expression of clostridium difﬁcile toxins a and b and ... · proteins induced at 37°c and thus...

INFECTION AND IMMUNITY, Apr. 2003, p. 1784–1793 Vol. 71, No. 40019-9567/03/$08.00�0 DOI: 10.1128/IAI.71.4.1784–1793.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Expression of Clostridium difficile Toxins A and B and Their SigmaFactor TcdD Is Controlled by Temperature

Sture Karlsson,1,2 Bruno Dupuy,3 Kakoli Mukherjee,4 Elisabeth Norin,2 Lars G. Burman,1and Thomas Akerlund1*

Department of Molecular Epidemiology and Biotechnology, Swedish Institute for Infectious Disease Control, S-17182Solna,1 and Microbiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm,2 Sweden; Unite de

Genetique Moleculaire Bacterienne, Institut Pasteur, 75724 Paris Cedex 15, France3; and AstraZeneca ResearchFoundation India, Malleswaram, 560003 Bangalore, India4

Received 26 August 2002/Returned for modification 30 October 2002/Accepted 30 December 2002

Growth temperature was found to control the expression of toxins A and B in Clostridium difficile VPI 10463,with a maximum at 37°C and low levels at 22 and 42°C in both peptone yeast (PY) and defined media. Theup-regulation of toxin A and B mRNA and protein levels upon temperature upshift from 22 to 37°C followedthe same kinetics, showing that temperature control occurred at the level of transcription. Experiments withClostridium perfringens using gusA as a reporter gene demonstrated that both toxin gene promoters weretemperature controlled and that their high activity at 37°C was dependent on the alternative sigma factorTcdD. Furthermore, tcdD was found to be autoinduced at 37°C. Glucose down-regulated all these responses inthe C. perfringens constructs, similar to its impact on toxin production in C. difficile PY broth cultures. C. difficileproteins induced at 37°C and thus coregulated with the toxins by temperature were demonstrated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as enzymes involved inbutyric acid production and as electron carriers in oxidation-reduction reactions. The regulation of toxinproduction in C. difficile by temperature is a novel finding apparently reflecting an adaptation of the expressionof its virulence to mammalian hosts.

Clostridium difficile is a gram-positive, spore-forming anaer-obic bacterium that is associated with diarrhea and colitis inhumans (26, 39). A reduction of the microbial flora in thecolon, usually caused by antibiotic therapy, allows overgrowthof C. difficile, with symptoms ranging from mild self-limitingdiarrhea to life-threatening pseudomembranous colitis. Clini-cally significant strains have a pathogenicity locus (PaLoc) in-tegrated into the chromosome (6, 12, 39). In most toxin-pro-ducing strains, the PaLoc includes both the enterotoxin (toxinA) and the cytotoxin (toxin B) genes, as well as three addi-tional open reading frames (ORFs), tcdD, tcdE, and tcdC (16).There is polymorphism in the toxin A and B genes amongclinical isolates of C. difficile (42), and virulent strains produc-ing only one of the toxins have been isolated (2, 8). The C.difficile toxins A and B belong to a class of proteins called largeclostridial cytotoxins that share the same general structure andenzymatic function (46). Thus, both toxins are monoglucosyltransferases that modify small regulatory GTP-binding pro-teins of the Rho family in the eucaryotic cell (18, 19). Theglucosylated target proteins are rendered inactive, resulting ina collapse of the actin cytoskeleton and death of colonic en-terocytes. In addition to the direct cytotoxic effect, toxin A alsoaffects neurons in the enteric nerve system to release substanceP (7). Substance P is then thought to activate mast cells, re-sulting in the recruitment of neutrophils and increased intes-tinal secretion, contributing to mucosal inflammation (47).

The functions of the three additional PaLoc ORFs (tcdD, tcdE,and tcdC) are less well studied. One breakthrough in the under-standing of toxin regulation came when tcdD was demonstratedto up-regulate toxin synthesis (35), and another occurred whenit was shown in a heterologous Clostridium perfringens reportergene system that tcdD encodes an alternative sigma factorinvolved in transcription of the toxin genes (28). The tcdD-dependent toxin expression was recently also confirmed in C.difficile (29), supporting the notion that C. perfringens is a goodmodel organism for studying C. difficile toxin regulation. Thefunctions of tcdE and tcdC remain poorly understood. TcdEhas similarities to holins, cytolytic proteins encoded by certainbacteriophages (45). Expression of tcdC is highest during ex-ponential growth (low toxin expression) and has been sug-gested to act as a negative regulator of the toxin genes (16).

