Vibrio cholerae is among the most intensively studied of those bacteria pathogenic for humans including its genetics, physiology and ecology (Faruque et al., 1998). Chitin, composed of β-1,4-linked N-acetylglucosamine (GlcNAc) residues, is one of the most abundant biopolymers in nature and, perhaps, the most abundant in the marine environment (Gooday, 1990). The V. cholerae connection with chitin is an extensively documented phenomenon and, for microbial ecology, one of the best examples of a successful bacteria–substrate interaction, with a complex and profound influence on the lifestyle of this bacterium, inside and outside its human host, as well as its ecological function in the natural environment (Colwell, 2002). Emergent properties deriving from V. cholerae–chitin interactions can be detected at multiple levels in an hierarchical scale in the environment, essentially comprising a biological system from the cell to the global environment. These properties, each linked one to the other, include cellular physiological responses (chemotaxis, chitin utilization, cell multiplication, induction of competence), multicellular organization, participation in carbon (C) and nitrogen (N) cycling in the aquatic ecosystem and, not the least, pathogenicity for humans (Fig. 1). In this review we integrate the most recent findings in the literature on V. cholerae–chitin interactions, providing a global perspective and emphasizing the multiple lifestyle of V. cholerae.
Binding to chitin
V. cholerae strains possess multiple strategies for surface colonization depending upon the presence and expression of both conserved and variables genes (Mueller et al., 2007). Binding to chitin is a complex process involving hydrophobic and ionic bonds, forces responsible for the primary reversible phase of attachment and specific cell ligands that are responsible for subsequent firm anchoring to substrate (Table 1).
Table 1. V. cholerae ligands involved in adhesion to chitin-containing substrates and mammalian intestine.
|V. cholerae adhesins||Chitin particles||Crustacea esoskeleton||Intestine||References|
|MSHA||+||+||+/−||Finn et al. (1987), Watnick et al. (1999), Tacket et al. (1998), Chiavelli et al. (2001), Meibom et al. (2004)|
|ChiRP||+||n.t.||−||Meibom et al. (2004)|
|TCP||−a||−||+||Herrington et al. (1988), Attridge et al. (1996), Thelin and Taylor (1996), Tacket et al. (1998), Kirn et al. (2000), Reguera and Kolter (2005)|
|HA||+b||n.t.||n.t.||Sasmal et al. (1992), Datta-Roy et al. (1989)|
|CBP||+||+||+||Singh et al. (1994), Tarsi and Pruzzo (1999), Zampini et al. (2005)|
|GbpA||+||+||+||Kirn et al. (2005)|
The presence of pili is associated with the ability of bacterial cells to colonize surfaces. Mannose-sensitive haemagglutinin (MSHA) is a type 4 pilus produced by V. cholerae O1 El Tor and O139; V. cholerae O1 classical strains express MshA pilin subunit, but neither assemble MSHA pili on the bacterial cell surface, nor display a MSHA-dependent haemagglutination phenotype (Jonson et al., 1990; Marsh et al., 1996). The role of MSHA in colonization of the human intestine remains under debate (Finn et al., 1987; Tacket et al., 1998), but MSHA is involved in colonization and subsequent biofilm formation on nutritive (cellulose) and non-nutritive (borosilicate glass) surfaces (Watnick et al., 1999). Furthermore, MSHA mediates association with, and killing by, Mytilus galloprovincialis hemocytes (Pruzzo et al., 2005), and promotes adherence to both chitin beads (Meibom et al., 2004) and crustaceans (Chiavelli et al., 2001; Meibom et al., 2004). MSHA appears to be of particular relevance with respect to the adhesive properties of encapsulated V. cholerae O139 strains, where it extends beyond capsular material that may mask other surface ligands (Chiavelli et al., 2001). Using chitin particles on a glass support, Watnick and colleagues (1999) did not find any difference in chitin binding by a wild-type V. cholerae O1 El Tor compared to a mshA mutant. In contrast, Chiavelli and colleagues (2001) showed that deletion of mshA in V. cholerae O1 El Tor or O139 resulted in significant decrease in adherence of the cells on exoskeletons of the crustacean zooplankton, Daphnia pulex, when assayed under a variety of conditions. This discrepancy might be related to the difference between serving as a binding substrate for chitin particles versus the chitinaceous exoskeleton, covered by the epicuticule. It has been suggested that adherence to chitin is enhanced by MSHA, independently from surface chemistry (Meibom et al., 2004).
