Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 Nterminal residues, the other mutated residues are essential for catalysis. Variations in K m values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis

Examining Bdellovibrio bacteriovorus cell division processes and their metabolic cues during predation

Bdellovibrio bacteriovorus is a predatory bacterium that invades and digests other Gram-negative bacteria in its host dependent (HD) lifestyle. This predator traverses the outer membrane and digests a pore in the cell wall of the prey cell; it then enters the inner periplasm, sealing the pore and outer membrane, and establishing itself. Through modification of the host cell wall, the prey is rounded forming a bdelloplast. From the inner periplasm, B. bacteriovorus digest the host biomass sequentially and uses it to fuel their own growth, undergoing filamentous growth in the bdelloplast. Once prey nutrients have been consumed, the filament undergoes septation to create a variable number of progeny. This number is dependent on the available resources from the bdelloplast and can be odd or even. Following on from former PhD student David Milner’s work, my study focussed on the interaction partners of DivIVA, using pairwise Bacterial Two Hybrid (BTH) assays and then constructing a BTH library. Additionally, I investigated the divIVA operon with bioinformatics and fluorescent microscopy. Finally, while shielding from COVID-19, I bioinformatically analysed the division and cell wall (dcw) cluster of B. bacteriovorus and produced a phylogenetic tree for a study on its deacetylases, which includes lysozymes specific to prey entry and exit. DivIVA is a protein initially studied in Firmicutes, such as Bacillus subtilis. Its homologues have been implicated in regulating sporulation, cell morphology, apical growth, and several other processes in multiple, mainly Gram-positive, bacteria. Milner previously showed that DivIVA in B. bacteriovorus had roles in cell morphology and, potentially, septal site selection. I continued work on this protein by testing for interacting partners of DivIVA using pairwise BTH. This revealed a potential network of interactions that connect the roles of DivIVA with amino acid and cofactor Pyridoxal 5’-Phosphate (PLP) homeostasis, as well as chromosome partitioning. This involved proteins transcribed from neighbouring genes bd0466 and bd0465; YggS, for PLP homeostasis, and pyrroline 5-carboxylate reductase, for proline synthesis. Further interactions were found between Bd0465 and the canonical chromosome partitioning protein ParA3. This led to me investigating unknown interaction partners through the construction and use of a BTH library. This found several potential interacting proteins. A TrmJ homologue suggests crosstalk between DivIVA and the oxidative stress response, a link that has previously been found in Mycobacterium tuberculosis and Streptococcus suis. A MenE homologue was also identified as a potential interactor; this functions in menaquinone biosynthesis, a compound used in the electron transport chain. In Gram-negative bacteria, it is used for respiration in low oxygen environments, which could be emulated by the bdelloplast. During the pandemic and shielding from COVID-19, I analysed the dcw cluster of B. bacteriovorus. In rod shaped bacteria, the dcw cluster is a highly conserved region of the genome containing an operon encoding division, septation and cell wall synthesis proteins, including FtsZ. Both the genes and the order in which they are transcribed is conserved among bacteria, however, B. bacteriovorus have fifteen genes inserted into the cluster, fragmenting the ancestral operon. Investigating these genes shows varying roles for the encoded proteins. These include amino acid and nucleotide synthesis and homeostasis, stress response and DNA repair, and outer membrane lipid synthesis proteins. Finally, I produced a phylogenetic tree for a publication on the family of deacetylases that target deacetylated GlcNac. B. bacteriovorus modify the prey cell wall upon invasion, deacetylating GlcNAc. This serves to soften the wall and prevent other B. bacteriovorus from invading. Three deacetylases, which target the cell wall, were identified and one, DslA, was shown to lyse the bdelloplast at the end of the HD cycle. My phylogenetic analysis shows that DslA is related to lysozymes in several α-proteobacteria, including some plant root symbiotes, as well as some β- and γ-proteobacteria. Altogether, these results show complex regulation of division and septation in this predatory bacterium. This study primes further investigation into the crosstalk between division and other systems during the growth phase of B. bacteriovorus in the bdelloplast, while also identifying several novel metabolic interactions of DivIVA that can be further studied

Examining Bdellovibrio bacteriovorus cell division processes and their metabolic cues during predation

