Hypothetical protein gene 1038 contributes to colistin resistance in Aeromonas hydrophila

Inhibition of vital respiratory enzymes, such as NADH: ubiquinone oxidoreductase (complex I), type II NADH-quinone oxidoreductases (NDH-2), and malate: quinone oxidoreductase, in the inner membrane, is a secondary antibacterial mechanism of colistin (1–3). However, colistin resistance mechanisms associated with this secondary mode of action of colistin have rarely been reported. Herein, we confirmed that the hypothetical protein gene 1038 was associated with colistin resistance in Aeromonas hydrophila by reducing antibiotic function in the inner membrane, providing novel knowledge on the generation of colistin resistance.

Ubiquinone Binding and Reduction by Complex I—Open Questions and Mechanistic Implications

NADH: ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chain. Complex I is a redox-driven proton pump that contributes to the proton motive force that drives ATP synthase. The structure of complex I has been analyzed by x-ray crystallography and electron cryo-microscopy and is now well-described. The ubiquinone (Q) reduction site of complex I is buried in the peripheral arm and a tunnel-like structure is thought to provide access for the hydrophobic substrate from the membrane. Several intermediate binding positions for Q in the tunnel were identified in molecular simulations. Structural data showed the binding of native Q molecules and short chain analogs and inhibitors in the access pathway and in the Q reduction site, respectively. We here review the current knowledge on the interaction of complex I with Q and discuss recent hypothetical models for the coupling mechanism.

Translational Proteomics for Transfusion Medicine: Resolution of the IVIG Proteomes of Different Geographically Sourced and Prepared IVIG Immunotherapies

Intravenous Immunoglobulin’s (IVIG’s) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at ≥ 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

Shared evolutionary footprints suggest mitochondrial oxidative damage underlies multiple complex I losses in fungi

Oxidative phosphorylation is among the most conserved mitochondrial pathways. However, one of the cornerstones of this pathway, the multi-protein complex NADH : ubiquinone oxidoreductase (complex I) has been lost multiple independent times in diverse eukaryotic lineages. The causes and consequences of these convergent losses remain poorly understood. Here, we used a comparative genomics approach to reconstruct evolutionary paths leading to complex I loss and infer possible evolutionary scenarios. By mining available mitochondrial and nuclear genomes, we identified eight independent events of mitochondrial complex I loss across eukaryotes, of which six occurred in fungal lineages. We focused on three recent loss events that affect closely related fungal species, and inferred genomic changes convergently associated with complex I loss. Based on these results, we predict novel complex I functional partners and relate the loss of complex I with the presence of increased mitochondrial antioxidants, higher fermentative capabilities, duplications of alternative dehydrogenases, loss of alternative oxidases and adaptation to antifungal compounds. To explain these findings, we hypothesize that a combination of previously acquired compensatory mechanisms and exposure to environmental triggers of oxidative stress (such as hypoxia and/or toxic chemicals) induced complex I loss in fungi.

Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°′ ∼−450 mV) using NADH (E°′ −320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°′ −80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.