The toxin yield in C. difficile is dependent on the nutrientlevels in the growth medium, and much data indicates thattoxin synthesis is turned on as a response to a shortage ofsugars and certain amino acids (9, 13, 21, 22, 23, 24, 37, 48, 50).In addition, growth-limiting levels of the vitamin biotin leads tohigh toxin production (49). In complex media, but not in de-fined media, the presence of rapidly metabolizable carbonsources lowers the toxin yields (9, 23, 37). Among amino acids,cysteine is particularly potent in down-regulating toxin synthe-sis (23, 24). Cysteine concomitantly down-regulates other pro-teins, including enzymes involved in the formation of butyricacid and butanol, and these metabolic end products affect toxinyields when added to C. difficile cultures (23).

A transition from ambient temperature to the range 36 to38°C, i.e., the body temperature of mammals, correlates with adramatic up-regulation of the expression of virulence determi-

* Corresponding author. Mailing address: Swedish Institute for In-fectious Disease Control, Department of Molecular Epidemiology andBiotechnology, S-171 82 Solna, Sweden. Phone: 46 8 4572467. Fax: 468 302566. E-mail: [email protected].

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nants in many pathogens (25, 33). In this paper, we show that37°C is the optimum temperature for toxin synthesis by C.difficile VPI 10463. Furthermore, the temperature control oftoxins A and B was found to occur at the level of transcriptionand to depend on the alternative sigma factor TcdD. The newfinding that the toxins are regulated by temperature adds to thecomplexity of their control and suggests a host-specific adap-tation of virulence expression in C. difficile.

MATERIALS AND METHODS

C. difficile strains and growth media. C. difficile VPI 10463 and the serogrouptype strains A (CCUG 37779), C (CCUG 37766), G (CCUG 37783), and H(CCUG 37784) were from the Culture Collection, University of Goteborg, Gote-borg, Sweden. C. difficile strains were grown in peptone yeast (PY) broth or thedefined medium SDM as described previously (23). Viable counts of C. difficilecultures were performed on blood agar.

Protein sampling, measurements of toxin, and short-chain fatty acid produc-tion. One-milliliter samples of C. difficile cultures were collected and eitherstored immediately at �20°C or separated into a cellular pellet and supernatantby centrifugation at 4,000 � g for 5 min before storage at �20°C. Sonication,protein measurements, short-chain fatty acid assays by gas-liquid chromatogra-phy, and determination of toxins (A and B) by enzyme immunoassay wereperformed as described previously (23).

Toxin stability assay. Cell culture supernatant containing toxin (10,000 U/ml)was obtained from a 48-h VPI 10463 culture grown at 37°C in PY broth withoutcysteine. VPI 10463 was also grown at 22, 37, and 42°C for 48 h in PY broth, and2.5-ml aliquots of the cultures were sonicated twice for 60 s each time in orderto stop growth and release intracellular components. The cell sonicates and thetoxin preparations were mixed 1:2 and incubated anaerobically at the respectivetemperatures (22, 37, and 42°C), and the toxin levels in the mixtures weredetermined at time zero and after 24 h. Each experiment was performed intriplicate.

Western blots of toxins A and B. Cellular proteins (1.5 �g) of C. difficile wereseparated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamidegels (7.5%). The proteins were transferred to polyvinylidene difluoride mem-branes (Millipore) using the Pharmacia Novablot transfer equipment and acontinuous buffer system (39 mM glycine, 48 mM Tris, 0.0375% [wt/vol] SDS,20% [vol/vol] methanol) according to the Multiphor II manual. The membraneswere dried and blocked overnight with 0.5% (vol/vol) Tween 20 at 4°C. Theblotted membranes were incubated for 1 h at room temperature with 0.2 �g ofantibodies against toxin A (PCG-4; r-Biopharm) or toxin B (2CV; r-Biop-harm)/ml in TST buffer (0.05 M Tris, 0.5 M NaCl, 0.1% Tween 20, pH 9). Afterthree washes in TST, the membranes were incubated with horseradish peroxi-dase-conjugated anti-mouse antibodies (DAKOPATTS; diluted 1:15,000 in TST)for 1 h and finally washed three times in TST. A chemiluminiscent signal (ECL Plus;Amersham) was used to detect the bands. The relative amounts of toxins weremeasured on scanned X-ray films using Molecular Analyst software (Bio-Rad).

