A Vibrio Cholerae Protease Needed for Killing of Caenorhabditis Elegans Has a Role in Protection from Natural Predator Grazing

A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing

Karolis Vaitkevicius*, Barbro Lindmark*, Gangwei Ou†, Tianyan Song*, Claudia Toma‡, Masaaki Iwanaga‡, Jun Zhu§, Agneta Andersson¶, Marie-Louise Hammarstro¨ m†, Simon Tuckʈ, and Sun Nyunt Wai*,**

Departments of *Molecular Biology and ¶Ecology and Environmental Science, and ʈUmeå Center for Molecular Pathogenesis, Umeå University, SE-90187 Umeå, Sweden; †Department of Clinical Microbiology, Division of Immunology, Umeå University, SE-90185 Umeå, Sweden; ‡Division of Bacterial Pathogenesis, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan; and §Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved May 2, 2006 (received for review March 2, 2006) Vibrio cholerae is the causal bacterium of the diarrheal disease of the gene encoding the HapR regulatory protein (12–14). The cholera, and its growth and survival are thought to be curtailed by QS regulatory cascade appears to be central to a number of bacteriovorous predators, e.g., ciliates and flagellates. We ex- virulence-related phenotypes, including CT production, biofilm plored Caenorhabditis elegans as a test organism after finding that formation, and protease secretion. QS may also be important in V. cholerae can cause lethal infection of this nematode. By reverse the environmental phase of the V. cholerae life cycle (12). genetics we identified an extracellular protease, the previously Extracellular proteases are considered as putative virulence uncharacterized PrtV protein, as being necessary for killing. The factors in several microbial diseases, including those caused by killing effect is associated with the colonization of bacteria within Pseudomonas aeruginosa (15) and V. cholerae (16, 17). Recently, the Caenorhabditis elegans intestine. We also show that PrtV is Matz et al. (18) observed that a QS mutant of P. aeruginosa had essential for V. cholerae in the bacterial survival from grazing by a significantly reduced antipredator fitness compared with iso- the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena genic WT strains. pyriformis. The PrtV protein appears to have an indirect role in the Although cholera is commonly considered to be a noninflam- interaction of V. cholerae with mammalian host cells as judged matory secretory disease, there are indications of some inflam- from tests with tight monolayers of human intestinal epithelial matory component(s) to the disease (19). In clinical trials most cells. Our results demonstrate a key role for PrtV in V. cholerae V. cholerae vaccine candidates still exhibit reactogenicity (20). interaction with grazing predators, and we establish Caenorhab- The mechanisms of reactogenicity caused by V. cholerae vaccine ditis elegans as a convenient organism for identification of V. candidates lacking e.g., CT gene, however, are still not known. cholerae factors involved in host interactions and environmental Levine and Noriega (21) suggested that an unidentified entero- persistence. toxin could cause reactogenic symptoms in the absence of CT but whose existence had been masked by the presence of CT. cholera ͉ host interactions ͉ environmental persistence The nematode Caenorhabditis elegans has been used success- fully as an invertebrate infection model to screen for virulence holera continues to be a major public and individual health factors of several human pathogens, e.g., P. aeruginosa, Salmo- Cproblem, especially in those regions of the world where it is nella enterica, and Serratia marcescens (22–28). endemic. Colwell (1) first hypothesized that coastal waters were Here, we establish that Caenorhabditis elegans is a useful an important reservoir of Vibrio cholerae. Huq et al. (2) reported model system for identifying and assessing factors other than CT that V. cholerae O1 cells could be observed to be attached to a from V. cholerae that may contribute to bacterial survival and variety of phytoplankton and zooplankton species. The incidence persistence in the environment and thereby can be important for and severity of epidemics have been linked to salinity, water pathogenesis and damage to host organisms. temperature, turbidity, and plankton blooms (3, 4). Cholera epidemics occur in a regular seasonal pattern. It has been Results and Discussion suggested that during interepidemic periods V. cholerae exists in V. cholerae Kills Caenorhabditis elegans upon Colonization of the an unexplained ecological association with aquatic organisms Intestinal Tract. We first tested the ability of V. cholerae Ol El Tor (5). During the environmental phase, V. cholerae resides in Inaba strain C6706 to kill Caenorhabditis elegans. L4 hermaph- diverse aquatic environments, often in association with marine rodites raised on OP50, a standard Escherichia coli strain used plankton (6). The association of V. cholerae with zooplankton has for cultivating