A Novel High-Content Phenotypic Screen To Identify Inhibitors of Mitochondrial DNA Maintenance in Trypanosomes.

Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. The survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here, we report the development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof of concept, we screened a diverse set of ∼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound.

Pharmacological Inhibition of the Vacuolar ATPase in Bloodstream-Form Trypanosoma brucei Rescues Genetic Knockdown of Mitochondrial Gene Expression.

Trypanosomatid parasites cause diseases in humans and livestock. It was reported that partial inhibition of the vacuolar ATPase (V-ATPase) affects the dependence of Trypanosoma brucei on its mitochondrial genome (kinetoplast DNA [kDNA]), a target of the antitrypanosomatid drug isometamidium. Here, we report that V-ATPase inhibition with bafilomycin A1 (BafA) provides partial resistance to genetic knockdown of mitochondrial gene expression. BafA does not promote long-term survival after kDNA loss, but in its presence, isometamidium causes less damage to kDNA.

The genetic basis of Escherichia coli pathoadaptation to macrophages.

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.

Increased survival of antibiotic-resistant Escherichia coli inside macrophages.

Mutations causing antibiotic resistance usually incur a fitness cost in the absence of antibiotics. The magnitude of such costs is known to vary with the environment. Little is known about the fitness effects of antibiotic resistance mutations when bacteria confront the host's immune system. Here, we study the fitness effects of mutations in the rpoB, rpsL, and gyrA genes, which confer resistance to rifampin, streptomycin, and nalidixic acid, respectively. These antibiotics are frequently used in the treatment of bacterial infections. We measured two important fitness traits-growth rate and survival ability-of 12 Escherichia coli K-12 strains, each carrying a single resistance mutation, in the presence of macrophages. Strikingly, we found that 67% of the mutants survived better than the susceptible bacteria in the intracellular niche of the phagocytic cells. In particular, all E. coli streptomycin-resistant mutants exhibited an intracellular advantage. On the other hand, 42% of the mutants incurred a high fitness cost when the bacteria were allowed to divide outside of macrophages. This study shows that single nonsynonymous changes affecting fundamental processes in the cell can contribute to prolonged survival of E. coli in the context of an infection.

Evolution of Escherichia coli to Macrophage Cell Line.

The genomes of species of Escherichia coli (E. coli) show an extraordinary amount of diversity, which include commensal strains and strains belonging to different pathovars. Many strains of E. coli, which can cause mild or severe pathologies in humans, have a commensal ancestor. Understanding the evolutionary changes that can lead to a transition from commensal to pathogen is an important task, which requires integration of different methodologies. One method is experimental evolution of bacteria, in controlled environments, that mimic some of the selective pressures, likely to be important during the transition to pathogenesis. The success of such a transition will depend, at least partially, on ability of E. coli to adapt to the presence of cells of the immune system. Here, we describe a protocol for performing experimental evolution of a commensal strain of E. coli, a derivative of the well studied K12, under the constant selective pressure imposed by cells of the innate immune system, specifically RAW 264.7 murine macrophage cell line.

Fitness Measurements of Evolved Escherichia coli.

Bacteria can adapt very rapidly to novel selective pressures. In the transition from commensalism to pathogenicity bacteria have to face and adapt to the host immune system. Specifically, the antagonistic interaction imposed by one of the first line of defense of innate immunity cells, macrophages, on commensal bacteria, such as Escherichia coli (E. coli), can lead to its rapid adaptation. Such adaptation is characterized by the emergence of clones with mutations that allow them to better escape macrophage phagocytosis. Here, we describe how to quantify the amount of fitness increase of bacterial clones that evolved under the constant selective pressure of macrophages, from a murine cell line RAW 264.7. The most widely used assay for measuring fitness changes along an evolutionary laboratory experiment is a competitive fitness assay. This assay consists of determining how fast an evolved strain outcompetes the ancestral in a competition where each starts at equal frequency. The strains compete in the same environment of the evolution experiment and if the evolved strain has acquired strong beneficial mutations it will become significantly overrepresented in repeated competitive fitness assays.