PHASE VARIATION OF CLOSTRIDIUM DIFFICILE VIRULENCE FACTORS

Clostridium difficile is a Gram-positive spore-forming anaerobe and the leading cause of antibiotic-associated diarrheal disease in the United States. C. difficile produces two toxins, TcdA and TcdB, that are necessary for diarrheal disease symptoms. Colonization of the intestine is a necessary prerequisite to diarrheal disease symptoms. C. difficile produces flagella that aid not only in bacterial motility, but adherence to intestinal tissue. SigD, a flagellar alternative sigma factor in the early stage flagellar (flgB) operon, indirectly activates expression of the tcdA and tcdB genes. Both flagella and toxins are C. difficile virulence factors that synergistically promote diarrheal disease symptoms, pathology, and inflammation. Therefore, factors that regulate expression of the flgB operon affect not only motility, but toxin production and the virulence of C. difficile. The main objective of the research described in this dissertation was to identify and characterize a genetic mechanism controlling co-regulated flagellar and toxin gene expression in C. difficile. In Chapter 2, we identified a “flagellar switch” located upstream of the flgB operon, that mediates the phase variable production of flagella and toxins in C. difficile. Bacteria with the sequence in one orientation produced flagella, were motile and secreted the toxins ("flg ON"). Bacteria with the sequence in the inverse orientation were aflagellate and showed decreased toxin secretion ("flg OFF"). We determined that the tyrosine recombinase RecV is required for inversion of the flagellar switch in both directions. In Chapter 3, we found a single strain family, designated as “ribotype 012”, of C. difficile exhibits low frequency inversion of the flagellar switch in laboratory-adapted, environmental, and clinical isolates. In Chapter 4, we demonstrated that Rho factor is required for flagellar phase variation in C. difficile. We hypothesize that Rho factor directly terminates transcription in the leader RNA of the flgB operon in flg OFF bacteria. Future studies will assess the virulence contribution of flagellar and toxin phase variation to host infection dynamics, outcome, and transmission. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection.Doctor of Philosoph

Phase variation of Clostridium difficile virulence factors

Clostridium difficile is a leading cause of nosocomial infections, causing disease that ranges from mild diarrhea to potentially fatal colitis. A variety of surface proteins, including flagella, enable C. difficile colonization of the intestine. Once in the intestine, toxigenic C. difficile secretes two glucosylating toxins, TcdA and TcdB, which elicit inflammation and diarrheal disease symptoms. Regulation of colonization factors and TcdA and TcdB is an intense area of research in C. difficile biology. A recent publication from our group describes a novel regulatory mechanism that mediates the ON/OFF expression of co-regulated virulence factors of C. difficile, flagella and toxins. Herein, we review key findings from our work, present new data, and speculate the functional consequence of the ON/OFF expression of these virulence factors during host infection

Characterization of flagellum and toxin phase variation in Clostridioides difficile ribotype 012 isolates

Clostridioides difficile causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. C. difficile produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum expression and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a flagellar switch upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in C. difficile strain R20291. However, we were unable to detect flagellar switch inversion in C. difficile strain 630, a ribotype 012 strain commonly used in research labs, suggesting that the strain is phase locked. To determine whether a phase-locked flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple C. difficile ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase-off, nonmotile, and attenuated for toxin production. We isolated low-frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase-on derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among C. difficile ribotype 012 strains and perhaps other ribotypes, which could impact disease progression and diagnosis

Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the “flagellar switch” upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5’ leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile

The orientation of the flagellar switch impacts the expression of the downstream flagellar genes.

<p>(A) Asymmetric PCR-digestion assay performed on <i>C</i>. <i>difficile</i> R20291 isolates with the flagellar switch in the published and inverse orientations respectively. (B) qRT-PCR was used to determine the abundance of representative flagellar gene transcripts in isolates with the flagellar switch in the published and inverse orientation. Four independent isolates were tested, and <i>Ct</i> values for each flagellar gene and the <i>codY</i> gene (non-regulated control) were normalized to those of the housekeeping gene <i>rpoC</i>; the published orientation samples were arbitrarily chosen as the reference condition. Shown are the means and standard deviations. * <i>p</i> < 0.05 by t-tests comparing mean transcript abundances between published and inverse samples, n = 4. (C) Visualization of flagella by transmission electron microscopy at 25,000X magnification. Size bars = 1 micron. Representative images of bacterial flagellar switch isolates are shown. Arrowheads indicate flagella. (D) Micrographs of enriched <i>flg</i> ON, <i>flg</i> OFF, and a <i>sigD</i> mutant transformed with the pP_<i>flgM</i>::<i>mCherryOpt</i> reporter. Channels used are indicated for each column; the fourth column images are a merge of the DIC, DAPI, and RFP. RFP positive and negative bacteria were visually enumerated relative to the DIC and DAPI channels, and quantifications are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.s007" target="_blank">S4 Fig</a>. White bars = 10 microns.</p

Evidence for DNA inversion at the flagellar switch.

