CdtC-Induced Processing of Membrane-Bound CdtA Is a Crucial Step in Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Holotoxin Formation

Abstract

is an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established an processing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA'), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA'. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA' and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA' showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formation is a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA' forms a complex with CdtC. Subsequent binding of CdtB to the CdtA'/CdtC complex results in CDT holotoxin formation.

Delivery, structure, and function of bacterial genotoxins

“…The holotoxin assembly in the PC occurs in a step-wise fashion ( Figure 2(a) ). CdtA crossed from the cytoplasm to the PC contains the conserved cysteine residue at its N -terminus to be lipidated in the PC by the action of a lipoprotein processing enzyme before it starts making a complex with CdtC [ 47 ] ( Figure 2(a) ). This conserved cysteine residue is part of the lipobox consensus sequence: (L) –3 -(A/S) –2 -(G/A) –1 -C +1 , where amino acid residues at the −2 and −1 positions can be any amino acid.…”

“…The lipoCdtA moves from the IM to the outer membrane (OM) within the PC, which recruits mature CdtC and forms a lipoCdtA and CdtC complex ( Figure 2(a) ). The lipoCdtA and CdtC complex is then freed from the OM via the N -terminal truncation of lipoCdtA, which forms a tripartite CDT holotoxin complex via its self-assembly with CdtB in the PC [ 47 ] ( Figure 2(a) ). The fully assembled CdtA/CdtB/CdtC holotoxin in the PC is ready to be secreted out to the extracellular environment.…”

Genome-Wide Identification of Host Genes Required for Toxicity of Bacterial Cytolethal Distending Toxin in a Yeast Model

“…AaCDT holotoxin complex is assembled and processed in the periplasm for secretion (Tsuruda et al, 2018). AaCDT is also found in the outer membrane vesicles (OMVs), which could deliver several bacterial proteins into host cells (Thay et al, 2014).…”