Abstract
Although proteolytic degradation of salivary proteins by oral bacteria has been known for some time, the relevance of proteolytic activity as a requirement for growth of bacteria in health and disease is poorly understood and often focuses on glycolysis as the major metabolic pathway. This thesis concentrated on studying protease production and metabolic activity by oral bacteria in health and in dental erosion.The first results chapter of this thesis assessed salivary proteins as key nutrients for oral bacterial growth in vitro in the absence of glucose – i.e. during times of starvation in the mouth. Altering the incubation environment, from aerobic to anaerobic, induced bacterial proteolysis and increased the growth of oral biofilms. This demonstrates that bacteria will readily produce proteases in biofilms from healthy individuals. The proline-rich proteins were the most degraded salivary proteins, and with this degradation came an increase in proline positively correlated to protease, and subsequently peptidase, activity.
The connection between protease activity and proline production was explored further.
Proline was shown to inhibit salivary protein degradation in saliva-inoculated saliva-grown biofilms, which has not been reported before. Proline also decreased lactate production in these biofilms – this is an important metabolite associated with enamel dissolution in dental caries. Novel 13C nuclear magnetic resonance tracked the degradation of proline to 5-aminopentanoate, butyrate, propionate and gamma-aminobutyrate (GABA).
Following on from this, a more reductionist approach was taken to determine which bacterial species within a supragingival defined consortium model would utilise proline, and how this affected their metabolism. All of the 8 species that were investigated consumed proline to some extent (Neisseria subflava, Streptococcus mutans NCTC 10449, Streptococcus gordonii NCTC 7865, Streptococcus oralis NCTC 7864, Veillonella dispar NCTC 11831, Prevotella nigrescens ATCC 33563, Fusobacterium nucleatum subspecies polymorphum ATCC 10953 and Lactobacillus rhamnosus ATCC 7469). There were visible differences in the metabolomic profiles of the bacteria that were supplemented with proline compared to the same bacteria that were not. Again, a reduction in lactate was witnessed by bacteria that consumed proline. The effects of proline and glucose on Streptococcus mutans, a well characterised bacterial species, was investigated – mainly to determine whether proline could elicit similar effects to arginine on the pH of the growth environment. On its own, proline did not change the pH of the growth environment.
Finally, a preliminary investigation into the role of bacteria in dental erosion was performed using 5 participants with dental erosion and 6 healthy controls. Analysis of the salivary microbiome by 16S rRNA gene sequencing showed that there were differences in the alpha and beta diversity between the two groups – participants with dental erosion had a less diverse microbiome than healthy controls. Two key proteins present in the enamel pellicle, prolactin inducible protein and zinc alpha-2-glycoprotein, were shown to be degraded by bacteria in biofilms grown from the saliva of individuals with dental erosion. This was also witnessed in some participants from the healthy group, thus this may suggest the association with disease but that this may not be a fundamental factor. Participants that demonstrated this degradation had an increased relative abundance of Prevotella; a bacterial species that has been described previously as proteolytic.
Collectively, the data in this thesis has shown that bacteria have complex nutritional networks and that they will express proteases and peptidases to utilise proteins and amino acids as an energy source, both in health and in disease. The data in this thesis also puts forward evidence that bacteria may play a role in dental erosion which has not been reported previously.
| Date of Award | 1 Apr 2022 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Guy Carpenter (Supervisor) & Rebecca Moazzez (Supervisor) |