Bdellovibrio bacteriovorus is a predatory bacterium that invades and digests other Gram-negative bacteria in its host dependent (HD) lifestyle. This predator traverses the outer membrane and digests a pore in the cell wall of the prey cell; it then enters the inner periplasm, sealing the pore and outer membrane, and establishing itself. Through modification of the host cell wall, the prey is rounded forming a bdelloplast. From the inner periplasm, B. bacteriovorus digest the host biomass sequentially and uses it to fuel their own growth, undergoing filamentous growth in the bdelloplast. Once prey nutrients have been consumed, the filament undergoes septation to create a variable number of progeny. This number is dependent on the available resources from the bdelloplast and can be odd or even. Following on from former PhD student David Milner’s work, my study focussed on the interaction partners of DivIVA, using pairwise Bacterial Two Hybrid (BTH) assays and then constructing a BTH library. Additionally, I investigated the divIVA operon with bioinformatics and fluorescent microscopy. Finally, while shielding from COVID-19, I bioinformatically analysed the division and cell wall (dcw) cluster of B. bacteriovorus and produced a phylogenetic tree for a study on its deacetylases, which includes lysozymes specific to prey entry and exit. DivIVA is a protein initially studied in Firmicutes, such as Bacillus subtilis. Its homologues have been implicated in regulating sporulation, cell morphology, apical growth, and several other processes in multiple, mainly Gram-positive, bacteria. Milner previously showed that DivIVA in B. bacteriovorus had roles in cell morphology and, potentially, septal site selection. I continued work on this protein by testing for interacting partners of DivIVA using pairwise BTH. This revealed a potential network of interactions that connect the roles of DivIVA with amino acid and cofactor Pyridoxal 5’-Phosphate (PLP) homeostasis, as well as chromosome partitioning. This involved proteins transcribed from neighbouring genes bd0466 and bd0465; YggS, for PLP homeostasis, and pyrroline 5-carboxylate reductase, for proline synthesis. Further interactions were found between Bd0465 and the canonical chromosome partitioning protein ParA3. This led to me investigating unknown interaction partners through the construction and use of a BTH library. This found several potential interacting proteins. A TrmJ homologue suggests crosstalk between DivIVA and the oxidative stress response, a link that has previously been found in Mycobacterium tuberculosis and Streptococcus suis. A MenE homologue was also identified as a potential interactor; this functions in menaquinone biosynthesis, a compound used in the electron transport chain. In Gram-negative bacteria, it is used for respiration in low oxygen environments, which could be emulated by the bdelloplast. During the pandemic and shielding from COVID-19, I analysed the dcw cluster of B. bacteriovorus. In rod shaped bacteria, the dcw cluster is a highly conserved region of the genome containing an operon encoding division, septation and cell wall synthesis proteins, including FtsZ. Both the genes and the order in which they are transcribed is conserved among bacteria, however, B. bacteriovorus have fifteen genes inserted into the cluster, fragmenting the ancestral operon. Investigating these genes shows varying roles for the encoded proteins. These include amino acid and nucleotide synthesis and homeostasis, stress response and DNA repair, and outer membrane lipid synthesis proteins. Finally, I produced a phylogenetic tree for a publication on the family of deacetylases that target deacetylated GlcNac. B. bacteriovorus modify the prey cell wall upon invasion, deacetylating GlcNAc. This serves to soften the wall and prevent other B. bacteriovorus from invading. Three deacetylases, which target the cell wall, were identified and one, DslA, was shown to lyse the bdelloplast at the end of the HD cycle. My phylogenetic analysis shows that DslA is related to lysozymes in several α-proteobacteria, including some plant root symbiotes, as well as some β- and γ-proteobacteria. Altogether, these results show complex regulation of division and septation in this predatory bacterium. This study primes further investigation into the crosstalk between division and other systems during the growth phase of B. bacteriovorus in the bdelloplast, while also identifying several novel metabolic interactions of DivIVA that can be further studied

Antibiotic Resistance and Cell-Wall Recycling in Pseudomonas aeruginosa

The threat of antibiotic resistance and the global rise of pan-resistant bacteria is a serious concern at present. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is frequently associated with multi and pan-drug resistant infections. This research delves into the mechanism of resistance to a class of drugs known as the β-lactams. AmpC β-lactamase encoded chromosomally in P. aeruginosa is one of the predominant causes of resistance to many β-lactams. Previous research on this pathway identified the AmpC regulatory protein - AmpR and elaborated on its regulon in P. aeruginosa. In this dissertation, further investigation in the mechanisms associated with AmpR regulation of AmpC and its connection with the cell-wall recycling pathway is explored. Cell-wall recycling, a common phenomenon in both Gram-positive and negative bacteria is investigated in some detail in P. aeruginosa for the first time. The identity of the cell-wall recycling products or muropeptides in P. aeruginosa is elucidated. Around 20 distinct muropeptides were identified through liquid chromatography/mass spectrometry analyses of bacterial extracts. Furthermore, iv the muropeptide effector of AmpR that is instrumental in the activation of this transcription factor is identified. The role of two permeases AmpG and AmpP in antibiotic resistance and cell-wall recycling are also investigated by comparing antibiotic susceptibility and muropeptide profile of the isogenic mutants of ampG and ampP with the wild-type PAO1. Along with investigating permeases, the role of a putative N-acetylglucosaminidase FlgJ is also investigated. Finally, keeping in mind the broad role of AmpR in regulating P. aeruginosa virulence and antibiotic resistance, we try to identify small -molecule inhibitors for AmpR. In our effort to identify inhibitors, a novel reporter-based screening assay is developed. In summary, this dissertation increases our understanding of cell-wall recycling and mechanisms of β-lactam resistance and attempts at establishing novel-antibacterial targets and inhibitors