Temporal Quorum-Sensing Induction Regulates Vibrio cholerae ...

INFECTION AND IMMUNITY, Jan. 2007, p. 122–126 0019-9567/07/$08.00⫹0 doi:10.1128/IAI.01190-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 1

Temporal Quorum-Sensing Induction Regulates Vibrio cholerae Biofilm Architecture䌤 Zhi Liu, Fiona R. Stirling, and Jun Zhu* Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received 28 July 2006/Returned for modification 8 September 2006/Accepted 22 October 2006

Vibrio cholerae, the pathogen that causes cholera, also survives in aqueous reservoirs, probably in the form of biofilms. Quorum sensing negatively regulates V. cholerae biofilm formation through HapR, whose expression is induced at a high cell density. In this study, we show that the concentration of the quorum-sensing signal molecule CAI-1 is higher in biofilms than in planktonic cultures. By measuring hapR expression and activity, we found that the induction of quorum sensing in biofilm-associated cells occurs earlier. We further demonstrate that the timing of hapR expression is crucial for biofilm thickness, biofilm detachment rates, and intestinal colonization efficiency. These results suggest that V. cholerae is able to regulate its biofilm architecture by temporal induction of quorum-sensing systems. Vibrio cholerae is a gram-negative, facultative pathogen that is the causative agent of cholera, a devastating diarrheal disease that affects millions of people in the developing world each year (5). Between epidemics, V. cholerae organisms live in marine, estuarine, and freshwater environments in association with zooplankton, phytoplankton, crustaceans, insects, and plants (3, 12). Various studies have suggested that biofilm-mediated attachment to abiotic and biotic surfaces may be important for V. cholerae survival in the environment (28, 29, 33). Biofilm formation in V. cholerae is a multistep developmental process that is controlled by several regulatory pathways (28). The surface attachment of V. cholerae activates the transcription of the vps (Vibrio polysaccharide synthesis) genes that are responsible for synthesis of the VPS exopolysaccharide, the major component of the biofilm matrix (13, 24, 33). The regulation of VPS synthesis has been partially elucidated through the work of several groups. Environmental signals, such as monosaccharides, nucleosides, and bile salts, have been identified as activators of vps gene transcription and biofilm formation (10, 11, 13). VpsT, VpsR, and VieA are additional regulators of biofilm formation that respond to as-yet-unidentified environmental signals (2, 26, 31). In addition, quorum sensing also negatively regulates biofilm formation by repressing the expression of the vps operon (9, 34). Quorum sensing is a signaling process by which single-celled bacteria are able to produce and respond to small diffusible molecules called autoinducers, which accumulate as cell density increases and regulate the expression of a range of genes that control various physiological functions (6, 20, 27). The quorum-sensing system in V. cholerae has been shown to respond to at least two autoinducer molecules (21, 23, 34): CAI-1 and AI-2. CAI-1, whose structure is yet to be solved, is produced by CqsA and plays a major role in the regulation of biofilm formation. AI-2

is a furanosyl borate diester synthesized by LuxS that is also produced by many other bacteria (30). In contrast to CAI-1, AI-2 is largely dispensable in biofilm regulation (30). The accumulation of these autoinducers modulates the activity of a central regulator, LuxO, via the membrane receptors CqsS and LuxPQ (21). At low cell densities, LuxO actively represses the expression of another key quorum-sensing regulator, HapR, by activating the expression of a set of small RNAs which destabilize hapR mRNA (15, 16). At high cell densities, LuxO is inactivated and, thus, hapR expression is activated. Quorumsensing-deficient mutants, such as the hapR and cqsA mutants, form thicker biofilms than do their wild-type isotypes (9, 34). These quorum-sensing-deficient mutants also experience difficulty in detaching from biofilm structures, and it was hypothesized that this may result in the decreased colonization efficiency observed in these strains (34). MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. The Escherichia coli and V. cholerae strains used in this study are listed in Table 1 and were propagated in LB containing appropriate antibiotics at 37°C, unless otherwise noted. The cqsA-lacZ transcriptional fusion reporter was constructed by cloning the cqsA promoter region and lacZ of V. cholerae into pBBR1-MCS4 (14), and the construct was subsequently introduced into V. cholerae C6706lacZ. The inducible hapR plasmid was constructed by cloning the hapR coding sequence into pBAD24 (8), resulting in pZL37. The plasmids were then transformed into V. cholerae strains. To induce hapR, 0.1% arabinose was added to the medium at different time points. The hapR-Kmr transcriptional reporter was constructed by cloning a PCR-amplified 5⬘-end fragment of hapR overlapped with the promoterless kanamycin (Km) resistance gene into pGP704. The resulting plasmid, pJZ235, was then introduced into V. cholerae and screened for homologous recombination events. Biofilm formation assays. V. cholerae strains were grown on LB agar plates overnight and resuspended in LB broth at an optical density at 600 nm of ⬃0.6. Five milliliters of a 1:100 dilution of this suspension was then inoculated into 50-ml Falcon tubes containing 22-by-22-mm coverslips. Biofilms were formed on the coverslips at the air-liquid interface by allowing these cultures to stand for the time indicated at room temperature, which is close to the temperature experienced by V. cholerae in natural reservoirs (28). At the time points indicated, the coverslips were taken out and washed in phosphate-buffered saline buffer. Biofilm structures were disrupted by vortexing the coverslips in phosphate-buffered saline in the presence of glass beads (1 mm), and bacterial number was determined by serial dilutions. Biofilm development was quantified by crystal violet as described previously (35). Biofilm volume was estimated by measuring the aver-

* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. Phone: (215) 573-4104. Fax: (215) 898-9557. E-mail: junzhu @mail.med.upenn.edu. 䌤 Published ahead of print on 30 October 2006. 122

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VOL. 75, 2007 TABLE 1. Strains and plasmids used in this study Strain or plasmid

Relevant characteristic(s)

V. cholerae strains C6706 C6706lacZ MM194 MM920 JZV256 Plasmids pBAD24 pBBR1-MCS4 pGP704 pBB1 pJZ318 pJZ239 pZL37 pJZ235 pJZ241

Source or reference

El Tor, wild type lacZ deletion hapR deletion cqsA luxQ; CAI-1 bioassay strain PhapR-Kmr integrated in hapR locus

25 35 35 21

Cloning vector Cloning vector R6K origin vector, mob RP4 luxCDABE of V. harveyi on a cosmid Pvps(vc0930)-luxCDABE Promoterless lacZ of V. cholerae in pBBR1-MCS4 PBAD-hapR in pBAD24 PhapR-Kmr in pGP704 PcqsA-lacZ in pJZ239

8 14 22 21

This study

11 This study This study This study This study

age depth of biofilms by confocal scanning laser microscopy using an MRC-1024 confocal microscope (Bio-Rad) according to preciously reported protocols (1). Detachment assays were performed as described previously (34) except for using coverslips instead of glass beads. CAI-1 production assays. Biofilms were formed as described above, and at different time points, coverslips were withdrawn from the tubes and rinsed briefly with fresh LB twice. The coverslips were then placed in tubes containing 1 ml of fresh LB and disrupted using glass beads. These samples, together with planktonic cultures, were then subjected to CAI-1 production assays performed as previously described (21, 34). Luminescence assays. V. cholerae strains containing either luxCDABE from Vibrio harveyi on a cosmid (pBB1) or a plasmid harboring Pvps-lux (pJZ318) were allowed to form biofilms as described above. At different time points, both planktonic and disrupted biofilm-associated cells were collected for luminescence measurements using a Bio-Tek Synergy HT spectrophotometer and CFU counting. Relative light units are defined as 106 light units/CFU ml⫺1. ␤-Galactosidase activity assays. Biofilms of C6706lacZ containing the cqsAlacZ reporter (pJZ241) were formed and disrupted as described above. Biofilmassociated and planktonic cells were collected and assayed for ␤-galactosidase activity, which was normalized against the optical density at 600 nm and reported as Miller units as previously described (19). Temporal hapR expression using the hapR-Kmr reporter. Strain JZV256 that carries a chromosomal hapR-Kmr fusion was allowed to form biofilms as described above. At the various time points indicated, coverslips were withdrawn from the cultures and rinsed briefly with LB. Coverslips were then placed in a tube containing 1 ml of fresh LB and disrupted by vortexing with glass beads. Samples of planktonic and disrupted biofilm-associated cells were then added into fresh LB in the absence or presence of 500 ␮g/ml kanamycin and further incubated for 10 min at 37°C. This treatment is sufficient to kill 100% of V. cholerae cells that do not carry any Kmr gene. After the kanamycin treatment, the numbers of surviving cells were determined by serial dilution. hapR expression was defined as the percentage of Km-surviving cells versus non-Km-treated cells. Infant mouse colonization assays. Biofilms of C6706lacZ and MM194 containing pZL37 were formed on microglass beads (50 to 100 ␮m; Polysciences) as previously described (34), except that 0.1% arabinose was added at the different time points indicated. The biofilms on beads were used as an inoculum, and the infant mouse colonization assay was performed as previously described (7).