Caenorhabditis elegans (29), were transferred onto proven to be a key factor in deciphering the global nature of the lawns of V. cholerae strain C6706. The nematodes died within cholera epidemics (7). In such natural bacterioplankton com- Ϸ5 days, indicating that V. cholerae exerted a slow killing effect munities V. cholerae and other bacteria are also at the base of the (Fig. 1). Routinely the parental worms were moved to a new pelagic microbial food web (8). Bacterial growth and survival are plate with V. cholerae every 2 days to distinguish between parents subject to constraint by bacteriovorous predators, e.g., protozoa and progeny. To examine whether diffusible compounds were such as ciliates and flagellates (9, 10). Little has been known responsible for the killing effect, V. cholerae strain C6706 was about mechanisms and adaptations of bacteria to reduce grazing grown on nitrocellulose filters (pore diameter 0.2 ␮m) covering mortality compared with adaptations toward abiotic factors the surface of the nematode growth medium plates. After (substrate, temperature, pH, etc.) (11). V. cholerae expresses well characterized factors to establish and cause disease in the mammalian host, including cholera toxin Conflict of interest statement: No conflicts declared. (CT) and toxin-coregulated pili (Tcp). It has been shown that This paper was submitted directly (Track II) to the PNAS office. quorum sensing (QS) plays a role in the regulation of virulence Abbreviations: CT, cholera toxin; Tcp, toxin-coregulated pili; QS, quorum sensing. in V. cholerae (12). At least three autoinducer signaling circuits **To whom correspondence should be addressed. E-mail: [email protected]. function through the action of LuxO, leading to the repression © 2006 by The National Academy of Sciences of the USA

9280–9285 ͉ PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601754103 Downloaded by guest on September 25, 2021 on E. coli (Fig. 1). To establish whether a continuous or a transient contact was necessary, worms were transferred to the lawn of V. cholerae C6706, allowed to feed for 24 h, and then, after three washes with sterile M9 buffer to remove surface- located bacteria, placed onto lawns of E. coli OP50. Locomotion, pharyngeal pumping, and egg-laying ability appeared reduced for the first 48–72 h. However, over the next 24–48 h, all of these activities gradually improved. Our results show that the killing effect requires direct and continuous contact with live bacteria, which shows resemblance to the case of P. aeruginosa infection in Caenorhabditis elegans as described (28). To determine whether the ability to kill Caenorhabditis elegans is a common property of different serotypes of V. cholerae strains, we tested several clinical isolates in the nematode killing Fig. 1. Characterization of the killing of Caenorhabditis elegans by V. cholerae. Kinetics of killing of worms (n ϭ 50) with V. cholerae strain C6706, assay. Representative strains of the serotypes Classical Inaba, E. coli OP50 (P Ͻ 0.0001), UV-killed C6706 (P Ͻ 0.0001), and a plate where Classical Ogawa, and non-O1 non-0139 were found to kill (Fig. C6706 had been grown on a ﬁlter and then removed (P Ͻ 0.0001). 7, which is published as supporting information on the PNAS web site). V. cholerae does not, however, seem to be inherently toxic to Caenorhabditis elegans because we also found some growth, the filters were removed and worms were placed on the strains that did not kill the nematodes (Fig. 7). plates. After Ϸ12 h on such a plate the worms were transferred To follow the fate of the bacteria upon ingestion by the worm, to the plate containing OP50. As seen in Fig. 1, no killing effect we used derivatives of the bacteria (Table 1, which is published was observed in this experiment, suggesting that the killing of as supporting information on the PNAS web site) expressing the Caenorhabditis elegans by V. cholerae requires direct contact with GFP. V. cholerae strain C6706͞pSMC2 was found to be as the bacteria. virulent as C6706 in the killing assay (data not shown). When L4 To investigate whether bacteria needed to be alive to cause worms were placed on the GFP-labeled V. cholerae for 12 h,

killing, L4 worms were placed on C6706 bacteria killed by UV intact V. cholerae curved rods were seen to accumulate in both MICROBIOLOGY irradiation. In this case, the worms survived as long as those fed the pharynx and the lumen of the intestine (Fig. 2 A–D).

Fig. 2. Visualization of V. cholerae colonizing the intestinal lumen of Caenorhabditis elegans. Nomarski (A, C, E, and G) and ﬂuorescence (B, D, F, and H) photomicrographs of worms fed for 12 h with V. cholerae C6706͞pSMC2 (A–D)orE. coli OP50͞pSMC2 (E–H). The white arrows in B–D show V. cholerae bacilli. Black arrows in C (numbered 1 and 2) show the apical membrane and the basal membrane of intestinal tract, respectively. Note that C6706͞pSMC2 bacteria were present both in the pharynx (B) and the intestinal lumen (D).

