Investigating galactose metabolism in Streptococcus pneumoniae

Streptococcus pneumoniae is a formidable human pathogen. Responsible for between 1 and 2 million deaths annually, the pneumococcus makes a major contribution to global morbidity and mortality. In order to cause disease, the pneumococcus must first colonise the human nasopharynx. This colonisation is typically asymptomatic and provides the ideal niche from which the pneumococcus can transmit itself to new hosts. However, in some cases, the pneumococcus will undergo a ‘switch’ from harmless coloniser to invasive pathogen, transiting to deeper, usually sterile niches in the body and causing invasive disease. A key determinant of successful colonisation of the nasopharynx is the ability to metabolise the different carbon sources that are available. While the pneumococcus typically prefers to metabolise glucose, this carbon source is actively eliminated from the human nasopharynx in an attempt to maintain airway sterility. In the absence of glucose, galactose is the predominant sugar in this niche. Galactose can be metabolised by two pathways in the pneumococcus, the Leloir pathway and the tagatose-6-phosphate pathway. A study by Trappetti et al., in 2017 was the first to show a link between carbohydrate metabolism and cell-to-cell signalling in the pneumococcus, demonstrating that the quorum sensing molecule Autoinducer 2 (AI-2) is likely phosphorylated during import into the cell. Phosphorylated AI-2 is then proposed to either directly or indirectly phosphorylate GalR, the regulator of the Leloir pathway, driving an increase in galactose metabolism and a subsequent hypervirulent phenotype. They propose that this phosphorylation occurs at the putative phosphorylation sites identified by Sun et al., in 2010: Serine 317, Threonine 319 and Threonine 323. To better understand the role of these putative phosphorylation sites, a series of amino acid substitution mutants were generated in which each of the sites were replaced, either singly or in combination, with either the non-phosphorylatable residue alanine (A) or the phosphomimetic aspartic acid (D) or glutamic acid (E). While the use of phosphomimetic residues proved somewhat challenging, the use of non-phosphorylatable alanine residues revealed that each of the three putative phosphorylation sites are required for growth in galactose, successful activation of the Leloir pathway and disease progression in a murine model of infection. What became clear during this study was that despite having two functional pathways encoded for galactose metabolism, there was an inability for one pathway to rescue the other during times of metabolic distress. This indicated that these pathways may not be as discreet as once thought. To further investigate this potential interplay, a series of mutants were generated, deleting key genes from either the Leloir or the T6P pathways. This approach revealed that deleting genes from either pathway resulted in an inability to metabolise galactose, as well as transcriptional changes indicating that there is indeed interplay between these two pathways, with GalR possibly playing a key role as the central regulator of both pathways. Additionally, we found that there is differential accumulation of metabolites intracellularly as a result of these mutations, which may hold the key to deciphering exactly how these two pathways are linked. Finally, using dual in vivo RNA sequencing, we have revealed that GalR and its putative phosphorylation sites play an important role in virulence, leading to skewing of the immune response during infection. Collectively, the findings of this thesis have significantly advanced our understanding of pneumococcal galactose metabolism, particularly in terms of its regulation via GalR. Additionally, we have shed light on the interplay between the Leloir and T6P pathways, showing for the first time that there is a definitive link that requires both pathways to be present and functional in order to survive in the presence of galactose, much like what is found in the human nasopharynx. We have also shown that the putative GalR phosphorylation sites play a key role in pneumococcal galactose metabolism, pneumococcal virulence and the host response to infection. This project provides the foundation for further investigation into the regulation of pneumococcal galactose metabolism, and the wide-reaching impacts this pathway has on pneumococcal virulence and disease.

Author dc:creator

• McLean, Kimberley Taylor

Advisors dc:contributor.advisor

• Paton, James
• Trappetti, Claudia

• Streptococcus pneumoniae, galactose, metabolism, carbohydrate metabolism, Leloir pathway, TaQatose-6-phosphate pathway

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/2440/136167

OAI identifier oai:identifier

oai:digital.library.adelaide.edu.au:2440/136167