Journal of Bacteriology, March 2004, p . 1355-1361, Vol . 186, No . 5

Expression of Cholera Toxin under Non-AKI Conditions in Vibrio cholerae El Tor Induced by Increasing the Exposed Surface of Cultures

Joaquín Sánchez,^1* Gerardo Medina,^2, Thomas Buhse,³ Jan Holmgren,⁴ and Gloria Soberón-Chavez⁵

Facultad de Medicina,¹ Centro de Investigaciones Químicas, UAEM,³ Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca,² Instituto de Investigaciones Biomédicas, UNAM, Circuito Escolar, Ciudad Universitaria, México D . F., México,⁵ Department of Medical Microbiology and Immunology, Göteborg University, Göteborg, Sweden⁴

Received 26 August 2003/ Accepted 18 November 2003

 
The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin [CT] in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical . Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT . The AKI conditions involve biphasic cultures.In phase 1 the organism is grown in a still tube for 4 h . Inphase 2 the medium is poured into a flask to continue growthwith shaking . Virtually no expression of CT occurs if this protocolis not followed . Here we demonstrated that CT expression takesplace in single-phase still cultures if the volume-to-surface-arearatio is decreased, both under air and under an inert atmosphere.The expression of key genes involved in the regulation of CTproduction was analyzed, and we found that the expression patternclosely resembles the in vivo expression pattern.

 
Strains of Vibrio cholerae El Tor and classical biotypes are causative agents of cholera, and both biotypes produce the virulence factor cholera toxin [CT], which is a powerful enterotoxin considered the main toxin responsible for the profuse loss of fluid that characterizes the disease . Although the El Tor and classicalbiotypes are very similar, they have distinguishing propertiesthat include disparate needs for efficient in vitro productionof CT . The classical strains are relatively permissive, andthey produce CT under different conditions, but induction ofCT synthesis in the El Tor strains is more demanding.

In order to produce CT from the El Tor biotype, both for characterization of the toxin and for eventual use of the toxin in immunization, ways to induce the in vitro expression of the El Tor CT were investigated . The so-called AKI conditions were discovered through trial-and-error experimentation [10] . The AKI conditions involvebiphasic cultures; vibrios are first grown in a tube for 4 h, and then the culture is poured into a flask whose capacity is approximately 10-fold greater than that of the tube to continue growth with shaking . Why these conditions are required to induce production of CT and how they influence the complex regulatory cascade involved in CT expression are unknown . The aim of thepresent study was to contribute to the elucidation of thesematters.

The ctxAB genes code for the CT protein, which is made up of the CTA and CTB subunits . The regulation of ctxAB in V . cholerae is elaborate, and it involves a multistep cascade . In response to the external environment, diverse regulators can influence expression of ctxAB [17] . In a working model, in which the regulatorsat the core of the cascade are included, the control of ctxABis thought to occur as follows [Fig . 1] . The AphA and AphB proteinsactivate transcription of the tcpPH operon [12] that codes forthe TcpP and TcpH proteins [Fig . 1] . The TcpP-TcpH couple interacts with the ToxR protein [positive regulator], which acts in conjunction with the ToxS protein [ToxR-ToxS] [Fig . 1] . The TcpP-TcpH-ToxR-ToxScomplex then promotes transcription of toxT [3, 6-8] [Fig. 1],and this leads to production of the ToxT protein [Fig . 1] . TheToxT protein directly activates transcription of the ctxAB operon[2], and this results in the synthesis of CT [Fig . 1] . At the same time that ToxT activates expression of ctxAB, it also activatesexpression of the toxin-coregulated pilus [TCP] operon [25]; this operon codes for another virulence factor, designated the TCP, which is a surface structure involved in colonization ofthe intestine [30] . The first gene downstream from the ToxT-activatedpromoter is tcpA, and this gene codes for the TcpA protein [Fig.1] . The toxT gene lies downstream from tcpA and within the TCPoperon itself [not shown in Fig . 1] . Thus, upon induction ofthe TCP operon by ToxT, a transcript is produced that includesthe toxT mRNA, and from this transcript additional synthesisof ToxT can occur [1, 32] . In this study we monitored expressionof toxT from the nearby TcpP-TcpH-ToxR-ToxS-induced promoterand not from the distant ToxT-activated tcpA promoter.

