Anti-C. diphtheriae Diphtheria Toxin Polyclonal antibody

Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the hmuO promoter region relative to that of wildtype ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of hmuO and the hrtAB genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.

Diphtheria is a dreadful disease caused by Corynebacterium diphtheriae. Lysogenised bacteriophages carrying toxin gene in C. diphtheriae can make the strain toxigenic. However, such phage disseminates the toxin genes to other strains when it undergoes lytic phase. As little is known about the phage diversity in C. diphtheriae in India, the present study was undertaken to investigate the prophages integrated into the genome of 29 clinical isolates of C. diphtheriae using whole-genome shotgun sequencing. Amongst these isolates, 27 were toxigenic, while 2 were non-toxigenic strains. Of the 27 toxigenic strains, all harbored known phages carrying toxin gene and two other phages with unknown function. However, the two non-toxin strains did not harbour any of the phages in the genome. It is imperative to devise prevention strategies that hinder the dissemination of toxin by prophages, as it may increase the complications of diphtheria post-immunisation.