RESULTS AND DISCUSSION To further investigate the role that quorum sensing plays in the regulation of biofilm formation, we grew V. cholerae El Tor C6706 in LB medium at 22°C and assayed for the accumulation

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of the major quorum-sensing molecule CAI-1 in biofilms and planktonic cultures at different time points. Biofilm volume was estimated by measuring the average thickness of the biofilm structures using confocal laser microscopy (data not shown) (34). Figure 1A shows that over time, the concentration of CAI-1 produced by biofilm-associated cells was 104-fold higher than that in planktonic cultures. To test whether this higher concentration of CAI-1 was due to an increase in the transcription of the CAI-1 synthase gene cqsA in biofilms, we introduced a plasmid containing a cqsA-lacZ transcriptional fusion into V. cholerae and assayed the ␤-galactosidase activity of biofilm-associated and planktonic cells. No significant difference in cqsA activity between biofilm and planktonic cells was detected (Fig. 1B), suggesting that biofilm cells do not produce more CAI-1 per se; rather, as the volume of biofilms is very small, the cell density of biofilms is much higher than that of free-living cells. Alternatively, it is possible that the biofilm matrix may restrict the diffusion of these small molecules to achieve a localized high autoinducer concentration. We then investigated the consequences of a high concentration of CAI-1 in biofilm cells. In general, when autoinducers

FIG. 1. V. cholerae autoinducer CAI-1 production and CAI-1 synthase gene cqsA expression in biofilm and planktonic cells. (A) CAI-1 production. Bacteria were grown in LB at 22°C. Planktonic cells (triangles) and biofilm-associated cells (squares) were then separated and subjected to CAI-1 bioassays (21, 34). Biofilm volume was estimated using confocal laser microscopy, and the culture volume was used as the planktonic cell volume. (B) cqsA-lacZ expression. A strain containing a plasmid-encoded cqsA-lacZ transcriptional fusion (pJZ241) was grown in LB, and at different time points, planktonic (white bars) and biofilm (gray bars) cells were collected and assayed for ␤-galactosidase activity (19). The results are representative of three experiments ⫾ standard deviations (error bars).

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FIG. 2. Quorum-sensing activation in planktonic and biofilm states. (A) Light production. A strain containing the cosmid pBB1 was grown in LB at 22°C. At the times indicated, light production was measured from planktonic (triangles) and biofilm (squares) cells. The number of bacteria in each sample was determined by serial dilution and plating on LB plates with appropriate antibiotics. (B) hapR expression. V. cholerae isolates containing a chromosomally integrated hapR-Kmr transcriptional fusion, JZV256, were grown in LB at 22°C. Planktonic (triangles) and biofilm (squares) cells collected at various time points were treated with kanamycin (500 ␮g/ml) for 10 min, and viable cells were counted by plating on LB plates. The results are representative of three experiments ⫾ standard deviations.

accumulate to their threshold concentrations, the quorumsensing system is induced. Thus, it is possible that biofilmassociated cells reach their “quorum” earlier than do planktonic cells because of the faster accumulation of autoinducers in the biofilm. To test this possibility, we introduced a cosmid containing the V. harveyi luxCDABE operon, whose promoter can be activated by V. cholerae quorum-sensing systems (21), into V. cholerae and used light production as an indicator of quorum-sensing-dependent gene expression in planktonic and biofilm cells. Overnight cultures were diluted, and light production per cell was measured during subsequent growth. Figure 2A shows that planktonic cells produced light in a cell density-dependent pattern. This U-shaped curve is typical of a quorum-sensing-dependent phenotype, with the initial decrease in luminescence per cell resulting from the dilution of the culture causing a drop in the extracellular autoinducer levels to below the threshold concentration required for the stimulation of lux expression; subsequently, as cell density increases, lux is induced over time due to the accumulation of new autoinducers (21). Biofilm-associated cells, however, ex-