Cloning of toxin A and toxin B gene fragments. Chromosomal DNA from C.difficile was purified using a DNeasy tissue kit (Qiagen). Using the primer pairs5�-TGCTTCCAGGTATTCACTCTGA-3� plus 5�-ACACTGCCCTAAAGCGAAAGC-3� and 5�-TGCATTTTTGATAAACACATTGAA-3� plus 5�-GCAGCAGCTAAATTCCACCT-3�, respectively, gene fragments for toxins A (280 bp)and B (402 bp) were PCR amplified with Ampli-taq Gold (Perkin-Elmer) as aDNA polymerase. The fragments were subsequently cloned into the vectorPCR-Script Amp (Stratagene), creating the plasmids SK-A3 and SK-B3. Theintegrity of the cloned inserts was analyzed by DNA sequencing. The Escherichiacoli strain Epicuran Coli XL1-Blue (Stratagene) was used as the recipient for theplasmids. Cells were grown at 37°C in Luria-Bertani medium supplemented with50 �g of ampicillin/ml. SK-A3 and SK-B3 where then purified and used togenerate radiolabeled specific antisense RNA probes (see below) for the assay oftoxin A and toxin B mRNA levels. DNA restriction, ligation, agarose gel elec-trophoresis, and electroporation were carried out as described previously (30).

Synthesis of antisense RNA probes. In vitro transcription of the cloned toxinA and B fragments was generated using the MAXI script kit (Ambion) accordingto the recommendations provided by the manufacturer. Briefly, 1 �g of linear-ized plasmid (SK-A3 or SK-B3) was incubated for 2 h at room temperature in thepresence of T3 RNA polymerase and nucleotides. [�-32P]UTP (3 �M; Amer-sham Pharmacia Biotech) was used as the labeling nucleotide. The DNA tem-plate was degraded by incubating it for 15 min at 37°C in the presence of 2 U ofDNase I. The reaction was stopped by adding 1 �l of 0.5 M EDTA. The RNA

was denatured for 3 min at 80°C and separated on a 5% polyacrylamide–8 Murea gel. The full-length transcripts were gel purified, and the specific radioac-tivity of the eluted probes was determined using the Liquid Scintillation SystemLS 1801 (Beckman).

Preparation of C. difficile RNA. C. difficile was harvested by adding 10%(vol/vol) of a mixture of 95% ethanol–5% phenol to the cultures and snap-frozenin liquid nitrogen. Samples were stored at �70°C and thawed on ice. The cellswere pelleted by centrifugation for 5 min at 4,000 � g and 4°C, and RNA wasextracted with a Fast Prep 120 incubator (Bio 101) using a FastRNA kit, blue(Bio 101), as recommended by the manufacturer. The RNA concentration wasdetermined by spectrophotometry, and RNA integrity was determined by anal-ysis of the 16S/23S rRNA by gel electrophoresis.

RPA. RNase protection assays (RPA) were performed using the RPA III kit(Ambion) according to the manufacturer’s recommendations. Briefly, aliquots oftoxin A- and B-specific antisense RNA probes (5 � 104 cpm) were coprecipitatedwith 15 �g of C. difficile RNA and dissolved in 10 �l of hybridization buffer. Thesamples were heated for 3 min at 90°C and incubated at 42°C for at least 6 h andthen digested with RNase T1. Protected fragments were precipitated, denaturedfor 3 min at 90°C, and separated on a 5% polyacrylamide–8 M urea gel. The gelswere dried, and radioactivity was quantified using PhosphorImager ImageQanNT software (Molecular Dynamics).

Construction of reporter gene fusions for C. perfringens. The vector pTUM177,used for studying gene expression in C. perfringens, was constructed by introduc-ing the E. coli gusA gene into the C. perfringens-E. coli shuttle vector pJIR750 (4).The gusA gene was engineered to contain the C. difficile toxB (tcdB) ribosomebinding site upstream of the gusA start codon (28). The promoter and the firstcoding nucleotides of tcdA, tcdB, tcdD, and gdh (6, 9) were fused in frame withthe gusA gene in pTUM177, creating the plasmids pTUM181, pTUM182,pTUM183, and pTUM481, respectively (28, 29). The construction of pTUM307,a TcdD-expressing plasmid, was described previously (28). To test the effect oftemperature on toxA, toxB, and tcdD gene expression with tcdD in trans, plasmidpTUM181, pTUM182, or pTUM183 was introduced into electrocompetent cellsof C. perfringens strain SM101 (52) with or without pTUM307 present. C. per-fringens was grown in TY medium or TY medium supplemented with 1% glu-cose, and �-glucuronidase activity, representing reporter gene expression, wasassayed as described previously (9).

2-D SDS-PAGE. Protein samples of C. difficile cultures were collected at theappropriate times by centrifugation at 4,000 � g for 5 min at 4°C. The cells werewashed twice in ice-cold phosphate-buffered saline and stored at �70°C to awaitfurther analysis. The cells were disrupted by sonication, and 20 �g of C. difficileproteins was separated on two-dimensional (2-D) SDS-polyacrylamide gel elec-trophoresis (PAGE) and silver stained as described previously (23). The gelswere dried using Novex frames.

