School of Biotechnology, Gautam Buddha University, Greater Noida, Uttar Pradesh, 201306, India.
School of Vocational Studies and Applied Sciences, Gautam Buddha University, Greater Noida, Uttar Pradesh, 201306, India.
Microb Cell Fact. 2022 Jan 29;21(1):15. doi: 10.1186/s12934-022-01743-2.
Tuberculosis currently stands as the second leading cause of deaths worldwide due to single infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis.
We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins.
The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.
结核病目前是全球因单一感染源导致死亡的第二大原因，仅次于严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）。结核病耐药性的当前挑战凸显了迫切需要开发新的抗分枝杆菌化合物。在本研究中，我们报告了一种细菌实验室污染物（LC-1）的偶然发现，该污染物在接种结核分枝杆菌的琼脂平板上显示出生长抑制区。
我们利用微生物学、生物化学和生物物理方法来表征 LC-1 及其分泌物中的抗分枝杆菌化合物。基于 16S rRNA 测序和 BIOLOG 分析，LC-1 被鉴定为人葡萄球菌，一种人类细菌共生菌。抗分枝杆菌活性最初是在通过超滤培养滤液（CF）获得的 30 kDa 截留物中发现的。通过对 30 kDa 截留物的排阻色谱法获得的峰级分的 SDS-PAGE 分析证实存在高分子量（≥30 kDa）蛋白。峰级分-1（F-1）对牛分枝杆菌 BCG 表现出抑制活性，但对耻垢分枝杆菌、大肠杆菌和金黄色葡萄球菌没有抑制活性。活性级分 F-1 在用蛋白酶 K 和 α-糜蛋白酶处理时失活。然而，它在广泛的热和 pH 处理下保留其抗分枝杆菌活性。即使在 -20°C 下长时间储存（~12 个月），F-1 的抗分枝杆菌活性也得以维持。质谱分析表明，鉴定的肽质量与任何先前已知的细菌素都不匹配。
本研究强调了 LC-1 的 CF 中存在的高分子量蛋白（s）的抗分枝杆菌活性，这些蛋白（s）可能会进一步进行测试，以针对结核分枝杆菌。这些蛋白质的热和 pH 稳定性增加了它们作为治疗性蛋白质的特性，并可能有助于其长保质期。LC-1 作为一种人类共生菌，可以在未来进行测试，以评估其作为治疗结核病的益生菌的潜力。
