Characterisation of trehalose-6-phosphate phosphatases from bacterial pathogens.

The trehalose biosynthesis pathway has recently received attention for therapeutic intervention combating infectious diseases caused by bacteria, helminths or fungi. Trehalose-6-phosphate phosphatase (TPP) is a key enzyme of the most common trehalose biosynthesis pathway and a particularly attractive target owing to the toxicity of accumulated trehalose-6-phosphate in pathogens. Here, we characterised TPP-like proteins from bacterial pathogens implicated in nosocomial infections in terms of their steady-state kinetics as well as pH- and metal-dependency of their enzymatic activity. Analysis of the steady-state kinetics of recombinantly expressed enzymes from Acinetobacter baumannii, Corynebacterium diphtheriae and Pseudomonas stutzeri yielded similar kinetic parameters as those of other reported bacterial TPPs. In contrast to nematode TPPs, the divalent metal ion appears to be bound only weakly in the active site of bacterial TPPs, allowing the exchange of the resident magnesium ion with other metal ions. Enzymatic activity comparable to the wild-type enzyme was observed for the TPP from P. stutzeri with manganese, cobalt and nickel. Analysis of the enzymatic activity of S. maltophilia TPP active site mutants provides evidence for the involvement of four canonical aspartate residues as well as a strictly conserved histidine residue of TPP-like proteins from bacteria in the enzyme mechanism. That histidine residue is a member of an interconnected network of five conserved residues in the active site of bacterial TPPs which likely constitute one or more functional units, directly or indirectly cooperating to enhance different aspects of the catalytic activity.

Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.

A suicide inhibitor of nematode trehalose-6-phosphate phosphatases.

Protein-based drug discovery strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors. Currently, there are no known trehalose-6-phosphate phosphatase (TPP) inhibitors that possess reasonable inhibition constants and chemical scaffolds amenable to convenient modification. In the present study, we subjected recombinant TPPs to a two-tiered screening approach to evaluate several diverse compound groups with respect to their potential as TPP inhibitors. From a total of 5452 compounds tested, N-(phenylthio)phthalimide was identified as an inhibitor of nematode TPPs with apparent Ki values of 1.0 µM and 0.56 µM against the enzymes from the zoonotic roundworms Ancylostoma ceylanicum and Toxocara canis, respectively. Using site-directed mutagenesis, we demonstrate that this compound acts as a suicide inhibitor that conjugates a strictly conserved cysteine residue in the vicinity of the active site of nematode TPPs. The anthelmintic properties of N-(phenylthio)phthalimide were assessed in whole nematode assays using larvae of the ascaroids T. canis and T. cati, as well as the barber's pole worm Haemonchus contortus. The compound was particularly effective against each of the ascaroids with an IC50 value of 9.3 µM in the survival assay of T. cati larvae, whereas no bioactivity was observed against H. contortus.