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Applied and Environmental Microbiology, July 2001, p. 3220-3225, Vol. 67, No. 7
Department of Biology, Dartmouth
College,1 and Department of Microbiology
and Immunology, Dartmouth Medical School,2
Hanover, New Hampshire 03755
Received 22 January 2001/Accepted 11 April 2001
The bacterium Vibrio cholerae, the etiological agent
of cholera, is often found attached to plankton, a property that is
thought to contribute to its environmental persistence in aquatic
habitats. The V. cholerae O1 El Tor biotype and
V. cholerae O139 strains produce a surface pilus termed
the mannose-sensitive hemagglutinin (MSHA), whereas V.
cholerae O1 classical biotype strains do not. Although
V. cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the
other strains. Since V. cholerae O1 El Tor and V.
cholerae O139 have displaced V. cholerae O1
classical as the major epidemic strains over the last fifteen years, we
investigated the potential role of MSHA in mediating adherence to
plankton. We found that mutation of mshA in V.
cholerae O1 El Tor significantly diminished, but did not
eliminate, adherence to exoskeletons of the planktonic crustacean
Daphnia pulex. The effect of the mutation was more pronounced for V. cholerae O139, essentially eliminating
adherence. Adherence of the V. cholerae O1 classical
mshA mutant was unaffected. The results suggest that
MSHA is a factor contributing to the ability of V.
cholerae to adhere to plankton. The results also showed that
both biotypes of V. cholerae O1 utilize factors in addition to MSHA for zooplankton adherence. The expression of MSHA and
these additional, yet to be defined, adherence factors differ in a
serogroup- and biotype-specific manner.
Throughout history, aquatic
ecosystems have consistently been the focal points of cholera outbreaks
(7, 11, 32, 48), and the bacillus Vibrio
cholerae is now known to be endemic in aquatic environments,
present even in the absence of human inputs (7, 8, 12, 16, 17,
23, 32, 33, 66). Little is known about what regulates V. cholerae abundance in aquatic systems, but outbreaks in humans are
hypothesized to be correlated with seasonally high abundances (blooms)
of phytoplankton or zooplankton (6, 7, 11, 33). Survival
and abundance of V. cholerae in planktonic communities, and
therefore cholera outbreaks in humans, are believed to depend in part
on the ability of V. cholerae to attach to the surfaces of
phytoplankton and zooplankton (5, 10, 24-31, 33-35, 51,
53). Bacterial attachment to planktonic detrital particles has
been shown to increase bacterial productivity and is believed to be a
way to escape low-nutrient conditions (see, for example, reference
47). Considerably less is known about the role of living
plankton as a bacterial microhabitat, but it has been proposed for
V. cholerae that attachment to plankton increases survival
and growth by providing both a source of nutrition and a
microenvironment that provides protection from conditions which are
detrimental to V. cholerae (i.e., extremely low or high salinity) (43, 44, 62, 63, 75). Of additional medical concern, colonization of plankton surfaces may serve to spatially concentrate bacteria, making it easier for humans to consume infectious doses (7, 27).
Historically, strains of V. cholerae associated with
epidemic disease have been of the O1 serogroup, which is divided into two biotypes: classical and El Tor (4, 7, 61). A
distinguishing feature between these biotypes is the production of a
cell-associated, mannose-sensitive hemagglutinin (MSHA) by
strains of the El Tor biotype (15, 19). The
hemagglutinating activity is the result of the elaboration of a type 4 pilus, for which MshA is the major subunit (37). The gene
encoding the MSHA subunit, mshA, has been characterized and
is located in a large gene cluster involved in biogenesis of the pilus
structure (20, 38, 41, 42). Presence of pili on bacterial
cells is often associated with the ability to colonize surfaces. A
study with mshA mutant strains has demonstrated a role for
MSHA in colonization and subsequent biofilm formation on abiotic
surfaces (borosilicate glass) and biotic surfaces (cellulose)
(73). Another V. cholerae type 4 pilus, the
toxin-coregulated pilus (TCP), is necessary for colonization of the
mammalian intestine (3, 21, 65, 68, 69). There is no
apparent role for MSHA for V. cholerae colonization of the human digestive tract, as evaluated by using defined mshA
null mutants in animal models and adult volunteer studies (3, 65, 69), and, vice versa, there is no known role for TCP in the colonization of nonintestinal surfaces (73, 74). Thus,
MSHA might have a specific role in environmental survival for V. cholerae.
