The role of the N-glycolyl modification in Mycobacterial peptidoglycan synthesis and survival

Mycobacteria are acid fast bacilli responsible for the wide spread global diseases tuberculosis and leprosy. The increased persistence of multidrug resistant (MDR) mycobacterial strains has led to the focus on discovery of new and under-utilised cellular targets such as the cell wall. Peptidoglycan, the principle structural component of the bacterial cell wall is a heteropolymer comprised of alternating monosaccharides cross-linked by pentapeptide chains. The cell wall of mycobacteria are inherently resistant to antimicrobials and aid in evasion from host immune detection due to modifications to its composition. The hydroxylase enzyme NamH has been documented to play a role in the N-glycolylation of peptidoglycan monosaccharides, utilizing molecular oxygen during aerobic growth to convert N-acetylto N-glycolyl groups. This modification is found predominantly in Actinobacteria, except Mycobacterium leprae due to genomic reduction. The percentage incorporation of Nacetylated and N-glycolylated saccharides is dependent upon the environment and functional characterisation of the impact of each modification is vital to achieving a greater understanding into mycobacterial response to a range of factors including dormancy, resuscitation and intracellular propagation. The investigations described in this thesis concern the susceptibility of a M. smegmatis DnamH strain, the cell wall of which contains solely N-acetylated cell wall components towards: (a) selected hydrolytic enzymes, as a model of the survival of phagocytosed mycobacteria within the harsh conditions of the phagolysosome and; (b) new and existing antimicrobials commonly used as therapies against infection. The absence of the Nglycolylated sugar within the peptidoglycan cell wall led to consistently observed increases in susceptibility to a range of hydrolytic enzymes and antimicrobials, especially those which target the formation of peptidoglycan. Mycobacterial Mur ligases demonstrated increased catalytic bias towards N-glycolylated substrates to increase their inclusion into the wide peptidoglycan sacculus. Investigations were expanded to characterize the impact of newly discovered known cell wall active compounds against the peptidoglycan biosynthesis machinery

Biguanide iridium(III) complexes with potent antimicrobial activity

We have synthesized novel organoiridium(III) antimicrobial complexes containing a chelated biguanide, including the antidiabetic drug metformin. These 16- and 18-electron complexes were characterized by NMR, ESI-MS, elemental analysis, and X-ray crystallography. Several of these complexes exhibit potent activity against Gram-negative bacteria and Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus (MRSA)) and high antifungal potency toward C. albicans and C. neoformans, with minimum inhibitory concentrations (MICs) in the nanomolar range. Importantly, the complexes exhibit low cytotoxicity toward mammalian cells, indicating high selectivity. They are highly stable in broth medium, with a low tendency to generate resistance mutations. On coadministration, they can restore the activity of vancomycin against vancomycin-resistant Enterococci (VRE). Also the complexes can disrupt and eradicate bacteria in mature biofilms. Investigations of reactions with biomolecules suggest that these organometallic complexes deliver active biguanides into microorganisms, whereas the biguanides themselves are inactive when administered alone

Combatting AMR : photoactivatable ruthenium(ii)-isoniazid complex exhibits rapid selective antimycobacterial activity

The novel photoactive ruthenium(II) complex cis-[Ru(bpy)2(INH)2][PF6]2 (1·2PF6, INH = isoniazid) was designed to incorporate the anti-tuberculosis drug, isoniazid, that could be released from the Ru(II) cage by photoactivation with visible light. In aqueous solution, 1 rapidly released two equivalents of isoniazid and formed the photoproduct cis-[Ru(bpy)2(H2O)2]2+ upon irradiation with 465 nm blue light. We screened for activity against bacteria containing the three major classes of cell envelope: Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, and Mycobacterium smegmatis in vitro using blue and multi-colored LED multi-well arrays. Complex 1 is inactive in the dark, but when photoactivated is 5.5× more potent towards M. smegmatis compared to the clinical drug isoniazid alone. Complementary pump-probe spectroscopy measurements along with density functional theory calculations reveal that the mono-aqua product is formed in <500 ps, likely facilitated by a 3MC state. Importantly, complex 1 is highly selective in killing mycobacteria versus normal human cells, towards which it is relatively non-toxic. This work suggests that photoactivatable prodrugs such as 1 are potentially powerful new agents in combatting the global problem of antibiotic resistance

