A multiomic approach to defining the essential genome of the globally important pathogen Corynebacterium diphtheriae

Abstract

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.

The exoproteome and surfaceome of toxigenicCorynebacterium diphtheriae1737 and its response to iron-restriction and growth on human hemoglobin

“…Two of the three most abundant proteins were ribosomal protein L7/L12 (RplL) and elongation factor Tu (Tuf), which is consistent with a prior proteomic analysis of whole cell lysate from non-toxigenic C. diphtheriae strain ISS3319. 5 The second most abundant protein, a putative secreted protease (DIP2069), was predominantly found in the supernatant. The relative abundance of each protein in the different fractions was estimated by constructing its “localization profile” (see methods).…”

“…2 Both toxigenic (encoding diphtheria toxin, “DT”) and non-toxigenic Corynebacterium subspecies are capable of colonizing the upper respiratory tract in humans. 1,5 However, a global phylogenetic analysis suggests biotype gravis predominated in the Eastern European 1990s outbreak and was particularly lethal because it had re-acquired the DT gene ( tox ) from a β corynephage. 6 Genomic sequencing of the related NCTC13129 strain subsequently revealed the presence of pathogenicity islands that contain a number of genes that encode for surface and secreted virulence factors, including pili, 7,8 heme-acquisition machinery, 7,9,10 and antimicrobial-resistance genes.…”

Proteomics of Toxigenic Corynebacteria

“…Most of these targets identified via in silico approaches were verified. Transposon Directed Insertion Sequencing (TraDIS) of a high-density transposon mutant pool indicated the corresponding genes as essential when C. diphtheriae was grown in broth [ 23 ]. Today, none of these putative drug and vaccine targets are tested in clinical trials, since antibiotic resistances are rare in C. diphtheriae and the toxoid vaccine directed against the diphtheria toxin has been successfully applied for more than 100 years.…”