Vaitkevicius et al. PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 ͉ 9281 Downloaded by guest on September 25, 2021 V. cholerae Worm Killing Effect Is Mediated by LuxO-Regulated Genes in the QS Pathway. The expression of known virulence factors of V. cholerae is regulated in a coordinated fashion in response to QS (12). Transcription of the regulatory gene hapR leads to reduced biofilm formation, reduced CT expression, and increased protease expression. Intriguingly, the strain carrying a hapR gene deletion was strongly attenuated in its ability to kill worms (Fig. 3A). The attenuation was not caused by, e.g., a growth defect of hapR because we detected no difference in growth rate when we compared it with WT V. cholerae C6706. Transcomplementation of the hapR deficiency by introducing a plasmid (phapR) carrying a WT allele restored the killing effect (Fig. 3A). The luxO gene encodes a negative regulator of hapR transcription (12). In the absence of LuxO, hapR is constitutively transcribed, leading to increased production of several secreted proteins. We found that luxO mutant V. cholerae killed worms more quickly (within 3 days) than WT (Fig. 3A). A luxO hapR double mutant was attenuated, indicating that the effect of luxO depends on functional hapR (Fig. 3A). Similarly, we investigated the effect of mutations in the upstream part of the QS regulatory pathways. Mutations abolishing the synthesis of the receptors of the two known QS compounds in V. cholerae, i.e., the Cqs and AI-2 systems were tested (Fig. 3B). The cqsA (gene for synthesis of autoinducer 1) mutant bacteria were attenuated, whereas the luxQ (gene for receptor of autoinducer 2) mutant bacteria could cause lethal infection of worms as efficiently as WT. The result with the cqsA mutant is consistent with the hapR mutant results described above because it is known that a cqsA mutation results in strongly reduced hapR expression (30). When both systems were abolished (i.e., ⌬cqsA ⌬luxQ and ⌬cqsA ⌬luxS) the V. cholerae were completely attenuated.

V. cholerae Lethal Infection of the Caenorhabditis elegans Depends on a HapR-Regulated Protease. Because two processes known to be regulated by hapR, biofilm formation and CT expression, did not seem to be required for killing of Caenorhabditis elegans,we investigated whether proteases regulated by the LuxO–HapR Fig. 3. Effect of V. cholerae mutations on nematode killing. (A) The LuxO– pathway might be involved. Analysis of supernatants from broth HapR regulatory network inﬂuences V. cholerae nematode infection. V. cultures showed that several secreted proteins were more abun- cholerae mutants (n ϭ 50) ⌬hapR (P Ͻ 0.0001), ⌬luxO (P Ͻ 0.0001), ⌬hapR⌬luxO (P Ͻ 0.0001), tcpA::Km (P ϭ 0.2), ⌬toxR (P ϭ 0.0001), and dant in the case of the luxO mutant than from WT V. cholerae ⌬hapR͞phapR (P ϭ 0.7) were compared with the WT strain C6706 (n ϭ 50). The (Fig. 4). The luxO mutant also had clearly higher levels of ⌬ctx mutant strain E4 was compared with the otherwise isogenic WT strain protease activity (Fig. 9, which is published as supporting E7946 (n ϭ 50, P ϭ 0.3). (B) Kinetics of killing of Caenorhabditis elegans by V. information on the PNAS web site). Furthermore, SDS͞PAGE cholerae QS-deﬁcient mutants. The WT V. cholerae strain C6706 (n ϭ 50) was analyses indicated that the most abundant protein in the super- compared with mutant derivatives (n ϭ 50): ⌬cqsA (P Ͻ 0.0001), ⌬luxQ (P ϭ natant (from either luxO or WT) was HA͞P (Fig. 4). 0.1), ⌬cqsA⌬luxQ (P Ͻ 0.0001), ⌬cqsA⌬luxS (P Ͻ 0.0001), ⌬hapA (P ϭ 0.6), HA͞P (also denoted HapA) is encoded by the hapA gene, is ⌬prtV (P Ͻ 0.0001), and transcomplemented strain ⌬prtV͞pPrtV (P ϭ 0.06). regulated by hapR, and is the major extracellular protease (31). However, when hapA mutant V. cholerae were tested in the Caenorhabditis elegans killing assay we found no attenuation in However, in worms feeding on OP50͞pSMC2 we did not observe comparison with WT (Fig. 3B). The culture supernatant from any fluorescent bacteria (Fig. 2 E–H). the hapA mutant contained some proteins not found in the supernatants from the hapR mutant (Fig. 4). We identified the Killing of Caenorhabditis elegans by V. cholerae Does Not Depend on three most abundant putative HapR-regulated