In V. cholerae El Tor, a type 4 PilA-containing pilus, the expression of which is induced by chitin (also designated chitin-regulated pilus, ChiRP), has been reported to contribute to colonization of chitin (Meibom et al., 2004). It has been suggested that in chitin surfaces that have not been colonized, the random collision of planktonic V. cholerae with the surface leads to MSHA-mediated adherence (Meibom et al., 2004). In the case of those surfaces colonized by chitinolytic bacteria, the gradients of GlcNAc and (GlcNAc)2 direct bacteria to the chitin surface and induce synthesis of ChiRP, promoting colonization, followed by efficient digestion and assimilation of the polysaccharide (Meibom et al., 2004). More recently, the toxin-co-regulated pilus (TCP), that is required for intestinal colonization (Herrington et al., 1988; Attridge et al., 1996; Tacket et al., 1998; Thelin and Taylor, 1996; Kirn et al., 2000) and cholera toxin gene acquisition by phage infection (Waldor and Mekalanos, 1996), has also been shown to have a role in biofilm formation on chitin surfaces by V. cholerae El Tor, as discussed later in this review (Reguera and Kolter, 2005).
Bacterial ligands mediating adhesion to chitin by a specific binding to GlcNAc have been shown to be present in V. cholerae classical strains. A cell-associated GlcNAc-specific haemagglutinin (HA) was purified from V. cholerae by affinity chromatography on a chitin column, followed by BioGel filtration (Sasmal et al., 1992, 1996). Chitin binding proteins (CBPs) were found in several Vibrio species, both cell wall associated and extracellular (Montgomery and Kirchman, 1993; Pruzzo et al., 1996; Tarsi and Pruzzo, 1999; Kirn et al., 2005; Vezzulli et al., 2008). In V. cholerae, Tarsi and Pruzzo (1999) identified two surface associated CBPs (36 kDa and 53 kDa) involved in GlcNAc-sensitive adhesion of classical strains to chitin beads: the 53 kDa protein was also found to mediate adherence to the copepod Tigriopus fulvus (Zampini et al., 2005). A putative 53 kDa chitinase mediating adherence to chitin surfaces and Daphnia magna via GlcNAc binding, and termed GlcNAc-binding protein A (GbpA), has been described by Kirn and colleagues (2005). GbpA was shown to be a secreted protein, also found in the cell wall during transit across the envelope via EPS secretion (Kirn et al., 2005).
In spite of the acquired knowledge, the full contribution of each of the above ligands to persistence and survival of the various clones, serotypes and biotypes of V. cholerae in natural aquatic environments is still unclear, as well as their potential role in the replacement of the classical strain of V. cholerae O1 with V. cholerae O1 El Tor that has occurred in many geographical locations worldwide (Chiavelli et al., 2001).
