Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates

Abstract

The pathogenicity locus (PaLoc) of Clostridium difficile contains toxin A and B genes and three accessory genes, including tcdD and tcdC, which are supposed to code for the positive and negative regulators of toxin expression, respectively. Different studies have described variations in C. difficile toxin A and B genes, but little is known about C. difficile variants for the accessory genes. The PaLoc of several C. difficile clinical isolates was investigated by three different PCR methods with the aim to identify variant strains. Of the toxinogenic C. difficile strains examined, 25% showed variations. No correlation between C. difficile variant strains and key patient groups was found. Interestingly, all of these strains showed a variant tcdC gene. Three different tcdC alleles were identified, and one of these had a nonsense mutation which reduced the TcdC protein from 232 to 61 amino acids. It is possible that different TcdC variants affect toxin production differently, a hypothesis with important implications for the pathogenic potential of variant C. difficile strains.

FIG. 1.

Primers used in the PaLoc analysis. (A) Specificity and nucleotide sequences of primers and molecular sizes of the PCR products obtained for each pair of primers. (B) Location of PCR primers on a schematic representation of the PaLoc region. The small arrowheads indicate the orientation of primers.

FIG. 2.

Detection of PaLoc accessory genes cdd-3, tcdC, tcdD, tcdE, and cdu-2 by multiplex PCR in eight C. difficile strains with variant PaLoc genes identified in this study. Lane 1, Pd 5; lane 2, Pd7; lane 3, Pd13; lane 4, Pd16; lane 5, Pd53; lane 6, Pd 55; lane 7, M7; lane 8, Pd3; lane 9, DNA molecular weight marker IX; lane 10, C. difficile VPI10463; lane 11, ATCC 43597; lane 12, C. difficile 51377; lane 13, C. difficile 57267.

FIG. 3.

Toxinotyping of eight C. difficile strains with variant PaLoc genes and of the reference strains C. difficile VPI 10463, C. difficile 51377, and C. difficile 57267. (For descriptions of the other toxinotypes, see Rupnik et al. [21, 22]). The PCR-RFLP patterns of A3 and B1 fragments are shown for each strain. A3 fragments were digested with EcoRI (E), and B1 fragments were digested with HincII (H) and AccI (A). M, 100-bp DNA ladder (BioLabs). Pd7, Pd13, Pd16, and Pd55 (C. difficile strains), toxinotype V; Pd5 and Pd53, toxinotype VI; Pd3 and M7, toxinotype 0 (C. difficile M7 represents a new toxinotype, as demonstrated by PCR-RFLP analysis of the entire PaLoc). C. difficile VPI 10463, C. difficile 51377, and C. difficile 57267 are the reference strains for toxinotypes 0, VI, and VII, respectively.

FIG. 4.

Comparison of the TcdC nucleotide and amino acid sequences of the C. difficile reference strain VPI 10463 with those of the variant TcdC proteins identified in C. difficile clinical isolates examined in this study. Dots and dashes indicate identical bases and gaps, respectively, for the different tcdC alleles (tcdC-A, -B, and -C). The termination codon in tcdC-A is underlined. Only the amino acid changes are indicated for each TcdC variant. The eight 3-amino-acid repeats of the VPI 10463 TcdC are indicated by open (acidic in nature) and grey (basic in nature) boxes.