Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development

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

Génomique fonctionnelle des protéines de division cellulaire et du peptidoglycane : développement de nouveaux agents antibactériens

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2006-2007Cette thèse de doctorat présente la problématique de résistance aux antibiotiques parmi les pathogènes bactériens en émergence et en réémergence à travers le monde. En effet, le développement et la propagation des mécanismes de résistance compromet l’efficacité des traitements antibactériens disponibles et met en danger la vie des patients infectés. Cette thèse se concentre sur l’identification de nouvelles cibles antibactériennes et sur le développement de nouvelles classes d’agents antibactériens en utilisant le pathogène opportuniste Pseudomonas aeruginosa en tan que modèle d’étude. Le premier chapitre aborde l’exploitation des protéines de division cellulaire FtsZ et FtsA en tant que cibles antibactériennes. Suite à une revue de la littérature détaillée, deux articles scientifiques décrivent la synthèse et la sélection d’inhibiteurs contre FtsZ et FtsA. Ces inhibiteurs représentent des candidats prometteurs en vue du développement d’une nouvelle classe d’agents antibactériens. Le deuxième chapitre du corps de la thèse porte sur l’utilisation des amides ligases MurC, MurD, MurE et MurF essentielles à la biosynthèse de la paroi bactérienne en tant que cibles antibactériennes. Suite à une revue de la littérature sur la biologie de ces enzymes, trois articles scientifiques relatent la sélection d’inhibiteurs peptidiques par présentation phagique contre les enzymes MurD, MurE et MurF. Le mode d’action innovateur de ces inhibiteurs permet d’envisager le développement de nouveaux agents antibactériens par peptidomimétisme. Le dernier chapitre expose le pouvoir antibactérien des endolysines de bactériophages. Une revue de la littérature résume le mode d’action et la biologie des endolysines en tant qu’agents antibactériens efficaces ciblant l’intégrité de la paroi bactérienne. Par la suite, un article décrit la capacité de l’endolysine du phage ΦKZ à hydrolyser la paroi bactérienne des bactéries à Gram-négatif et à outrepasser les membranes bactériennes. Ainsi, cette enzyme possède un potentiel antibactérien fort intéressant. En conclusion, cette thèse fournit plusieurs pistes attrayantes afin de développer de nouvelles stratégies antibactériennes pour contrer la problématique de résistance aux antibiotiques.This thesis first presents the critical outcome of antibiotic resistance among emerging and re-emerging bacterial pathogens worldwide. The incessant increase and spread of antibiotic resistance mechanisms compromise the efficiency of available antibacterial therapies and increase the impact of bacterial infections on human mortality and morbidity. This thesis focuses efforts to identify new antibacterial targets in order to develop novel classes of antibacterial agents using the opportunistic pathogen Pseudomonas aeruginosa as a research model. The first chapter of this thesis reports the exploitation of the cell division proteins FtsZ and FtsA as antibacterial targets. A detailed scientific review is presented along with two articles reporting the synthesis and selection of inhibitors against FtsZ and FtsA. These inhibitors represent potent candidates to develop new classes of antibacterial agents targeting the bacterial cell division process. The second chapter describes the use of the essential bacterial cell wall biosynthesis enzymes MurC, MurD, MurE and MurF as antibacterial targets. A scientific review first summarises the biology of these amide ligase enzymes and three scientific articles report the selection of peptide inhibitors against MurD, MurE and MurF by phage display. The novel mode of action of these inhibitors against the unexploited Mur enzymes can be the basis for future development of antibacterial agents targeting the cell wall biosynthesis pathway by peptidomimetism. The last chapter exposes the antibacterial potential of the phage-encoded endolysin enzymes. A review describes the mode of action and the biology of endolysins as efficient antibacterial agents targeting the integrity of the bacterial cell wall layer. Finally, an article presents the peptidoglycan hydrolytic activity of the P. aeruginosa phage ΦKZ gp144 lytic transglycosylase. This endolysin is able to pass through the bacterial membranes and thus represents a strong candidate for developing new antibacterial therapies against Gram-negative bacteria. In conclusion, this thesis provides various attractive ways to develop new antibacterial strategies and face the problem of antibiotic resistance

Unravelling the reaction mechanism of glutamate amidation in Staphylococcus aureus peptidoglycan