Despite the fact that O1 strains of the El Tor biotype were not
associated with epidemic cholera until the beginning of the seventh
pandemic in 1961, they have displaced classical biotype strains during
the past 15 years to become the most prevalent epidemic strains at the
present time (4, 7, 59, 61). During this time, there was
also a transient outbreak caused by strains of the previously
unrecognized O139 serogroup (14, 49, 59). Strains of the
O139 serogroup share many characteristics with O1 El Tor strains and
are thought to be derived from O1 El Tor (18, 36, 55, 71).
The reasons for the predominance of El Tor and the appearance of the
highly related O139 strains are probably multifactorial and likely
include parameters associated with interactions of the organism with
the human host as well as with the environment. Since MSHA is a factor
that is expressed by O1 strains of the El Tor biotype and O139 strains
and is not expressed by strains of the previously predominant O1
classical biotype (1, 19), we investigated whether MSHA
might provide an advantage for environmental persistence of El Tor
biotype strains by mediating the ability to colonize planktonic hosts.
Specifically, we have investigated whether MSHA mediates attachment of
V. cholerae to the chitinous exoskeletons of the crustacean
zooplankton Daphnia pulex (V. cholerae is found
on Daphnia in Bangladesh [23]) by O1 El Tor,
classical, and O139 strains. For the wild-type and mutant El Tor
strains, we also examined two additional factors hypothesized to
increase the tendency of V. cholerae to colonize plankton:
low-nutrient conditions and the presence of a mucilaginous surface on
the host plankton.
Bacterial strains and plasmids.
V. cholerae
strains used in this study were serogroup O1 El Tor biotype strain
C6706 str2 (69), serogroup O139 vaccine strain CVD 112 (64), and serogroup O1 classical strain O395 Sm
(68) and their respective isogenic mshA mutant
derivatives KHT46 (69), KHT37 (69), and JM69
(39). These strains were labeled with green fluorescent
protein (GFP) by electroporation with the GFP expression plasmid,
pVSP61TIR (46), followed by growth on Luria-Bertani (LB)
agar plates supplemented with kanamycin (45 µg/ml) to select for
acquisition of the plasmid. Bacteria were grown in either LB medium or
minimal medium (M9) supplemented with 0.2% (wt/vol) glucose
(45).
Daphnia source and culture methods.
Daphnia
pulex (Crustacea: Cladocera) used in this study were cultured from
a single clone originating from a pond located in Gunnison National
Forest, Colorado. Daphnia were cultured in filtered pond
water (Storrs Pond, New Hampshire) at 20°C with a 14 h-10 h
light-dark cycle and were fed the alga Cryptomonas erosa.
Adult Daphnia molted every 2.5 days under our culture
conditions, and whole, newly molted exoskeletons (<48 h) were used as
a substrate for the colonization assays.
Adherence assays.
Cultures of V. cholerae
expressing GFP were grown in LB or M9 liquid medium supplemented with
kanamycin (45 µg/ml) for 18 h at 37°C with aeration. Twenty
milliliters of fresh LB or M9 containing three D. pulex
exoskeletons was inoculated with V. cholerae from these
cultures at a 1:1,000 dilution corresponding to
Quantification of V. cholerae
adherence.
For all experiments, the inoculation density of
V. cholerae cells was determined by serial dilution and
colony counts. Adherence to D. pulex exoskeletons by
GFP-expressing V. cholerae was quantified from digitally
acquired images of photographic slides with an Optimas macro
(Optimas, version 6.5; Media Cybernetics) to count attached
V. cholerae cells, measure the surface area examined, and
then determine the number of attached cells per square millimeter. We
then standardized attached-cell density among assays by multiplying actual cell density by a correction factor accounting for differences in inoculation densities, which were determined by plate counts. With
one exception (noted in Results), attached bacteria were counted on the
same segment of the second antennae in all assays to control for an
apparent systematic variation in the number of bacteria attached to
different parts of the exoskeleton. Standardized attached-cell
density in different treatments was compared by analysis of variance
(ANOVA), with different exoskeletons serving as replicates. Where
necessary, densities were log transformed to equalize variance among treatments.