proteins detected CT or Biofilm Formation. The ctx and tcpA mutant strains were able in the supernatant from the hapA mutant V. cholerae by mass Materials and Methods to kill Caenorhabditis elegans worms as efficiently as the WT spectrometry (see ). This analysis revealed that the protein bands corresponded to proteins encoded by the bacteria (Fig. 3A), indicating that CT and Tcp are not absolutely ORFs VCA0812, VCA0813, and VCA0223 (32). The protein required for lethal infection. In the case of the toxR mutant, products are a leucine aminopeptidase-related protein, leucine altered in the regulatory gene affecting both ctx and tcpA aminopeptidase (Lap) (33), and the PrtV protease (34), respec- expression, there was a small, but significant, reduction in killing. tively. We refer to the ORF VCA0812 as lapX. Recent tran- The ability to form biofilm has been associated with V. scriptome analyses indicated that prtV gene transcription is cholerae pathogenicity in some systems (12). We found, however, affected by a hapR mutation (30). that some of the biofilm-deficient mutants that we had obtained To determine whether any of the identified proteins above in a separate study with the Ol El Tor Inaba strain A1552 were played a role in worm killing we tested ⌬prtV, ⌬lap, and ⌬lapX able to kill Caenorhabditis elegans with WT efficiency (Fig. 8, mutants in the Caenorhabditis elegans assay. The ⌬prtV mutant which is published as supporting information on the PNAS web showed complete attenuation in comparison with the WT (Fig. site). 3B; P Ͻ 0.0001). To assess whether the attenuation effect

9282 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601754103 Vaitkevicius et al. Downloaded by guest on September 25, 2021 Fig. 4. Identification of secreted V. cholerae proteases. Coomassie brilliant blue-stained gel after SDS͞PAGE analysis of secreted proteins from different derivatives of the V. cholerae strain C6706: ⌬hapA, WT, ⌬prtV, ⌬luxO, ⌬luxO ⌬prtV, ⌬luxO ⌬hapR, and ⌬hapR strains. Equal amounts of trichloracetic acid-precipitated samples of supernatants from bacterial cultures at the same OD were tested. Arrows indicate proteins subjected to mass spectrometric analysis. Molecular size markers are shown in lane 8. The identity of the HapA protein was confirmed by immunoblot analysis (data not shown) using anti- HA͞P polyclonal antiserum (33). MICROBIOLOGY depended on the prtV mutant locus per se, we performed a transcomplementation test by introducing a plasmid (pKVA232) Fig. 5. Survival of ciliates and flagellates grazing on WT and mutant V. harboring the cloned WT allele of the prtV gene. The comple- cholerae. The ciliate T. pyriformis (A) and flagellates Cafeteria roenbergensis mentation restored the WT killing effect (Fig. 3B), ruling out (B) were allowed to graze on different V. cholerae C6706 strains, and the that the mutation indirectly caused attenuation by polar effects protozoa viability was monitored after 4 days as described in Materials and from the prtV deletion mutation. Neither of the ⌬lap and ⌬lapX Methods. mutants showed significant attenuation (Fig. 10, which is published as supporting information on the PNAS web site). The PrtV Is Important for V. cholerae Inhibition of Natural Bacteriovorous ⌬ prtV mutation was also found to abolish most of the killing Predators. To test whether predator grazing, and thereby V. ⌬ effect of the luxO derivative. Furthermore, the cloned prtV cholerae persistence͞survival, would be influenced by alteration gene resulted in an increased killing effect by the WT V. cholerae of prtV expression we carried out studies with bacteriovorous strain C6706, and it restored a significant killing effect in the case unicellular organisms typically found in marine plankton com- of the hapR mutant derivative (Fig. 10). We conclude that this munities. We used a series of mutant bacterial strains in tests reverse genetics approach identified the PrtV protein as a factor with the flagellate Cafeteria roenbergensis and the ciliate Tetra- required for the V. cholerae lethal infection of Caenorhabditis hymena pyriformis. The results clearly indicated that V. cholerae elegans. caused a reduction in protozoan activity and survival in a fashion This PrtV protein is one of the