Protein expression analysis. Analyzer version 6.1 software (BioImage) wasused for protein spot detection, gel matching, quantification of spot intensities,and estimation of the isoelectric points (pIs) and molecular masses of proteins.Images were normalized by the total intensity of all matched spots for each gel.For each temperature experiment, gels from two independent cultures were usedto create a reference image representing the average protein expression. Spotswith a difference in intensity of fivefold or more between temperature experi-ments were selected. To minimize errors in spot intensity caused by overexposuredue to the nonlinearity of protein staining by silver, the intensities of the selectedproteins were converted to their Gaussian values. Finally, the selected spots wereverified with those of the original gels by ocular inspection. Using C. difficileproteins previously identified (23) and known proteins loaded onto the gels asmarkers, the pIs and molecular masses of the proteins were calculated.

N-terminal amino acid sequencing and database analysis of temperature-regulated proteins. 2-D SDS-PAGE-separated C. difficile proteins were trans-ferred to polyvinylidene difluoride membranes, stained with Coomassie brilliantblue, destained, washed, and dried before the proteins of interest were excisedand subjected to amino-terminal sequencing by Edman degradation at the Pro-tein Analysis Center, Karolinska Institute, Stockholm, Sweden. The amino acidsequence was matched and identified in the genome sequence database for C.difficile strain 630 at the Sanger Center. Further characterization of the proteinswas made using ORF-finder and the BLAST search algorithm at the NationalCenter for Biotechnology Information website.

RESULTS

Impact of growth temperature on C. difficile toxin expres-sion. The toxin yields from C. difficile strain VPI 10463, ahigh-level toxin-producing isolate belonging to serogroup G,

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were found to be highly dependent on the growth temperature.Toxin yields in 72-h PY broth cultures were maximal at 37°C(2,100 U/ml) but low at 22 (50 U/ml) and 42°C (� 0.2 U/ml)(Fig. 1). The final culture optical densities (OD) were 0.80, 1.1,0.66, 0.65, and 0.43 for the tested growth temperatures (22 to42°C) (Fig. 1), and viable counts at 42°C were about half thoseat 37°C after 72 h. Toxin stability experiments, in which adefined amount of C. difficile toxin was added to C. difficilecultures grown at different temperatures, demonstrated thatthe toxin levels changed only 5% after 24 h of incubation (seeMaterials and Methods). Accordingly, the results from thegrowth and toxin stability experiments showed that the lowtoxin levels in cultures grown at 22 or 42°C could not beexplained simply by poor growth or poor viability of the cells orproteolysis of the toxins at these temperatures. Toxin yields infour C. difficile reference strains, representing the serogroupsA, C, H, and G, ranged from 10 to 60 U/ml at 37°C and thuswere generally much lower than those in strain VPI 10463. Theserogroup G reference strain showed evident temperature de-pendence, with the highest toxin yields at 37°C. This was ob-served to some extent in the serogroup H strain, whereas theserogroup A and C strains had low toxin yields (0 to 10 U/ml)regardless of temperature (data not shown).

Temperature regulation of C. difficile toxin expression oc-curs at the level of transcription. Shifts of C. difficile VPI 10463PY broth cultures from 22 to 37 or 42°C resulted in an ex-pected increase in the growth rate (Fig. 2A), but only the 37°Cculture showed a major induction of toxin as determined byenzyme immunoassay (Fig. 2B). Similar results were obtainedin defined medium lacking biotin, a medium known to promotehigh toxin production (50), showing that the temperature reg-ulation of toxin production in C. difficile VPI 10463 was notmedium dependent (Fig. 2A and B, insets). Western blotsconfirmed that both toxins were under temperature control

(Fig. 2C and D). The amount of toxin A, but not toxin B, wassmaller at 1,300 min than at 300 min, which may indicatedegradation of toxin A (Fig. 2C). However, no additionalbands corresponding to degradation products appeared on theWestern blots, and the reason for the smaller amount of toxinA at 1,300 min is not known. Toxin A mRNA and toxin BmRNA increased continuously upon a shift to 37°C, showingthat both toxins were controlled by temperature at the level oftranscription (Fig. 2E and F). For toxin A, the mRNA levelswere 15- and 5-fold lower at 22 and 42°C than at 37°C at 300min, and the corresponding differences for toxin B mRNAwere 7- and 6-fold (Fig. 2E and F).