Alkaline phosphatase assays.
Expression of mshA
was monitored by assaying alkaline phosphatase activity of O1 El Tor
mshA-phoA fusion strain JM191 after overnight growth in
either M9 or LB as previously described (41). Assays were
performed on three separate occasions, and the result were averaged.
The values of independent assays varied by less than 10%.
Effects of mshA mutation and mucilage coating on
exoskeleton adherence by the V. cholerae
O1 El Tor biotype.
The contribution of MSHA to planktonic
adherence was examined by testing wild-type and mshA mutant
strains in a D. pulex attachment assay. The results of six
different assay experiments are presented in Table
1. In the first assay, we compared
adherence by the V. cholerae O1 El Tor biotype strain,
C6706, and isogenic mshA deletion mutant, KHT46. These
strains were incubated in LB medium with "clean" Daphnia
exoskeletons and with exoskeletons from Daphnia that had
been completely covered with an epibiotic alga, Colacium vesiculosum. C. vesiculosum cells attach to zooplankton by using a
mucilaginous polysaccharide (40, 58, 72). When the
exoskeleton is molted, the epibiotic cells detach to search for a new
host (40, 57, 70), leaving the exoskeleton coated with
algal mucilage. Mucilage is a substance suggested to be a preferred colonization substrate for V. cholerae because of its
nutritional and protective properties (22, 30-34, 53). We
found that there was significantly greater colonization of the
wild-type O1 El Tor strain in comparison to the mshA mutant
(ANOVA, P < 0.0001) and that colonization was
significantly greater on the mucilage-coated exoskeletons (ANOVA,
P = 0.026). There was a larger difference between the
wild type and mutant for the mucilage-coated exoskeletons (60-fold)
than for the clean exoskeletons (33-fold), but this difference was not
statistically significant (ANOVA, two-way interaction, P = 0.17). Mucilage-coated exoskeletons were not used
in any other experiments. An additional assay with the wild type O1 El
Tor and the isogenic mshA mutant strains was conducted in
which a different area of the exoskeleton (side of the carapace) was
examined (assay 6; Table 1). While colonization of both wild-type and mutant strains was less than that in assay 1, the reduction in colonization of the mshA mutant compared to colonization of
the O1 El Tor wild type was again significant (ANOVA, P = 0.008 [Table 1]).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3220-3225.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Mannose-Sensitive Hemagglutinin of
Vibrio cholerae Promotes Adherence to
Zooplankton
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
106 cells/ml, and the mixture was incubated at
23°C for 2 h. Exoskeletons were then rinsed serially three times
in 20 ml of KRT buffer (128 mM NaCl, 5.1 mM KCl, 1.34 mM
MgSO4, 2.7 mM CaCl2, 10 mM
Tris-HCl, pH 7.5) by gentle stirring and transfer with a wide-bore
pipette. Exoskeletons were slide mounted with Gel/Mount (Biomeda Corp., Foster City, Calif.), and fluorescent images were captured on Kodak
Ektachrome Elite II slide film (ASA 400) utilizing a Zeiss Axiophot
microscope and an HQ FITC filter set with an excitation wavelength of
490 nm and an emission wavelength of 520 nm.
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Results of V. cholerae attachment assays for
O1 El Tor and classical biotype and O139 strains and corresponding
isogenic mshA mutantsa
Effect of mshA mutation on exoskeleton adherence by
V. cholerae O139.
Several studies
suggest that V. cholerae O139 strains are derivatives of O1
El Tor strains that have acquired properties through one or more
horizontal gene transfer events that confer a different O antigen,
capsule production, and antibiotic resistance (18, 36, 55,
71). We were therefore interested in determining whether the
contribution of MSHA to exoskeleton adherence extended to this
serogroup. In assay 2, we compared adherence by the V. cholerae O139 strain, CVD 112, and adherence by its isogenic
mshA deletion mutant, KHT37, in LB medium. There was a
substantial number of attached cells of the
mshA+ O139 strain but essentially no
attachment of the mshA mutant cells (Fig.
