Characterization of Flagellum and Toxin Phase Variation in Clostridioides difficile Ribotype 012 Isolates

Abstract

causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum expression and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a flagellar switch upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in strain R20291. However, we were unable to detect flagellar switch inversion in strain 630, a ribotype 012 strain commonly used in research labs, suggesting that the strain is phase locked. To determine whether a phase-locked flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase-off, nonmotile, and attenuated for toxin production. We isolated low-frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase-on derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among ribotype 012 strains and perhaps other ribotypes, which could impact disease progression and diagnosis. produces flagella that enhance bacterial motility and secretes toxins that promote diarrheal disease symptoms. Previously, we found that production of flagella and toxins is coregulated via a flippable DNA element termed the flagellar switch, which mediates the phase-variable production of these factors. Here, we evaluate multiple isolates of ribotype 012 strains and find them to be primarily flagellar phase off (-off state). Some, but not all, of these isolates showed the ability to switch between -on and -off states. These findings suggest heterogeneity in the ability of ribotype 012 strains to phase-vary flagellum and toxin production, which may broadly apply to pathogenic .

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile

“…In 630∆erm, which has a much higher percentage of TcdA‐ON cells because flgB is locked in the ON orientation (Fig. ; (Anjuwon‐Foster et al, )), the corresponding numbers were 83% and 84% TcdA‐ON for cells lacking or containing spores respectively (Table ). We also observed a small number of free spores that were red fluorescent.…”

“…). As will be explained in more detail below, this difference likely reflects much higher levels of σ D production in CD630Δerm (Anjuwon‐Foster and Tamayo, ; Anjuwon‐Foster et al, ). σ D increases toxin production by activating tcdR transcription (McKee et al, ; Anjuwon‐Foster and Tamayo, ).…”

The impact of pH onClostridioides difficilesporulation and physiology

“…Because toxin expression and motility are both regulated by SigD, the flagellar alternative sigma factor (40–42), we next investigated the impact of pH on C. difficile motility. To test this, we analyzed the motility of C. difficile on soft agar plates at a range of pH conditions between 6.2 and 8.2.…”

“…Differences in phenotypes for cells grown on solid and liquid medium have been observed for B. cereus , with plate-grown cells displaying increased gamma radiation resistance and a more developed S-layer compared to cells grown in liquid (70). Other studies of C. difficile found substantial differences in gene expression profiles between biofilms grown in broth and grown on plates (71), as well as differences in the expression of phase variation genes and the orientation of invertible elements (42, 72). We anticipate that differences in gene expression for liquid and solid medium affected the survival and adaptation of C. difficile after an acid shock; however, this was not explored in this study.…”

“…As strain-dependent phenotypic differences are often described in C. difficile (34, 36, 37, 42, 45–47), we investigated the C. difficile pH response using the historical isolate 630∆ erm , which is commonly studied in laboratories, and the epidemic-associated strain R20291. We found that during growth in liquid culture, no strain-dependent differences were observed under different pH conditions (Fig.…”