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hibited an earlier and higher induction of Lux activity, indicating that the quorum-sensing system is activated earlier and to higher maximum levels in V. cholerae biofilms than in planktonic cultures. In V. cholerae, quorum-sensing regulation acts through HapR, the key positive quorum-sensing regulator. We therefore examined hapR expression during biofilm growth. We fused the hapR promoter with a kanamycin resistance gene and integrated this construct into the V. cholerae chromosome by homologous recombination. The percentage of kanamycinresistant cells during cell growth corresponded to hapR expression as judged by the hapR-lacZ reporter (data not shown). Moreover, both planktonic cells and biofilm-associated bacteria that were disrupted prior to kanamycin exposure, without the hapR-Kmr insertion, were readily killed by kanamycin during growth (data not shown), indicating that kanamycin resistance was not due to any spontaneous mutations. The advantage of using this reporter is that it is very sensitive in detecting gene expression at the single-cell level. As expected, the percentage of kanamycin-resistant biofilm cells increased rapidly and hapR expression reached its maximal level after 8 h of growth, while the percentage of planktonic cells expressing hapR was much lower than that of biofilm cells (Fig. 2B). Taken together, these data suggest that the V. cholerae quorum-sensing system is activated earlier and to higher levels in biofilms than in planktonic cultures. Previous studies have shown that quorum sensing negatively regulates biofilm formation in V. cholerae by repressing vps expression (9, 32, 34). To further investigate the impact of fast induction of quorum sensing in biofilm-associated cells on biofilm formation, we constructed a plasmid containing the hapR gene under the control of an arabinose-inducible PBAD promoter (8) and introduced this into a hapR deletion strain. The resulting strain, in addition to wild-type and hapR strains containing a vector control, was assayed for biofilm thickness after 18 h of growth. We added 0.1% arabinose to the medium at a number of different time points to artificially turn on quorumsensing regulation at various times during the 18 h of growth. hapR mutants formed much thicker biofilms than those of wild-type strains (Fig 3A), as previously demonstrated (9, 34). When hapR expression was induced at early time points (0 and 4 h) by the addition of arabinose, the resultant biofilms were similar to those formed by the wild-type strain (Fig. 3A). However, when hapR was induced in cultures after 8 h of growth, the biofilms formed were as thick as those observed with the hapR mutant, suggesting that the timing of hapR expression is critical for controlling biofilm formation. Interestingly, strains with early induction of hapR expression did not reduce biofilm formation, but rather formed wild-type-like biofilms. It is therefore possible that other cell density-dependent factors are involved in the process of HapR repression of biofilm formation. To further investigate the effect of the timing of hapR expression, we introduced a plasmid containing Pvps-lux (11) into the above-mentioned strains and investigated how the timing of hapR induction affected vps expression. Again, when hapR was induced by arabinose at early time points, vps expression in biofilms was similar to that observed in wild-type strains (Fig. 3B). When hapR was induced later than 8 h, the expression of vps was comparable to that of the hapR mutant. Quorum sensing negatively regulates biofilm formation in V. cholerae, preventing the formation of biofilms that are too

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FIG. 3. The timing of hapR expression affects biofilm formation and vps expression. hapR mutants containing either the empty vector pBAD24 (white bars) or pZL37 (containing PBAD-hapR) (gray bars) were grown in LB. Arabinose (0.1%) was added at the times indicated. After an 18-h total incubation, biofilm mass (A) and vps expression (B) were measured and compared to those of a wild-type strain containing the vector control grown under the same conditions. The results are representative of three experiments ⫾ standard deviations (error bars).

thick. It was hypothesized that thicker biofilms reduce the colonization efficiency of V. cholerae because it is difficult for bacteria to detach from overly thick biofilm structures and colonize new sites (34). As we have demonstrated that biofilm thickness is modulated by the timing of quorum sensing induction in V. cholerae, we predicted that the timing of hapR induction is also important for biofilm detachment and colonization efficiency. We thus measured the detachment rate of the hapR mutant containing a plasmid harboring PBAD-hapR (pZL37) from biofilms when arabinose is added at different time points. Biofilms with the induction of hapR at early time points (0 to 4 h) displayed detachment rates similar to that of the wild type, while biofilms with hapR induction at 8 h or later showed lower detachment rates similar to that exhibited by the hapR mutant (Fig. 4A). The lower detachment rate from biofilms by late induction of hapR also correlated with colonization efficiency (Fig. 4B). When we performed infant mouse competition assays using wild-type biofilms and biofilms with hapR induced at different time points, we found that biofilms with late induction of hapR showed a greater-than-10-fold reduction in colonization capacity, similar to that of hapR biofilms (34). This suggests that bacterial cells may disperse more

