Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers.

We studied membrane activity of the bacterial peptide TisB involved in persister cell formation. TisB and its analogs form multi-state ion-conductive pores in planar lipid bilayers with all states displaying similar anionic selectivity. TisB analogs differing by ±1 elementary charges show corresponding changes in selectivity. Probing TisB pores with poly-(ethylene glycol)s reveals only restricted partitioning even for the smallest polymers, suggesting that the pores are characterized by a relatively small diameter. These findings allow us to suggest that TisB forms clusters of narrow pores that are essential for its mechanism of action.

Pharmacological validation of Trypanosoma brucei phosphodiesterases B1 and B2 as druggable targets for African sleeping sickness.

Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report, we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei , the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogues, show modest inhibition of TbrPDEB1 and B2 and quickly kill the bloodstream form of the subspecies T. brucei brucei . We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.

An alternative pathway for reduced folate biosynthesis in bacteria and halophilic archaea.

Whereas tetrahydrofolate is an essential cofactor in all bacteria, the gene that encodes the enzyme dihydrofolate reductase (DHFR) could not be identified in many of the bacteria whose genomes have been entirely sequenced. In this communication we show that the halophilic archaea Halobacterium salinarum and Haloarcula marismortui contain genes coding for proteins with an N-terminal domain homologous to dihydrofolate synthase (FolC) and a C-terminal domain homologous to dihydropteroate synthase (FolP). These genes are able to complement a Haloferax volcanii mutant that lacks DHFR. We also show that the Helicobacter pylori dihydropteroate synthase can complement an Escherichia coli mutant that lacks DHFR. Activity resides in an N-terminal segment that is homologous to the polypeptide linker that connects the dihydrofolate synthase and dihydropteroate synthase domains in the haloarchaeal enzymes. The purified recombinant H. pylori dihydropteroate synthase was found to be a flavoprotein.

Interactions of glutaredoxins, ribonucleotide reductase, and components of the DNA replication system of Escherichia coli.

A strain of Escherichia coli missing three members of the thioredoxin superfamily, thioredoxins 1 and 2 and glutaredoxin 1, is unable to grow, a phenotype presumed to be due to the inability of cells to reduce the essential enzyme ribonucleotide reductase. Two classes of mutations can restore growth to such a strain. First, we have isolated a collection of mutations in the gene for the protein glutaredoxin 3 that suppress the growth defect. Remarkably, all eight independent mutations alter the same amino acid, methionine-43, changing it to valine, isoleucine, or leucine. From the position of the amino acid changes and their effects, we propose that these alterations change the protein so that its properties are closer to those of glutaredoxin 1. The second means of suppressing the growth defects of the multiply mutant strain was by mutations in the DNA replication genes, dnaA and dnaN. These mutations substantially increase the expression of ribonucleotide reductase, most likely by altering the interaction of the regulatory protein DnaA with the ribonucleotide reductase promoter. Our results suggest that this increase in the concentration of ribonucleotide reductase in the cell allows more effective interaction with glutaredoxin 3, thus restoring an effective pool of deoxyribonucleotides. Our studies present direct evidence that ribonucleotide reductase is the only essential enzyme that requires the three reductive proteins missing in our strains. Our results also suggest an unexpected regulatory interaction between the DnaA and DnaN proteins.

Functions of thiol-disulfide oxidoreductases in E. coli: redox myths, realities, and practicalities.

A large family of enzymes contributes to the thiol-disulfide redox environment of the cells of most organisms. These proteins belong to pathways that carry out a variety of reactions, including the promotion of disulfide bond formation in extracytoplasmic proteins, the isomerization of proteins with incorrect disulfide bonds, and the reduction of disulfide bonds in the active sites of cytoplasmic proteins. Although the redox activities of these proteins measured in vitro often is consistent with the role (oxidant or reductant) these proteins perform in vivo, this is not always the case. The measured redox potentials can even suggest a function for a protein opposite of that which it carries out in the cell. Structural features of such proteins can contribute to a direction of electron transfer inconsistent with the redox potential. Furthermore, the environment in which such proteins are found may determine the protein's physiological role. Detailed analysis of these proteins in Escherichia coli provides strains that are useful for biotechnological purposes. Increasing the activity of certain of these proteins in the cell envelope or altering the thiol-disulfide redox environment of the cytoplasm to make it more oxidizing enhances the yield of useful disulfide bond-containing proteins such as tissue plasminogen activator and immunoglobulins.

Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene.

So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A DeltapyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.

Genetic evidence for a novel thymidylate synthase in the halophilic archaeon Halobacterium salinarum and in Campylobacter jejuni.

A search of the complete genome sequence of the halophilic archaeon Halobacterium salinarum failed to identify a gene homologous to the thymidylate synthase (thyA) gene present in the closely related Haloferax volcanii. To understand the source of thymidine synthesis in Hbt. salinarum, a genomic library of Hbt. salinarum was constructed and used to complement a Hfx. volcanii thyA deletion mutation. The Hbt. salinarum ORF that complemented the thyA mutation shares sequence homology with ORFs found in numerous microorganisms that lack a thyA gene, including the recently discovered thyX of Helicobacter pylori. We also show that a homolog of the Hbt. salinarum ORF is present in Campylobacter jejuni and is able to complement an Escherichia coli thyA mutant under oxygen-limiting conditions.