The physiological response of the cell in the hierarchical scale
Binding to chitin in the environment may be either a casual phenomenon or promoted by chitin and/or chitin oligomers. Chemotaxis of V. cholerae toward chitin oligosaccharides has been reported by Li and Roseman (2004). Based on previous studies of V. furnissii by Bassler and colleagues (1989; 1991), these authors inferred that the secreted chitinase of starving cells comes into contact with chitin in the microenvironment and generates a disaccharide and/or a (GlcNAc)n gradient, causing the cells to swim up the gradient to the cuticle or chitin (besides inducing ChiRP pilus synthesis, as reported above, Meibom et al., 2004). The complete genome sequence of V. cholerae has revealed a relatively large number of genes with homology to known chemotaxis-related genes (Heidelberg et al., 2000). In total, there are 68 V. cholerae open reading frames (ORFs) that have been annotated as putative chemotaxis-related genes, with 22 ORFs coding for homologues of che genes and 46 ORFs encoding possible membrane-spanning methyl-accepting chemotaxis proteins (MCPs). Most of the ORFs encoding the different che homologues are clustered in three different regions located on both chromosomes (Heidelberg et al., 2000; Boin et al., 2004).
Once attached, V. cholerae cells are able to utilize chitin as a source of C and N (Colwell, 1970; Colwell and Spira, 1992). It was reported early on that V. cholerae multiplies efficiently on chitinous fauna, including crabs, shrimp and zooplankton (Nalin et al., 1979). Its was also observed that V. cholerae O1, grow readily when incubated at 37°C in chitin suspensions, and that the Vibrio mean counts changed little after incubation with chitin at 19°C for 24 h, but fell sharply in the absence of chitin (Kaneko and Colwell, 1974; Nalin et al., 1979). Results of several studies suggested that the surface and gut of zooplankton are ecosystems that may deter the onset of a non-culturable state and/or provide for improved growth of these bacteria (Huq et al., 1983).
Microarray expression profiling and mutational studies of V. cholerae growing on a natural chitin surface has led to a stage-specific model of the V. cholerae chitin utilization programme (Meibom et al., 2004). Expression profiling studies identified three classes of chitin-regulated genes, a part of which come under control of a membrane-bound chitin-sensing histidine chinase, termed ChiS (Li and Roseman, 2004). In the presence of chitin sources and/or gradients of GlcNAc and (GlcNAc)2, full induction of the ChiS-dependent gene (ChiS-regulon) occurs. The involved molecular mechanisms are described in detail by Meibom and colleagues (2004), who showed that (i) ChiS regulates expression of the (GlcNAc)2−6 gene set, including a (GlcNAc)2 catabolic operon, two extracellular chitinases named ChiA-1 (Connel et al., 1998) and ChiA-2 (Meibom et al., 2004), a chitoporin, and ChiRP that mediates colonization of a chitin surface by bacteria, including those that might have previously adhered through the constitutively expressed MSHA (see above), (ii) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc and (iii) the glucosamine dimer (GlcN)2 a product of chitin decomposition and N-deacetylation, induces genes required for the transport and catabolism of nonacetylated chitin residues. Furthermore, it has been shown that ChiA-1, an 88 kDa endochitinase encoded by the chiA gene of V. cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway, a mechanism which also transports cholera toxin (Connell et al., 1998).
A recently discovered consequence of chitin binding on the physiology of V. cholerae is the induction of the competence state, i.e. the capability for acquiring exogenous genetic material during growth on chitin via transformation Meibom and colleagues (2004; 2005) showed that chitin induced the production of a protein appendage, known as a type IV pilus, a structure sometimes required for uptake of DNA in bacteria. They also observed that chitin oligosaccharides, as well as natural chitin from crab shells, stimulated transformation of bacteria to antibiotic resistance and restored their ability to synthesize amino acids. These studies led to the conclusion that competence requires a type IV pilus assembly complex, a putative DNA binding protein and three controlling environmental determinants: the presence of chitin; increasing cell density; and nutrient limitation, growth deceleration, or stress (Meibom et al., 2004). The discovery of chitin-induced DNA transformation in V. cholerae provides a new mechanism for this microorganism effectively to acquire those genes useful in adapting to the aquatic habitat or infecting its human host (Bartlett and Azam, 2005), in parallel with other well-known mechanisms, such as bacterial conjugation and transduction (Faruque and Mekalanos, 2003).
