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A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

ABSTRACT: In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

SUBMITTER: Reardon-Robinson ME

PROVIDER: S-EPMC9974433 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

Reardon-Robinson Melissa E ME Nguyen Minh Tan MT Sanchez Belkys C BC Osipiuk Jerzy J Rückert Christian C Chang Chungyu C Chen Bo B Nagvekar Rahul R Joachimiak Andrzej A Tauch Andreas A Das Asis A Ton-That Hung H

Proceedings of the National Academy of Sciences of the United States of America 20230214 8

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontan ...[more]

PMID: 36787356

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Project description:Corynebacterium diphtheriae is the etiological agent of diphtheria, a disease caused by the presence of the diphtheria toxin. However, an increasing number of records report non-toxigenic C. diphtheriae infections. Here, a C. diphtheriae strain was recovered from a patient with a past history of bronchiectasis who developed a severe tracheo-bronchitis with multiple whitish lesions of the distal trachea and the mainstem bronchi. Whole-genome sequencing (WGS), performed in parallel with PCR targeting the toxin gene and the Elek test, provided clinically relevant results in a short turnaround time, showing that the isolate was non-toxigenic. A comparative genomic analysis of the new strain (CHUV2995) with 56 other publicly available genomes of C. diphtheriae revealed that the strains CHUV2995, CCUG 5865 and CMCNS703 share a lower average nucleotide identity (ANI) (95.24 to 95.39%) with the C. diphtheriae NCTC 11397T reference genome than all other C. diphtheriae genomes (>98.15%). Core genome phylogeny confirmed the presence of two monophyletic clades. Based on these findings, we propose here two new C. diphtheriae subspecies to replace the lineage denomination used in previous multilocus sequence typing studies: C. diphtheriae subsp. lausannense subsp. nov. (instead of lineage-2), regrouping strains CHUV2995, CCUG 5865, and CMCNS703, and C. diphtheriae subsp. diphtheriae subsp. nov, regrouping all other C. diphtheriae in the dataset (instead of lineage-1). Interestingly, members of subspecies lausannense displayed a larger genome size than subspecies diphtheriae and were enriched in COG categories related to transport and metabolism of lipids (I) and inorganic ion (P). Conversely, they lacked all genes involved in the synthesis of pili (SpaA-type, SpaD-type and SpaH-type), molybdenum cofactor and of the nitrate reductase. Finally, the CHUV2995 genome is particularly enriched in mobility genes and harbors several prophages. The genome encodes a type II-C CRISPR-Cas locus with 2 spacers that lacks csn2 or cas4, which could hamper the acquisition of new spacers and render strain CHUV2995 more susceptible to bacteriophage infections and gene acquisition through various mechanisms of horizontal gene transfer.

Project description:An increasing number of isolations of Corynebacterium diphtheriae has been observed in recent years in the archipelago of New Caledonia. We aimed to analyze the clinical and microbiological features of samples with C. diphtheriae. All C. diphtheriae isolates identified in New Caledonia from May 2015 to May 2019 were included. For each case, a retrospective consultation of the patient files was conducted. Antimicrobial susceptibility phenotypes, tox gene and diphtheria toxin expression, biovar, and the genomic sequence were determined. Core genome multilocus sequence typing (cgMLST), 7-gene MLST, and search of genes of interest were performed from genomic assemblies. Fifty-eight isolates were included, with a median age of patients of 28 years (range: 9 days to 78 years). Cutaneous origin accounted for 51 of 58 (87.9%) isolates, and C. diphtheriae was associated with Staphylococcus aureus and/or Streptococcus pyogenes in three-quarters of cases. Half of cases came either from the main city Noumea (24%, 14/58) or from the sparsely populated island of Lifou (26%, 15/58). Six tox-positive isolates were identified, associated with recent travel to Vanuatu; 5 of these cases were linked and cgMLST confirmed recent transmission. Two cases of endocarditis in young female patients with a history of rheumatic fever involved tox-negative isolates. The 58 isolates were mostly susceptible to commonly used antibiotics. In particular, no isolate was resistant to the first-line molecules amoxicillin or erythromycin. Resistance to tetracycline was found in a genomic cluster of 17 (29%) isolates, 16 of which carried the tetO gene. There were 13 cgMLST sublineages, most of which were also observed in the neighboring country Australia. Cutaneous infections may harbor nontoxigenic C. diphtheriae isolates, which circulate largely silently in nonspecific wounds. The possible introduction of tox-positive strains from a neighboring island illustrates that diphtheria surveillance should be maintained in New Caledonia, and that immunization in neighboring islands must be improved. Genomic sequencing uncovers how genotypes circulate locally and across neighboring countries. IMPORTANCE The analysis of C. diphtheriae from the tropical archipelago of New Caledonia revealed a high genetic diversity with sublineages that may be linked to Polynesia, Australia, or metropolitan France. Genomic typing allowed confirming or excluding suspected transmission events among cases and contacts. A highly prevalent tetracycline-resistant sublineage harboring the tetO gene was uncovered. Toxigenic isolates were observed from patients returning from Vanuatu, showing the importance of improving vaccination coverage in settings where it is insufficient. This study also illustrates the importance for diphtheria surveillance of the inclusion of isolates from cutaneous sources in addition to respiratory cases, in order to provide a more complete epidemiological picture of the diversity and transmission of C. diphtheriae.

Dataset Information

A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

Publications

A cryptic oxidoreductase safeguards oxidative protein folding in <i>Corynebacterium diphtheriae</i>.

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