Systems analysis of the transcriptional response of human ileocecal epithelial cells to Clostridium difficile toxins and effects on cell cycle control.
BACKGROUND
Toxins A and B (TcdA and TcdB) are Clostridium difficile's principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure.


RESULTS
We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis.


CONCLUSIONS
This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.
Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908, USA.
Kevin M
KM
D'Auria
Gina M
GM
Donato
Mary C
MC
Gray
Glynis L
GL
Kolling
Cirle A
CA
Warren
Lauren M
LM
Cave
Michael D
MD
Solga
Joanne A
JA
Lannigan
Jason A
JA
Papin
Erik L
EL
Hewlett
D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D002432:Cecum / Q000166:cytology*; D002453:Cell Cycle / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004768:Enterotoxins / Q000633:toxicity*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D007082:Ileum / Q000166:cytology*; D046228:Microarray Analysis; D049490:Systems Biology / Q000379:methods; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile