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

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.

Neonatal bacterial sepsis and meningitis are severe, life-threatening infections that are difficult to control by prophylaxis; further, they do not always respond to aggressive combination antibiotic therapy. One of the major causative agents, Escherichia coli K1, is acquired at birth, colonizes the immature neonatal GI tract and may translocate into the blood circulation prior to invasion of the central nervous system. Many aspects of E. coli K1 pathogenesis are unclear, but there is strong evidence that systemic infection is determined by the capacity of the neuropathogen to escape from the lumen of the gut into the circulation. Key features of the infection can be replicated in the neonatal rat. In contrast to infection-resistant animals, the physical and chemical barriers to translocation in the GI tract of infection-susceptible neonatal rats are not fully formed: protective mucin layers in the small intestine and colon are less substantial and the population of alpha-defensin-secreting Paneth cells is small. I have recently obtained evidence that colonizing E. coli K1 bacteria do not stimulate the synthesis of the alpha-defensins Defa24 and Defa-rs1 in susceptible rats but do so in non-susceptible animals. Further, the expression of Trefoil factor 2 (Tff2), a peptide associated with maturation and stability of the mucin barrier, is substantially down-regulated by E. coli K1 in susceptible neonates. I will evaluate the hypothesis that dysregulation of functional barrier development in the neonate by colonizing E. coli K1 allows translocation of the neuropathogen from the gut to the circulation and is the basis of susceptibility to systemic infection. As oral administration of trefoil factor peptides accelerates repair of GI mucosal lesions, infection-susceptible rat neonates will be dosed with recombinant Tff2 to determine if this intervention can provide protection against systemic infection and form the basis of a novel approach to prophylaxis of the disease.

Industry, policy, the public: The industrial pharmaceutical sector in the UK is world-class and for many years has been at the leading edge of R&D, resulting in the introduction of many successful product launches in key therapeutic areas. However, like many large concerns worldwide, the bigger companies in the UK sector have considerably reduced their activities in the anti-infective area, due to the perception that antibiotic R&D is at the fringe of economic viability, that antibacterial therapeutics perform poorly in the marketplace compared with drugs for chronic conditions and that their Net Present Value is below the considered threshold of viability. The number of cases of NBM and sepsis in the UK is in the few thousands and does not represent a viable market for protein therapeutic products but the at-risk cohort, linked to pre-term birth and low socioeconomic status, is considerably larger and niche companies may show interest in products that could be developed as fast-track entities. A more likely alternative for the development of a Tff2 formulation, should results warrant such a consideration, would be an international medical charity that could provide the necessary support to realize a Tff2 product. For these reasons I hope to evolve a work programme with the MRC Unit in The Gambia and to approach the Gates Foundation if deemed appropriate. I believe the approach to the prevention of these infections to be novel and a successful outcome to this work could have a substantial impact on policy with respect to the prevention of common infections in the developing world.
Training: This proposal provides an opportunity for postdoctoral training in an interdisciplinary project involving whole animal biology, molecular microbiology, histology, immunohistochemistry and prophylactic interventions in an area with great practical potential, coupled with state-of-the-art technologies. The PDRA will be embedded in laboratory with a focus on novel therapeutics with the opportunity to work in a world-class laboratory providing complimentary insights into structural and biochemical aspects of the GI tract. This will provide an opportunity to interact with a broad range of successful scientists and the opportunities for cross-fertilisation of ideas and technologies are very high.