Characterisation of the conserved mycobacterial penicillin binding protein LpqF

Thomas, Clare ORCID: 0000-0001-6426-5890 (2025). Characterisation of the conserved mycobacterial penicillin binding protein LpqF. University of Birmingham. Ph.D.

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Abstract

Tuberculosis is a global disease affecting humans, animals, and marine life. In humans, it is primarily caused by the bacterium Mycobacterium tuberculosis and a few closely related species. M. tuberculosis was responsible for 1.6 million deaths in 2022, equating to one death every 20 seconds. Antibiotic resistance in M. tuberculosis is a major concern and a significant driver of mortality. The Mycobacterium genus has a unique, impermeable cell wall built on a foundation of peptidoglycan, which provides tensile strength and resists osmotic pressure. Peptidoglycan, an extensive cell wall polymer of alternating N-acetylglucosamine and N-acetylmuramic acid residues, is cross-linked by short peptides. Peptidoglycan-targeting enzymes, such as D,D-carboxypeptidases, are tightly regulated to enable growth and division.

Building on the observation that the loss of LpqF leads to altered colony morphology in M. marinum, this thesis characterises the function of this protein. LpqF is a low molecular weight penicillin-binding protein (PBP) conserved in mycobacteria. Our data support classifying LpqF as a D,D-carboxypeptidase that is inhibited by meropenem. These data show that LpqF has a unique N-terminal NTF2-like domain that binds to peptidoglycan, suggesting a positioning mechanism for the catalytic domain to access its substrate. Additionally, two of eight mutants (D85A and R98A) designed in the N-terminal NTF2-like domain further support this hypothesis by showing that these residues likely have roles in anchoring LpqF to peptidoglycan. Furthermore, this thesis presents evidence that Δlpqf causes an accumulation of the fluorescent D-amino acid (FDAA) 3-[7-nitrobenzofurazan]-carboxamide-D-alanine (NADA) along the lateral cell wall in M. marinum, indicating the involvement of LpqF in peptidoglycan incorporation.

This work provides a strong foundation for further analysis and elucidation of D,D-carboxypeptidases. Understanding the function and regulation of LpqF could offer valuable insights into developing inhibitors targeting proteins involved in peptidoglycan biosynthesis, potentially aiding in the creation of new therapeutics against drug-resistant strains of M. tuberculosis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):