Characterisation of the ATP-dependent ligase, Lig E: An investigation into its significance in Neisseria gonorrhoeae

Abstract

The rapid rise in multidrug-resistant Neisseria gonorrhoeae isolates has caused major public concern for the treatment of its sexually transmitted disease, gonorrhoea. Previously easily treatable, gonorrhoea is now spreading at alarming rates due to the bacterium’s ability to take up DNA from its environment without regulation. With approximately 106 million new cases annually, thorough investigations into novel gonococcal pathways that can be targeted to limit its survival and spread are crucial. One possible target is the disruption of its DNA repair systems, which include ligases that seal breaks in DNA, and would greatly discourage bacterial growth. Interestingly, in addition to its replicative ligase, N. gonorrhoeae expresses a minimal ATP-dependent ligase, Ligase E (Lig E), which contains a signal peptide that indicates a likely periplasmic location as opposed to the cytosolic location of genomic DNA. Hence, I hypothesise that Lig E is working on repairing fragmented extracellular DNA which has a role in both biofilm formation and bacterial competence, both of which would be greatly beneficial for the persistence and acquisition of antibiotic resistance genes in N. gonorrhoeae.

In this thesis, I focussed on characterising Lig E from N. gonorrhoeae (Ngo-Lig E) to determine its contribution to gonococcal phenotype, as well as its structure, function and cellular location. To analyse its biological role, several ngo-lig E mutants were created in vivo and their growth, stress response, biofilm formation as well as infection and adhesion to a human cell line were studied. I showed that Ngo-Lig E is not essential for normal planktonic N. gonorrhoeae growth and survival; however, it is important for its biofilm formation and subsequent adhesion onto human cells. Following this, I further investigated the potential role of Ngo-Lig E on biofilm formation by looking at its morphology via scanning electron microscopy and confocal laser scanning microscopy after growing them under constant shear forces with a continual supply of fresh media. Data from this supported earlier indications that Ngo-Lig E is important for N. gonorrhoeae in biofilm formation, as well as the extent of damage the bacterium can cause to human tissue. Taking its natural competence into account, I also investigated the potential role of Ngo-Lig E in the uptake of fragmented DNA during transformation and found a decreased ability of N. gonorrhoeae to repair and take up nicked DNA when ngo-lig E was absent. Investigations into the potential location of Ngo-Lig E showed that it is likely transported into both the periplasmic and extracellular space in N. gonorrhoeae, where it may perform the aforementioned dual-role in biofilm formation and DNA uptake. Finally, I solved the three-dimensional structure of Ngo-Lig E, highlighting its minimal architecture and lack of complete encirclement around its substrate DNA.

The work presented in this thesis provides the first report of a solved structure of Lig E from a human pathogen, the first report of in vivo characterisation of this enzyme, as well as the first report of attempting to characterise its role and location. Collectively, this thesis has allowed for the establishment of important characteristics of Ngo-Lig E that were previously unknown. Although further research into this enzyme is necessary, the results presented here bring to light a potential novel pathway that may be targeted by future drug developments to tackle the emerging threat of multidrug-resistant N. gonorrhoeae in our community.