The multicellular level in the hierarchical scale: biofilm formation on chitin-containing surfaces
Chitin interactions at the cellular level such as those described above, in turn can lead to the formation of multicellular complexes, e.g. biofilms (Costerton et al., 1999). In the aquatic environment, diverse surfaces are available for biofilm formation, including suspended mineral particulates, of which the negatively charged silicates are a major component, plants whose surfaces include organic polymers such as cellulose, and the exoskeletons of crustaceans (including zooplankton organisms), which are composed primarily of chitin (Watnick et al., 1999). Environmental studies have clearly shown that attachment to chitin surfaces is an integral part of the aquatic lifestyle of V. cholerae (Tamplin et al., 1990), and biofilm formation constitutes a successful survival mechanism (Watnick and Kolter, 1999; Heithoff and Mahan, 2004; Hall-Stoodley and Stoodley, 2005).
On the basis of previous reports conducted using both artificial and natural substrates (Watnick and Kolter, 1999; Yildiz and Schoolnik, 1999; Watnick et al., 2001; Haugo and Watnick, 2002; Kierek and Watnick, 2003), Moorthy and Watnick (2004) proposed a general model for V. cholerae biofilm development, comprising unique patterns of transcription in the planktonic, monolayer and biofilm stages. Free-swimming planktonic cells are characterized by the presence of flagella and the flagellar genes are actively transcribed at this stage. Transient interactions with a surface are observed in the planktonic stage and these might be mediated by MSHA. Surface association leads to repression of flagellar gene transcription and this, in turn, leads to permanent attachment of cells to the surface in a monolayer. Once formed, this permanent attachment is distinguished from transient attachment sensitive to inhibition by mannose or by α-methylmannoside, by resistance to the action of these sugars. A microbial monolayer is then formed on the surface that degrades the polysaccharide polymers, providing a carbohydrate-rich environment and signals via the quorum sensing circuit (Hammer and Bassler, 2003) to V. cholerae to progress to the next phase, characterized by decreased flagellar gene expression and increased transcription of those genes required for intercellular adhesion and EPS production (Yildiz and Schoolnik, 1999; Watnick et al., 2001; Haugo and Watnick, 2002; Kierek and Watnick, 2003; Moorthy and Watnick, 2004). This results in formation of a mature biofilm consisting of pillars of bacteria attached to the surface (Watnick and Kolter, 1999; Yildiz and Schoolnik, 1999). A rugose morphotype of V. cholerae O1 El Tor requiring a chromosomal locus vps, can arise after prolonged incubation during a biofilm assay and confer increased growth and optimal biofilm development for the organism (Yildiz and Schoolnik, 1999; Hammer and Bassler, 2003). The predominace of the rugose variant allows for the cells, as biofilms, to be predation resistant (Matz and Kjelleberg, 2005; Matz et al., 2005). In addition, the production of biofilm-specific compounds inhibitory to grazing protozoans also appears to be a key defence mechanism, and possibly contributes greatly to the maintenance of V. cholerae on surfaces, including those of chitin material (Matz et al., 2005).
In order to gain insight into the genetic basis of biofilm formation on chitin-containing substrates, Reguera and Kolter (2005) carried out an unbiased genetic screen to identify Tn10 insertion mutants of a gfp-tagged pandemic V. cholerae El Tor strain (N16961) with defects in colonization of chitin. Among the c. 4400 Tn10 insertion mutants screened, they identified 126 with colonization defects, including several carrying transposon insertions in genes implicated in the biosynthesis and assembly of TCP, such as tcpB, tcpE, tcpG and tcpT, suggesting an as yet unrecognized role for TCP in the colonization of chitin surfaces. Using scanning electron microscopy to visualize development of wild-type and TCP mutant biofilms on the surfaces of chitinaceous squid pens, it was found that, although TCP does not function as a bacterial adhesin, it mediates bacterial interactions required for biofilm differentiation on chitinaceous surfaces (Reguera and Kolter, 2005).
