Identification of an immunodominant ABC transporter in methicillin-resistant Staphylococcus aureus infections
Identification of an immunodominant ABC transporter in methicillin-resistant Staphylococcus aureus infections
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DOI：
10.1128/iai.68.6.3200-3209.2000
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发表时间：
2000-06-01
影响因子：
3.1
通讯作者：
Hodgetts, SJ
中科院分区：
文献类型：
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作者：
Burnie, JP;Matthews, RC;Hodgetts, SJ
26例因耐甲氧西林金黄色葡萄球菌（EMRSA-15）流行毒株导致败血症的患者（其中6人死亡）的免疫印迹血清显示，EMRSA-15抗原在61 kDa处具有免疫优势。生存与61-kDa带免疫球蛋白G有统计学显著相关（P < 0.001）。通过筛选来自败血症发作后收集的患者血清的EMRSA-15基因组表达文库，通过测序获得阳性克隆来鉴定抗原。在免疫blots上与61 kda抗原反应，通过检索金黄色葡萄球菌NCTC 8325和MRSA菌株COL数据库获得该蛋白的氨基末端，并在大肠杆菌TOP 10F'中表达，得到的氨基酸序列与ABC transporters同源，具有Walker A和Walker B配对基序，与枯草芽孢杆菌的YkpA同源性为73%。EMRSA-15败血症患者血清中衍生氨基酸序列的表位定位描绘了7个表位。其中三个表位，以肽1 （KIKVYVGNYDFWYQS）、2 （TVIVSHDRHFLY -NNV）和3 （TETFLRGFLGRMLFS）为代表，被合成并用于从噬菌体抗体展示库中分离人重组抗体，在小鼠感染模型中，与对照组相比，针对肽1和2的重组抗体使器官集落计数呈对数减少。
Immunoblotting sera from 26 patients with septicemia due to an epidemic strain of methicillin-resistant Stapylococcus aureus (EMRSA-15), 6 of whom died, revealed an immunodominant EMRSA-15 antigen at 61 kDa. There was a statistically significant correlate (P < 0.001) between survival and immunoglobulin G to the 61-kDa band. The antigen was identified by sequencing positive clones obtained by screening a genomic expression library of EMRSA-15 with pooled sera from patients taken after the septicemic episode. fluted antibody reacted with the 61-kDa antigen on immunoblots, The amino terminus was obtained by searching the S. aureus NCTC 8325 and MRSA strain COL databases, and the whole protein was expressed in Escherichia coli TOP 10F', The derived amino acid sequence showed homology with ABC transporters, with paired Walker A and Walker B motifs and 73% homology to YkpA from Bacillus subtilis. Epitope mapping of the derived amino acid sequence with sera from patients who had recovered from EMRSA-15 septicemia delineated seven epitopes. Three of these epitopes, represented by peptides 1 (KIKVYVGNYDFWYQS), 2 (TVIVSHDRHFLY -NNV), and 3 (TETFLRGFLGRMLFS) were synthesized and used to isolate human recombinant antibodies from a phage antibody display library, Recombinant antibodies against peptides 1 and 2 gave logarithmic reductions in organ colony counts, compared with control groups, in a mouse model of the infection, This study suggests the potential role of an ABC transporter as a target for immunotherapy.