MurT-GatD is the bi-enzymatic complex responsible for catalysing the amidation of peptidoglycan in Gram-positive bacteria, ensuring the correct assembly of their cell wall. MurT-GatD is essential in antibiotic resistant human pathogens such as Staphylococcus aureus, Streptococcus pneumoniae and Mycobacterium tuberculosis, and therefore, constitutes a promising target for the development of new antimicrobial agents. Peptidoglycan amidation is achieved through the glutamine amidotransferase activity of the MurT-GatD complex, requiring the presence of glutamine, ATP and magnesium. The main goal of this thesis is to unravel the reaction mechanism of peptidoglycan amidation through an integrative approach that combines structural methods with functional assays, in order to characterize the structure-function relationship of the S. aureus MurT-GatD complex. The crystal structure of isolated S. aureus GatD was solved at 1.9 Å of resolution (PDB 5N9M) and provided the first structural insights into the MurT-GatD complex. GatD adopts the overall fold of glutaminase proteins and shows the nucleophilic cysteine and the polarizing histidine commonly associated with glutamine hydrolysis. 1H-NMR experiments showed that GatD C94A or H189A abolished the glutaminase activity of the complex. Similarly, GatD R128 also proved to be an essential residue for catalysis, likely by capturing glutamine to the active site. The crystal structure of S. aureus MurT-GatD was solved at 2.9 Å of resolution (PDB 7Q8E), showing a heterodimer in an extended conformation. GatD adopts the same glutaminase-like fold as in its isolated form, while MurT displays two distinct domains: the central domain containing, a cysteine-rich insertion and the ATP binding site, and the C-terminal domain that interacts with GatD. The overall structure of S. aureus MurT-GatD is very different from S. pneumoniae MurT-GatD, which adopts a compact conformation. The two complexes also adopt different conformations in solution, as observed through Small-Angle X-ray Scattering (SAXS) studies, and showed different in vitro enzymatic activities, suggesting that the extended conformation of S. aureus MurT-GatD is catalytically less competent. The structural data of MurT-GatD from S. aureus and S. pneumoniae were combined to identify the molecular determinants involved in the glutaminase and amidotransferase active sites of the complex, as well as, in protein-protein interactions. The work carried out in this thesis contributed to the clarification of the reaction mechanism of peptidoglycan amidation, which can be explored for the development of new drugs with antimicrobial activity.MurT-GatD é o complexo bi-enzimático que catalisa a amidação do peptidoglicano em bactérias Gram-positivas, garantindo a correta organização da sua parece celular. MurT-GatD é essencial em bactérias resistentes a antibióticos que são patogénicas a humanos, como Staphylococcus aureus, Streptococcus pneumoniae e Mycobacterium tuberculosis, e, consequentemente constitui um alvo promissor a ter em conta no desenvolvimento de novos agentes antimicrobianos. A amidação do peptidoglicano é realizada pela actividade de glutamina amidotransferase do complexo MurT-GatD, requerendo a presença de glutamina, ATP e magnésio. O principal objectivo desta tese é revelar o mecanismo reaccional da amidação do peptidoglicano através de uma abordagem integrativa que combina métodos estruturais com ensaios funcionais, de modo a caracterizar a relação estrutura-função do complexo MurT-GatD de S. aureus. A estrutura cristalina da proteína GatD de S. aureus foi resolvida a 1.9 Å de resolução (PDB 5N9M) e forneceu as primeiras evidências estruturais sobre o complexo MurT-GatD. GatD adopta uma organização 3D semelhante a glutaminases, apresentando uma cisteína nucleofílica e uma histidina polarizadora comummente associadas à hidrólise de glutamina. Experiências de 1H-NMR mostraram que os mutantes C94A ou H189A da GatD aboliram a actividade de glutaminase do complexo. De forma similar, R128 da GatD também provou ser um resíduo essencial na catálise, provavelmente por capturar glutamina para o centro activo. A estrutura cristalina do complexo MurT-GatD de S. aureus foi resolvida a 2.9 Å de resolução (PDB 7Q8E), mostrando um heterodímero numa conformação estendida. A GatD adopta a mesma organização estrutural característica de glutaminases, enquanto a MurT apresenta dois domínios distintos: o domínio central que contém uma inserção rica em cisteínas e o local de ligação ao ATP, e o domínio C-terminal que interage com a GatD. A estrutura global do complexo MurT-GatD de S. aureus é muito diferente do complexo MurT-GatD de S. pneumoniae, tendo em conta que este adopta uma conformação compacta. Os dois complexos também adoptam diferentes conformações em solução, como observado através de estudos de Dispersão de raios X a Baixo Ângulo (SAXS), tendo demonstrado diferentes actividades enzimática in vitro, sugerindo que a conformação estendida de MurT-GatD de S. aureus é cataliticamente menos competente. Os dados estruturais sobre o complexo MurT-GatD de S. aureus e S. pneumoniae foram combinados para identificar os determinantes moleculares envolvidos nos centros activos de glutaminase e de amidotransferase do complexo, assim como, nas interacções proteína-proteína. O trabalho desta tese teve uma importante contribuição para a elucidação do mecanismo reaccional da amidação do peptidoglicano, o que pode vir a ser explorado no desenvolvimento de novos fármacos com actividade antimicrobiana

