Background: The protease family, a group of enzymes produced by the host and bacterial species, play important roles in maintaining the homeostasis of the intestinal barrier. The concentrations of major gastrointestinal proteases including; pepsin, trypsin, and chymotrypsin, have been measured in preterm neonates. However, their longitudinal development is not well described. This thesis aims to investigate the dynamics of proteases, relate activity to disease and bacterial colonisation and measure their impact in vitro in a neonatal cohort.

Methods: Daily faecal and ileostomy samples were collected from birth until discharge or death in 66 preterm neonates born before 32 weeks’ gestation. Substrate and inhibitor-based assays were applied to profile total protease activity (ng/µL trypsin equivalent (TE)) in 20 control neonates, three surgically confirmed necrotising enterocolitis (NEC) cases, one spontaneous intestinal perforation (SIP) case, one intestinal atresia case, and three culture confirmed septicaemia cases. Postnatal colonisation of the intestinal tract was followed in 15 neonates by metataxonomic analysis. The proteolytic impact on the intestinal barrier was assessed in a Caco-2 cell model.

Results: Control, NEC/SIP and septicaemia neonates were characterised by patterns of high and low proteolytic activities. Activity increased with postmenstrual age only in control neonates (p<0.05). Inhibitor analysis revealed faecal and ileostomy samples were characterised by serine and cysteine proteases. Colonisation was irregular and dominated by genera that were not influenced by postnatal age, mode of delivery or feeding regimen. Correlation analysis revealed positive associations between Clostridium sensu stricto and Corynebacterium genera and faecal and ileostomy proteolytic activity (p <0.05). Caco-2 cell exposure to samples with low protease activity (< 250 ng/µL TE) led to a greater decrease in transepithelial electrical resistance.

Conclusions: Having established that proteases are present and active during the early neonatal period; this thesis provides the scope to continue investigating their associations with bacteria and their roles in neonatal diseases.