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. 2011 Jun 1;203(11):1613-20.
doi: 10.1093/infdis/jir112.

Clostridium difficile transcriptome analysis using pig ligated loop model reveals modulation of pathways not modulated in vitro

Joy Scaria  1 , Tavan Janvilisri, Susan Fubini, Robin D Gleed, Sean P McDonough, Yung-Fu Chang
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Clostridium difficile transcriptome analysis using pig ligated loop model reveals modulation of pathways not modulated in vitro (V体育官网)

Joy Scaria et al. J Infect Dis. .

Abstract

A pig ligated loop model was used to analyze the in vivo transcriptome response of Clostridium difficile. Bacterial RNA from the loops was retrieved at different times and was used for microarray analysis. Several virulence-associated genes and genes involved in sporulation cascade were differentially expressed (DE). In concordance with observed upregulation of toxin genes in microarray, enzyme-linked immunosorbent assay estimation of total toxin showed high amounts of toxin in the loops. Several genes that were absent in primary annotation of C. difficile 630 but annotated in a secondary annotation were found to be DE. Pathway comparison of DE genes in vitro and in vivo showed that when several pathways were expressed in all conditions, several of the C. difficile pathways were uniquely expressed only in vivo. The pathways observed to be modulated only in this study could be targets of new therapeutic agents against C VSports手机版. difficile infection. .

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Figures

Figure 1.
Figure 1.
Projection of differentially expressed genes at various time points on C. difficile 630 genome. At various time points, those genes showing at least 1.75-fold change at 2% significance were selected. From outside to inside: ring 1, molecular clock of C. difficile genome; rings 2 and 3, cluster of orthologous (COG) gene categories; ring 4, DE genes at 12 h after injection; ring 5, DE genes at 8 h after injection; ring 6, DE genes at 4 h after injection. In rings 4–6, red color indicates up-regulation and green color indicates down-regulation of genes.
Figure 2.
Figure 2.
Comparison of DE genes and toxin levels at different time points. A, From list of significantly expressing genes obtained by ANOVA at 2% significance level, total number of genes showing at least 1.75-fold change at different time points are compared as a Venn diagram. B, Fold change pattern of JCVI unique genes that were found to be modulated at all time points. C, Total toxin A/B levels in the loops at different time points was estimated using ELISA.
Figure 3.
Figure 3.
Comparison of C. difficile pathways modulated in vitro and in vivo: DE genes in vitro and in vivo were mapped to pathways using pathway tools software, and significantly modulated pathways in each condition were compared. Pathways are color coded as follows: blue, pathways predicted in C. difficile 630 but not found expressing in any of the conditions compared; red, pathways modulated in vitro; orange, pathways modulated during Caco2 cell infection; green, pathways modulated at 4 h after injection in loops; pink, pathways modulated at 4 h after injection in loops; brown, pathways modulated at 12 h after injection in loops.
Figure 4.
Figure 4.
Validation of microarray data by qRT-PCR. Gene expression changes in ligated loop compared with control, measured by microarray analysis or qRT-PCR. Data are plotted as fold-change differences of microarray data compared with those of qRT-PCR.
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References (VSports手机版)

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