Untersuchung zur antibakteriellen Wirkung und zum Biosynthese-Gencluster des Peptidantibiotikum Feglymycin

Feglymycin ist ein aus Streptomyces sp. DSM 11171 isoliertes, lineares 13mer-Peptid, das zu einem hohen Anteil aus den nicht-proteinogenen Aminosäuren Hpg (4-Hydroxyphenylglycine) und Dpg (3,5-Dihydroxyphenylglycine) besteht. Zudem besitzt es eine interessante, alternierende Abfolge von D- und L- Aminosäuren und strukturelle Ähnlichkeiten mit den Glycopeptiden der Vancomycin-Gruppe von Antibiotika und den Glycodepsipeptid-Antibiotika Ramoplanin und Enduracidin. Außerdem besitzt Feglymycin eine interessante Bioaktivität. Es wirkt in vivo antibakteriell gegen MRSA-Stämme (multi-resistente Staphylococcus aureus Stämme) und inhibitiert in vitro die Replikation von HIV-Viren im Zellkulturtest. Aufgrund seiner molekularen Masse und strukturellen Ähnlichkeit mit bekannten Zellwandbiosynthese-Inhibitoren wie z.B. Vancomycin und Ramoplanin, wurde auch Feglymycin als Zellwandbiosynthese-Inhibitor getestet. In diesen Tests zeigte Feglymycin keinen Effekt auf die membran-gebundenen zweite und die dritte Stufe der Peptidoglycanbiosynthese. Jedoch deuteten die Experimente auf eine Inhibition der früheren Biosyntheseschritte hin. Ziel dieser Arbeit war es, die antibakterielle Wirkung von Feglymycin auf die bakterielle Zellwandbiosynthese im Detail zu untersuchen und das biologische Target zu identifizieren. Zusätzlich wurde das Feglymycin Biosynthese-Gencluster untersucht. In LC-MS „one-pot assays“ wurde die Wirkung von Feglymycin auf die isolierten E. coli-Enzyme MurA-F getestet. Hierbei konnte reproduzierbar gezeigt werden, das Feglymycin die Enzyme MurA (Enopyruvyl-UDP-GlcNAc Synthase) und MurC (UDP-N-Acetyl-muramyl-L-alanin Ligase) inhibiert. In spektrophotometischen Assays mit den E. coli-Enzymen MurA und MurC konnte ein Ki-Wert von 0.33 +/- 0.04 μM für das MurC Enzym und ein Ki-Wert von 3.4 +/- 1.1 μM für das MurA Enzym bestimmt werden. Weitere Untersuchungen zeigten, dass Feglmycin auch die MurA (IC50 = 3.5 +/- 1.3 μM) und MurC (IC50 = 1.0 +/- 0.6 μM) Enzyme des gram-positiven Bakteriums Staphylococcus aureus inhibiert. Feglymycin zeigte dabei eine nicht-kompetitive Inhibition gegenüber der Bindung der Substrate des MurA Enzyms PEP (Phosphoenolpyruvat) und UDP-GlcNAc (UDP-N-Acetylglucosamin) und der Substrate des MurC Enzyms UDP-MurNAc (UDP-N-Acetylmuramat), ATP (Adenosintriphosphat) and L-Alanin. Feglmycin ist daher der erste Naturstoff der das MurC Enzym inhibiert. Zudem zeigt Feglymycin einen nicht-kompetitive Inhibitionstyp. Circulardichromismus (CD) Experimente mit den isolierten E. coli-Enzymen MurA und MurC und Feglymycin deuten einen möglichen allosterischen Effekt des Inhibitors auf die Enzyme an. Zusätzlich wurde die Feglmycinproduktion durch den Stamm Streptomyces sp. DSM 11171 und die Feglymycin-Detektion mittels LC-MS optimiert. Durch die Sequezierung des Genoms von Streptomyces sp. DSM 11171 konnte das Feglymycin Biosynthese-Gencluster identifiziert werden. Bei der Annotation des Genclusters zeigte sich, dass es sich bei Feglymycin um ein nicht-ribosomal synthetisiertes Peptid (NRPS) handelt dessen Biosynthese der Biosynthese der Glycopeptidantibiotika der Vancomycin-Gruppe von Antibiotika ähnelt. Zudem konnten im Streptomyces sp. DSM 11171 Genom weitere