Abstract
Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.
Highlights
Clostridioides difficile, a spore-forming bacterial pathogen, is the primary cause of antibiotic-associated diarrhea, an infection usually acquired during stays in healthcare facilities
We tested the virulence of strain LB-CD16, a codY null mutant [44] of strain UK1, a ribotype 027 isolate from the Stoke-Mandeville outbreak of 2003 [45]. (The mutation was created by insertion within codY of an intron containing an erythromycin-resistance determinant and is noted as codY::intron::erm.) In a mouse model of infection in which mice are fed a cocktail of antibiotics before infection [46], strain UK1 and the codY null mutant (LB-CD16) induced diarrhea and weight loss in all mice infected at a dose of 105 spores per mouse (Fig 1A and Table 1); 10% of the mice died after infection by UK1, 20% after infection by the codY null mutant
The results obtained with strains expressing mutant forms of CodY with different levels of residual activity imply that some metabolism genes are considerably derepressed when CodY activity is only slightly reduced, some genes are only significantly derepressed when CodY activity is more reduced and other genes are only derepressed when CodY activity is eliminated
Summary
Clostridioides difficile, a spore-forming bacterial pathogen, is the primary cause of antibiotic-associated diarrhea, an infection usually acquired during stays in healthcare facilities. Spore formation appears to be the primary factor responsible for the unusual frequency of recurrent C. difficile infection; the spores released from the GI tract are difficult to kill, survive in the environment and are able to cause a new round of infection in patients who have completed their antibiotic treatment. To determine the number of codY-containing Tn916 insertions per C. difficile genome, chromosomal DNA was extracted as previously described (72). The correlation coefficient (R2) for the line was 0.99 or greater This plot was used as a standard curve for extrapolating the relative concentration levels of the codY gene in strain LB-CD16 carrying Tn916 embedded with various versions of the codY gene. Strain ND-CD13 was later found to have two copies of the codY gene (S2 Table)
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