Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator

Abstract

Clostridium difficile binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, here designated cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR(+) and cdtR-negative strains of C. difficile, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.

FIG. 1.

Relevant shuttle plasmids. Schematic representations of the CDT binary toxin expression plasmid pJIR3107 (A) and the cdtR complementation plasmid pJIR3394 (B) are shown. The position and orientation of the pCD6 plasmid replication genes (repA and orfB) are shown, as is the transfer origin (oriT), the selectable markers (ermB or catP), the CDT binary toxin genes (cdtA and cdtB), and the cdtR gene.

FIG. 2.

Analysis of binary toxin production by complemented strains. (A) CDTa Western immunoblot using cross-reactive Ia-specific antibodies and precipitated supernatant proteins from the strains indicated. (B) CDTb Western immunoblot using cross-reactive Ib-specific antibodies and precipitated supernatant proteins. (C) ADP-ribosyltransferase activity assay using purified rabbit skeletal muscle actin, [³²P]NAD and precipitated supernatant proteins. The data are the mean values and standard deviations from three independent experiments. CD196, binary toxin-positive reference strain; JIR8094, binary toxin-negative strain; CD37, nontoxigenic strain; V, pMTL9361Cm vector plasmid; CDT, pJIR3107, which carries the wild-type cdtAB genes. The arrows indicate the 48-kDa CDTa protein and 75-kDa CDTb protein. The higher-molecular-mass products are most likely higher-order aggregates and the smaller protein bands breakdown products.

FIG. 3.

Schematic representation of the CDT region and flanking genes. The regions from the nontoxigenic isolate CD37 (A), the binary toxin-negative isolate strain 630 (B), and the binary toxin-positive isolate QCD-32g58 (C) are shown. The positions of the 5′ flanking genes CD2601 and CD2602, the 3′ flanking gene trpS, the response regulator gene cdtR, and the CDT binary toxin-encoding genes cdtAB, or their pseudogenes, are shown. For each variant of the CDT region the positions of the 5′ and 3′ conserved boundaries are shown, and the size of the entire CdtLoc is indicated. The unique 68-bp sequence that is present in CD37 and other nontoxigenic isolates in place of the CdtLoc is shown in bold, and the nucleotide boundaries of the CDT region that are conserved in all three variants are underlined.

FIG. 4.

Alignment of CdtR orthologs. The predicted CdtR protein from C. difficile strain 630 and predicted AgrA orthologs of S. aureus (ABB17535), Staphylococcus epidermidis (AAO25552) and Clostridium acetobutylicum (AAK78066) were aligned using CLUSTALW (58). Identical amino acid residues are shaded with black, and strongly similar amino acid residues are shaded with gray.

FIG. 5.

Analysis of binary toxin production in derivatives of CD37. (A) CDTa Western immunoblot using cross-reactive Ia specific antibodies and precipitated supernatant proteins from the strains indicated. (B) CDTb Western immunoblot using cross-reactive Ib specific antibodies and precipitated supernatant proteins. (C) ADP-ribosyltransferase activity assayed using purified rabbit skeletal muscle actin, [³²P]NAD and precipitated supernatant proteins. The data are the mean values and standard deviations from three independent experiments CD196, binary toxin-positive reference strain; M7404, binary toxin-positive NAP1/027 strain; CD37, nontoxigenic strain; pJIR3107, encodes the wild-type cdtAB genes (shown as + in the CDT rows); V, pMTL9361Cm vector plasmid (shown as − in the CDT rows); pJIR3394, encodes the wild-type cdtR gene (shown as + in the cdtR rows); pJIR3395, encodes the ΔcdtR gene (shown as ΔcdtR in the cdtR rows). The arrows indicate the 48-kDa CDTa protein and 75-kDa CDTb protein.