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首页> 外文期刊>Applied Microbiology >Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools
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Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

机译：通过生物信息学和基于抗体的蛋白质检测工具鉴定单核细胞增生李斯特菌的表面蛋白质生物标记

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The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes , with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples.IMPORTANCE L. monocytogenes is traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface of L. monocytogenes strains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolate L. monocytogenes from food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of the L. monocytogenes serotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range of L. monocytogenes isolates.
机译：革兰氏阳性单核细胞增生性李斯特菌在人类食源性疾病中造成很大比例的死亡。在选择性富集培养条件下，在广泛的单核细胞增生李斯特菌血清型中特异性表达的表面蛋白可作为潜在的生物标记，通过基于抗体的方法检测和分离这种病原体。我们的研究旨在鉴定此类生物标志物。对单核细胞增生李斯特氏菌血清型4b菌株F2365基因组的询问确定了130个假定的或已知的表面蛋白。由于四种单核细胞增生李斯特氏菌菌株之间存在保守区域（在其他李斯特菌中有所不同），由于存在保守区域，因此将四个表面蛋白LMOf2365_0578，LMOf2365_0581，LMOf2365_0639和LMOf2365_2117的同源物评估为生物标记。尽管通过PCR检测了生物中所有四种蛋白质的mRNA转录本，但针对四种重组蛋白的兔多克隆抗体显示在血清型4b菌株LI0521细胞表面仅LMOf2365_0639的表达。针对LMOf2365_0639的35种单克隆抗体（MAb）中的三种，单克隆抗体M3643，M3644和M3651，特异性识别了在非选择性脑心脏输注培养基中生长的46种单核细胞增生李斯特菌谱系I和II的42种（91.3％）。 M3644和M3651与17种单核细胞增生李斯特氏菌谱系I和II分离株中的14至15（82.4至88.2％）反应，而M3643与选择性富集培养基中生长的24种谱系I，II和III分离株中的22（91.7％）反应（ UVM1，改良的Fraser，Palcam和UVM2介质）。与在选择性富集培养基中生长的其他一些李斯特菌属物种相比，这三种MAb仅表现出较弱的反应性（在414 nm处的光密度接近临界值）。总体而言，数据表明LMOf2365_0639在特定单克隆抗体的帮助下可作为单核细胞增生李斯特菌的表面生物标记物，用于临床，环境和食品样品中的病原体检测，鉴定和分离。至少12种血清型。当前，没有可用的单克隆抗体（MAb）能够与代表所有12种血清型的单核细胞增生李斯特菌菌株的表面结合。这样的抗体将是有用的，并且是开发从食物样品中检测和分离单核细胞增生李斯特氏菌的方法所需要的。在我们的研究中，我们旨在鉴定在各种血清型之间具有氨基酸序列保守的区域的表面蛋白，然后将它们用作开发MAb的抗原靶标（生物标记）。通过生物信息学和蛋白质表达分析，我们鉴定了由单核细胞增生李斯特菌血清型4b菌株F2365的基因组编码的四个假定的表面蛋白候选物LMOf2365_0639中的一种，是有用的表面生物标记。在我们的研究中，针对35个针对LMOf2365_0639的单克隆抗体进行了广泛的评估，结果发现了三种单克隆抗体（M3643，M3644和M3651）可以识别多种单核细胞增生李斯特菌。

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