Groß Rüdiger, Bauer Richard, Krüger Franziska, Rücker-Braun Elke, Olari Lia-Raluca, Ständker Ludger, Preising Nico, Rodríguez Armando A, Conzelmann Carina, Gerbl Fabian, Sauter Daniel, Kirchhoff Frank, Hagemann Benjamin, Gačanin Jasmina, Weil Tanja, Ruiz-Blanco Yasser B, Sanchez-Garcia Elsa, Forssmann Wolf-Georg, Mankertz Annette, Santibanez Sabine, Stenger Steffen, Walther Paul, Wiese Sebastian, Spellerberg Barbara, Münch Jan
Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.
Institute of Medical Microbiology and Hygiene, Ulm University Medical Center, Ulm, Germany.
Front Microbiol. 2020 Apr 6;11:508. doi: 10.3389/fmicb.2020.00508. eCollection 2020.
The placenta acts as physical and immunological barrier against the transmission of viruses and bacteria from mother to fetus. However, the specific mechanisms by which the placenta protects the developing fetus from viral and bacterial pathogens are poorly understood. To identify placental peptides and small proteins protecting from viral and bacterial infections, we generated a peptide library from 10 kg placenta by chromatographic means. Screening the resulting 250 fractions against Herpes-Simplex-Virus 2 (HSV-2), which is rarely transmitted through the placenta, in a cell-based system identified two adjacent fractions with significant antiviral activity. Further rounds of chromatographic purification and anti-HSV-2 testing allowed to purify the bioactive peptide. Mass spectrometry revealed the presence of a 36-mer derived from the C-terminal region of the hemoglobin β subunit. The purified and corresponding chemically synthesized peptide, termed HBB(112-147), inhibited HSV-2 infection in a dose-dependent manner, with a mean IC in the median μg/ml range. Full-length hemoglobin tetramer had no antiviral activity. HBB(112-147) did not impair infectivity by direct targeting of the virions but prevented HSV-2 infection at the cell entry level. The peptide was inactive against Human Immunodeficiency Virus Type 1, Rubella and Zika virus infection, suggesting a specific anti-HSV-2 mechanism. Notably, HBB(112-147) has previously been identified as broad-spectrum antibacterial agent. It is abundant in placenta, reaching concentrations between 280 and 740 μg/ml, that are well sufficient to inhibit HSV-2 and prototype Gram-positive and -negative bacteria. We here additionally show, that HBB(112-147) also acts potently against strains (including a multi-drug resistant strain) in a dose dependent manner, while full-length hemoglobin is inactive. Interestingly, the antibacterial activity of HBB(112-147) was increased under acidic conditions, a hallmark of infection and inflammatory conditions. Indeed, we found that HBB(112-147) is released from the hemoglobin precursor by Cathepsin D and Napsin A, acidic proteases highly expressed in placental and other tissues. We propose that upon viral or bacterial infection, the abundant hemoglobin precursor is proteolytically processed to release HBB(112-147), a broadly active antimicrobial innate immune defense peptide.
胎盘作为一种物理和免疫屏障，可防止病毒和细菌从母体传播给胎儿。然而，胎盘保护发育中的胎儿免受病毒和细菌病原体侵害的具体机制尚不清楚。为了鉴定能保护胎儿免受病毒和细菌感染的胎盘肽和小蛋白质，我们通过色谱方法从10千克胎盘中生成了一个肽库。在基于细胞的系统中，用很少通过胎盘传播的单纯疱疹病毒2（HSV-2）对所得的250个馏分进行筛选，鉴定出两个相邻的具有显著抗病毒活性的馏分。通过进一步的色谱纯化和抗HSV-2测试，得以纯化出生物活性肽。质谱分析显示存在一种源自血红蛋白β亚基C末端区域的36聚体。纯化后的以及相应化学合成的肽，称为HBB(112 - 147)，以剂量依赖的方式抑制HSV-2感染，平均IC50在中μg/ml范围内。全长血红蛋白四聚体没有抗病毒活性。HBB(112 - 147)不是通过直接靶向病毒粒子来损害其感染性，而是在细胞进入水平阻止HSV-2感染。该肽对1型人类免疫缺陷病毒、风疹和寨卡病毒感染无活性，表明其具有特定的抗HSV-2机制。值得注意的是，HBB(112 - 147)先前已被鉴定为广谱抗菌剂。它在胎盘中含量丰富，浓度在280至740μg/ml之间，足以抑制HSV-2以及典型的革兰氏阳性和阴性细菌。我们在此还表明，HBB(112 - 147)也以剂量依赖的方式对多种菌株（包括一种多重耐药菌株）有强效作用，而全长血红蛋白则无活性。有趣的是，HBB(112 - 147)在酸性条件下抗菌活性增强，酸性条件是感染和炎症状态的一个特征。实际上，我们发现HBB(112 - 147)是由组织蛋白酶D和天冬氨酸蛋白酶A从血红蛋白前体中释放出来的，这两种酸性蛋白酶在胎盘和其他组织中高度表达。我们提出，在病毒或细菌感染时，丰富的血红蛋白前体被蛋白水解加工以释放HBB(112 - 147)，这是一种具有广泛活性的抗菌先天免疫防御肽。