<p>(A) Nucleotide sequences corresponding to the 5’ UTR of the <i>flgB</i> operon from genome sequences available for PCR ribotype 027 strains were aligned using Clustal Omega. Shown are the regions corresponding to the putative flagellar switch and flanking imperfect inverted repeats. For strain BI1 “inverse”, the alignment was repeated after replacing the putative switch with its reverse complement. Identical nucleotides are indicated with blue shading. (B) Diagram of the PCR strategy used to detect the putative flagellar switch orientation. The primer names and sequences used for each strain are listed in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.s002" target="_blank">S2 Table</a>. The predicted product sizes are based on R20291 sequence. (C) Orientation-specific PCR products for the flagellar switch from three <i>C</i>. <i>difficile</i> strains representing three ribotypes (R20291, 027, NCBI Accession No FN545816.1; ATCC43598, 017, NCBI sequence read archive SRX656590 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.ref115" target="_blank">115</a>]; 630Δ<i>erm</i>, 012, NCBI Accession No. EMBL:LN614756 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.ref116" target="_blank">116</a>]).</p

A genetic switch controls the production of flagella and toxins in <i>Clostridium difficile</i>

<div><p>In the human intestinal pathogen <i>Clostridium difficile</i>, flagella promote adherence to intestinal epithelial cells. Flagellar gene expression also indirectly impacts production of the glucosylating toxins, which are essential to diarrheal disease development. Thus, factors that regulate the expression of the <i>flgB</i> operon will likely impact toxin production in addition to flagellar motility. Here, we report the identification a “flagellar switch” that controls the phase variable production of flagella and glucosylating toxins. The flagellar switch, located upstream of the <i>flgB</i> operon containing the early stage flagellar genes, is a 154 bp invertible sequence flanked by 21 bp inverted repeats. Bacteria with the sequence in one orientation expressed flagellum and toxin genes, produced flagella, and secreted the toxins (“<i>flg</i> phase ON”). Bacteria with the sequence in the inverse orientation were attenuated for flagellar and toxin gene expression, were aflagellate, and showed decreased toxin secretion (“<i>flg</i> phase OFF”). The orientation of the flagellar switch is reversible during growth <i>in vitro</i>. We provide evidence that gene regulation via the flagellar switch occurs post-transcription initiation and requires a <i>C</i>. <i>difficile</i>-specific regulatory factor to destabilize or degrade the early flagellar gene mRNA when the flagellar switch is in the OFF orientation. Lastly, through mutagenesis and characterization of flagellar phase locked isolates, we determined that the tyrosine recombinase RecV, which catalyzes inversion at the <i>cwpV</i> switch, is also responsible for inversion at the flagellar switch in both directions. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection.</p></div

Identification of the recombinase that mediates inversion of the flagellar switch.

<p>(A,B) Orientation-specific PCR (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.g001" target="_blank">Fig 1B</a>) assays to identify the conserved recombinase that catalyzes inversion at the flagellar switch from ON to OFF (A) and OFF to ON (B). The gene name or R20291 locus tag numbers for the eight conserved recombinases are shown. A 375 bp product indicates the ON orientation; a 281 bp product indicates the OFF orientation. (C) Orientation-specific PCR for the flagellar switch to determine whether the <i>recV cwpV</i> locked ON and OFF mutants were locked for the flagellar switch. (D) Orientation-specific PCR for the flagellar switch in complemented <i>recV</i> mutants (pP_<i>tet</i>::<i>recV</i>) and controls.</p

The orientation of the flagellar switch impacts toxin production.

<p>(A) qRT-PCR was used to determine the abundance of the indicated transcripts in <i>flg</i> ON and OFF isolates of <i>C</i>. <i>difficile</i> R20291. Four independent isolates were tested, and C<i>t</i> values for each gene were normalized to those of the housekeeping gene <i>rpoC</i>; the <i>flg</i> ON samples were arbitrarily chosen as the reference condition. Shown are means and standard deviations. * <i>p</i> < 0.05 by t-tests comparing mean transcript abundances between <i>flg</i> ON and OFF samples, n = 4. (B) TcdA protein levels in cell lysates of <i>flg</i> ON and OFF isolates were evaluated by western blot. Shown is a representative image for three independent experiments each with at least three replicates of each <i>flg</i> phase. (C) The <i>flg</i> ON and OFF isolates, as well as the <i>sigD</i> mutant control, were grown to stationary phase in TY medium. The supernatants were serially diluted and applied to Vero cells for 24 hours. Cell viability was assessed using the CellTiter Glo assay. Data are combined from two independent experiments each with four replicates of <i>sigD</i> mutant and <i>flg</i> phase variants, and means and standard deviations are shown. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by one-way ANOVA comparing the means for each dilution.</p

The orientation of the flagellar switch controls flagellar gene expression post-transcription initiation.

<p>(A) Diagram of the reporter gene fusions (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.s003" target="_blank">S1 Methods</a>). The known <i>flgB</i> operon promoter, the Cd1 c-di-GMP riboswitch, and the orientation of the flagellar switch are indicated, if present. (B,C) The fusions in A were integrated into the <i>C</i>. <i>difficile</i> R20291 chromosome (left) or the <i>B</i>. <i>subtilis</i> BS49 chromosome via Tn916 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.ref112" target="_blank">112</a>]. Alkaline phosphatase (AP) activity was measured as described previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006701#pgen.1006701.ref077" target="_blank">77</a>]. Means and standard deviations are shown. *** <i>p</i> < 0.001 by one-way ANOVA and Bonferroni’s multiple comparisons test, n = 8. n.s. = not significant. (D) Northern blot detection of the <i>phoZ</i>-containing transcripts from <i>C</i>. <i>difficile</i> R20291 bearing fusions 3 or 4. The full length (FL) transcript of ~2400 nt is indicated. 5S RNA served as the loading control. The image is representative of three biological replicates for each strain.</p