 
The molecular bases of the differential expression of CT byEl Tor and classical strains have essentially been detemined.First, it was demonstrated that there is biotype-specific controlover expression of toxT [4] . Then it was shown that the functionalities of the classical and El Tor TcpP and TcpH were not substantially different and thus that regulatory differences between the two biotypes were not due to the activity of these proteins butto their levels of expression [22, 23] . Subsequently, it wasshown that differential expression of tcpP and tcpH in the twobiotypes was due to an affinity of the AphB activator that waslower for the El Tor tcpP promoter than for the classical tcpPpromoter [13, 14] . This difference in affinity was apparently the result of a single-nucleotide mutation [13, 14] . Notwithstandingthis mutation, the affinity of AphB for the El Tor tcpP promoteris thought to be sufficient for activation of tcpP, but onlyunder permissive [i.e., AKI] conditions . Under nonpermissiveconditions the mutation is critical and the affinity of AphBfor the tcpP promoter is not great enough for induction . Withoutproduction of TcpP the regulatory cascade is not activated,and no expression of CT occurs.

When one examines how the AKI conditions might work, one natural assumption regarding the shaking phase is that it could functionby increasing oxygen availability . In preliminary experiments [unpublished data] we explored this and other possibilities.When we replaced air with nitrogen or helium in the shakingculture phase, we found that CT synthesis was not reduced asexpected if oxygen or even another component of air was actingas an inducer . We then investigated whether mechanical motionwithout aeration induced expression of CT, but expression didnot occur . Subsequently, we explored whether an increase inthe surface area of the exposed culture stimulated productionof CT and observed stimulation under these conditions.

In this paper we describe experiments which demonstrated that,in the El Tor vibrios, CT synthesis is induced in single-phasestill cultures if the surface area of the exposed culture isincreased . These results partially explain why CT is producedunder AKI conditions . To determine the effect of these new conditionson the CT genetic regulatory cascade, we examined the transcriptionof tcpP, toxT, ctxA, and tcpA at 1-h time intervals by primer extension . The patterns of expression of these genes were found to closely resemble those observed under in vivo conditions[18, 19].

 
Strain and cultures. V . cholerae El Tor strain E7946 was used in this study . Thisstrain was maintained at -70°C in Luria-Bertani medium containing20% glycerol . For cultures the strain was plated onto Luria-Bertaniagar directly from the frozen glycerol stock . After overnightgrowth at 37°C under an air atmosphere, a suspension ofbacteria in saline solution [0.9% NaCl], adjusted to an opticaldensity at 600 nm [OD₆₀₀] of 1.0, was produced . Two microlitersof this suspension per 10 ml of medium was used to start eachliquid culture, which was incubated at 37°C . All bacterial suspensions were fresh and used within 10 min after preparation. The medium used for all cultures was AKI [1.5% Bacto-Peptone [Difco], 0.4% yeast extract, 0.5% NaCl] without sodium bicarbonate[11, 20].

