þ T cells, serum IL-6, and TNF-a, after induction of sepsis by inoculated with 6 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. Blood samples were taken at the indicated time points. A. CD4þ/CD8þ ratio. B. Serum IL-6 levels. C. Serum IL-6 levels. The means SD of ten mice are shown. * ¼ p < 0.05, p-value was obtained by 2-tailed t-test.
reduction in a mouse model of infection [18]. Jonsson IM et al. have demonstrated that mice that were inoculated with the wildtype Newman strain displayed significantly higher rate of mortality, showed a less favorable development of weight and displayed higher frequency and severity of arthritis than mice inoculated with the S. aureus mutant lacking mgrA gene expression in a murine model of hematogenously spread S. aureus arthritis [19]. Our results showed that significantly faster and more severe progression of sepsis in mice inoculated with the
wild-type strain Newman than mice inoculated with mgrA knockout strain. Cytokines are a group of proteins that are expressed by several cell types, act as immune mediators and regulators [20]. Levels of several important cytokines can easily be measured in the peripheral circulation, and therefore, cytokines have long been proposed as potential markers. The most commonly cytokines are tumor necrosis factor (TNF)-a and interleukin (IL)-6. Enhanced TNF-a and IL-6 levels evidently correlate positively with the
Fig. 4. Comparative bacteria loads in blood, liver, spleen, and kidney after mice death or sacrifice with infection inoculated with 3 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. A: blood bacterial load. B: organs bacterial load. The means SD of ten mice are shown. ns ¼ not significant. p-value was obtained by 2-tailed t-test.
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Fig. 5. Representative tissue sections of BALB/c mice at day 2: A: Liver of a mice inoculated with wild-type Newman showed moderate sinusoidal engorgement and focal lobular necrosis. B: Liver of a mice inoculated with mgrA knockout showed slight sinusoidal engorgement and focal lobular necrosis. C: Spleen of a mice inoculated with wild-type Newman showed Mast cell and moderate hemorrhagic necrosis. D: Spleen of a mice inoculated with mgrA knockout showed slight hemorrhagic necrosis. E: Kidney of a mice inoculated with wild-type Newman showed slight renal tubular epithelial cell swelling. F: Kidney of a mice inoculated with mgrA knockout showed no significant histopathology. Original magnification: 400. Hematoxylin-eosin staining was used.
incidence of sepsis [21]. Our results revealed that wild-type strain inoculated mice released significantly higher serum levels of TNF-a and IL-6, along with more severe sepsis progression, than mice inoculated with mgrA knockout strain. T cells secretes IL-2, IFNgand TNF-b, IL-4, IL-9, IL-10 and IL-13 et al. After antigen stimulation, CD4þ T cells are traditionally thought to differentiate into either Th1 or Th2 subsets. Each subset is characterized by its distinct cytokine production profile, mediation of cellular (Th1) or humoral (Th2) immunity. Previous studies have shown that CD4þ/ CD8þ T cells significantly changes in the inflammatory response
[11,12]. In this study, CD4þ/CD8þ T cells ratio was significantly lower in mgrA knockout strain inoculated mice than that wild-type strain inoculated mice at 24 h, 48 h, and 72 h. In vivo survival of S. aureus is also dependent on the ability to evade killing by host macrophages. Most S. aureus clinical isolates express a thin microcapsular layer that is composed of serotype 5, serotype 8 or serotype 336 capsular polysaccharide (CP). Expression of type 5 and type 8 capsule is associated with increased virulence in animal infection models. In vitro phagocytosis assays revealed that the presence of the capsule reduced the uptake of
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cells by neutrophils in the presence of normal serum opsonins, indicating that capsule is anti-opsonic [22,23]. MgrA can upregulate capsular polysaccharide encoding genes, and a lack of the mgrA gene can lead to diminished expression of CPs and thereby to decreased bacterial resistance to phagocytosis and intracellular killing [19]. Indeed, Our results showed that mice inoculated with wild-type strain had significantly more bacterial load in blood and tissue than that mgrA knockout strain inoculated mice. We concluded that mgrA knockout strain had a reduced expression of CP8 due to lack of mgrA gene which lead to decreased bacterial resistance to phagocytosis. In addition, we demonstrated that wildtype strain inoculated mice had faster and more severe histopathology than that mgrA knockout inoculated mice, suggesting that mgrA regulates virulence factors which impact on tissue damage. Once S. aureus invades the tissue, it produces a large number of cell surface-associated components and secreted products that include adhesins, enzymes, toxins, capsular polysaccharides, and other gene products that facilitate tissue colonization, tissue destruction, or immune evasion [24,25]. Our results demonstrated that mice inoculated with mgrA knockout presented significantly lower bacteria load in kidney and liver than that mice inoculated with wild-type strain. Jonsson IM et al. [22] have also reported significantly lower mgrA mutant bacteria load in kidneys both at early and late stages of infection. Previous study have showed [26,27] sortase A was more expressed in the wild-type than in the mgrA mutant strain, and an upregulation of sortase A in vitro results in increased expression of proteins like protein A, clumping factor, and collagen adhesin and thereby efficient adherence to host tissues by wild-type staphylococci. Mutants that lack the mgrA will thus have difficulties to colonize the host, suggesting that MgrA also regulates virulence factors that are of importance for colonization in tissue. In summary, the current study verified previous findings on the role of mgrA as a global regulator in S. aureus, this study showed that mgrA increased mortality and accelerate the onset and development of sepsis, the BALB/c mice inoculated with wild-type strain Newman showed more intense inflammatory response, more blood and tissue bacteria load, and more severe histopathological changes than the mice inoculated with mgrA knockout strain. 