Temperature regulation of toxin A and B promoters is de-pendent on TcdD. As temperature affected C. difficile toxingene expression at the mRNA level (Fig. 2E and F), we inves-tigated the possible role of the alternative sigma factor TcdD inthis regulation. Due to a lack of genetic tools in C. difficile, weused C. perfringens as the host for the different gusA fusionplasmids that we made (see Materials and Methods).pTUM181 (PtcdA-gusA), pTUM182 (PtcdB-gusA), or pTUM183(PtcdD-gusA) was introduced into C. perfringens strain SM-101lacking or carrying pTUM307, encoding TcdD (28). The C.perfringens strains were grown to stationary phase at varioustemperatures, and �-glucuronidase activity was used to moni-tor promoter activity. In the absence of tcdD, little or low gusAexpression was observed from the toxin A and B promoters,regardless of temperature (Fig. 3A and B). The presence oftcdD in trans dramatically increased expression from both toxinpromoters at 37°C but not at 22 or 42°C (Fig. 3A and B).Interestingly, tcdD was found to be positively autoregulated,but again only at 37°C (Fig. 3C). Similar to toxin expression inC. difficile (9, 24), the activity of the tcdA, tcdB, and tcdDpromoters was suppressed by adding 1% glucose to the growthmedium (Fig. 3A to C). Control experiments using the pro-moter of gdh, known to be under glucose control (20), wereperformed by introducing pTUM481 (Pgdh-gusA) into C. per-fringens with or without the TcdD-encoding plasmid pTUM307present. The expression of gusA from pTUM481 was affectedlittle by temperature or by tcdD but was down-regulated byglucose (Fig. 3D), showing that the constructs in C. perfringenswere stable and functional at all temperatures tested. Thus, theresults presented in Fig. 3A to C were not caused by nonspe-cific effects of 22 and 42°C temperatures on gusA expression.Finally, control experiments using the gusA plasmid pTUM177lacking any promoter yielded negligible �-glucuronidase activ-ity irrespective of temperature or the presence of tcdD in trans(Fig. 3E). Western blots and immunoprecipitation of proteinsfrom 37°C cultures of C. difficile VPI 10463 or C. perfringensstrains carrying tcdD using polyclonal mouse or rabbit anti-TcdD antibodies failed to detect TcdD (data not shown), im-plying that TcdD is normally produced at low levels and/or isunstable.

In summary, the pattern of temperature regulation of toxingene expression in C. difficile VPI 10463 was also present in theC. perfringens model system, but only in the presence of tcdD.Furthermore, the results showed that TcdD was autoinducedspecifically at 37°C.

Identification of temperature-regulated C. difficile proteins.Previous studies showed that the patterns of protein expressionin C. difficile differ significantly under growth conditions yield-

FIG. 1. Toxin yields determined by enzyme immunoassay in C.difficile VPI 10463 grown for 72 h in PY broth at indicated tempera-tures. The means and standard errors of three independent experi-ments are shown. The asterisk indicates that the toxin yield was belowthe detection limit of 0.2 U/ml.

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FIG. 2. Bacterial growth (A), toxin yields determined by enzyme immunoassay (B) and Western blotting (C and D), and toxin mRNA levelsdetermined by RPA (E and F) in C. difficile VPI 10463 after temperature upshifts from 22 to 37 or 42°C in PY broth. Data from correspondingexperiments in biotin-limited defined medium (SDM) are shown in insets in panels A and B. The mRNA expression at 22 and 42°C is indicatedas a single point at 300 min in panels E and F. Histograms (C and D) and mRNA curves (E and F) show the percentage of maximum band intensityfor each experiment. The results are representative of two independent experiments.



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FIG. 3. (A to C) Expression of �-glucuronidase in C. perfringens strain SM101 carrying plasmids with gusA as the reporter gene fused to thepromoters of tcdA (A), tcdB (B), and tcdD (C) with or without a plasmid carrying tcdD in trans. The cells were grown at 22, 37, or 42°C to stationaryphase in TY medium with or without 1% glucose. (D and E) Control experiments with gusA fused to the promoter of the glutamate dehydrogenasegene (gdh) (D) or promoterless gusA (E) with or without tcdD in trans.



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ing high and low toxin production (see the introduction). Toinvestigate whether this also applied to temperature, we ana-lyzed changes of protein expression in C. difficile VPI 10463 PYbroth cultures upon temperature upshifts. The average toxinyields 300 min after temperature shifts to 37 and 42°C were 530and 58 U/ml, compared to 36 U/ml at 22°C. Twenty-eightproteins were found by 2-D SDS-PAGE to have their highest

expression at 37°C, and seven of these were identified by N-terminal sequencing (Fig. 4A and C, no. 1 to 7, and Table 1).Several of these proteins matched other clostridial proteinsknown to be involved in reductive-oxidative metabolism lead-ing to the formation of butyric acid (see Fig. 6).