Growth under different atmospheres. Nitrogen, helium, and air atmospheres were used for cultures.In all cases the cultures were grown in vials or Erlenmeyerflasks fitted with gas-tight rubber caps [catalog no . DX3043CA [Daigger & Co . Inc., Linconshire, Ill.] or catalog no.Z10145-1 [Aldrich Chemical Co., St . Louis, Mo.]] . The air initiallypresent in vials and flasks was replaced by another atmosphereby repetitive displacement . This was done by passing two needlesthrough the cap, one for filling and the other for emptying. Filling was done by using a small latex balloon that contained the gas being assayed [50 to 200 ml], which was connected viaa syringe to the filling needle . After repetitive displacementthe filling needle and balloon were left in situ . The low positive pressure exerted by the balloon ensured that there was constant replenishment of gas and guarded against the potential entryof external air in the event of minor leaks . The purity of thenitrogen and helium used [Praxair Technology, Inc., Danbury,Conn.] was 99.999% . The initial tests to determine the effectsof the variable volumes on CT expression were performed in vials.For these tests 15-ml cultures were first grown under the typicalAKI conditions . Screw-cap vials [19 by 70 mm] were filled tothe top with medium and then inoculated . Following inoculationthe vials were incubated under still conditions for 4 h . Afterthis, 200-, 400-, 600-, 800-, or 1,600-µl aliquots ofthe growing cultures were transferred to 7.5-ml vials [15 by60 mm] . The atmosphere in the vials was then quickly replaced[with air or nitrogen], and the vials were incubated under stillconditions for 3 h . In the experiments in which there was a constant exposed surface there was no pregrowth under AKI conditions, and cultures were started directly in 125-ml Erlenmeyer flasks [single-phase cultures] . For these single-phase cultures a constant volume [3 ml] of preinoculated medium was used . After the preinoculated medium was placed in the flasks, the atmosphere was replaced with air, nitrogen, or helium . Once the atmosphere was replaced,the flasks were incubated under still conditions for 3, 4, 5,6, or 7 h.

To obtain an improved appreciation of the culture conditions,we estimated surface areas, depths, and volume-to-surface ratios.For the 7.5-ml vials the calculated exposed surface area was1.54 cm² . In these vials the 1.6-ml cultures were approximately1 cm deep, while the 0.2-ml cultures were approximately 1.25mm deep . The estimated volume-to-surface-area ratios for the1.6-ml cultures were approximately 1 ml/cm², while the estimated volume-to-surface-area ratios for the 0.2-ml cultures were 0.13ml/cm² . In the 125-ml flasks the calculated exposed culturesurface area was 26 cm², the depth of the cultures [3 ml] was1.2 mm, and the volume-to-surface-area ratio was 0.12 ml/cm².

RNA isolation, primer extension analysis, and DNA sequencing. Total RNA was isolated with the TRIZOL reagent from GIBCO-BRL [Bethesda, Md.] . The concentrations of RNA were adjusted tothe same value by using the OD₂₆₀ and were verified by visual estimation of the intensity of the 16S and 23S RNA bands inethidium bromide-stained UV-illuminated agarose gels . In general,duplicate cultures were prepared for each sample time, and thebacterial cell pellets from these duplicate cultures were pooledbefore isolation of RNA . However, for the shorter times [3 and4 h] pooling of pellets from up to five replicate cultures wasnecessary to obtain sufficient amounts of RNA . To ensure thatconditions were as similar as possible for replicate cultures,the preparation of inocula, the inoculation time, the incubationperiod, and the point at which cultures were harvested wereall done with prearranged time delays . In addition, the OD₆₀₀of each replicate culture was verified to ensure that the valueswere within the expected range.

Primer extension reactions were performed by using the C . therm. polymerase for reverse transcription [Roche Diagnostics, Indianapolis, Ind.] according to the manufacturer's instructions . The following primers were used: tcpP oligo [GCA TAA TAG ACT TGA TTA GTG CAT TC], toxT oligo [5' CCA ATC ATT GCG TTC TAC TCT GAA G 3'], tcpA oligo [5' CGA GTA ATG TCA TAC CCT CTT GAC 3'], and ctxA oligo [5' CTG CCC GAT ATA ACT TAT CAT CAT TTG C 3'].

For DNA sequencing reactions a ThermoSequenase kit [AmershamLife Sciences Corp., Cleveland, Ohio] was used with [-³²P]ATP [Amersham International] as the substrate for end labeling.

Enzyme-linked immunosorbent assay. The GM1 enzyme-linked immunosorbent assay [28] was used for semiquantitative determination of CT in culture supernatants;whole CT [Calbiochem, Inc., Darmstadt, Germany] at a concentrationof 1 µg/ml was used as a standard . Monoclonal antibodyLT39 directed towards the B subunit of CT [29], kindly provided by A.-M . Svennerholm [Göteborg University, Göteborg,Sweden], was used for detection.