The community in the hierarchical scale: association with chitinous organisms in the aquatic environment
In the late 1970s and early 1980s, evidence rapidly accumulated showing unequivocally that pathogenic Vibrio species, including V. cholerae, are naturally occurring in the aquatic environment (Colwell et al., 1977; Blake et al., 1980). Notably, Colwell and colleagues (1980) reported isolation of V. cholerae from plankton samples from both Bangladesh and Chesapeake Bay water, and proposed that a global association between V. cholerae and chitinous plankton existed. This hypothesis was subsequently confirmed by further studies (Huq et al., 1983), namely that strains of V. cholerae, both O1 and non-O1 serogroups, were found to attach to the surfaces of live copepods maintained in natural water samples collected from the Chesapeake Bay and Bangladesh environs. The specificity of attachment of V. cholerae to live copepods was confirmed by scanning electron microscopy, which revealed that the oral region and egg sac were the most heavily colonized areas of the copepods. In addition, survival of V. cholerae in water was extended in the presence of live copepods. The presence of V. cholerae O1 year-round via its commensal association with plankton was established by Colwell and co-workers using direct detection methods (Huq et al., 1990). Since then, other studies have further documented the association between V. cholerae and chitinous plankton in both the marine (Huq et al., 1995; Gil et al., 2004; Huq et al., 2005; Baffone et al., 2006) and freshwater (Shukla et al., 1995) environments, including results of genetic techniques, such as polymerase chain reaction (PCR) and gene probes to detect Vibrio directly in environmental samples (Gil et al., 2004; Huq et al., 2005; Baffone et al., 2006). It was shown by Heidelberg and colleagues (2002) that the relative proportion of V. cholerae, compared with other bacteria, such as γ-proteobacteria and Vibrio/Photobacterium, was higher in the case of V. cholerae associated with zooplankton (especially zooplankton > 200 μm) than in water samples alone. These results suggested that V. cholerae outcompetes other bacterial taxa associated with zooplankton and is commensal with copepods. Plankton organisms and especially zooplankton (i.e. copepods) thus serve as a reservoir of V. cholerae that, by adhering to external surfaces and the gut of copepods, can survive in seawater longer than free living planktonic cells (Colwell, 1996). Huq and colleagues (1983) first proposed that, once cells of V. cholerae attach to zooplankton, they are protected from the external environment and begin to proliferate, taking advantage of the increased surface area and improved conditions of nutrition, the latter derived from the disintegration of phytoplankton and release of nitrogenous products into the water. They also showed that V. cholerae associated with living copepods remained culturable at least 10 days or longer than V. cholerae associated with dead copepods, indicating that living copepods confer on vibrios some form of protection.
Adhesion of vibrios to copepods was reported to be less efficient than their attachment to chitin particles (Kaneko and Colwell, 1975; Vezzulli et al., 2008), perhaps because the copepod esoskeleton contains a wax epicuticle which prevents close contact with the chitin until bacterial enzymatic activities (e.g. lipase) have digested it. The presence of the epicuticule also suggests that some of the recognized adhesins promote V. cholerae adherence to crustacean, directly binding to the epicuticle covering the natural chitin surfaces. Accordingly, the external layer on the external surface of planktonic species may affect V. cholerae colonization (Chiavelli et al., 2001). Recently, laboratory microcosm experiments were used to test for differences in colonization of the copepod species, and different life stages, by V. cholerae O1 and O139. Results showed that V. cholerae O1 consistently achieved higher abundances than V. cholerae O139, when colonizing adults of copepods Acartia tonsa and Eurytemora affinis as well as the multiple life stages of E. affinis (Rawlings et al., 2007).