We also measured protein expression in C. difficile strainVPI 10463 cultures after temperature uphifts in defined me-

FIG. 4. (A and B) 2-D SDS-PAGE of proteins expressed by C. difficile VPI 10463 during growth in PY broth (A) and in defined medium (SDM)without biotin (B). The cultures were incubated at 22°C to an OD at 600 nm (OD600) of 0.2 to 0.3 (PY broth) or an OD420 of 0.1 to 0.2 (SDM),shifted to 37 or 42°C or left at 22°C, and harvested 300 (PY broth) or 600 (SDM) min after the temperature shift. The arrowheads indicate proteinswith higher expression at 37 than at 22 and 42°C. (C) Enlarged sections of the 22, 37, and 42°C 2-D SDS-PAGE gels highlighting N-terminalsequenced proteins (see also proteins 1 to 9 in Table 1).



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dium with or without biotin, supporting low and high toxinexpression, respectively. Twelve proteins matched the criteriafor having the highest expression at 37°C during biotin limita-tion, and thus high toxin production, and two of these wereidentified (Fig. 4B and C, no. 8 and 9, and Table 1). TheN-terminal sequences of these two proteins were identical, andthe proteins showed similarity to iron-sulfur-containing pro-teins involved in reduction-oxidation processes.

Relation between growth temperature and butyric acid-bu-

tanol metabolism. As the above-mentioned experiments indi-cated that butyric acid production pathways were temperatureregulated like toxin production, we monitored the productionof butyric acid during a temperature upshift of a C. difficile VPI10463 culture in PY broth. The rise of butyric acid levels in themedium at 37°C (Fig. 5A) paralleled that of the toxin levels(Fig. 2B) and reached a maximum of 4 mM at 1,300 min,whereas the butyric acid levels in the 22 and 42°C cultures werethree- to fourfold lower. The production of other short-chain

FIG. 5. (A) Levels of butyric acid produced in cultures of C. difficile VPI 10463 grown in PY broth after a temperature upshift from 22 to 37or 42°C. The growth curves and toxin production during these experiments were analogous to those in Fig. 2A and B (data not shown). The valuesare means of two independent experiments. (B) Toxin yields, determined by enzyme immunoassay, from C. difficile VPI 10463 grown for 48 h atthe indicated temperatures in PY broth or PY broth with 15 mM butyric acid or butanol added. The means and standard errors of threeindependent experiments are shown. The asterisks indicate that toxin levels were below the detection limit of 0.2 U/ml.

TABLE 1. Selected proteins from C. difficilea

No.Relative intensityb

kDa pI N-terminalsequenced Protein Ide Sif Organism

22°C 37°C 42°C

1 1.6 11 6.9 56 5.7 MKVLIIGGVA NADH oxidase 33 50 C. perfringens2 0.3 4.2 3.7 51 5.5 MEKAVENFED Succinate-semialdehyde

dehydrogenase66 82 C. kluyveri

3 0.4 2.3 1.2 42 5.1 MKFYVYKAPD Putative hydrolase 51 69 Salmonella enterica serovarTyphimurium

4 0.3 3.8 1.4 34 6.3 MKLAVIGSXT 3-hydroxybutyryl-CoAdehydrogenase

100 100 C. difficile

5 6.9 22 15 32 6.3 MKLAVIGSXT 3-hydroxybutyryl-CoAdehydrogenase

100 100 C. difficile

6 0.2 1.1 0.7 23 5.1 MHXIFINKDL Nitrate reductase 47 62 C. perfringens7 10 18 13 19 NDc DKVEIPPEEN No match in database ND ND ND8 4.6 14 4.2 22 5.0 MKKFVXTVXG Rubrerythrin 76 85 C. acetobutylicum9 5.7 12 3.9 22 5.1 MKKFVXTVXG Rubrerythrin 76 85 C. acetobutylicum

a Relative amounts, physical properties, and identities of selected proteins from C. difficile VPI 10463 specifically induced concomitantly with its toxins at 37°C duringgrowth in PY broth (Fig. 4A, spots 1 to 7) and in biotin-limited defined medium (Fig. 4B, spots 8 and 9). Genes were identified by searching for a high-score matchof the N-terminal sequence in the Sanger Center C. difficile strain 630 sequence database (http://www.sanger.ac.uk/Projects/C difficile/) using the BLAST algorithm. Thegenome segments found were further analyzed using ORF-finder (http://www.ncbi.nlm.nih.gov/), and the ORFs in the contigs were identified using the BLASTalgorithm and the nonredundant database at the National Center for Biotechnology Information.

b Relative spot intensity is the calculated Gaussian value (see Materials and Methods).c ND, not determined.d X, unidentified amino acid.e Id, percentage of identical amino acids found in the identified protein and the corresponding homologue.f Si, percentage of similar amino acids found in the identified protein and the corresponding homologue.