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FIG. 4. The timing of hapR expression affects biofilm detachment and colonization efficiency. (A) Wild-type (wt) V. cholerae and the hapR mutant containing either pBAD24 or pZL37 (containing PBADhapR) were grown in LB. Arabinose (0.1%) was added at the time points indicated. After an 18-h total incubation, biofilm-associated cells were transferred to fresh LB medium and the number of bacterial cells entering the planktonic phase after 30 min was measured (34). The results are representative of three experiments ⫾ standard deviations (error bars). (B) Eighteen-hour biofilms of hapR mutants containing pZL37 that were grown in LB with arabinose added at the times indicated were mixed with wild-type biofilms at an approximately 1:1 ratio and intragastrically inoculated into 6-day-old infant CD-1 mice to perform in vivo competition assays (7). After a 20-h period of colonization, intestinal homogenates were collected, and the ratio of mutant-to-wild-type bacteria was determined.

slowly from thicker biofilm structures in vivo, thus resulting in reduced colonization efficiency. This hypothesis remains to be proven. In this study, we have shown that the V. cholerae quorumsensing system was activated faster in biofilm-associated cells than in free-living bacteria, due to higher autoinducer concentration in biofilms. This action results in the formation of normal biofilm structures from which bacteria can rapidly disperse in order to efficiently colonize the host when necessary. This process could be critical since V. cholerae may enter hosts from environmental reservoirs in the form of biofilms. Evidence to support this process is provided by a study showing that the number of cholera cases in a Bangladeshi village

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dramatically declined when V. cholerae-associated macroparticles were removed from drinking water using a crude method of filtration through sari cloth (4). Furthermore, free-living V. cholerae are highly sensitive to low pH, while V. cholerae biofilms are more acid resistant, resulting in the hypothesis that this increased resistance to acid may promote survival during passage through the stomach (34). Upon reaching the intestine, it may be critical for V. cholerae to leave the biofilm structure in order to colonize the intestinal surface, as failure to do so reduces colonization efficiency (34). Therefore, an early induction of the quorum-sensing machinery in biofilms to tightly control biofilm thickness might be advantageous in promoting this process. Quorum-sensing systems in V. cholerae are tightly regulated. In addition to autoinducer-regulated LuxO activity, other components are also involved in regulating quorum-sensing systems, including the VarS/VarA two-component sensory system that comprises an additional quorum-sensing-dependent regulatory input (15), the regulator VqmA that modulates hapR expression (18), and HapR autorepression (17). Our findings of early quorum sensing induction in biofilms may represent yet another regulatory mechanism to ensure appropriate temporal and spatial gene expression in V. cholerae. ACKNOWLEDGMENTS We thank our lab members for helpful discussion and critically reviewing the manuscript. This study was supported by the NIH/NIAID K22 award (AI060715), the MOE Major Fund (306009), and the McCabe award. REFERENCES 1. Bomchil, N., P. Watnick, and R. Kolter. 2003. Identification and characterization of a Vibrio cholerae gene, mbaA, involved in maintenance of biofilm architecture. J. Bacteriol. 185:1384–1390. 2. Casper-Lindley, C., and F. H. Yildiz. 2004. VpsT is a transcriptional regulator required for expression of vps biosynthesis genes and the development of rugose colonial morphology in Vibrio cholerae O1 El Tor. J. Bacteriol. 186:1574–1578. 3. Colwell, R. R., and A. Huq. 1994. Environmental reservoir of Vibrio cholerae. The causative agent of cholera. Ann. N. Y. Acad. Sci. 740:44–54. 4. Colwell, R. R., A. Huq, M. S. Islam, K. M. Aziz, M. Yunus, N. H. Khan, A. Mahmud, R. B. Sack, G. B. Nair, J. Chakraborty, D. A. Sack, and E. Russek-Cohen. 2003. Reduction of cholera in Bangladeshi villages by simple filtration. Proc. Natl. Acad. Sci. USA 100:1051–1055. 5. Faruque, S. M., M. J. Albert, and J. J. Mekalanos. 1998. Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Microbiol. Mol. Biol. Rev. 62:1301–1314. 6. Fuqua, W. C., S. C. Winans, and E. P. Greenberg. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176:269–275. 7. Gardel, C. L., and J. J. Mekalanos. 1996. Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression. Infect. Immun. 64:2246–2255. 8. Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121–4130. 9. Hammer, B. K., and B. L. Bassler. 2003. Quorum sensing controls biofilm formation in Vibrio cholerae. Mol. Microbiol. 50:101–104. 10. Haugo, A. J., and P. I. Watnick. 2002. Vibrio cholerae CytR is a repressor of biofilm development. Mol. Microbiol. 45:471–483.

Editor: A. Camilli

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