Examination of the Clostridioides (Clostridium) difficile VanZ ortholog, CD1240

Abstract

Clostridioides (Clostridium) difficile causes severe diarrheal disease that is directly associated with antibiotic use and resistance. Although C. difficile demonstrates intrinsic resistance to many antimicrobials, few genetic mechanisms of resistance have been characterized in this pathogen. In this study, we investigated the putative resistance factor, CD1240 (VanZ1), an ortholog of the teicoplanin resistance factor, VanZ, of Enterococcus faecium. In C. difficile, the vanZ1 gene is located within the skin element of the sporulation factor σ^K, which is excised from the mother cell compartment during sporulation. This unique localization enabled us to create a vanZ1 deletion mutant by inducing excision of the skin element. The Δskin mutant exhibited moderately decreased resistance to teicoplanin and had small effects on growth in some other cell-surface antimicrobials tested. Examination of vanZ1 expression revealed induction of vanZ1 transcription by the antimicrobial peptide LL-37; however, LL-37 resistance was not impacted by VanZ1, and none of the other tested antimicrobials induced vanZ1 expression. Further, expression of vanZ1 via an inducible promoter in the Δskin mutant restored growth in teicoplanin. These results demonstrate that like the E. faecium VanZ, C. difficile VanZ1 contributes to low-level teicoplanin resistance through an undefined mechanism.

Keywords: Antimicrobial resistance; Clostridium difficile; Glycopeptides; Teicoplanin; Vancomycin; vanZ.

Figure 1. Alignment of VanZ-family proteins

A) Unrooted phylogenetic tree relating the VanZ of E. faecium to CD1240 (VanZ1) and three additional VanZ-like proteins (CD2015, CD2520 and CD2160) was created using Phylo.io version 1.0k (http://phylo.io/)(51). B) Protein sequences for C. difficile VanZ1 (CD1240, WP_009889012.1) and E. faecium VanZ (YP_006374628.1) were aligned using MAFFT version 7.388 (52). An asterisk below the sequence indicates identical residues, colons indicate similar residues, and dots indicate low similarity.

Figure 2. vanZ1 genetic context and deletion of the skin^cd element (Δskin mutant)

A) Graphical representation of the vanZ1 gene (green), within the sigK skin^cd element of strain 630Δerm. sigK (CD1230) is depicted in blue, with the skin^cd element delineated in the light gray box. Additional annotated skin^cd genes are depicted in grey, and pseudogenes are shown in white. B) Confirmation of skin^cd deletion and complementation. PCR products were generated using primers flanking the excised skin^cd element (280 bp, primers QRTBD326/IMV833), CD1234 (300 bp, primers oMC1609/oMC1610), or vanZ1 (564 bp, primers oMC1397/oMC1398). Genomic DNA from 630Δerm (control), Δskin (MC1041), Δskin + vector control (MC1260), or Δskin + pP_cprA::vanZ1 (MC1261) were used as template.

Figure 3. Growth of the Δskin mutant in cell surface-acting antimicrobials

Active cultures of strains 630Δerm and Δskin (MC1041) were diluted in A) BHIS, or BHIS supplemented with B) 60 μg/ml cefoperazone, C) 0.15 μg/ml teicoplanin, D) 10 μg/ml nisin, E) 1 μg/ml vancomycin, or F) 2.5 μg/ml LL-37. Graphs represent the mean OD₆₀₀ readings from three independent replicates with error bars showing the standard error of the mean. Data were analyzed by Student’s t-test comparing 630Δerm to Δskin at each time point with Holm-Sidak correction for multiple tests. No statistically significant differences were observed for growth in any antibiotic at any time point.

Figure 4. Deletion of vanZ1 does not alter gross cell morphology

Phase contrast micrographs of strain 630Δerm (A, C, E, G, I, K) and Δskin (B, D, F, H, J, L) grown to mid-log phase in BHIS alone (A, B), 0.125 μg/ml teicoplanin (Teic; C, D), 1.0 μg/ml vancomycin (Van; E, F), 60 μg/ml cefoperazone (Cef; G, H), 10 μg/ml nisin (Nis; I, J), or 2.5 μg/ml LL-37 (K, L) are shown. Scale bars indicate 10 μm.

Figure 5. Addition of vanZ1 restores growth of the Δskin mutant in teicoplanin

Active cultures of strains 630Δerm pMC211 (vector control, MC282), 630Δerm pMC632 (pPcprA::vanZ1, MC907), Δskin + pMC211 (vector control, MC1260), and Δskin pMC632 (pPcprA::vanZ1, MC1261) were diluted in A) BHIS, or with supplementation with B) 0.15 μg/ml teicoplanin, or C) 1.0 μg/ml vancomycin. All cultures also contained 2 μg/ml thiamphenicol to maintain the plasmid and 0.5 μg/ml nisin to induce expression from PcprA. Graphs represent the mean OD₆₀₀ readings from three independent replicates (Figure S1) with error bars indicating the standard error of the mean. Data were analyzed by two-way ANOVA with Dunnett’s multiple comparison test, comparing to 630Δerm vector control at the same time point. * indicates adjusted P-value < 0.05, ** indicates ≤ 0.001.