Biguanide Iridium(III) Complexes with Potent Antimicrobial Activity

We have synthesized novel organoiridium­(III) antimicrobial complexes containing a chelated biguanide, including the antidiabetic drug metformin. These 16- and 18-electron complexes were characterized by NMR, ESI-MS, elemental analysis, and X-ray crystallography. Several of these complexes exhibit potent activity against Gram-negative bacteria and Gram-positive bacteria (including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)) and high antifungal potency toward <i>C. albicans</i> and <i>C. neoformans</i>, with minimum inhibitory concentrations (MICs) in the nanomolar range. Importantly, the complexes exhibit low cytotoxicity toward mammalian cells, indicating high selectivity. They are highly stable in broth medium, with a low tendency to generate resistance mutations. On coadministration, they can restore the activity of vancomycin against vancomycin-resistant <i>Enterococci</i> (VRE). Also the complexes can disrupt and eradicate bacteria in mature biofilms. Investigations of reactions with biomolecules suggest that these organometallic complexes deliver active biguanides into microorganisms, whereas the biguanides themselves are inactive when administered alone

Biguanide Iridium(III) Complexes with Potent Antimicrobial Activity

We have synthesized novel organoiridium­(III) antimicrobial complexes containing a chelated biguanide, including the antidiabetic drug metformin. These 16- and 18-electron complexes were characterized by NMR, ESI-MS, elemental analysis, and X-ray crystallography. Several of these complexes exhibit potent activity against Gram-negative bacteria and Gram-positive bacteria (including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)) and high antifungal potency toward <i>C. albicans</i> and <i>C. neoformans</i>, with minimum inhibitory concentrations (MICs) in the nanomolar range. Importantly, the complexes exhibit low cytotoxicity toward mammalian cells, indicating high selectivity. They are highly stable in broth medium, with a low tendency to generate resistance mutations. On coadministration, they can restore the activity of vancomycin against vancomycin-resistant <i>Enterococci</i> (VRE). Also the complexes can disrupt and eradicate bacteria in mature biofilms. Investigations of reactions with biomolecules suggest that these organometallic complexes deliver active biguanides into microorganisms, whereas the biguanides themselves are inactive when administered alone

Biguanide Iridium(III) Complexes with Potent Antimicrobial Activity

We have synthesized novel organoiridium­(III) antimicrobial complexes containing a chelated biguanide, including the antidiabetic drug metformin. These 16- and 18-electron complexes were characterized by NMR, ESI-MS, elemental analysis, and X-ray crystallography. Several of these complexes exhibit potent activity against Gram-negative bacteria and Gram-positive bacteria (including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)) and high antifungal potency toward <i>C. albicans</i> and <i>C. neoformans</i>, with minimum inhibitory concentrations (MICs) in the nanomolar range. Importantly, the complexes exhibit low cytotoxicity toward mammalian cells, indicating high selectivity. They are highly stable in broth medium, with a low tendency to generate resistance mutations. On coadministration, they can restore the activity of vancomycin against vancomycin-resistant <i>Enterococci</i> (VRE). Also the complexes can disrupt and eradicate bacteria in mature biofilms. Investigations of reactions with biomolecules suggest that these organometallic complexes deliver active biguanides into microorganisms, whereas the biguanides themselves are inactive when administered alone

Biguanide Iridium(III) Complexes with Potent Antimicrobial Activity

We have synthesized novel organoiridium­(III) antimicrobial complexes containing a chelated biguanide, including the antidiabetic drug metformin. These 16- and 18-electron complexes were characterized by NMR, ESI-MS, elemental analysis, and X-ray crystallography. Several of these complexes exhibit potent activity against Gram-negative bacteria and Gram-positive bacteria (including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)) and high antifungal potency toward <i>C. albicans</i> and <i>C. neoformans</i>, with minimum inhibitory concentrations (MICs) in the nanomolar range. Importantly, the complexes exhibit low cytotoxicity toward mammalian cells, indicating high selectivity. They are highly stable in broth medium, with a low tendency to generate resistance mutations. On coadministration, they can restore the activity of vancomycin against vancomycin-resistant <i>Enterococci</i> (VRE). Also the complexes can disrupt and eradicate bacteria in mature biofilms. Investigations of reactions with biomolecules suggest that these organometallic complexes deliver active biguanides into microorganisms, whereas the biguanides themselves are inactive when administered alone

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture