Biology subjects

Gennis, R. B.

Publications and source records attributed to Gennis, R. B..

3 recordsLinked to original sources

bioRxiv2025

Anti-Parasitics with a Triple Threat: Targeting Parasite Enzymes, the Proton Motive Force, and Host Cell-Mediated Killing

We investigated the effects of the tuberculosis drug candidate SQ109 (8a) and of its analog MeSQ109 (8b) against Leishmania mexicana in promastigote and amastigote forms, as well as against host cell macrophages finding potent activity (1.7 nM) for MeSQ109 against the intracellular forms, as well as low toxicity ([~]61 {micro}M) to host cells, resulting in a selectivity index of [~]36,000. We then investigated the mechanism of action of MeSQ109 finding that it targeted parasite mitochondria, collapsing the proton motive force, as well as targeting acidocalcisomes, rapidly increasing the intracellular Ca2+ concentration. Using an E. coli inverted membrane vesicle assay, we investigated the pH gradient collapse for SQ109 and 17 analogs finding that there was a significant correlation (on average R=0.67, p[~]0.008) between pH gradient collapse and cell growth inhibition in Trypanosoma brucei, T. cruzi, L. donovani and Plasmodium falciparum. We also investigated pH gradient collapse with other anti-leishmanial agents: azoles, antimonials, benzofurans, amphotericin B and miltefosine. The enhanced activity against intracellular trypanosomatids is seen with Leishmania spp. grown in macrophages but not with Trypanosoma cruzi in epithelial cells and is proposed to be due in part to host-based killing, based on the recent observation that SQ109 is known to convert macrophages to a pro-inflammatory (M1) phenotype.

microbiology↗

bioRxiv2021

Diversity and evolution of nitric oxide reduction

Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- [->] NO2- [->] NO [->] N2O [->] N2)1. Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone-depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we use phylogenomics to identify a number of previously uncharacterized HCO families and propose that many of them (eNOR, sNOR, gNOR, and nNOR) perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from Rhodothermus marinus, verifying that it performs nitric oxide reduction both in vitro and in vivo. These newly identified NORs exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.

biochemistry↗

bioRxiv2021

Evolution of the cytochrome-bd type oxygen reductase superfamily and the function of cydAA in Archaea

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify 3 families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA is the first isoform of cytbd containing only b-type hemes shown to be active when isolated, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.

biochemistry↗