 
In preliminary experiments [unpublished data] aimed at discriminating between the various potential factors that could induce expressionof CT in AKI cultures, we discovered that the exposed surfacesof cultures were a key stimulatory factor under AKI conditions. Following up on these results, we found that in still culturesand when the size of the container was constant, the productionof CT increased as the volume of the culture was reduced underair [Fig. 2A], nitrogen [Fig . 2B], and helium [data not shown]atmospheres . Expression under anaerobic atmospheres indicatesthat the shaking phase of AKI cultures induces production primarilybecause it involves an increase in the relative culture surfacearea and not because of increased aeration.

 
How these conditions induce expression of CT is not clear, butit is possible that vibrios are somehow able to sense a relativeincrease in the gas-liquid interphase . It could be that theincrease in the exposed culture surface area simply promotesthe loss of some volatile metabolite that inhibits inductionof CT production . Devising practical ways to determine if vibrioscan respond to gas-liquid [or analogous] interphases and analysesto detect and characterize volatile metabolites are experimentsthat remain to be done.

Progressively reducing the culture volume in vials that werea constant size had a dual effect because as the levels of CT increased, the bacterial densities of cultures also increased[Fig. 2] . To estimate the contribution of the progressively larger numbers of bacteria to the concentrations of CT, [CT]/optical density ratios were calculated . The results [Fig . 2] demonstratedthat the [CT]/optical density ratios increased continually andtherefore that there was induction of CT production in responseto the reduction in culture volume.

Based on the results described above, a constant volume of 3ml in a 125-ml flask was used for subsequent experiments . Theseproportions were approximately equivalent to those of the 200-µlcultures in the 7.5-ml vials . Single-phase still cultures werethen incubated under air, nitrogen, and helium atmospheres,and CT expression was monitored at 1-h intervals . Under thethree atmospheres increases in CT occurred in a time-relatedor growth-associated manner [Fig. 3], just as they did underother culture conditions . The expression of CT in these timecourse experiments may also be attributed to induction becauseas the growth curve progressed, the [CT]/OD₆₀₀ ratios also increased[data not shown].

 
The use of single-phase still cultures to induce expressionof CT may represent a step forward for gene expression studiesof V . cholerae El Tor because this system is simpler than thebiphasic AKI system . Moreover, as discussed below, the geneexpression patterns suggest that this type of culture may havemore potential to mimic the in vivo situation.

Under our culture conditions expression under a helium or nitrogen atmosphere was higher than expression under an air atmosphere.To profit from this effect and at the same time avoid potential contributions of the used atmosphere, we used the inert gashelium for subsequent analyses . We also reasoned that an atmospherelacking oxygen would be more similar to the microaerophilicintestinal milieu and therefore that the data obtained couldeventually be more useful for comparisons with in vivo results.

To determine how the classical strains responded to these culture conditions, the O395 strain was grown under the optimal stimulating conditions and under a helium atmosphere . The time course CT expression pattern for the O395 strain was practically the sameas that for the El Tor E7946 strain, although the CT concentrationswere approximately threefold higher [data not shown] . Theseresults suggest that there was an equivalent response of thegenetic regulatory cascade . Therefore, our culture system mayprovide the opportunity to compare side by side and under exactlythe same growth conditions the gene expression patterns of theclassical and El Tor strains.

To survey how the regulatory molecular cascade controlling CT expression in El Tor strain E7946 was affected by the new conditions, we analyzed the patterns of expression of tcpP, tcpA, toxT, and ctxA by the primer extension method . In these experiments the transcriptional start point [TSP] was also determined . The TSP for the tcpP gene in El Tor strains has been mapped, and our data coincided with the previously reported site [23] . OurTSPs for tcpA and ctxA also coincided with the previously proposedEl Tor TSPs [21, 25, 31] . However, the TSP for the toxT genefrom El Tor strains has not been reported, and our TSP did not coincide with a previously proposed TSP for the classical O395 strain [7]; our TSP was located 42 bases upstream [Fig. 4].This was not an additional TSP because close inspection of theregion where the classical TSP was expected revealed no detectablebands at any of the sample times [data not shown] . The DNA sequenceupstream from our TSP was found to be identical to the classicalsequence . Therefore, the TSP identified does not appear to bethe result of a new promoter generated by mutation, at leastnot in the near vicinity . We are currently experimentally reviewingthe TSPs for classical and El Tor strains under various in vitrogrowth conditions.

 
In agreement with the observed induction of CT [Fig . 2 and 3]and as proposed by the regulatory cascade model [Fig . 1], wedetected expression of tcpP under our conditions . Transcriptionof tcpP took place early in the culture and at h 3 [Fig . 5A],but the expression was ephemeral, so that at subsequent timesthe tcpP message became undetectable [Fig . 5A] . Repeat experiments with alternate primers and/or overexposure of film to reveal potential weak signals [data not shown] suggested that thisresult was not due to technical reasons but to true suppressionof tcpP expression . Induction of tcpP may have started almostexactly at 3 h because sampling at 2.5 h did not result in adetectable signal . Therefore, tcpP transcription appeared totake place in the form of a single pulse that lasted about 1h.

 
The expression of toxT is positively affected by TcpP [Fig. 1], and therefore, it was important to determine how the ephemeralexpression of tcpP affected toxT transcription . In addition,the pattern of expression of toxT was of interest because wehave previously found that under AKI conditions toxT is expressedin a transient manner and only during an approximately 2-h timewindow [20] . In the present experiments toxT was not expressedtransiently but was expressed continuously and from the earliesttime sampled [Fig. 5B] . This pattern of expression was alsofound under in vivo conditions; under these conditions toxT transcription was estimated to start at h 3 and to be continuous after this [19] . In brief, unlike expression of toxT under theAKI conditions, expression of toxT under our conditions wascontinuous instead of transient, and the expression patternclosely resembled the in vivo expression pattern.

The continuous expression of toxT, despite the ephemeral transcription of tcpP, is an intriguing observation given that activation of toxT is thought to depend on the TcpP protein . To explain continuous expression of toxT in the apparent absence of TcpP, direct induction of toxT by ToxR could be postulated . However, this possibility seems improbable because ToxR appears to act by providing the promoter recognition function for a productive interaction of TcpP with the RNA polymerase rather than by directly inducing toxT [15, 16] . One alternative explanation is thatthe regulatory cascade is primed by the transient presence ofTcpP and that an additional regulator[s] is responsible forthe continuous expression of toxT . It is also possible thatafter priming by TcpP the H-NS protein, which normally repressestoxT [24] [Fig . 1], no longer binds to the promoter region andthat this causes constitutive transcription of toxT . Whateverthe conditions responsible for continuous expression of toxT,the presence of ToxR seems to be essential because ToxR-negativemutants are highly defective for toxT expression [3] . The hypothesis that ToxR is crucial for the expression of toxT is also supported by the in vivo observation that a double tcpPH toxR mutant wasincapable of inducing tcpA [18], most likely because in theabsence of ToxR no ToxT was produced . A theoretical scenarioin which the constant presence of TcpP [unlike ToxR] is notstrictly necessary for induction of toxT is consistent withthe observation that a tcpPH mutant was able to induce [in vivo]both ctxA and tcpA [18] . This scenario presupposes the presenceof ToxT in the cell and hence provides evidence of TcpPH-independent induction of the toxT gene, most likely mediated by ToxR . It seems possible that in the absence of continuous synthesis ofTcpP, constant expression of toxT can occur.

The regulator ToxT directly activates the ctxAB operon [Fig. 1] . To monitor this effect, we examined transcription of ctxA.Expression of ctxA was found to start at h 4 and to be essentiallyconstant after this, although the level of expression at thestarting point was higher [Fig . 5C] . Under AKI conditions expressionof ctxA was similar except that transcription started 1 h laterand when the cultures were in the shaking phase [20] . When thepattern obtained under in vivo conditions was compared withour ctxA expression pattern, the patterns coincided totallyin terms of timing because in the mouse model expression ofctxA also occurred at h 4 of growth [18].