1). Attachment was zero on the
Daphnia body part routinely examined (precluding statistical
comparison), although a very few cells were observed to be attached to
other areas of the exoskeletons.
|
Effect of mshA mutation on exoskeleton adherence by the V. cholerae O1 classical biotype. V. cholerae O1 strains of the classical biotype express MshA in an extracytoplasmic location, as judged by mshA-phoA gene fusion analysis, but fail to assemble functional MSHA pili on the bacterial surface (41). Therefore, it was of interest to determine whether a classical biotype strain could adhere to D. pulex exoskeletons, assuming that expression of MshA that is not assembled into a pilus could not contribute to this process. In assays 3 and 4, we compared attachment of the O1 classical strain, O395, and its mshA mutant, JM69, in LB medium in two experiments. In assay 3, there was a 1.8-fold decrease in attachment of the mutant, but this was not statistically significant (ANOVA, P = 0.16), while in assay 4, the mshA mutant actually had a significantly higher (ANOVA, P = 0.0003) attachment density, but again by less than a twofold difference. Given the opposite results of assays 3 and 4 and the small numerical difference in attached-cell density compared with the much larger effects of the other two mutant strains, it is likely that the result of assay 4 is not biologically significant (real effect) but rather is a statistical artifact due to the unusually low variance among replicate exoskeletons for the JM69 strain (Table 1).
Effect of nutrient growth conditions on exoskeleton adherence. In a recent study by Watnick et al. (73), V. cholerae was found to have altered attachment to various surfaces depending on the MSHA status of the bacteria and whether growth prior to the assay was in rich medium (LB) or minimal medium (M9). Assay 5 was conducted with M9 minimal medium to examine the effect of nutrient-limiting conditions on exoskeleton binding for the wild-type O1 El Tor and the mshA mutant. Again, attachment was significantly less for the mutant than for the wild-type strain (ANOVA, P = 0.0078), although the magnitude of difference between the two strains was only fivefold, much less than that seen in LB medium. For both the wild-type and mutant strains, the density of colonized cells was approximately an order of magnitude greater in the nutrient-limiting medium than was seen in LB (Table 1). This difference was not contributed to by greater MshA expression in nutrient-limiting medium since MshA expression was actually slightly decreased under this growth condition as determined by measurement of the alkaline phosphatase activity produced by mshA-phoA fusion strain JM191. The specific activity was 1,215 ± 22.6 (standard error) units in LB and 1,087 ± 28.8 units in M9 (ANOVA, P = 0.025).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Role of MSHA in crustacean exoskeleton adherence for different strains of V. cholerae. The mshA structural subunit gene is essential for the production of MSHA type 4 surface pili present on O1 El Tor and O139 strains of V. cholerae associated with human cholera epidemics. Deletion of mshA to create mutant strains of O1 El Tor (KHT46) or O139 (KHT37) resulted in a significant decrease in adherence of V. cholerae cells on exoskeletons of the crustacean zooplankton D. pulex when assayed under a variety of conditions. These findings suggest that the MSHA type 4 pilus may be an important mediator of O1 El Tor and O139 adherence in the aquatic environment since V. cholerae is found attached to zooplankton in this setting (5, 24-31, 33-35, 50, 53). Our results with respect to the role of MSHA in mediating attachment to the chitinous exoskeleton of D. pulex differ from those reported by Watnick et al. (73), who used chitin particles on a glass support. In that study no difference was seen in the chitin binding by a wild-type O1 El Tor strain versus that by a mshA mutant. However, these results were based on a qualitative rather than a quantitative assessment and so may not have been able to distinguish between attachment levels of the wild-type and mutant strains. In addition, this discrepancy might reflect a difference between the use of a natural chitinous exoskeleton versus the use of chitin particles as the binding substrate.
