Defining the Roles of TcdA and TcdB in Localized Gastrointestinal Disease, Systemic Organ Damage, and the Host Response during Clostridium difficile Infections

Abstract

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, a significant animal pathogen, and a worldwide public health burden. Most disease-causing strains secrete two exotoxins, TcdA and TcdB, which are considered to be the primary virulence factors. Understanding the role that these toxins play in disease is essential for the rational design of urgently needed new therapeutics. However, their relative contributions to disease remain contentious. Using three different animal models, we show that TcdA(+) TcdB(-) mutants are attenuated in virulence in comparison to the wild-type (TcdA(+) TcdB(+)) strain, whereas TcdA(-) TcdB(+) mutants are fully virulent. We also show for the first time that TcdB alone is associated with both severe localized intestinal damage and systemic organ damage, suggesting that this toxin might be responsible for the onset of multiple organ dysfunction syndrome (MODS), a poorly characterized but often fatal complication of C. difficile infection (CDI). Finally, we show that TcdB is the primary factor responsible for inducing the in vivo host innate immune and inflammatory responses. Surprisingly, the animal infection model used was found to profoundly influence disease outcomes, a finding which has important ramifications for the validation of new therapeutics and future disease pathogenesis studies. Overall, our results show unequivocally that TcdB is the major virulence factor of C. difficile and provide new insights into the host response to C. difficile during infection. The results also highlight the critical nature of using appropriate and, when possible, multiple animal infection models when studying bacterial virulence mechanisms.

Importance: Clostridium difficile is a leading cause of antibiotic-associated diarrhea and an important hospital pathogen. TcdA and TcdB are thought to be the primary virulence factors responsible for disease symptoms of C. difficile infections (CDI). However, the individual contributions of these toxins to disease remain contentious. Using three different animal models of infection, we show for the first time that TcdB alone causes severe damage to the gut, as well as systemic organ damage, suggesting that this toxin might be responsible for MODS, a serious but poorly understood complication of CDI. These findings provide important new insights into the host response to C. difficile during infection and should guide the rational development of urgently required nonantibiotic therapeutics for the treatment of CDI.

FIG 1

Toxin B is the primary mediator of fulminant C. difficile disease. (A to D) Monash mice were infected with C. difficile strains. Results for the strains are indicated by different colors as follows: black, wild-type strain M7404 [TcdA⁺B⁺ (WT)] (n = 19); light blue, tcdB mutant 1 (TcdA⁺B⁻1) (n = 14); dark blue, tcdB mutant 2 (TcdA⁺B⁻2) (n = 10); pink, tcdA mutant 1 (TcdA⁻B⁺1) (n = 13); purple, tcdA mutant 2 (TcdA⁻B⁺2) (n = 10); green, tcdA tcdB double mutant (TcdA⁻B⁻) (n = 15); light orange, cdtA mutant 1 (CDT⁻1) (n = 10); dark orange, cdtA mutant 2 (CDT⁻2) (n = 10); and gray, mock-infected control (mock) (n = 5). Results are shown for weight loss (A), survival (B), activity and appearance (C), and colon length (D) of infected Monash mice. (E) Survival of Sanger mice infected with C. difficile strains indicated by different colors as follows: black, wild-type TcdA⁺ TcdB⁺ strain [TcdA⁺B⁺ (WT)] (n = 16); light blue, TcdA⁺ TcdB⁻ mutant 1 (TcdA⁺B⁻1) (n = 16); pink, TcdA⁻ TcdB⁺ mutant 1 (TcdA⁻B⁺1) (n = 13); light orange, cdtA mutant 1 (CDT⁻1) (n = 15); and gray, mock infected (n = 15). (F) Days from infection to death of Hines hamsters infected with C. difficile strains indicated by different colors as follows: black, wild-type TcdA⁺ TcdB⁺ strain [TcdA⁺B⁺ (WT)] (n = 8); light blue, TcdA⁺ TcdB⁻ mutant 1 (TcdA⁺B⁻1) (n = 9); pink, TcdA⁻ TcdB⁺ mutant 1 (TcdA⁻B⁺1) (n = 9); and light orange, cdtA mutant 1 (CDT⁻1) (n = 10). Data represent the mean results ± standard deviations (SD).

FIG 2

Toxin B causes severe local histopathological damage. Histopathological analyses of tissues collected from mice infected with the wild-type strain M7404 [TcdA⁺B⁺ (WT)], tcdA mutant 1 (TcdA⁻B⁺), tcdB mutant 1 (TcdA⁺B⁻), tcdA tcdB double mutant (TcdA⁻B⁻), or cdtA mutant 1 (CDT⁻1) or mock infected with PBS were performed. (A) Representative images of hematoxylin-and-eosin-stained tissues from Monash mice on day 2 postinfection and from Sanger mice on day 4 postinfection are shown. Scale bars are shown (200 µm). (B) Histopathological scoring of damage to tissues from Monash mice infected with the wild-type and mutant C. difficile strains. Scores are shown for tissues from mice infected with the TcdA⁺ TcdB⁺ (WT) strain (n = 12), TcdA⁺ TcdB⁻ mutant 1 (n = 6), TcdA⁺ TcdB⁻ mutant 2 (n = 5), TcdA⁻ TcdB⁺ mutant 1 (n = 6), TcdA⁻ TcdB⁺ mutant 2 (n = 6), TcdA⁻ TcdB⁻ double mutant (n = 6), CDT⁻ mutant 1 (n = 5), and CDT⁻ mutant 2 (n = 5) and from mock-infected mice (n = 6). All values are mean results ± SD. (C) Histopathological scoring of damage to tissues in Sanger mice infected with wild-type and mutant C. difficile strains. Scores are shown for tissues from mice infected with the TcdA⁺ TcdB⁺ (WT) strain (n = 12), TcdA⁺ TcdB⁻ mutant 1 (n = 12), TcdA⁻ TcdB⁺ mutant 1 (n = 8), and CDT⁻ mutant 1 (n = 12) and from mock-infected mice (n = 12). All values are mean results ± SD.

FIG 3

C. difficile infection with TcdB-producing strains is associated with multiorgan damage. (A) Organs were collected from Monash mice and assessed for damage compared to the state of control tissues, and the data collated. (B) Representative images of organ tissues collected from Monash CDI mice infected with wild-type strain M7404 [TcdA⁺B⁺ (WT)], tcdA mutant 1 (TcdA⁻B⁺), tcdB mutant 1 (TcdA⁺B⁻), or tcdA tcdB double mutant (TcdA⁻B⁻) or mock infected with PBS. Note the loss of structure in medullary and cortical regions in the thymus (M, medulla, C, cortex) and in the germinal centers (Gc) of the sagittal lymph nodes (SG Lns). Scale bars are shown (thymus, 500 µm; spleen, 200 µm; SG Lns and kidney, 100 µm).

FIG 4

Transcriptomic analysis of the host response during C. difficile infection. Heat maps of the transcriptomes of colonic tissues collected from mice following infection with the wild-type strain M7404 [TcdA⁺B⁺ (WT)], tcdB mutant 1 (TcdA⁺B⁻), or tcdA mutant 1 (TcdA⁻B⁺) or mock infection with PBS using the Monash mouse model (A) or the Sanger mouse model (B) of CDI. Scaled expression values within the heat maps are color coded according to the color range shown, with red being the lowest and green being the highest level of transcription. The dendrogram shown above each heat map depicts hierarchical clustering of the transcriptomic response of each mouse following infection and was derived using a Pearson hierarchical-clustering algorithm. The infecting strains are indicated underneath the individual heat maps.