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In JoVE (1)
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Articles by Ching Wen Tseng in JoVE
JoVE Immunology and Infection
تحت الجلد من العدوى المكورات العنقودية المذهبة المقاومة للميثيسيلين (MRSA)
Department of Pediatrics, Cedars-Sinai Medical Center
ويستخدم الجلد الناعمة والفئران نموذج عدوى الأنسجة لتقييم وظيفة ضراوة المكورات العنقودية الذهبية المقاومة للميثيسيلين (MRSA) والاستجابات المناعية المضيف. هنا ، وقدمنا نموذجا العدوى تحت الجلد للجلد والأنسجة اللينة العدوى.
Other articles by Ching Wen Tseng on PubMed
Accessory Gene Regulator Control of Staphyloccoccal Enterotoxin D Gene Expression
The quorum-sensing system of Staphylococcus aureus, the accessory gene regulator (Agr) system, is responsible for increased transcription of certain exoprotein genes and decreased transcription of certain cell wall-associated proteins during the postexponential phase of growth. This regulation is important for virulence, as evidenced by a reduction in virulence associated with a loss of the Agr system. The enterotoxin D (sed) determinant is upregulated by the Agr system. To define the Agr-regulated cis element(s) within the sed promoter region, we utilized promoters not regulated by Agr to create hybrid promoters. Hybrid promoters were created by using sed sequences combined with the enterotoxin A (sea) promoter or the S. aureus lac operon promoter sequences. The results obtained indicated that the Agr control element of the sed promoter resides within the -35 promoter element and at the Pribnow box to the +1 site of the promoter. At these positions of the sed promoter, a directly repeated 6-bp sequence was found. This repeat is important for overall promoter activity, and maximal regulation of the promoter activity requires both repeat elements. Furthermore, Agr control of sed promoter activity was found to be dependent upon the presence of a functional Rot protein. Therefore, the postexponential increase in sed transcription results from the Agr-mediated reduction in Rot activity rather than as a direct effect of the Agr system.
Rot Repression of Enterotoxin B Expression in Staphylococcus Aureus
The accessory gene regulator (Agr) system is a quorum-sensing system of Staphylococcus aureus responsible for upregulation of certain exoprotein genes and downregulation of certain cell-wall associated proteins during the post-exponential phase of growth. The enterotoxin B (seb) determinant is upregulated by the Agr system. Agr-regulated cis elements within the seb promoter region were examined by deletion analyses of the seb promoter by a hybrid promoter approach utilizing the staphylococcal lac operon promoter. To identify the regulatory pathway for enterotoxin B expression, the seb promoter fused to the chloramphenicol acetyltransferase reporter gene was introduced into mutants of S. aureus lacking agr or different members of the Sar family of transcriptional regulators. Agr control of seb promoter activity was found to be dependent upon the presence of a functional Rot protein, and Rot was shown to be able to bind to the seb promoter. Therefore, the Agr-mediated post-exponential-phase increase in seb transcription results from the Agr system's inactivation of Rot repressor activity.
Regulation of Rot Expression in Staphylococcus Aureus
Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in Staphylococcus aureus. The function of Rot, but not the transcription of rot, is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In addition, the alternative sigma factor (sigma(B)) has a repressive effect on rot expression during the postexponential phase of growth. The transcriptional profiles of Rot in sigma(B)-positive and sigma(B)-negative strains in the postexponential and stationary phases of growth were compared. An upregulation of rot expression was observed during the stationary phase of growth, and this upregulation occurred in a sigma(B)-dependent manner. The effects of other staphylococcal transcriptional factors were also investigated. Electrophoretic mobility shift assays revealed that proteins present in staphylococcal lysates retarded the mobility of the rot promoter fragment and that the effect was reduced, but not eliminated, with lysates from strains lacking a functional SarS protein. A modest upregulation of rot expression was also observed in sarS-negative strains. Affinity purification of proteins binding to the rot promoter fragment, followed by N-terminal protein sequencing, identified the SarA and SarR proteins. Primer extension analysis of the rot promoter revealed a number of discreet products. However, these RNA species were not associated with identifiable promoter activity and likely represented RNA breakdown products. Loss of Rot function during the postexponential phase of growth likely involves degradation of the rot mRNA but not the inhibition of rot transcription.
Staphylococcus Aureus Panton-Valentine Leukocidin Contributes to Inflammation and Muscle Tissue Injury
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens public health worldwide, and epidemiologic data suggest that the Panton-Valentine Leukocidin (PVL) expressed by most CA-MRSA strains could contribute to severe human infections, particularly in young and immunocompetent hosts. PVL is proposed to induce cytolysis or apoptosis of phagocytes. However, recent comparisons of isogenic CA-MRSA strains with or without PVL have revealed no differences in human PMN cytolytic activity. Furthermore, many of the mouse studies performed to date have failed to demonstrate a virulence role for PVL, thereby provoking the question: does PVL have a mechanistic role in human infection? In this report, we evaluated the contribution of PVL to severe skin and soft tissue infection. We generated PVL mutants in CA-MRSA strains isolated from patients with necrotizing fasciitis and used these tools to evaluate the pathogenic role of PVL in vivo. In a model of necrotizing soft tissue infection, we found PVL caused significant damage of muscle but not the skin. Muscle injury was linked to induction of pro-inflammatory chemokines KC, MIP-2, and RANTES, and recruitment of neutrophils. Tissue damage was most prominent in young mice and in those strains of mice that more effectively cleared S. aureus, and was not significant in older mice and mouse strains that had a more limited immune response to the pathogen. PVL mediated injury could be blocked by pretreatment with anti-PVL antibodies. Our data provide new insights into CA-MRSA pathogenesis, epidemiology and therapeutics. PVL could contribute to the increased incidence of myositis in CA-MRSA infection, and the toxin could mediate tissue injury by mechanisms other than direct killing of phagocytes.
Staphylococcus Aureus Panton-Valentine Leukocidin Targets Muscle Tissues in a Child with Myositis and Necrotizing Fasciitis
The incidence of myositis has been increasing since the advent of the epidemic of community-associated methicillin-resistant Staphylococcus aureus infection, and Panton-Valentine leukocidin has been implicated as a factor contributing to more-severe muscle injury. We report a case of severe myositis accompanying septic osteomyelitis and necrotizing fasciitis caused by a Panton-Valentine leukocidin-positive S. aureus strain. Immunostaining showed strong binding of the Panton-Valentine leukocidin toxin to necrotic muscle tissues.