A fairly hot topic deserving further study is the question of how and to what extent the interaction between bacteria and crustaceans is regulated by the physical and chemical conditions of natural aquatic ecosystems. Several studies have shown that nutrient levels, temperature, salinity and pH affect V. cholerae colonization of chitin surfaces (Huq et al., 1984; Hood and Winter, 1997; Islam et al., 1999; Watnick and Kolter, 1999; Chiavelli et al., 2001; Pruzzo et al., 2003). Interestingly, Pruzzo and colleagues (2003) showed that in harsh growth conditions vibrios do not lose adhesive properties towards zooplankton, even if in the VBNC state.
As a further example of the impact of the V. cholerae connection to chitin at the community level, it was recently reported that chironomids (non-biting midges) constituted a new important reservoir of V. cholerae in the environment (Broza and Halpern, 2001). Although the bacterium has been found associated mainly with egg masses, attachment to the adult midge was also observed (Broza et al., 2005). Using green fluorescent protein (GFP)-tagged V. cholerae, it was shown that most bacteria remained attached to the midge chitin of the adults (Broza et al., 2005). Simulated field and laboratory experiments showed that V. cholerae aerial transfer by flying chironomids, a process largely depending upon the interaction with chitin, may play an additional important role in the dissemination of V. cholerae in nature (Broza et al., 2005).
The ecosystem in the hierarchical scale: chitin cycling
Chitin utilization is important at the ecosystem level by contributing to both C and N recycling. Chitin is one of the most abundant and important sources of nutrients and energy in the marine environment (Gooday, 1990). It is distributed throughout all kingdoms, as it is a crucial component of the cell walls of moulds, yeasts, fungi and certain green algae, and is a major component of the cuticles and exoskeletons of worms, molluscs and arthropods (Jeuniaux, 1982). Annual chitin production is estimated to be > 1011 t in the aquatic biosphere. It has been estimated that the huge production of chitin by copepods and other organisms results in a continuous ‘rain’ of particulate organic matter down through the water column (Yu et al., 1991). From an ecological point of view, chitin plays a key role in the biogeochemical cycles of both C and N, and the rates of chitin production and degradation influence C and N pools and their availability (Poulicek et al., 1998). Chitin is, however, rapidly recycled in most environments and the accumulation of chitin in sediment is low (Gooday, 1990). Poulicek and Jeuniaux (1991) demonstrated that about 90% of the chitin produced in the water column in marine environments was digested within 150 h; in sediments, chitin digestion took longer than in the open water, i.e. at least 1 year. It has been shown that microorganisms, e.g. chitinolytic bacteria, that are ubiquitous in the marine environment play a major role in chitin recycling in the ocean (Kirchner, 1995; Poulicek et al., 1998). Without bacterial activity returning the insoluble polysaccharide to the ecosystem in a biologically useful form, the oceans would be depleted of C and N in a relatively short time (Yu et al., 1991). Adhering bacteria are able to metabolize chitin more efficiently than free-living bacteria, thereby increasing the rate of chitin mineralization in the natural environment (Yu et al., 1991). For complete hydrolysis of chitin to N-acetylglucosamine (GlcNAc), the concerted action of chitinase and β-N-acetylglucosaminidase is essential (Gooday, 1990). Chitinases are enzymes that randomly cleave glycosidic linkages of GlcNAc to produce soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by β-N-acetylglucosaminidases. Among these enzymes, the chitinases play a central role in degradation by bacteria, although the process is extremely complex (Hood and Meyers, 1977). As all Vibrio species, including V. cholerae, produce an active chitinase, it is suggested that a primary role of vibrios is the colonization of chitin and initiation of its degradation in aquatic ecosystems. Such a role was postulated by Hood and Meyers (1977), who demonstrated the role of Vibrio species in chitin turnover and in the metabolism of a marine crustacean.
The global scale: cholera pandemics
At the highest hierarchical level of consideration, the interaction of V. cholerae with chitin plays a role in cholera epidemics and pandemics worldwide.
