Chemical synergy between ionophore PBT2 and zinc reverses antibiotic resistance

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you."Lisa Bohlmann, David M. P. De Oliveira, Ibrahim M. El-Deeb, Erin B. Brazel, Nichaela Harbison-Pric

Rescuing tetracycline class antibiotics for the treatment of multidrug-resistant Acinetobacter baumannii pulmonary infection

Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients, and antibiotic treatment is compromised by multidrug-resistant strains resistant to β-lactams, carbapenems, cephalosporins, polymyxins, and tetracyclines. Among COVID-19 patients receiving ventilator support, a multidrug-resistant A. baumannii secondary infection is associated with a 2-fold increase in mortality. Here, we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break the resistance of A. baumannii to tetracycline class antibiotics. In vitro, the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multidrug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. PBT2 and zinc disrupted metal ion homeostasis in A. baumannii, increasing cellular zinc and copper while decreasing magnesium accumulation. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multidrug-resistant A. baumannii infections. Importance: Within intensive care unit settings, multidrug-resistant (MDR) Acinetobacter baumannii is a major cause of ventilator-associated pneumonia, and hospital-associated outbreaks are becoming increasingly widespread. Antibiotic treatment of A. baumannii infection is often compromised by MDR strains resistant to last-resort β-lactam (e.g., carbapenems), polymyxin, and tetracycline class antibiotics. During the on-going COVID-19 pandemic, secondary bacterial infection by A. baumannii has been associated with a 2-fold increase in COVID-19-related mortality. With a rise in antibiotic resistance and a reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. Rescuing the efficacy of existing therapies for the treatment of MDR A. baumannii infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.David M.P. De Oliveira, Brian M. Forde, Minh-Duy Phan, Bernhard Steiner, Bing Zhang, Johannes Zuegg, Ibrahim M. El-deeb, Gen Li, Nadia Keller, Stephan Brouwer, Nichaela Harbison-Price, Amanda J. Cork, Michelle J. Bauer, Saleh F. Alquethamy, Scott A. Beatson, Jason A. Roberts, David L. Paterson, Alastair G. McEwan, Mark A.T. Blaskovich, Mark A. Schembri, Christopher A. McDevitt, Mark von Itzstein, Mark J. Walke

Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria

Purple non-sulfur phototrophic bacteria, exemplifed by Rhodobacter capsulatus and Rhodobacter sphaeroides, exhibit a remarkable versatility in their anaerobic metabolism. In these bacteria the photosynthetic apparatus, enzymes involved in CO2 fixation and pathways of anaerobic respiration are all induced upon a reduction in oxygen tension. Recently, there have been significant advances in the understanding of molecular properties of the photosynthetic apparatus and the control of the expression of genes involved in photosynthesis and CO2 fixation. In addition, anaerobic respiratory pathways have been characterised and their interaction with photosynthetic electron transport has been described. This review will survey these advances and will discuss the ways in which photosynthetic electron transport and oxidation-reduction processes are integrated during photoautotrophic and photoheterotrophic growth

Dimethylsulfoxide enhances phototrophic growth of Rhodobacter sphaeroides in blue light

Dimethylsulfoxide enhanced the phototrophic growth of Rhodobacter sphaeroides in blue light by enabling cells to grow to a higher culture density. This enhanced growth with dimethylsulfoxide was not due to redox poising of the cyclic photosynthetic electron transfer chain and did not involve respiration. It was demonstrated that carotenoids and cyclic electron transfer were obligatory for the effect of dimethylsulfoxide suggesting that this molecule enhances the harvesting of blue light. The enhancement of blue light-dependent phototrophic growth by dimethylsulfoxide was shown to occur at very low concentrations of this molecule and this may have some significance for the growth of phototrophic bacteria in some environments. Copyright (C) 1998 Federation of European Microbiological Societies

