[1]谭利,王轶男,陈江,等.金黄色葡萄球菌RNAⅢ敲除株对γ-溶血素表达调控的影响[J].第三军医大学学报,2020,42(01):45-49.
 TAN Li,WANG Yinan,CHEN Jiang,et al.Effect of deletion of Staphylococcus aureus RNAⅢ on γ-hemolysin expression[J].J Third Mil Med Univ,2020,42(01):45-49.
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金黄色葡萄球菌RNAⅢ敲除株对γ-溶血素表达调控的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第01期
页码:
45-49
栏目:
基础医学
出版日期:
2020-01-15

文章信息/Info

Title:
Effect of deletion of Staphylococcus aureus RNAⅢ on γ-hemolysin expression
作者:
谭利 王轶男陈江 姜北喻胜鹏黎庶胡晓梅
陆军军医大学(第三军医大学):基础医学院微生物学教研室;第一附属医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室;第一附属医院骨科,全军矫形外科中心
Author(s):
TAN Li WANG Yinan CHEN Jiang JIANG Bei YU Shengpeng LI Shu HU Xiaomei

Department of Microbiology, College of Basic Medical Sciences, State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burns, Department of Orthopedics, Center of Plastic Surgery, First Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
金黄色葡萄球菌RNAⅢ&gamma-溶血素基因敲除
Keywords:
Staphylococcus aureus RNAⅢ &gamma-hemolysin homologous recombination  
分类号:
R378.11;R394-33;R394.2
文献标志码:
A
摘要:

目的 利用同源重组技术构建金黄色葡萄球菌Newman RNAⅢ基因敲除株,探究RNAⅢ敲除对金葡菌γ-溶血素表达调控的影响。方法 分别扩增RNAⅢ基因的上下游同源臂,根据同源重组原理,构建pBT2基因敲除载体,利用同源重组技术,构建RNAⅢ敲除株。并对野生株和敲除株γ-溶血素的转录水平和蛋白水平进行研究。结果 实时荧光定量 PCR结果显示RNAⅢ敲除株γ-溶血素A/B/C亚基的转录水平较野生株显著降低, Western blot检测结果也显示敲除株γ-溶血素蛋白表达水平较野生株明显减少。结论RNAⅢ敲除可显著影响金葡菌γ-溶血素的表达。

Abstract:

Objective To construct a RNAⅢ gene knockout mutant of Staphylococcus aureus (S. aureus) and evaluate the effect of RNAⅢ on the expression of γ-hemolysin. Methods  In order to construct temperature-sensitive S. aureus knock-out vectors, 2 homologous sequences around RNAⅢ were cloned and inserted into pBT2 vector to construct knock-out plasmid pBT2∷RNAⅢ, and then the obtained plasmids RNAⅢ were transferred into S. aureus strain Newman, finally Newman∷ΔRNAⅢ mutant were prepared with homologous recombination. The transcriptional level of γ-hemolysin hlgA/B/C was detected by quantitative real-time PCR (RT-PCR) and the protein levels in the culture supernatants of Newman wild type strain and Newman∷ΔRNAⅢ were analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and  Western blot analysis. ResultsNewman∷ΔRNAⅢ mutant was constructed successfully. RT-PCR analysis revealed that the expression of γ-hemolysin in the Newman∷ΔRNAⅢ mutant was significantly decreased as compared to that of Newman wild type strain. Western blotting also showed the transcription level of γ-hemolysin was significantly reduced in the mutant strain. ConclusionThe knockout of RNAⅢ has a great effect on the expression of γ-hemolysin in S. aureus.

参考文献/References:

[1]KONG E F, JOHNSON J K, JABRA-RIZK M A. Community-associated methicillin-resistant Staphylococcus aureus: an enemy amidst us[J]. PLoS Pathog, 2016, 12(10): e1005837. DOI:10.1371/journal.ppat.1005837.
[2]YAMASHITA K, KAWAI, TANAKA Y, et al. Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components[J]. Proc Natl Acad Sci USA, 2011, 108(42): 17314-17319. DOI:10.1073/pnas.1110402108.
[3]ZIMMERMANN-MEISSE G, PRVOST G, JOVER E. Above and beyond C5a receptor targeting by staphylococcal leucotoxins: retrograde transport of panton-valentine leucocidin and γ-hemolysin[J]. Toxins (Basel), 2017, 9(1): E41. DOI:10.3390/toxins9010041.
[4]BRONESKY D, WU Z F, MARZI S, et al. Staphylococcus aureus RNAⅢ and its regulon link quorum sensing, stress responses, metabolic adaptation, and regulation of virulence gene expression[J]. Annu Rev Microbiol, 2016, 70: 299-316. DOI:10.1146/annurev-micro-102215-095708.
[5]OLIVEIRA D, BORGES A, SIMES M. Staphylococcus aureus toxins and their molecular activity in infectious diseases[J]. Toxins (Basel), 2018, 10(6): E252. DOI:10.3390/toxins10060252.
[6]TOMITA N, ABE K, KAMIO Y, et al. Cluster-forming property correlated with hemolytic activity by staphylococcal γ-hemolysin transmembrane pores[J]. FEBS Lett, 2011, 585(21): 3452-3456. DOI:10.1016/j.febslet.2011.09.041.
[7]DUMONT A L, YOONG P, DAY C J, et al. Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1[J]. Proc Natl Acad Sci USA, 2013, 110(26): 10794-10799. DOI:10.1073/pnas.1305121110.
[8]SPAAN A N, HENRY T, VAN ROOIJEN W J M, et al. The staphylococcal toxin panton-valentine leukocidin targets human C5a receptors[J]. Cell Host Microbe, 2013, 13(5): 584-594. DOI:10.1016/j.chom.2013.04.006.
[9]PREVOST G, COUPPIE P, PREVOST P, et al. Epidemiological data on Staphylococcus aureus strains producing synergohymenotropic toxins[J]. J Med Microbiol, 1995, 42(4): 237-245. DOI:10.1099/00222615-42-4-237.
[10]SPAAN A N, VRIELING M, WALLET P, et al. The staphylococcal toxins γ-haemolysin AB and CB differentially target phagocytes by employing specific chemokine receptors[J]. Nat Commun, 2014, 5: 5438. DOI:10.1038/ncomms6438.
[11]MALACHOWA N, WHITNEY A R, KOBAYASHI S D, et al. Global changes in Staphylococcus aureus gene expression in human blood[J]. PLoS ONE, 2011, 6(4): e18617. DOI:10.1371/journal.pone.0018617.
[12]YAMAZAKI K, KATO F, KAMIO Y, et al. Expression of gamma-hemolysin regulated by sae in Staphylococcus aureus strain Smith 5R[J]. FEMS Microbiol Lett, 2006, 259(2): 174-180. DOI:10.1111/j.1574-6968.2006.00236.x.
[13]XU T, WANG X Y, CUI P, et al. The agr quorum sensing system represses persister formation through regulation of phenol soluble modulins in Staphylococcus aureus[J]. Front Microbiol, 2017, 8: 2189. DOI:10.3389/fmicb.2017.02189.
[14]VANDERPOOL C K, BALASUBRAMANIAN D, LLOYD C R. Dual-function RNA regulators in Bacteria[J]. Biochimie, 2011, 93(11): 1943-1949. DOI:10.1016/j.biochi.2011.07.016.
[15]LIU Y, MU C H, YING X M, et al. RNAⅢ activates map expression by forming an RNA-RNA complex in Staphylococcus aureus[J]. FEBS Lett, 2011, 585(6): 899-905. DOI:10.1016/j.febslet.2011.02.021.
[16]GUPTA R K, LUONG T T, LEE C Y. RNAⅢ of  the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA[J]. Proc Natl Acad Sci USA, 2015, 112(45): 14036-14041. DOI:10.1073/pnas.1509251112.

更新日期/Last Update: 2020-01-07