NRPS und Polyketidsynthase (PKS) Gencluster identifiziert und annotiert werden.Feglymycin is a linear 13-mer peptide produced by Streptomyces sp. DSM 11171 containing largely the non-proteinogenic Hpg (4-hydroxyphenylglycine) and the non-proteinogenic Dpg (3,5-dihydroxyphenylglycine) amino acids and an interesting alternation of D and L amino acids. It shows structural homogies to the glycopeptides of the vancomycin group of antibiotics and the glycodepsipeptide antibiotics ramoplanin and enduracidin. Feglymycin additionally shows an interesting biological activity. It possesses antibiotic activity against MRSA (multi-resistant Staphylococcus aureus) strains in vivo and inhibits syncytium formation in HIV infection in vitro. Due to its molecular mass and structural analogies to known inhibitors of the cell-wall biosynthesis, i.e. vancomycin and ramoplanin, feglymycin was tested as cell-wall biosynthesis inhibitor. In these tests feglymycin showed no effect on the membrane-bound second and third step of the peptidoglycan biosynthesis but the experiments indicated an inhibition of earlier biosynthetic steps. Aim of this work was to investigate the antibacterial activity of feglymycin on the bacterial cell-wall biosynthesis in more detail and to identify the biological target. Additionally the feglymycin biosynthesis gene cluster was investigated. Feglymycin was tested in a LC-MS one-pot assay against the isolated enzymes MurA-F from E. coli. Dereplication revealed that feglymycin specifically inhibits the enzymes MurA (enolpyruvyl-UDP-GlcNAc synthase) and MurC (UDP-N-acetyl-muramyl-L-alanine ligase). In in vitro assays with the enzymes MurA and MurC from gram-negative E. coli, a Ki value of 0.33 +/- 0.04 μM was determined for the MurC enzyme and a Ki value of 3.4 +/- 1.1 μM for the MurA enzyme. Further investigations showed that feglymycin also inhibits the MurA (IC50 = 3.5 +/- 1.3 μM) and MurC (IC50 = 1.0 +/- 0.6 μM) enzyme from gram-positive Staphylococcus aureus. The inhibition mode of feglymycin was found to be non-competitive with the binding of PEP (phosphoenolpyruvate) and UDP-GlcNAc (UDP-N-acetylglucosamine) in case of the MurA enzyme and non-competitive with binding of UDP-MurNAc (UDP-N-acetylmuramic acid), ATP (adenosine-triphosphate) and L-alanine in case of the MurC enzyme. Feglymycin is therefore the first natural compound found to inhibit the MurC enzyme showing a non-competitive inhibition type. Circular dichroism (CD) experiments with the isolated enzymes MurA and MurC from E. coli and feglymycin indicated a possible allosteric effect of feglymycin. Furthermore the feglymycin production by Streptomyces sp. DSM 11171 and feglymycin detection by LC-MS were optimized. Sequencing of the genome of Streptomyces sp. DSM 11171 allowed the idenfitication of the feglymycin biosynthesis gene cluster. Annotation of the gene cluster showed that feglymycin is a non-ribosomal synthesized peptide (NRPS) closely related to the glycopeptides of the vancomycin group of antibiotics. Additionally further NRPS and polyketide synthase (PKS) gene clusters were identified in the Streptomyces sp. DSM 11171 genome and annotated

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

ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A-102395 AND DISCOVERY OF NOVEL ACTINOMYCINS