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fatty acids (acetic, propionic, isobutyric, valeric, isovaleric, ca-proic, and isocaproic [for details, see reference 23]) did notshow any temperature dependence (data not shown).

Addition of butyric acid to C. difficile cultures growing at 37and 22°C induced toxin yields twofold (Fig. 5B). At 42°C, notoxin was detected even if butyric acid was added to the me-dium. The addition of butanol decreased toxin production 10-fold both at 37°C (920 to 110 U/ml) and at 22°C (51 to 5.5U/ml) (Fig. 5B). No apparent impact on growth due to theaddition of butyric acid or butanol compared to the control wasobserved (data not shown). Thus, although the toxin level washigher at 37°C than at 22°C, the relative change in toxin pro-duction by butyric acid or butanol was the same at both tem-peratures. In conclusion, butyric acid, butanol, and tempera-ture apparently affected toxin production independently ofeach other, and the results also suggested that the low toxinproduction at 22 and 42°C was due to different mechanisms.

DISCUSSION

We have described temperature-dependent production oftoxins A and B in C. difficile strain VPI 10463, controlled at thetranscriptional level with maximum expression at 37°C. Amongfour different serotype reference strains tested, the tempera-ture effect was most prominent in serogroup G (which includesstrain VPI 10463). Interestingly, the dominant strain amongclinical C. difficile isolates from hospitalized patients in the

United Kingdom is related to serogroup G (43). The alterna-tive sigma factor TcdD (28), encoded by the gene locateddirectly upstream of tcdB on the PaLoc, was required for thetemperature-dependent activity of the toxin promoters, asshown using a gusA reporter system in C. perfringens. Further-more, tcdD was autoinduced strictly at 37°C. Adding glucose tothe medium resulted in loss of the TcdD-dependent expressionfrom the promoters of tcdA, tcdB, and tcdD and showed thatthe nutrient effect on toxin production observed in C. difficile isalso present in C. perfringens.

Limiting the levels of cysteine leads to up-regulation of toxinproduction in C. difficile and also to the induction of the keyenzyme in butyric acid production, 3-hydroxybutyryl coenzymeA (CoA) dehydrogenase (23). Its gene is clustered with thoseencoding other enzymes involved in butyric acid-butanol pro-duction on both the Clostridium acetobutylicum and the C.difficile chromosomes (Fig. 6) (36). Similar to the toxins, ex-pression of 3-hydroxybutyryl-CoA dehydrogenase here wasfound to be highest at 37°C, and the largest difference (�10-fold) was found between the 22 and 37°C cultures. This tem-perature response was also observed for succinate-semialde-hyde dehydrogenase, another enzyme involved in butyric acidformation but via a different pathway (Fig. 6). The succinatepathways in C. difficile share temperature control and down-regulation by glucose (S. Karlsson, L. G. Burman, and T.Åkerlund, unpublished data), similar to the transcriptionalcontrol of the toxin genes and tcdD. The corresponding operon

FIG. 6. Operon structure of genes in C. difficile involved in butyric acid production compared to those in other clostridia (A) and thecorresponding metabolic pathways (B). The arrows numbered 1 to 8 in panel B correspond to genes and enzymes in panel A.



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structure is conserved in C. difficile, Clostridium aminobutyri-cum, and Clostridium kluyveri (Fig. 6) (11). An induced succi-nate pathway due to the absence of glucose may reflect anongoing fermentation, and subsequent limiting levels, of cer-tain amino acids. This may impose a metabolic stress thattriggers toxin production and that can be reversed by supple-menting PY broth with these amino acids or glucose (24).Many enzymatic reactions in the butyric acid production path-ways are NADH dependent, and also, NADH oxidase wasinduced at 37°C in C. difficile. Interestingly, virulence regula-tion by amino acids and temperature is also found in Bordetellapertussis, where the toxin PtX is specifically induced at 37°Cand is influenced by cysteine metabolism (5, 40).

