Deciphering Adaptation Strategies of the Epidemic <i>Clostridium difficile</i> 027 Strain during Infection through <i>In Vivo</i> Transcriptional Analysis

<div><p><i>Clostridium difficile</i> is responsible for a wide spectrum of infection from asymptomatic carriage to severe, relapsing colitis. Since 2003, <i>C</i>. <i>difficile</i> infections have increased with a higher morbidity and mortality due to the emergence of epidemic and hypervirulent <i>C</i>. <i>difficile</i> strains such as those of the epidemic lineage 027/BI/NAP1. To decipher the hypervirulence and epidemicity of 027 strains, we analyzed gene expression profiles of the R20291 027 strain using a monoxenic mouse model during the first 38h of infection. A total of 741 genes were differentially expressed during the course of infection. They are mainly distributed in functional categories involved in host adaptation. Several genes of PTS and ABC transporters were significantly regulated during the infection, underlying the ability of strain R20291 to adapt its metabolism according to nutrient availability in the digestive tract. In this animal model, despite the early sporulation process, sporulation efficiency seems to indicate that growth of R20291 vegetative cells versus spores were favored during infection. The bacterial mechanisms associated to adaptability and flexibility within the gut environment, in addition to the virulence factor expression and antibiotic resistance, should contribute to the epidemicity and hypervirulence of the <i>C</i>. <i>difficile</i> 027 strains.</p></div

Kinetics of sporulation rate in <i>C</i>. <i>difficile</i> associated mice.

<p>Mice were orally challenged with 1x10⁸ CFUs of vegetative cells. Vegetative cells were enumerated on BHI agar plates and spores after a heat shock treatment on BHI containing 0.1% of taurocholate sodium salt. </p

Regulation of sporulation and germination genes during the kinetics of infection.

<p>Regulation of sporulation and germination genes during the kinetics of infection.</p

Validation of microarray data by qRT-PCR.

<p>Fold changes in in vivo gene expression at 4, 6, 14 and 38h post-infection, compared to the in vivo expression at 8h post-infection, were measured by microarray and qRT-PCR. Data are plotted as log₂ ratios of microarrays data (<i>x</i>-axis) compared with those of qRT-PCR (<i>y</i>-axis).</p

Regulation of toxin genes during the kinetics of infection.

<p>Regulation of toxin genes during the kinetics of infection.</p

Gel mobility retardation of <i>tcdR</i> promoters with <i>E. coli</i> RNA polymerase core enzyme and SigD.

<p>A DNA fragment containing the <i>C. difficile </i><i>tcdR</i> promoter region (P<i>tcdR</i>) was incubated with SigD, <i>E. coli</i> SigA RNA polymerase (200nM) or <i>E. coli</i> RNA polymerase core enzyme alone (200nM) or after pre-incubation with SigD protein. Increasing concentrations of RNA polymerase containing SigD are indicated in the figure (from 50 to 200nM).</p

Effect of <i>flgM</i> overexpression on motility, flagellar and toxin genes expression.

<p>A: Quantitative RT-PCR analysis of <i>flgM, sigD</i>, <i>fliC</i>, <i>tcdR</i>, <i>tcdA</i> and <i>tcdB</i> expression was performed in <i>C. difficile</i> 630∆<i>erm</i>+pMTL007 and <i>C. difficile</i> 630Δ<i>erm</i> + pMTL::P<i>CD2767</i>-<i>flgM</i>, grown in BHI medium. Results are expressed as relative expression normalized by the 16S rRNA housekeeping gene. Error bars correspond to standard deviation from at least 3 biological replicates. B: Western blot analysis of FlgM, TcdA, FliC and SigD proteins from crude extracts of <i>C. difficile</i> 630∆<i>erm</i> + pMTL007 and <i>C. difficile</i> 630Δ<i>erm</i> + pMTL::P<i>CD2767</i>-<i>flgM</i>. SigA antibodies were used as an internal control. The results are representative from at least three biological replicates. C: Motility assay in agar soft tubes (0.175%) showing the loss of motility following <i>flgM</i> overexpression. </p

Effect of <i>sigD</i> inactivation on toxins expression during stationary phase.

<p>A: Quantitative RT-PCR analysis of <i>tcdA</i>, <i>tcdB</i> and <i>tcdR</i> expression in strains 1: 630<i>∆erm</i> + pRPF185, 2: <i>sigD</i>::<i>erm</i>+ pRPF185, 3: <i>sigD</i>::<i>erm</i> + pRPF-<i>sigD</i> and 4: <i>sigD</i>::<i>erm</i> + pRPF-<i>sigD</i> to <i>CD0272</i> grown in TY medium. Results are expressed as relative expression normalized by the 16S rRNA housekeeping gene. Error bars correspond to standard deviation from 3 biological replicates. B: Western blot analysis of TcdA from crude proteins extracts of <i>C. difficile</i> 630<i>∆erm</i> + pRPF185, <i>sigD</i>::<i>erm</i>+ pRPF185, <i>sigD</i>::<i>erm</i> + pRPF-<i>sigD</i> and <i>sigD</i>::<i>erm</i> + pRPF-<i>sigD</i> to <i>CD0272</i> strains grown in TY medium. SigA antibodies were used as an internal control. C: TcdA and TcdB expression levels in supernatants of <i>C. difficile</i> 630<i>∆erm</i> + pRPF185, <i>sigD</i>::<i>erm</i>+ pRPF185, <i>sigD</i>::<i>erm</i> + pRPF-<i>sigD</i> and <i>sigD</i>::<i>erm</i> + pRPF-<i>sigD</i> to <i>CD0272</i> Strains were quantified using ELISA test after 10 and 24 hours growth in TY medium. Error bars correspond to standard deviation from at least three biological replicates.</p

<i>sigD</i>, <i>flgM</i> and <i>fliC</i> transcriptional expression and protein level during growth in BHI medium.

<p>A: Quantitative RT-PCR analysis of <i>sigD</i>, <i>flgM</i> and <i>fliC</i> expression. Results are expressed as relative expression of <i>sigD</i>, <i>flgM</i> and <i>fliC</i> normalized by the 16S rRNA housekeeping gene. Error bars correspond to standard deviation from three biological replicates. B: Western blot analysis of SigD, FlgM and FliC protein levels. SigA antibodies were used as an internal control. The results are representative from at least three biological replicates.</p

Identification of the SigD-dependent promoter sequence by RACE-PCR.

<p>SigD-dependent transcription start sites upstream of start codons of genes involved in motility (<i>CD0226</i>, <i>flgM, fliC</i>), membrane transport (CD3527) and virulence (tcdR). The transcriptional start sites are indicated in bold and underlined. The -35 and -10 boxes corresponding to SigD-dependent promoters are indicated in bold.</p