The increase in antibiotic resistance of many bacterial strains including multidrug-resistant tuberculosis (MDR-TB) due to over- and misuse of antibiotics is a serious medical and economical problem. Therefore discovery and development of new antibiotics are urgently needed. Two projects were undertaken to address the need for new anti-tuberculosis antibiotics. 1. Discovery of new chemical entities. A-102395, a new nucleoside inhibitor of bacterial MraY (translocase I, EC 2.7.8.13) that is essential for bacterial survival, was isolated from the culture broth of Amycolatopsis sp. SANK 60206 in 2007. Although A-102395 is a potent inhibitor of translocase I with IC50 of 11 nM, it contradictingly does not have any antibiotic activity. A-102395 is a derivative of capuramycin with a unique aromatic side chain. A semisynthetic derivative of capuramycin is currently in clinical trials as an anti-tuberculosis antibiotic, suggesting potential for using A-102395 as a starting point for antibiotic discovery. The biosynthetic gene cluster of A-102395 was identified, including 35 putative open reading frames responsible for biosynthesis and resistance. A series of gene inactivation abolished the A-102395 production, indicating those genes within the cluster are essential for A-102395 biosynthesis. Functional characterization of Cpr17, which has sequence similarity to aminoglycoside phosphotransferases, revealed that it functions as a phosphotransferase conferring self-resistance by using GTP as phosphate donor. Furthermore the enzyme is characterized by low substrate specificity, as Cpr17 was capable of modifying a large series of natural or semi-synthesized analogues of capuramycins. A series of organism-specific high-throughput screening models for potential antibacterial agents targeting on bacterial cell wall synthesis have been established, including Escherichia coli and Mycobacterium tuberculosis. For this screen ten enzymes were successfully used to reconstitute cell wall biosynthesis in vitro. This screening is expected to allow us to identify the targets of novel antibiotics rapidly and in a cost-efficient manner. 2. Rediscovering old antibiotics. As part of our long term goal of discovering and developing novel anti-tuberculosis antibiotics, four novel actinomycins were isolated from the scale-up fermentation of Streptomyces sp. Gö-GS12, and their structures were characterized using mass spectrometry and 1D and 2D NMR. Their antibacterial activity against Gram-positive and Gram-negative strains were determined, as well as their cytotoxicity

Genome-Based Construction of the Metabolic Pathways of Orientia tsutsugamushi and Comparative Analysis within the Rickettsiales Order

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that belongs to the order of Rickettsiales. Recently, we have reported that O. tsutsugamushi has a unique genomic structure, consisting of highly repetitive sequences, and suggested that it may provide valuable insight into the evolution of intracellular bacteria. Here, we have used genomic information to construct the major metabolic pathways of O. tsutsugamushi and performed a comparative analysis of the metabolic genes and pathways of O. tsutsugamushi with other members of the Rickettsiales order. While O. tsutsugamushi has the largest genome among the members of this order, mainly due to the presence of repeated sequences, its metabolic pathways have been highly streamlined. Overall, the metabolic pathways of O. tsutsugamushi were similar to Rickettsia but there were notable differences in several pathways including carbohydrate metabolism, the TCA cycle, and the synthesis of cell wall components as well as in the transport systems. Our results will provide a useful guide to the postgenomic analysis of O. tsutsugamushi and lead to a better understanding of the virulence and physiology of this intracellular pathogen

Orientia tsutsugamushi and Comparative Analysis within the Rickettsiales Order

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that belongs to the order of Rickettsiales. Recently, we have reported that O. tsutsugamushi has a unique genomic structure, consisting of highly repetitive sequences, and suggested that it may provide valuable insight into the evolution of intracellular bacteria. Here, we have used genomic information to construct the major metabolic pathways of O. tsutsugamushi and performed a comparative analysis of the metabolic genes and pathways of O. tsutsugamushi with other members of the Rickettsiales order. While O. tsutsugamushi has the largest genome among the members of this order, mainly due to the presence of repeated sequences, its metabolic pathways have been highly streamlined. Overall, the metabolic pathways of O. tsutsugamushi were similar to Rickettsia but there were notable differences in several pathways including carbohydrate metabolism, the TCA cycle, and the synthesis of cell wall components as well as in the transport systems. Our results will provide a useful guide to the postgenomic analysis of O. tsutsugamushi and lead to a better understanding of the virulence and physiology of this intracellular pathogen