Serine Protease(s) Secreted by the Nematode <em>Trichuris muris</em> Degrade the Mucus Barrier

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a TH2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).肠道黏液屏障的聚合型黏蛋白组分在线虫感染过程中发生动态改变，其不仅可提供物理保护，还能直接作用于致病线虫，并辅助宿主驱逐寄生虫。尽管存在上述变化，但此前尚未有研究探讨线虫与黏蛋白及黏液屏障之间的直接相互作用。本研究采用成熟的鼠鞭虫（Trichuris muris）感染模型，探究线虫分泌的复杂免疫原性蛋白混合物——即排泄/分泌产物（excretory/secretory products，简称ESPs）——对黏蛋白的影响。我们设置不同的鼠鞭虫感染方案，以模拟慢性（低剂量）感染与急性（高剂量）感染两种模型。对从小鼠及人类肠道细胞系LS174T中分离得到的黏液与黏蛋白样本施以ESPs处理。实验结果证实，线虫分泌的丝氨酸蛋白酶可改变黏液屏障的特性：通过降解黏液凝胶中的黏蛋白组分，使屏障孔隙度升高。具体而言，该丝氨酸蛋白酶作用于主要肠道黏蛋白Muc2的N端聚合结构域，导致Muc2聚合物发生解聚。值得注意的是，在肠道中诱导表达、对蠕虫驱逐至关重要的呼吸/胃黏蛋白Muc5ac，可免受ESPs介导的解聚作用影响。此外，在抵抗慢性感染的小鼠体内，能够保护黏蛋白（尤其是Muc2）免受解聚的丝氨酸蛋白酶抑制剂（Serpins）呈现高表达水平。综上，本研究证实线虫可通过分泌丝氨酸蛋白酶降解黏液屏障中的黏蛋白，这或许能够修饰寄生虫的生存微环境，从而阻止宿主清除寄生虫，或帮助与黏液屏障紧密接触的寄生虫后端完成高效交配与产卵。然而，在TH2介导的蠕虫驱逐反应中，Serpins、Muc5ac以及高水平表达的Muc2可保护黏液屏障免受线虫分泌蛋白酶的降解。