less abundant of the secreted that depended on the QS regulon (Fig. 5 and Table 2, which is proteases, and little is known about its activity. The measure- published as supporting information on the PNAS web site). WT ments of proteolytic activity against azocasein indicated that V. cholerae caused a marked reduction in the number of viable 10–20% of total activity in culture supernatants was abolished by protozoa during prolonged cocultivation, whereas the bacterial the ⌬prtV mutation (Fig. 9). In accordance with HapA being density remained virtually unaltered. Furthermore, V. cholerae most abundant of the secreted proteases, abolishment of HapA with mutations abolishing the hapR or prtV genes appeared reduced the total protease activity to Ϸ10% of the WT level. It strongly attenuated in the predator grazing test, and most of the is possible that the different proteases might affect the activity bacteria were consumed by the predators within 3 days. In the or stability of each other. To test how protease levels might be case of the luxO mutant bacteria, on the other hand, the effect influenced and to analyze the possible synergistic effect of was more detrimental to the protozoa than that seen with the proteases on worm killing, a series of multiple deletion mutants WT V. cholerae strain (Fig. 5). was constructed. The ⌬lap ⌬lapX and ⌬hapA ⌬lap ⌬lapX com- Our findings also provide a feasible explanation for the binations resulted in a slight, barely significant, attenuation (Fig. observation recently reported by Matz et al. (35). Their results 10; P ϭ 0.04, P ϭ 0.004). The ⌬hapA ⌬lap ⌬lapX ⌬prtV mutant suggested that the V. cholerae HapR regulon had a distinctive was completely attenuated. environmental role for the production of some unidentified Also the above-mentioned V. cholerae isolates that did not kill antiprotozoal factor(s) inhibiting flagellate plankton activity and Caenorhabditis elegans (Fig. 7) carried the prtV gene locus as thus protecting the bacterial biofilm from grazing. The antipro- determined by PCR (data not shown). However, the preliminary tozoal factor(s) appeared to be some secreted product because studies of PrtV protein production by Western blot analysis cell-free supernatants of WT V. cholerae significantly reduced (unpublished data) indicated that the level of secreted PrtV was the feeding activity of predators. Our results show that the PrtV much reduced in comparison with that of strain C6706 in two of protein is such a factor. three cases. Whether or not PrtV protease activity might differ A recent report by Pukatzki et al. (36) demonstrated that some among isolates will require further biochemical studies. other secreted, not yet identified, V. cholerae protein(s) may

Vaitkevicius et al. PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 ͉ 9283 Downloaded by guest on September 25, 2021 contributing to the bacterial viability and persistence in such competitive environmental niches. How the PrtV protease evolved in V. cholerae to allow for survival in predators is not known, but evidently it is part of the HapR regulon that coordinates expression of several genes involved in environmental adaptation. It is an intriguing, albeit speculative, possibility that selection for PrtV in the environmental niche by virtue of its role in preventing predator grazing is matched by a role in keeping reactogenicity low in mammalian infections. Materials and Methods Bacterial Strains, Culture Conditions, and Plasmids. The bacterial strains and plasmids used are listed in Table 1. Bacteria were grown overnight at 37°C in LB broth supplemented, as appro- priate, with kanamycin (30 ␮g͞ml) or ampicillin (50 ␮g͞ml). Mutagenesis of the V. cholerae strain C6706 in the luxO, hapR, hapA, prtV, lap, and lapX loci were constructed by making Fig. 6. IL-8 secretion in tight monolayers of intestinal epithelial T84 cells after exposure to V. cholerae culture supernatants. Tight monolayers of T84 in-frame deletions of the entire reading frame in each case by cells (transepithelial resistance Ͼ2,000 ohm͞cm2) were tested with sterile using procedures as described (12, 38). Oligonucleotide primers ﬁltered supernatants from different V. cholerae derivatives for 18 h at which used are listed in