New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc₁ complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC₅₀, 30 nM) and cysts (IC₅₀, 4 μM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC₅₀, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc₁ complex Q[subscript]i site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites

A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex

AbstractIn anticipation of the structure of the bc1 complex which is now imminent, we present here a preliminary compilation of all available cytochrome b mutants that have been isolated or constructed to date both in prokaryotic and eukaryotic species. We have briefly summarized their salient properties with respect to the structure and function of cytochrome b and to the Qo and Qi sites of the bc1 complex. In conjunction with the high resolution structure of the bc1 complex, this database is expected to serve as a useful reference point for the available data and help to focus and stimulate future experimental work in this field

The primary pathway for lactate oxidation in Desulfovibrio vulgaris

International audienceThe ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant 426-28 was highly disrupted on D-lactate, whereas the growth of mutant 432-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species

A Tad-like apparatus is required for contact-dependent prey killing in predatory social bacteria

International audienceMyxococcus xanthus , a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contact with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages

Molar growth yield for the four strains under various growth conditions.

<p>Molar growth yield on lactate for the wild-type and deletion mutant strains cultured under anaerobic conditions (black bars) and with constant 0.02% O₂ gas mixture sparging (striped bars). Molar growth yields are expressed in 10¹² cells/mole of substrate.</p

Variability in the cell length of the various <i>Dv</i>H strains.

<p>Comparison of the distribution of the cell length in cultures under anaerobic conditions (black bars) and with constant 0.02% O₂ gas mixture sparging (striped bars). The average cell length was determined from two independent cultures (>200 cells were counted in each experiment). Statistical analysis using One-way ANOVA were performed to reveal significant differences between distributions (p<0.05), which were mentioned by an asterisk.</p

Protected PFOR rate in the four strains.

<p>Quantification of the rate of protected PFOR during the exponential growth phase under anaerobic (black bars) and with constant 0.02% O₂ gas mixture sparging (striped bars). Statistical analysis using Mann and Whitney test was used to compare means. Significant differences (p< 0.05) are indicated by an asterisk. All values are related to the PFOR protected rate in the wild-type strain under anaerobic conditions (value arbitrary set to 1).</p

Lactate consumption and acetate production in the <i>Dv</i>H strains.

<p>Quantification of consumed lactate (A) and produced acetate (B) after 40h growth under anaerobic conditions (black bars) or with constant 0.02% O₂ gas mixture sparging (striped bars) in medium C (with 37mM of lactate as initial concentration).</p

Quantification of cells able to divide and average division parameters of the wild-type and deletion mutant strains after oxygen exposure.

<p>Strains were cultured with constant 0.02% O₂ gas mixture sparging followed by a switch to anaerobic condition. Data are mean values of three independent experiments ± SD. Statistical analysis using t-test was used to compared means.</p><p>^{a, b, c}: Significant differences.</p><p>Quantification of cells able to divide and average division parameters of the wild-type and deletion mutant strains after oxygen exposure.</p