The high resolution crystal structure of DMSO reductase in complex with DMSO

The crystal structure of the molybdenum enzyme dimethylsulphoxide reductase (DMSOR) has been determined at 1.9 Å resolution with substrate bound at the active site. DMSOR is an oxotransferase which catalyses the reduction of dimethylsulphoxide (DMSO) to dimethylsulphide (DMS) in a two stage reaction which is linked to oxygen atom transfer and electron transfer. In the first step, DMSO binds to reduced (Mo((IV))) enzyme, the enzyme is oxidised to Mo((VI))) with an extra oxygen ligand and DMS is released. Regeneration of reduced enzyme is achieved by transfer of two electrons, successively from a specific cytochrome, and release of the oxygen as water. The enzyme, purified under aerobic conditions, is in the oxidised (Mo((VI))) state. Addition of a large excess of DMS to the oxidised enzyme in solution causes a change in the absorption spectrum of the enzyme. The same reaction occurs within crystals of the enzyme and the crystal structure reveals a complex with DMSO bound to the molybdenum via its oxygen atom. X-ray edge data indicate that the metal is in the Mo((IV)) state. The DMSO ligand replaces one of the two oxo groups which ligate the oxidised form of the enzyme, suggesting very strongly that this is the oxygen which is transferred during catalysis. Residues 384 to 390, disordered in the oxidised enzyme, are now clearly seen in the cleft leading to the active site. The side-chain of Trp388 forms a lid trapping the substrate/product

Manganous ions suppress photosynthesis gene expression in Rhodobacter sphaeroides

The effect of manganous ions [Mn(II)] and ferrous ions [Fe(II)] on expression of photosynthesis genes in Rhodobacter sphaeroides was investigated. The presence of Mn(II) during phototrophic (anaerobic) and chemotrophic (aerobic) growth of R. sphaeroides caused a decrease in the amount of bacteriochlorophyll and carotenoid pigments which were synthesized and this was associated mainly with a decrease in the level of light-harvesting complex II. Mn(II) was shown to cause a decrease in expression of the puc operon, which encodes the polypeptides of light-harvesting complex II. Expression of the puc operon is controlled by the central repressor of photosynthesis gene expression, PpsR. In a ppsR mutant there was no effect of Mn(II) on photosynthesis gene expression. It is concluded that Mn(II) may act as a co-repressor in the action of PpsR or act via an as yet uncharacterized protein that interacts with PpsR. In contrast to the effects of Mn(II), Fe(II) was required for high levels of photosynthesis gene expression. This requirement for Fe(II) was shown to be related to the regulation of hemA, a gene under the control of the transcriptional regulator, FnrL. Mn(II) did not affect FnrL-dependent gene expression

Thermodynamic characterization of the redox centers within dimethylsulfide dehydrogenase

Dimethylsulfide (DMS) dehydrogenase is a complex heterotrimeric enzyme that catalyzes the oxidation of DMS to DMSO and allows Rhodovulum sulfidophilum to grow under photolithotrophic conditions with DMS as the electron donor. The enzyme is a 164 kDa heterotrimer composed of an α-subunit that binds a bis(molybdopterin guanine dinucleotide)Mo cofactor, a polyferredoxin β-subunit, and a γ-subunit that contains a b-type heme. In this study, we describe the thermodynamic characterization of the redox centers within DMS dehydrogenase using EPR- and UV-visible-monitored potentiometry. Our results are compared with those of other bacterial Mo enzymes such as NarGHI nitrate reductase, selenate reductase, and ethylbenzene dehydrogenase. A remarkable similarity in the redox potentials of all Fe-S clusters is apparent

Isolation and characterization of a strain of Rhodobacter sulfidophilus: A bacterium which grows autotrophically with dimethylsulphide as electron donor

A marine photosynthetic bacterium (strain SH1) was isolated after enrichment under phototrophic conditions in media containing dimethylsulphide (DMS) and bicarbonate (HCO) as potential carbon sources. Analysis of culture medium using nuclear magnetic resonance spectrometry showed that during phototrophic and chemotrophic growth of strain SH1 on DMS/HCO dimethylsulphoxide (DMSO) was produced from DMS. These results indicate that strain SH1 grew autotrophically with DMS serving as an electron donor in photosynthesis and respiration, but not as a carbon source. Biochemical characterization and 16S rRNA analysis indicated that the isolate was a strain of Rhodobacter sulfidophilus. An assay for the enzyme catalysing the oxidation of DMS (DMS:acceptor oxidoreductase) was developed by measuring electron transfer from DMS to 2,6-dichlorophenolindophenol (DCPIP). This reaction was dependent on phenazine ethosulphate to mediate electron transfer from DMS:acceptor oxidoreductase to DCPIP. DMS:acceptor oxidoreductase was found to have a periplasmic location in strain SH1 as was a reduced methylviologen:DMSO oxidoreductase activity. Zymogram staining patterns of periplasmic fractions indicated that DMS:acceptor oxidoreductase and DMSO reductase were distinct enzymes. This was confirmed by resolution of the two activities by gel filtration