Table 3, which is published as supporting time the tissue culture medium at the basolateral side was collected and information on the PNAS web site. analyzed for the concentration of IL-8 as described in Materials and Methods. The transepithelial resistance was decreased to 1,672 and 609 ohm͞cm2 in the Caenorhabditis elegans Maintenance. Caenorhabditis elegans WT case of the WT and ⌬prtV strain, respectively. strain Bristol N2 was routinely maintained at 20°C on nematode growth medium agar plates seeded with E. coli OP50 by standard methods (29). Antibiotics were included for bacterial strains cause cytotoxicity toward the Dictyostelium discoideum amoe- carrying plasmids. bae. The VAS secretion system, which is not involved in PrtV secretion, was shown to be required for amoebae killing. Taken Caenorhabditis elegans Killing Assays. An overnight LB broth together our findings indicate that there are different bacterial culture (100 ␮l) of the test bacterial strain was spread on a factors that contribute to how V. cholerae survives predation by 5-cm-diameter nematode growth medium agar plate and incu- different bacteriovorous organisms. bated at 30°C for 24 h before the bacterial lawn was seeded with 50 L4 stage worms of Caenorhabditis elegans WT strain Bristol Lack of PrtV May Cause Enhanced Reactogenicity to V. cholerae. The N2. Plates were incubated at room temperature (23°C) and PrtV protein did not appear to influence bacterial colonization scored for live worms every day. E. coli OP50 was used as a during infection of a mammalian host as judged by an in vivo negative control. A worm was considered dead when it no longer colonization assay with the infant mouse model. The results responded to touch. Any worms that died as a result of getting showed that the prtV mutant colonizes infant mice to the same stuck to the wall of the plate were excluded from the analysis. extent as WT (ref. 34 and unpublished data). It has been Data were subjected to statistical analyses and plotted according suggested that there may be some inflammatory component to to a Kaplan–Meier survival graph by using the program PRISM, the disease caused by V. cholerae in humans, and recently it was version 4.0 (GraphPad, San Diego). Multiple experiments (three shown that bacterial supernatants can stimulate production of to four worm-killing assays per bacterial strain) were done, and IL-8 from intestinal epithelial T84 cells in vitro (37). Some the data presented are from a representative experiment. Sur- IL-8-stimulating factor from the bacteria may thereby play a role vival curves are considered significantly different from a V. in the reactogenicity seen with V. cholerae vaccine candidates. cholerae strain C6706 when P Ͻ 0.05. We tested whether the prtV gene product might be involved directly or indirectly as an IL-8 stimulator by using bacterial Microscopy. Nematode intestinal tracts and the presence of supernatants added to the apical side of tight monolayers of the bacteria were examined by Nomarski differential interference T84 cell line. The results showed that the amount of IL-8 contrast microscopy and fluorescence microscopy with a Zeiss released to the basolateral side was significantly higher in the Axioplan microscope. case of supernatants lacking the PrtV protease (Fig. 6). About 2-fold higher IL-8 levels were seen when the supernatants from SDS͞PAGE and Western Blot Analyses. To monitor proteases, su- ⌬prtV mutant derivatives were compared with supernatants from pernatants from V. cholerae were concentrated by trichloracetic ϩ otherwise isogenic prtV strains. That the effect is PrtV- acid precipitation and separated by SDS͞PAGE (39). Western dependent was verified by the transcomplementation test of the blot analyses were performed as described (40), and detection ⌬prtV mutation (using the plasmid clone pKVA232) that led to was done by using the ECLϩ chemiluminescence system (Am- restored lower (i.e., similar to PrtV WT) levels of IL-8. Analyses ersham Pharmacia Biotech). of IL-8 mRNA levels also revealed an increase in the case of the prtV mutant (Fig. 11, which is published as supporting informa- Human Intestinal Epithelial Cell Tight Monolayer Assay. Tight mono- tion on the PNAS web site), and we conclude