Modulation of innate defences in the neonatal gastrointestinal tract by colonizing neuropathogenic Escherichia coli

新生儿细菌性脑膜炎(NBM)在全球范围内对死亡率和神经功能障碍有很大影响。在发达国家，大约每3000名新生儿中就有一名感染NBM。由于感染进展非常迅速，抗生素治疗可能并不总是有效的，现在更常见的是抗生素耐药细菌。发展中国家的风险要严重得多，由于早产儿存活率较高，加上获得药物的机会有限，疾病发病率正在增加。在非洲各地区，这种疾病的发病率可能高达每1000名活产儿中就有6名，而且这种疾病具有很高的死亡率。幸存者经常遭受严重后果，如脑瘫、智力低下、癫痫、癫痫和听力障碍。与NBM有关的新生儿败血症的发生率甚至更高。一旦感染发生后采取预防而不是治疗的策略将有明显的好处，但疫苗接种通常能有效地控制细菌感染，但不能用来保护新生儿免受NBM的影响，NBM的主要原因之一是大肠杆菌。这种细菌会产生一种名为K1的保护性胶囊，它可以无害地定植在母亲的肠道中。有时，婴儿在出生期间或出生后不久就会从母亲那里感染细菌。K1细菌在新生儿的胃肠道(GI)定居，其原因尚不清楚，可能会重新定位到血液中，并传播到其他器官，包括大脑。这些潜在的致命感染往往发生在生命的前四周内。人类感染的主要特征，特别是强烈的年龄依赖性，可以在实验大鼠身上复制。出生两天(P2)的大鼠在定居后对感染高度敏感，而九日龄的动物则完全抵抗。我想了解这种年龄依赖的基础，因为这可能为保护新生儿的新的预防措施提供一个平台。我的合作者最近的研究表明，小肠受到一层薄薄的粘液和高浓度的多肽的保护，这些多肽保护着肠道的这一区域。在结肠中，保护主要由一层非常厚的、有结构的粘液提供。我的小组最近表明，在较年轻的新生儿(P2)中，感染的两种屏障都不是很好。此外，进入的K1定殖者似乎抑制粘液层的成熟，并且不会刺激抗菌肽的分泌。这些物理和化学屏障使细菌与肠壁保持安全距离；允许它们接近细胞表面可能会支持它们重新定位到血液中。我们现在希望确定这些因素是否对感染的年龄依赖性负责，并确定感染过程中的关键步骤。我们将在胃肠道中建立K1细菌逃逸到循环中的位置；这对于制定防止这种重新定位的策略将是重要的。“防御素”多肽是由小肠中的潘氏细胞产生的；在P2大鼠中只有少量存在。我们将通过评估K1定植对多肽分泌的影响以及通过化学干扰多肽分泌来检验这些细胞的作用。我们将跟踪肠道粘液层在生命的头两周的发展，并确定K1定植如何影响这种成熟。如果我们能确定K1细菌绕过这些屏障是感染易感性的基础，我们将给K1定植的P2大鼠注射一种名为Tff2的蛋白质，它驱动保护性粘液屏障的组装。如果Tff2能通过促进粘液层成熟来减少或预防易感幼鼠的感染，则可作为预防该病的新途径。

Neonatal bacterial meningitis (NBM) contributes substantially to mortality and neurological disability worldwide. In developed countries, NBM affects approximately one in 3,000 newborn infants. Antibiotic therapy may not always be effective as the infection progresses very rapidly and antibiotic resistant bacteria are now more commonly found. The risks are far worse in developing countries and disease incidence is increasing due to higher survival rates of premature infants coupled with limited access to medicines. The incidence may be as high as six per 1,000 live births in regions in Africa and the disease carries significant mortality. Survivors often suffer severe consequences such as cerebral palsy, mental retardation, seizures, epilepsy and hearing impairment. Rates of neonatal sepsis, which is linked to NBM, are even higher. A strategy of prevention rather than treatment once the infection has occurred would have clear benefit but vaccination, often effective in controlling bacterial infections, cannot be employed to protect the newborn from NBM.One of the major causes of NBM is Escherichia coli. This bacterium produces a protective capsule, termed K1, and it can harmlessly colonize the mother's intestinal tract. Sometimes the infant acquires the bacterium from the mother during or shortly after birth. The K1 bacterium colonizes the gastrointestinal (GI) tract of the newborn infant and for reasons that are poorly understood may relocate to the blood and disseminate to other organs, including the brain. These potentially lethal infections tend to occur within the first four weeks of life. Key features of the human infection, particularly the strong age dependency, can be replicated in the experimental rat. Two-day-old (P2) rats are highly susceptible to infection after colonization whereas nine-day-old animals are completely resistant. I wish to understand the basis of this age dependency as this may provide a platform for novel preventative measures to protect the newborn infant. Recent work by my collaborator has established that the small intestine is protected by a thin layer of mucus and a high concentration of peptides that protect this region of the gut. In the colon, protection is primarily afforded by a very thick, structured layer of mucus. My group has very recently shown that in the younger (P2) neonates neither of the barriers to infection is well-formed. In addition, the incoming K1 colonizers appear to suppress maturation of the mucus layers and do not provoke secretion of the antibacterial peptides. These physical and chemical barriers keep bacteria at a safe distance from the gut wall; allowing them to get close to cell surfaces may support their relocation to the blood. We now wish to determine if these factors are responsible for the age dependency of the infection and to define key steps in the infection process. We will establish the site in the GI tract from which K1 bacteria escape into the circulation; this will be important in formulating strategies for preventing this relocation. The "defensin" peptides are produced by Paneth cells in the small intestines; these are present in only small numbers in the P2 rats. We will examine the role of these cells by assessing the impact of K1 colonization on peptide secretion and by chemically interfering with peptide secretion. We will track the development of the intestinal mucus layer over the first two weeks of life and determine how K1 colonization affects this maturation. If we can establish that circumvention of these barriers by K1 bacteria is the basis of susceptibility to infection, we will dose K1-colonized P2 rats with a protein, termed Tff2, which drives the assembly of the protective mucus barrier. If Tff2 can reduce or prevent infection in susceptible rat pups by promoting mucus layer maturation, it could be used as a new approach to the prevention of this disease.