Expression of tcpA was found to occur synchronously with expression of ctxA and with a basically identical pattern, including higher expression at the initial time of induction [Fig . 5D] . Verysimilar expression patterns for ctxA and tcpA would be in fullagreement with the proposal that ToxT directly activates bothgenes [Fig . 1] . As for ctxA, there was a 1-h disparity in thetime of expression of tcpA when the data were compared withthe data obtained under AKI conditions . When the data were comparedwith the data obtained under in vivo conditions, the expressionpattern of tcpA also coincided; however, the similarities couldbe more limited in this case because in vivo transcription oftcpA was bimodal, with a weak expression peak at h 1 and a muchstronger peak at h 4 [18] . The latter peak exactly coincidedwith our tcpA induction time, but we could not test for expressionof this gene at 1 h due to the low bacterial densities at thispoint.

When we compared the expression patterns of ctxA, tcpA, and toxT under our conditions, transcription of ctxA and tcpA occurredabout 1 h later than transcription of toxT [Fig. 5] . Analogousdelays have been observed under AKI conditions [20] and in thein vivo model [18, 19] . Our culture procedure is new; therefore,the persistent occurrence of a delay suggests that this couldbe an obligatory step common to the three systems . Perhaps inevery case and soon after synthesis, ToxT is mainly quiescent,and activation of the protein is required for induction of itstarget genes . This hypothesis could be supported by the observationthat the activity of ToxT can be modulated by environmentalsignals [27].

We believe that our culture system could help identify the natural external stimuli that induce [or suppress] expression of CT,as well as other virulence factors, in V . cholerae . This maybe true because except for the potential absence of the early[1-h] peak of expression of tcpA, the El Tor in vivo temporalexpression patterns and our expression patterns basically coincidedperfectly . It is therefore possible that our system furnishessignals that, at least to some extent, successfully mimic thein vivo situation . This may have been partly due to the factthat we used anaerobic atmospheres, which presumably are conditionsthat are similar to in vivo conditions . Although air allowedexpression of CT, we noticed that the toxin levels were lower,and this suggests that the conditions were unfavorable . A positiveeffect of low-oxygen conditions would agree with the natureof AKI conditions because cultures are grown first in tubes,and this creates a microaerophilic environment, especially afterdissolved oxygen is depleted by bacterial growth . These similaritiescould explain why both systems successfully induce CT production.

Obviously, until fully perfected, in vitro systems will necessarily lack stimuli present in the in vivo models . However, to obtain partial yet potentially highly relevant responses, our methodand variations of this method could offer alternatives for studyinggene expression in V . cholerae . In this respect, it may be important that shallow standing cultures rather than shaking cultures lead to differential expression of at least 45 proteins in Mycobacterium bovis [5] . For V . cholerae gene expression studies and to betterimitate the bacterial surroundings during infection, our invitro conditions could be combined with in vivo stimuli, suchas intestinal fluid [26].

 
J.S . acknowledges financial support provided by SILANES, S.A.de C.V., México . G.S.-C., T.B., and J.S . acknowledgemembership in SNI, CONACyT, México . T.B . gratefully acknowledgesfinancial support provided by CONACyT México [researchgrant 34236-E].

We gratefully acknowledge Leticia López Escobar and DianaDiaz de Anda for skillful and dedicated technical assistance.

 
* Corresponding author . Mailing address: Facultad de Medicina, Av . Universidad 1001, Cuernavaca, Morelos, CP 62210, Mexico . Phone: 52-777-3297913 . Fax: 52-777-3297913 . E-mail: joaquin.sanchez@microbio.gu.se.

Present address: Universidad Autónoma de Baja California,Mexicali, México.

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What Is Fermentation?, What Is Growth Medium?, What Is Prokaryote?, What Is Bioassay?, What Is Yeast?, s, Bacteria, s, Microbiology, e, Microbes, a, Microbe, a, Microorganisms, e, Cholera, i, Edwardsiella, a, Suspension cells, o, Escherichia coli, s, Antibiotics, c, Candida albicans, e, Escherichia coli, n, Antibiotics, n, Microorganisms, e, Microbial, o, Escherichia coli, a, Bacillus, r, Streptococci, o, Escherichia coli, c, Staphylococcus aureus, c, Proteus, o, Bacteria, n, Salmonella, s, Shigella, e, Salmonella, o, Staphylococcus




 

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