V. cholerae O1 strains of the classical biotype express the MshA pilin subunit but do not assemble MSHA pili on the bacterial cell surface and do not display a MSHA-dependent hemagglutination phenotype (37, 41). As expected based on this property, the O1 classical mshA mutant strain did not show less adherence to zooplankton exoskeletons than the wild-type strain. However, the classical strain was able to attach. This finding indicates that surface factors other than MSHA pili are responsible for O1 classical attachment to chitinous exoskeletons. These factors may also be present on El Tor strains, since the O1 El Tor mshA mutant strain still showed substantial attachment to exoskeletons. An interesting aspect of the study was the greater contribution of MSHA to adherence by the O139 strain than to the highly related O1 El Tor strain. Attachment of the O139 mshA mutant was virtually undetectable. Perhaps this is due to the presence of a capsule on O139 strains. The MSHA pilus can likely protrude from the cell surface, extending out beyond the capsular material. In contrast, outer membrane factors that might participate in colonization by O1 El Tor could be shielded by the O139 carbohydrate capsule. These factors may include membrane-associated chitin binding proteins such as those that have been identified in V. alginolyticus and V. cholerae (54, 67). Our results suggest that analogous factors provide the sole mechanism for zooplankton adherence by O1 classical strains. In contrast, O139 strains appear to utilize MSHA alone, whereas O1 El Tor strains utilize a combination of MSHA and additional factors.Mucilaginous surface and nutrient-limiting conditions enhance adherence of V. cholerae to exoskeletons. A number of studies have found that V. cholerae is more commonly isolated from the surface of algal species that produce a mucilaginous sheath than from the surface of those that do not (30-34, 53). However, it is not clear whether this difference is due to enhanced adherence to such surfaces or to the increased growth due to the protective and nutritious microhabitat within the mucilage. In our work, the presence of a mucilaginous biofilm on the Daphnia exoskeletons significantly enhanced exoskeleton attachment by both the wild type and the mutant O1 El Tor, suggesting that the presence of mucilage on planktonic species may contribute to V. cholerae colonization in the environment. Our results suggest that both MSHA and additional undefined adherence factors have a role in this process.
We found that growth under the nutrient-limiting conditions of M9 minimal medium resulted in an order of magnitude increase in V. cholerae binding to the exoskeletons for both the wild type and the mshA mutant. Since MshA expression was actually slightly decreased under nutrient-limiting conditions, it is likely that nutrient deprivation of O1 El Tor V. cholerae resulted in increased exoskeleton binding by an MSHA-independent mechanism. Several other studies have found that harsh growth conditions such as low nutrient levels or nonoptimal salinity or pH enhance V. cholerae colonization of surfaces (22, 26, 35). The ability of some V. cholerae strains to produce chitinase and mucinase (9, 50, 51, 60) and to grow with chitin as the sole nutrient source in laboratory cultures (2) indicates a potential nutritional benefit to V. cholerae from colonization of zooplankton with chitinous exoskeletons and mucilage-sheathed phytoplankton. An additional benefit to the bacteria may be afforded by the physical protection provided by existence within a surface biofilm. Specific disadvantages of an attached lifestyle have not been demonstrated for V. cholerae, but if there is a cost of colonization, perhaps due to increased expression of colonization factors, then increased expression of colonization factors only in response to harsh conditions would be adaptive. Key questions that emerge from this study are as follows. (i) To what degree do different plankton attachment factors contribute to persistence, growth, and survival of the various epidemiological strains in the natural aquatic environment? (ii) Does the possession of the MSHA pilus in addition to at least one additional attachment factor by the O1 El Tor strain have a role in its worldwide replacement of the classical strain in the seventh pandemic and its present epidemiological predominance over O139 in Bangladesh and India (4, 7, 11, 59, 61)? (iii) Are particular attachment factors specialized for different types of planktonic surfaces? (iv) To what degree is expression of attachment factors regulated by physical and chemical conditions in aquatic ecosystems? These are all questions which deserve further study because they may be crucial in refining the ongoing efforts to develop predictive models of cholera outbreaks based on the state of the aquatic ecosystems that V. cholerae inhabits (7, 52).| |
ACKNOWLEDGMENTS |
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We thank Jean Richardson for assistance with Optimas Image Analysis.
This work was supported by National Institutes of Health grant AI-25096 (R.K.T.). J.W.M. was the recipient of a predoctoral fellowship from the National Institutes of Health (training grant AI-07519). D.A.C. received financial support from the Dartmouth College Cramer Fund, the Dartmouth Center for Environmental Health, and the NIEHS Superfund ES07373 to C. L. Folt and S. Y. Chen. Assistance and supplies for Daphnia and C. erosa culturing were provided by the laboratory of C. L. Folt, Department of Biological Sciences.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755. Phone: (603) 650-1632. Fax: (603) 650-1318. E-mail: ronald.k.taylor{at}dartmouth.edu.
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Abstract
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Materials and Methods
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Discussion
References
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Applied and Environmental Microbiology, July 2001, p. 3220-3225, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3220-3225.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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