Toxigenic V. cholerae O1 and O139 are recognized to be the causative agents of epidemic and global pandemic cholera, a disease characterized by acute dehydrating diarrhoea (Kaper et al., 1995). The association of toxigenic V. cholerae with zooplankton, and their eggs that are dispersed in the water, has proven to be a key factor in deciphering the seasonal and geographic patterns of cholera epidemics and pandemics, as well as functioning as a carrier via ocean currents sweeping along coastal areas, translocating plankton and their bacterial passengers (Lipp et al., 2002). The difference in colonization of copepods species by V. cholerae O1 and O139 recently reported by Rawlings and colleagues (2007) (see above) may be significant in the general predominance of V. cholerae O1 in cholera epidemics especially in those regions where water supplies are taken directly from the environment.
V. cholerae O1 colonizing shrimp carapaces has an increased resistance to the effects of high temperature, low pH and desiccation (Nalin et al., 1979; Huq et al., 1983; Colwell, 1996; Dumontet et al., 1996; Castro-Rosas and Escartin, 2005). In addition, when grown on chitin surface as biofilms, V. cholerae cells enhance their viability, and biofilms, in turn, can act as a reservoir for these bacteria between epidemics (Alam et al., 2007). An important consequence of biofilm development on chitin containing surfaces is the fact that a colonized copepod may contain up to 104 cells of V. cholerae. Therefore, plankton blooms can provide the requisite infectious dose for clinical cholera (reported to be 103V. cholerae cells) when untreated water is ingested, as is the case in villages in Bangladesh, India, and many other cholera endemic countries (Colwell, 1996). Nalin and colleagues (1979) also suggested that binding to chitin may offer protection for the vibrios from gastric acid during stomach transit when infecting humans.
On the basis of such acquired knowledge Colwell and colleagues (2003) developed a simple filtration procedure whereby zooplankton, most of the phytoplankton, and particulates > 20 μm were removed from pond or river water by a rudimentary cloth filtering apparatus before household use. Effective deployment of the filtration procedure, from September 1999 to July 2002 in 65 villages of rural Bangladesh, of which the total population for the entire study comprised approximate by 133 000 individuals, yielded a 48% reduction in cholera, compared with control villages not practising filtration.
It is now known that the population dynamics of V. cholerae in the environment are strongly controlled by environmental factors, such as water, temperature, salinity and, as shown above, the presence of copepods, which are, in turn, controlled by larger-scale climate variability (Lipp et al., 2002). The relationship of cholera epidemics, climate and human health has been revealed in greater detail by using satellite remote sensing, and, thereby, has provided stronger evidence that cholera epidemics are climate-linked (Colwell, 1996; Lobitz et al., 2000).
The role of the environment in the cholera disease and, specifically, of the connection with chitin, is further supported by recent studies showing that some virulence factors used by pathogens during infection may derive from their role in their natural habitat. As mentioned above, the ligands produced by V. cholerae that are involved in intestinal colonization, TCP and MSHA (although the role of latter in cholera is less certain than that of former) (Finn et al., 1987; Herrington et al., 1988; Attridge et al., 1996; Thelin and Taylor, 1996; Tacket et al., 1998; Kirn et al., 2000), have been recently implicated in chitin binding and biofilm formation on chitin containing surfaces (Finn et al., 1987; Kaper et al., 1995; Tacket et al., 1998; Reguera and Kolter, 2005). These findings suggest that V. cholerae clones capable of colonizing the intestinal tract are likely to persist in biofilms in the environment (Alam et al., 2006). Hence, the evolution of virulence in bacteria, such as V. cholerae, may be dictated by factors outside the host and the mechanisms of virulence likely reflect adaptive mechanisms functioning in the environment (Reguera and Kolter, 2005).