that PrtV is not an layers of the human colon carcinoma cell line T84 were estab- IL-8 stimulator per se. Rather it appeared that PrtV may play lished in a transwell system by using permeable polycarbonate some role in modulating͞reducing the activity of the as-yet- membrane supports with 0.4-␮m-diameter pores (Costar) and a unidentified component(s) that may be the cause of V. cholerae 1:1 mixture of Dulbecco͞Vogt modified Eagle’s medium and reactogenicity. Ham’s F12 medium supplemented with 8% FCS, 15 mM Hepes We conclude that the QS regulon has an important role in the buffer, and antibiotics. Cells were cultured in a humidified encounter between V. cholerae and the grazing predators in incubator at 37°C in 5% CO2 until a confluent monolayer with bacterioplankton communities. Our findings demonstrate that in a transepithelial electrical resistance of Ͼ2,000 ohm͞cm2 was particular the prtV gene product should be considered as a factor obtained as measured by the Millicell Electrical Resistance

9284 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601754103 Vaitkevicius et al. Downloaded by guest on September 25, 2021 System (Millipore) (41). Then the tissue culture medium in the Overnight cultures of the V. cholerae strains were diluted in upper chamber was replaced by a 500-␮l culture supernatant 0.9% NaCl, and 2.5 ϫ 107͞ml bacteria were transferred into wells sample from the tested bacterial strain, and cells were incubated in tissue culture plates. Subsequently, T. pyriformis or Cafeteria for an additional 5 or 18 h at which time the culture medium from roenbergensis was added to the wells at a final concentration of the lower chamber was collected. Thereafter cells were collected 6 ϫ 103͞ml. Numbers of ciliates and flagellates were analyzed by for RNA extraction. direct inspection with an inverted light microscope over 4 days. Generally each treatment was performed in replicate wells of Analysis of Secreted IL-8. Tissue culture medium from the lower four. Protozoa cell numbers were counted by using an inverted compartment of the transwell cultures, i.e., the basolateral side light microscope (Zeiss). Samples (50 or 10 ␮l) were taken from of the T84 monolayers, was collected and thereafter the concentration of IL-8 was determined in duplicates of serial dilu- each well into counting chambers, and the numbers of motile tions of tissue culture medium by using a commercially available protozoa were recorded. To obtain the total number of protozoa ELISA (Endogen Human IL-8 ELISA Kit; Pierce). per sample, formaldehyde was added to a final concentration of 2% (42), and all fixed cells were counted. The survival (per- Grazing Experiments. Experiments investigating the viability of the centage of input) after 4 days was calculated from the direct ciliate protozoan T. pyriformis CCAP 1630͞1W (obtained from counts of actively motile organisms. Culture Collection of Algae and Protozoa Scottish Association for Marine Science Research Services, Argyll, Scotland) and the Protein Identification. Mass spectrometry, protein identification, flagellate Cafeteria roenbergensis (purchased from Culture Col- and database searches were carried out at the Wallenberg lection of Algae and Protozoa Scottish Association for Marine Consortium North Expression Proteomics Facility (Institutionen Science Research Services) were performed in 24-well tissue fo¨r Medicinsk Biokemi och Mikrobiologi, Uppsala University, culture plates. The axenic protozoan culture was grown and Uppsala, Sweden). maintained in CCAP protease peptone yeast extract medium (room temperature without shaking). To make them bacteria- We thank Dr. Bernt Eric Uhlin for helpful suggestions and critical free, the ciliate and flagellate cultures were treated with anti- reading of the manuscript and Daw Hla Hla Nyunt for encouragement. biotics (50 ␮g͞ml ampicillin; 30 ␮g͞ml kanamycin; 30 ␮g͞ml This work was supported by grants from the Swedish Research Council, polymyxin; 30 ␮g͞ml streptomycin; 25 ␮g͞ml chloramphenicol), the Swedish Foundation for International Cooperation in Research and and tests with LB medium and agar plates confirmed that they Higher Education, Cancerfonden, The Wallenberg Foundation, and the were axenic. Faculty of Medicine, Umeå University. MICROBIOLOGY