High-level toxin production in C. difficile also occurs duringslowed growth due to biotin limitation and can be reversed byadding the amino acid asparagine, glutamic acid, glutamine, orlysine to the growth medium (50). The effect of biotin limita-tion may be linked to starvation for glutamine and the subse-quent block of glutamine-dependent purine biosynthesis (27).The pattern of up-regulated proteins during biotin limitation,however, was different from that in PY broth at 37°C, indicat-ing that the metabolic stresses imposed on the bacteria toinduce toxin production differed under the two conditions.Proteins previously shown to be up-regulated during biotinstarvation (24) were found here to also be affected by temper-ature. These showed similarity to iron-sulfur binding proteinsinvolved in reduction-oxidation reactions. However, biotinstarvation leads to growth arrest at low cell densities (24), andthe differential expression of various proteins observed may berelated to changes in both growth phase and stress due tolimiting biotin levels. Nevertheless, the up-regulation of toxinsynthesis during biotin and glucose-amino acid limitation at 37but not 22 and 42°C shows that temperature regulation isgeneral and may act at a fundamental level.

In order for bacteria to effectively compete and survive intheir ecological niche(s), they must be able to sense environ-mental changes and respond accordingly. Several principles ofregulation of gene expression by temperature have been de-scribed in bacteria, e.g., supercoiling of DNA, secondary struc-ture of RNA, activities of proteins, and temperature monitor-ing by signal transduction via two-component regulatorysystems (17). Recently, fatty acids were suggested to act astemperature sensors in Bacillus and Synechocystis (1, 44). Thecold shock response comprises proteins in general metabolism,as well as RNA chaperones and fatty acid desaturases, result-ing in correct mRNA and membrane phospholipid structure,respectively (38). The heat shock response is triggered whencells are exposed to either cell-damaging agents or high tem-perature. The proteins induced are mainly involved in themaintenance of correct protein structure, e.g., chaperones,proteases, transcription factors, or ribosome binding proteins(3, 51). However, the heat shock protein GroEL has also beensuggested to be virulence associated in C. difficile (14). Up-regulation of virulence determinants in response to transitionfrom ambient temperature to that of warm-blooded animals isdifferent from heat or cold shock and has evolved in suchdifferent bacterial genera as Bordetella, Borrelia, Escherichia,Salmonella, Shigella, Vibrio, and Yersinia (25, 33).

The alternative sigma factor TcdD of the C. difficile PaLochas similarities to the regulatory proteins BotR in Clostridium

botulinum (31), TetR in Clostridium tetani (32) and UviA in C.perfringens (10), all suggested to be sigma factors (28). TcdDalso shows homology to extracellular function (ECF) sigmafactors (28). ECF sigma factors are controlled by their cognateanti-sigma factors, which often are membrane proteins capableof sequestering the ECF sigma factor. At a given stimulus, theanti-sigma factor releases the ECF sigma factor to the cytosol,enabling transcription of the respective target genes (15, 41).ECF sigma factors are thought to act as general stress media-tors responding to a variety of signals, such as envelope ormetabolic stress, and several ECF sigma factors are cotrans-cribed with their specific anti-sigma factors (34, 41). The ap-parent positive autoregulation of tcdD suggests that a negativefactor is involved to modulate TcdD activity. Two candidatesare TcdE and TcdC, encoded by the PaLoc of C. difficile, andTcdC has also been suggested to negatively regulate toxinexpression (16). However, expression of tcdD was blocked bothby altered temperature (22 and 42°C) and by glucose in C.perfringens. C. perfringens lacks homologues to tcdE and tcdC,suggesting that these genes are not required for modulatingTcdD activity. However, we cannot exclude the possibility thattcdE and/or tcdC has a regulatory role in C. difficile. In view ofthe various growth conditions affecting toxin production in C.difficile, TcdD activity may be modulated by a pleiotropic reg-ulator and/or by other sigma factors competing with TcdD forRNA polymerase binding.

In summary, this is the first report of up-regulation of viru-lence by host temperature in clostridia. The expression of C.difficile toxins A and B and the toxin-specific sigma factorTcdD was temperature dependent, with a maximum at 37°C.The results further support the notion that toxin regulation islinked to the induction of metabolic pathways involved in bu-tyric acid production in PY broth. The findings presented hereencourage further studies regarding the regulation of virulencein C. difficile with respect to TcdD, growth temperature, andmetabolism.

ACKNOWLEDGMENTS

This work was supported by grants from the Vardal Foundation,Stockholm, Sweden, and by a Ph.D. student grant provided by theMicrobiology and Tumor biology Center at the Karolinska Institute.Protein data were obtained at the Protein Analysis Center, KarolinskaInstitute. The sequence data were produced by the Clostridium difficileSequencing Group at the Sanger Center and can be obtained at http://www.sanger.ac.uk/Projects/C_difficile/.

We are grateful for the excellent technical support provided byAnna-Karin Persson. We thank Nagraj Mani for kindly providing allpTUM plasmids.

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