Quinoline Compounds Targeting the c-Ring of ATP Synthase Inhibit Drug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) is a Gram-negative, biofilm-forming bacterium and an opportunistic pathogen. The growing drug resistance of PA is a serious threat that necessitates the discovery of novel antibiotics, ideally with previously underexplored mechanisms of action. Due to their central role in cell metabolism, bacterial bioenergetic processes are of increasing interest as drug targets, especially with the success of the ATP synthase inhibitor bedaquiline to treat drug-resistant tuberculosis. Like Mycobacterium tuberculosis, PA requires F1Fo ATP synthase for growth, even under anaerobic conditions, making the PA ATP synthase an ideal drug target for the treatment of drug-resistant infection. In previous work, we conducted an initial screen for quinoline compounds that inhibit ATP synthesis activity in PA. In the present study, we report additional quinoline derivatives, including one with increased potency against PA ATP synthase in vitro and antibacterial activity against drug-resistant PA. Moreover, by expressing the PA ATP synthase in Escherichia coli, we show that mutations in the H+ binding site on the membrane-embedded rotor ring alter inhibition by the reported quinoline compounds. Identification of a potent inhibitor and its probable binding site on ATP synthase enables further development of promising quinoline derivatives into a viable treatment for drug-resistant PA infection.

Both CD8 and CD4 T cells contribute to immunosurveillance preventing the development of neoantigen-expressing autochthonous sarcomas.

The adaptive immune system plays an essential anti-tumor role through immunosurveillance and response to immunotherapies. Characterizing phenotypic features and mechanisms of dysfunction of tumor-specific T cell populations may uncover novel immunotherapeutic targets and biomarkers of response. To study tumor-specific T cell responses in vivo, a tumor model must express a known neoantigen. While transplant models with known neoantigen expression are widely available, autochthonous tumor models in which the tumor coevolves with the immune system are limited. In this study, we combined CRISPR/Cas9 and sleeping beauty transposase technology to develop an autochthonous orthotopic murine sarcoma model with oncogenic KrasG12D, functionally impaired p53, and expression of known MHCI and MHCII sarcoma neoantigens. Using MHC tetramer flow cytometry, we identified a tumor-specific immune response in the peripheral blood as early as 10 days after tumor induction leading to tumor clearance. Tumors developed at high penetrance after co-depletion of CD8 and CD4 T cells, but depletion of either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These results suggest that CD8 and CD4 T cells can independently contribute to immunosurveillance leading to clearance of sarcomas expressing MHCI and MHCII neoantigens.

Mutational analysis of a conserved positive charge in the c-ring of E. coli ATP synthase.

F1Fo ATP synthase is a ubiquitous molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The membrane-embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which together provide two aqueous half channels that facilitate proton translocation. Previous work has suggested that Arg50 and Thr51 on the cytoplasmic side of each subunit c are involved in the proton translocation process, and positive charge is conserved in this region of subunit c. To further investigate the role of these residues and the chemical requirements for activity at these positions, we generated 13 substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and passive H+ permeability activities, as well as the ability of mutants to carry out oxidative phosphorylation in vivo. While polar and hydrophobic mutations were generally tolerated in either position, introduction of negative charge or removal of polarity caused a substantial defect. We discuss the possible effects of altered electrostatics on the interaction between the rotor and stator, water structure in the aqueous channel, and interaction of the rotor with cardiolipin.