An additional recent finding, pointing to a common link between the persistence of V. cholerae in the environment and its infection of humans, comes from studies conducted by Zampini and colleagues (2005) who, starting from observations on the molecular mechanisms underlying bacterial binding to chitin, found a new relationship between the V. cholerae lifestyle inside and outside the human host. In fact, by using TnphoA mutants selected using a V. cholerae O1 classical strain, these authors have shown that the 53 kDa protein involved in bacterial attachment to chitin particles and copepods (Tarsi and Pruzzo, 1999), also mediates adherence to cultured intestinal epithelial cells. The interaction was found to occur via GlcNAc binding; in fact, this sugar, besides being a chitin monomer, also represents a common modification of glycoproteins and lipids present on the intestinal epithelium. These results are in agreement with previous observations by Datta-Roy et al. (1989) and Sasmal and colleagues (1992) who showed that adhesion to rabbit intestine of most V. cholerae O1 and non-O1 isolates was inhibited to various extent by GlcNAc and suggested a role for GlcNAc-sensitive ligands in mediating adherence to intestinal cells. From a more general point of view, these observations suggest the ability to use the same structure to interact with different substrates (in the environment and on the human host) may be a common property of pathogenic bacteria that have environmental reservoirs and may well represent a discriminating feature between the harmless and the potentially pathogenic environmental bacteria. The role of GlcNAc-sensitive ligands in intestinal adherence was confirmed by elegant, subsequent studies conducted by Kirn and colleagues (2005) who used a ΔgbpA mutant and a GbpA producing derivative of its (ΔgbpA, pGbpA-His strain), to show that the 53 kDa GbpA protein implicated in adherence of a V. cholerae classical strain to chitin surfaces, also mediates adherence to intestinal cells. In addition, they suggested that the GbpA ligand could prove to be a promising target for vaccine design. In fact, the LD50 in infant mice of the V. choleraeΔgbpA mutant was shown to be about 10 fold higher than that of the wild-type strain and the ΔgbpA, pGbpA-His strain. Interestingly, a TnphoA mutant not expressing a 53 kDa protein has previously been shown to be less toxigenic in vitro than the parent strain, and to have an LD50 in infant mice significantly higher than the parent strain (Singh et al., 1994). Accordingly, mice orally inoculated with wild-type V. cholerae bacteria mixed with antisera from rabbits immunized using a cloned 53 kDa protein showed significant survival compared to control mice (Kirn et al., 2005).
Chitin is an abundant polymer in the marine, estuarine and brackish water environments, and the V. cholerae–chitin connection has played a fundamental role in the ecology and evolution of both the bacterium and the disease cholera (Fig. 1). This interaction has provided the microorganism with a significant number of advantages, including food availability, adaptation to environmental nutrient gradients, tolerance to stress and protection from predators. The hierarchical perspective of scale used in the discussion of this interesting connection suggests the multiple lifestyles of V. cholerae and its pathogenicity-linked properties are a consequence of environmental selection in its primary aquatic habitat. If persistence inside its primary habitat (i.e. the aquatic environment) is paramount, then V. cholerae has been able to acquire those properties needed for survival, and its fitness and function outside the natural environment, for example in the human gut, largely depend on adapting environment-acquired properties to the new habitat. Strong evidence for such an evolution is provided by the findings of Reguera and Kolter (2005) regarding the role of TCP in mediating bacterial interactions required for biofilm differentiation on chitinaceous surfaces, and the recent discovery that chitin-binding molecular determinants are involved in both environmental persistence and virulence (Kirn et al., 2005; Zampini et al., 2005). A speculation can be tempted from this review of the literature that the mechanisms of cholera in the human gut derive from an adaptation by the bacterium to assist its host in osmoregulation, as the physiological responses of V. cholerae are optimized to the physical and chemical conditions in the marine, estuarine and brackish water environment. Thus, the interaction of V. cholerae with chitin, at multiple levels in an hierarchical scale in the environment (the global perspective), represents a useful model for the analysis of the role of primary habitat selection in the development of pathogenicity traits of bacteria whose primary (autochthonous) habit is the aquatic environment.