Verma Monika, Suryanarayana Nagendra, Tuteja Urmil, Thavachelvam Kulanthaivel, Rao M K, Bhargava Rakesh, Shukla Sangeeta
Microbiology Division, Defence Research & Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
Pharmacology and toxicology Division, Defence Research & Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, 474002, India.
Toxicon. 2017 Dec 1;139:58-65. doi: 10.1016/j.toxicon.2017.09.008. Epub 2017 Sep 14.
Anthrax associated causalities in humans and animals are implicated mainly due to the action of two exotoxins that are secreted by the bacterium Bacillus antharcis during the infection. These exotoxins comprise of three protein components namely protective antigen (PA), lethal factor (LF) and edema factor (EF). The protective antigen is the common toxin component required to form both lethal toxin (LeTx) and edema toxin (EdTx). The LeTx is formed, when PA combines with LF and EdTx is formed when PA combines with EF. Therapeutic interventions aiming to neutralize these key effectors of anthrax pathology would therefore, provide an effective means to counter the toxicity imposed by the anthrax toxins on the host. The present work describes the lethal toxin neutralization potential of polyclonal antisera developed against the individual domains of the protective antigen component of the anthrax toxin. The individual domains were produced as recombinant proteins in E. coli and validated with peptide mass fingerprinting by MALDI-TOF analysis and corresponding mice polyclonal antisera by western blotting. Each domain specific antibody titre and isotype was ascertained by ELISA. The isotyping revealed the predominance of IgG1 isotype. The toxin neutralizing potential of these domain specific antisera were evaluated by in-vitro cell viability MTT assay, employing J774.1 mouse macrophage cell line against LeTx (0.25 μg ml PA and 0.125 μg ml LF concentrations). Among the four domain specific antisera, the antiserum against PA domain IV could neutralize LeTx with high efficiency. No significant neutralization of LeTx was observed with other domain specific antibodies. Results indicate that antibodies to r-PA domain IV could be explored further as therapeutic anti toxin molecule along with appropriate antibiotic regimens against anthrax.
人类和动物中与炭疽相关的伤亡主要是由于感染期间炭疽芽孢杆菌分泌的两种外毒素的作用。这些外毒素由三种蛋白质成分组成，即保护性抗原（PA）、致死因子（LF）和水肿因子（EF）。保护性抗原是形成致死毒素（LeTx）和水肿毒素（EdTx）所需的共同毒素成分。当PA与LF结合时形成LeTx，当PA与EF结合时形成EdTx。因此，旨在中和炭疽病理这些关键效应物的治疗干预措施将提供一种有效手段来对抗炭疽毒素对宿主造成的毒性。本研究描述了针对炭疽毒素保护性抗原成分各个结构域产生的多克隆抗血清的致死毒素中和潜力。各个结构域作为重组蛋白在大肠杆菌中产生，并通过基质辅助激光解吸电离飞行时间（MALDI-TOF）分析的肽质量指纹图谱和蛋白质印迹法对相应的小鼠多克隆抗血清进行验证。通过酶联免疫吸附测定（ELISA）确定每个结构域特异性抗体的效价和亚型。亚型分析显示IgG1亚型占主导地位。通过体外细胞活力MTT测定法，使用J774.1小鼠巨噬细胞系针对LeTx（PA浓度为0.25μg/ml和LF浓度为0.125μg/ml）评估这些结构域特异性抗血清的毒素中和潜力。在四种结构域特异性抗血清中，针对PA结构域IV的抗血清能够高效中和LeTx。其他结构域特异性抗体未观察到对LeTx的显著中和作用。结果表明，针对重组PA结构域IV的抗体可与针对炭疽的适当抗生素方案一起进一步作为治疗性抗毒素分子进行探索。