1. Colwell, R. R. (1997) J. Ind. Microbiol. Biotechnol. 18, 302–307. 22. Aballay, A., Yorgey, P. & Ausubel, F. M. (2000) Curr. Biol. 10, 1539–1542. 2. Huq, A., Colwell, R. R., Rahman, R., Ali, A., Chowdhury, M. A., Parveen, S., 23. Alegado, R. A., Campbell, M. C., Chen, W. C., Slutz, S. S. & Tan, M. W. (2003) Sack, D. A. & Russek-Cohen, E. (1990) Appl. Environ. Microbiol. 56, 2370– Cell Microbiol. 5, 435–444. 2373. 24. Darby, C., Cosma, C. L., Thomas, J. H. & Manoil, C. (1999) Proc. Natl. Acad. 3. Huq, A., Sack, R. B., Nizam, A., Longini, I. M., Nair, G. B., Ali, A., Morris, Sci. USA 96, 15202–15207. J. G., Jr., Khan, M. N., Siddique, A. K., Yunus, M., et al. (2005) Appl. Environ. 25. Ewbank, J. J. (2002) Microbes Infect. 4, 247–256. Microbiol. 71, 4645–4654. 26. Labrousse, A., Chavet, S., Couillault, C., Kurz, C. L. & Ewbank, J. J. (2000) 4. Louis, V. R., Russek-Cohen, E., Choopun, N., Rivera, I. N., Gangle, B., Jiang, Curr. Biol. 10, 1543–1545. S. C., Rubin, A., Patz, J. A., Huq, A. & Colwell, R. R. (2003) Appl. Environ. 27. Kurz, C. L., Chauvet, S., Andres, E., Aurouze, M., Vallet, I., Michel, G. P. F., Microbiol. 69, 2773–2785. Uh, M., Celli, J., Filloux, A., de Betnzmann, S., et al. (2003) EMBO J. 22, 5. Colwell, R. R. & Huq, A. (1994) Ann. N. Y. Acad. Sci. 740, 44–54. 1451–1460. 6. Huq, A., Small, E. B., West, P. A., Huq, M. I., Rahman, R. & Colwell, R. R. 28. Tan, M. W., Rahme, L. G., Sternberg, J. A., Tompkins, R. G. & Ausubel, F. M. (1983) Appl. Environ. Microbiol. 45, 275–283. (1999) Proc. Natl. Acad. Sci. USA 96, 2408–2413. 7. Colwell, R. R. (1996) Science 274, 2025–2031. 29. Brenner, S. (1974) Genetics 77, 71–94. 8. Fenchel, T. (1988) Annu. Rev. Ecol. Syst. 19, 19–38. 30. Zhu, J. & Mekalanos, J. J. (2003) Dev. Cell 5, 647–656. 9. Sherr, E. B. & Sherr, B. F. (2002) Antonie Van Leeuwenhoek 81, 293–308. 31. Benitez, J. A., Silva, A. J. & Finkelstein, R. A. (2001) Infect. Immun. 69, 10. Matz, C. & Kjelleberg, S. (2005) Trends Microbiol. 7, 302–307. 6549–6553. 11. Roszak, D. B. & Colwell, R. R. (1987) Microbiol. Rev. 51, 365–379. 32. Peterson, J. D., Umayam, L. A., Dickinson, T., Hickey, E. K. & White, O. 12. Zhu, J., Miller, M. B., Vance, R. E., Dziejman, M., Bassler, B. L. & Mekalanos, (2001) Nucleic Acids Res. 29, 123–125. J. J. (2002) Proc. Natl. Acad. Sci. USA 99, 3129–3134. 33. Toma, C. & Honma, Y. (1996) Infect. Immun. 64, 4495–4500. 13. Kovacikova, G. & Skorupski, K. (2002) Mol. Microbiol. 46, 1135–1147. 34. Ogierman, M. A., Fallarino, A., Riess, T., Williams, S. G., Attridge, S. R. & 14. Miller, M. B., Skorupski, K., Lenz, D. H., Taylor, R. K. & Bassler, B. L. (2002) Manning, P. A. (1997) J. Bacteriol. 179, 7072–7080. Cell 110, 303–314. 35. Matz, C., McDougald, D., Moreno, A. M., Yung, P. Y., Yildiz, F. H. & 15. Malloy, J. L., Veldhuizen, R. A., Thibodeaux, B. A., O’Callaghan, R. J. & Kjelleberg, S. (2005) Proc. Natl. Acad. Sci. USA 102, 16819–16824. Wright, J. R. (2005) Am. J. Physiol. 288, L409–L418. 36. Pukatzki, S., Ma, A. T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, 16. Silva, A. J. & Benitez, J. A. (2004) J. Bacterial. 186, 6374–6382. W. C., Heidelberg, J. F. & Mekalanos, J. J. (2006) Proc. Natl. Acad. Sci. USA 17. Stewart-Tull, D. E., Bleakley, C. R. & Galloway, T. S. (2004) Vaccine 22, 103, 1528–1533. 3026–3034. 37. Zhou, X., Gao, D. Q., Michalski, J., Benitez, J. A. & Kaper, J. B. (2004) Infect. 18. Matz, C., Bergfeld, T., Rice, S. & Kjelleberg, S. (2004) Environ. Microbiol. 6, Immun. 72, 389–397. 218–226. 38. Skorupski, K. & Taylor, R. K. (1996) Gene 169, 47–52. 19. Kaper, J. B., Morris, J. G. & Levine, M. M. (1995) Clin. Microbiol. Rev. 8, 39. Laemmli, U. K. (1970) Nature 227, 680–685. 48–86. 40. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 20. Tacket, C. O., Losonsky, G., Nataro, J. P., Comstock, L., Michalski, J., 4350–4354. Edelman, R., Kaper, J. B. & Levine, M. M. (1995) J. Infect. Dis. 172, 883–886. 41. Shieh, J. T. & Bergelson, J. M. (2002) J. Virol. 76, 9474–9480. 21. Levine, M. M. & Noriega, F. (1995) PNG Med. J. 38, 325–331. 42. Matz, C. & Jurgens, K. (2001) Appl. Environ. Microbiol. 67, 814–820.

Vaitkevicius et al. PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 ͉ 9285 Downloaded by guest on September 25, 2021