Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora

Abstract

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors

“…Many ADPRTs, however, share less than 10% protein sequence similarity (Kumar et al, 2019), making identification of new family members challenging. Further, bioinformatic searches assume that novel ADPRTs are exclusively present in pathogens (Tremblay et al, 2020). There is also a bias toward pathogenic bacteria in the number of genomes publicly available, although this has been rapidly changing with large microbiome sequencing efforts of commensal genomes (Almeida et al, 2021 .…”

Erratum: Tremblay, O., et al. Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora. Toxins 2020, 12, 792

“…In the original paper [ 1 ], Jennifer Geddes-McAlister was not included as an author in the published article. Jennifer Geddes-McAlister also contributed to investigation, formal analysis, supervision for this research.…”

A Structural Approach to Anti-Virulence: A Discovery Pipeline

“…The cellular targets for mART toxins are often key regulators of cell function and include (i) GTPases, (ii) actin, (iii) kinase regulators, (iv) elongation factors, and (v) RNA-recognition motifs, and even DNA (target genes) [22][23][24][25][26][27][28]. We developed a data-mining strategy based on fold-recognition methods [29][30][31] that unearths new mARTs as targets for disease intervention using the anti-virulence or anti-infective approach (Figure 1).…”

“…The targets for mART toxins are often key regulators of cell function and include (i) G (ii) actin, (iii) kinase regulators, (iv) elongation factors, and (v) RNA-recognition and even DNA (target genes) [22][23][24][25][26][27][28]. We developed a data-mining strategy based o recognition methods [29][30][31] that unearths new mARTs as targets for disease interv using the anti-virulence or anti-infective approach (Figure 1). Step 1: Known mART toxin sequences are used (input) to provide several thousands (10,000-20,000) of candidate toxin sequences arising from iterative search tools such as HHblits and HHsenser.…”

“…Our efforts to date have led to the discovery of 30 new mARTs with rigorous characterization of several toxins accompanied by more than 50 crystal structures, including ExoA (P. aeruginosa) [35][36][37][38][39][40][41][42][43][44][45][46][47], Cholix (V. cholerae) [34,[48][49][50][51][52][53][54][55], Certhrax (B. cereus) [56], Photox (P. luminescens) [57,58], VahC/VsdC (A. hydrophila) [59], C3larvinA, Plx2A (P. larvae) [60][61][62][63], Vis (V. splendidus) [64], Scabin (Streptomyces scabies) [23,[65][66][67], and Vorin (E. amylovora) [31].…”