4. Material and methods 4.1. Mice Male BALB/c mice (6e8 week old, weight 18e22 g) were maintained separately at the Animal Facility of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, under controlled conditions (specific pathogen free, 22 C, 55% humidity, and 12 h light/dark cycle). All experimental procedures on animals used in this study were performed under a protocol approved by the Huazhong University of Science and Technology Animal Care and Use Committee (Permit Number:SYXK2009-0049). 4.2. Bacterial strains and construction of mgrA knockout strain S. aureus wild-type strain Newman (Biovector Science Lab, Inc, China) and its isogenic mgrA knockout strain were used in this study. To construct mgrA mutant, we amplified by PCR two fragments that flanked the left and right sides of mgrA gene sequence targeted for deletion. The two resulting PCR fragments have a 16base complementary region to facilitate annealing followed by a second round of PCR amplification with outside primers to obtain a single fragment. The fusion product was purified, digested with SmaI, and ligated into shuttle vector pMAD. The resulting
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recombinant pMAD plasmids were transformed first into S. aureus RN4220 and then into Newman strain by electroporation [28,29]. Construction and selection of the putative mgrA mutant was performed as described previously [30]. The plausible allelic replacement mutants were confirmed by PCR and Southern blots with the appropriate probes. The bacterial strains were cultured in Brain-Heart Infusion (BHI) broth at 37 C in air with shaking to log phase. Bacteria were centrifuged at 5000 g for 10 min and washed twice with sterile phosphate buffered saline (PBS). Then, the bacterial suspension was resuspended to an OD600 of 0.4. To verify the correct dilution, an aliquot was serially diluted on Tryptic Soy Agar (TSA) plates and colonies were counted after a 24 h incubation at 37 C. 4.3. Experimental infection of mice Four separate experiments were performed in this study. In all experiments, the mice were inoculated i.v. into tail vein with 0.2 ml suspension of S. aureus, and weighed regularly during the course of experiments. In the first experiment we assessed the impact of mgrA mutation on mortality, ten mice per group were inoculated with a dose of 6.0 106 CFU of S. aureus, a bacteria dose known to induce sepsis (up to 14 days). In the second experiment, mice were inoculated with a dose of 6.0 106 CFU of S. aureus, blood were analyzed for CD4þ/CD8þ T cells, and serum was analyzed IL-6, and TNF-a levels (up to 120 h). In the third experiment, mice were inoculated with a dose of 3.0 106 CFU of S. aureus for assessment of weight change (up to 14 days). In the fourth experiment, mice were inoculated with a dose of 3.0 106 CFU of S aureus for examination of blood and tissue bacteria load and histological analyses (up to 14 days). 4.4. Bacterial counts in blood and tissues The blood added to broth was serially diluted 1:10 in 0.9% NaCl. A 0.2 ml aliquot of each dilution was plated onto Blood Agar. The number of viable cells was estimated after counting on plates and multiplying by the appropriate dilution factor. The number of viable bacteria was expressed as Log10CFU/mL. Tissue segments of spleen, liver and kidney were weighted and homogenized; a 0.1 ml aliquot was then serially diluted 1:10 in sterile sodium chloride. Another aliquot of 0.1 ml of each dilution was plated onto Blood Agar. Plates were incubated at 35 C and the number of viable colonies were counted at each dilution and multiplied by the appropriate dilution factor. The number of viable bacteria was expressed as Log10CFU/g. 4.5. Flow cytometric analysis Blood was collected into heparin containing tubes. Erythrocytes were removed by cell lysis in BD FACS lysing solution (BD Biosciences, Heidelberg, Germany). After washing two times with FACS buffer (phosphate-buffered saline, 0.5% bovine serum albumin, 0.1% sodium azide. BD Biosciences), FITC-anti mouse CD4 and FITC-anti mouse CD8a (Biolegend, California, USA) were added to cell suspensions. All of these cell suspensions were incubated for 30 min at 4 C. After two washes with FACS buffer, the pellets were resuspended in 300 ml FACS buffer followed by analysis with FACScan flow cytometer (Becton Dickinson). 4.6. Enzyme-linked immunosorbent assay (ELISA) for IL-6 and TNFa At the indicated time points after induction of sepsis, approximately 0.5 ml blood were collected by eye enucleated, placed on ice
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and allowed to clot before centrifugation at 3000 g for 10 min. The serum levels of cytokines were measured by a standard sandwich cytokine ELISA procedure, according to the instructions of mouse IL-6 and TNF-a kits (R&D Systems, Minneapolis, MN, USA). 4.7. Histological analyses For evaluation of the extent of tissue damage and cellular response, histological analyses were performed. For the two experimental groups, three mice per group for every time point were submitted to histopathology examination after 1, 2, 4, 7, 14day after inoculation with 3 106 CFU of S. aureus wild-type Newman, or its isogenic mgrA knockout. The following organs were collected from each animal: liver, spleen and kidneys. Tissues were fixed with 4% paraformaldehyde, then stained with hematoxylineeosin. 4.8. Statistical analysis Statistical analysis of survival curves was estimated by Kaplane Meier analysis, log-rank test was used. To compare weight change, ManneWhitney U-test was used. Cytokine levels, blood and tissues bacterial counts were expressed as the mean SD, data sets were analyzed using two-tailed Student’s t test. Any p value
