Research Article: Vibrio cholerae Biofilms and Cholera Pathogenesis

Date Published: February 4, 2016

Publisher: Public Library of Science

Author(s): Anisia J. Silva, Jorge A. Benitez, Stephen Baker.

Abstract: Vibrio cholerae can switch between motile and biofilm lifestyles. The last decades have been marked by a remarkable increase in our knowledge of the structure, regulation, and function of biofilms formed under laboratory conditions. Evidence has grown suggesting that V. cholerae can form biofilm-like aggregates during infection that could play a critical role in pathogenesis and disease transmission. However, the structure and regulation of biofilms formed during infection, as well as their role in intestinal colonization and virulence, remains poorly understood. Here, we review (i) the evidence for biofilm formation during infection, (ii) the coordinate regulation of biofilm and virulence gene expression, and (iii) the host signals that favor V. cholerae transitions between alternative lifestyles during intestinal colonization, and (iv) we discuss a model for the role of V. cholerae biofilms in pathogenicity.

Partial Text: The water-borne diarrheal disease cholera is caused by the gram-negative and motile bacterium Vibrio cholerae of serogroup O1 and O139. V. cholerae, as other members of the Vibrionaceae family, are common inhabitants of aquatic ecosystems. In regions where cholera is endemic, occurrence of the disease follows a seasonal pattern that correlates with climatic changes [1–8]. Import of V. cholerae O1 into nonendemic areas with poor sanitation commonly results in rapid dissemination of the disease through a fast fecal–oral route that takes advantage of the transient hyperinfective stage of V. cholerae present in fresh cholera stool [9–12]. V. cholerae O1 can be divided into two biotypes, classical and El Tor, which differ in the severity of clinical symptoms and the expression and regulation of major virulence factors [13]. Humans have experienced seven cholera pandemics. The seventh and current pandemic is characterized by the predominance of the O1 serogroup of the El Tor biotype, with periodic emergence of serogroup O139, which originated from the El Tor biotype and exhibits a new lipopolysaccharide (LPS) and a capsule [14].

The two major virulence factors expressed by V. cholerae O1 and O139 are (i) cholera toxin (CT), an AB5 family ADP-ribosyltransferase responsible for the profuse rice-watery diarrhea typical of this disease [13], and (ii) the toxin-coregulated pilus (TCP), a type IV pilus that mediates adherence and microcolony formation and is required for intestinal colonization in neonate mice and humans [15–17]. The genes encoding the CT subunits ctxA and ctxB constitute an operon within the prophage form of the filamentous phage CTXΦ [18]. The genes required for TCP biogenesis form a large cluster known as the V. cholerae pathogenicity island (VPI) or TCP island [19]. Within this cluster, tcpA encodes the major pilus subunit.

V. cholerae has evolved to effectively colonize disparate ecological niches: the nutrient-rich human small intestine and aquatic environments. In the aquatic environment, Vibrios must withstand diverse physical, chemical, and biological stresses that include nutrient limitation, extreme temperatures, oxidative stress, bacteriophage predation, and protozoan grazing [27,28]. In the gastrointestinal tract, Vibrios are exposed to low pH, bile acids, elevated osmolarity, iron limitation, antimicrobial peptides, and intermittent nutrient deprivation [29]. Thus, both environments pose common and specific challenges to bacterial growth and multiplication. The human small intestine, nevertheless, provides a superior bounty of nutrients compared to aquatic environments. Consistently, V. cholerae can grow to high titers in the human gut, and cholera patients can shed 107–109 virulent Vibrios per mL in the rice-watery stool [12]. In order to reach high titers in the gut, V. cholerae must overcome as many stressful conditions as it requires to survive and persist outside the human host. Proof of this is that disruption of genes encoding the general stress response regulator RpoS (σS) or the RNA polymerase σE subunit (RpoE) that mediates the envelope stress response results in significant attenuation of V. cholerae virulence and its capacity to colonize the small intestine [30,31]. Thus, whether in the human host or in the aquatic environment, the cholera bacterium employs common survival strategies. These stratagems involve (i) the activation of general and specific stress responses, (ii) expression of flagellar motility and chemotaxis, (iii) attachment to surfaces, (iv) development of multicellular sessile communities, and (v) detachment. Particularly critical to V. cholerae survival in the host and estuarine waters is its ability to switch between motile (planktonic) and sessile (biofilm) lifestyles in response to chemical and physical changes in the extracellular milieu.

Biofilms are microbially derived sessile communities characterized by cells that are attached to a substratum, an interface, or to each other; are embedded in a self-produced matrix; and exhibit an altered phenotype with respect to growth rate and transcription profile [32,33]. This definition includes communities of Vibrios anchored to abiotic surfaces or to biotic substrata such as the human intestinal mucosa or the chitinous exoskeleton of crustaceans, Vibrio aggregates in suspension, floccules, and pellicles formed at the liquid–air interface of static cultures.

The regulation of virulence gene expression has been the subject of extensive research and recent reviews [97,98]. Expression of CT and TCP is regulated by a complex regulatory network (Fig 1). At the top of the Tox regulatory cascade, regulators AphA and AphB enhance the transcription of the transmembrane regulators TcpP and TcpH [99,100]. TcpP/H, in concert with transmembrane regulators ToxR/S [101,102], activate the expression of the soluble AraC-family regulator ToxT [103]. Finally, ToxT interacts with the ctxA and tcpA promoters to activate the production of CT and TCP [103]. The dependence of ctxAB and tcpA expression on the upstream regulators ToxR/S, TcpP/H, and ToxT was confirmed in vivo using the suckling mouse model and RIVET [104].

Numerous physical and chemical cues in the gut (i.e., temperature, pH, oxygen tension, osmolarity, bile salts, antimicrobial peptides) can impact the infective process. It is likely that all these factors, at least indirectly, influence virulence and biofilm formation. Compounds that perturb the cell envelope can generate additional stresses, resulting in elevated expression of RNA polymerase subunits σE and σS [134]. Intestinal bile exhibits these properties and has received abundant consideration as a host-specific signal that can potentially modulate Vibrio behavior in the gut. Intestinal bile is a complex mixture of bile acids, cholesterol, and unsaturated fatty acids and is subject to numerous chemical transformations in the gastrointestinal tract (i.e., removal of amino acid side chains, oxidation, hydroxylation, and dehydroxylation). Crude bile or sodium cholate was found to enhance biofilm formation in a VpsR-dependent manner [135]. This observation is consistent with the recent finding that a mixture of bile acids increased the intracellular c-di-GMP pool, an effect that was quenched in the presence of bicarbonate [136]. Surprisingly, the individual bile salt taurocholate was found to promote biofilm dispersal rather than formation [137]. These differences may reflect the limited capacity of commercial bile preparations to represent the properties of bile secreted into the intestinal lumen.

The small intestine commences at the pyloroduodenal junction and ends at the ileocaecal junction and comprises, successively, the duodenum, jejunum, and ileum. The mucosal side of the small intestine is composed of absorptive polarized epithelial cells (enterocytes) organized in the form of finger-like projections or villi and mucin-secreting goblet cells covered by a protective mucus barrier. The protective mucus coat consists of a firmly adherent inner layer overlaying the villi and a loosely attached outer layer [144]. The thickness and biophysical properties of the mucus barrier varies along the gastrointestinal tract and is determined by the balance between its secretion rate and its erosion through enzymatic degradation and mechanical shear. The total mucus layer thickness is estimated to be 170–123 μM in the duodenum and jejunum and 480 μM in the ileum [145].

In the context of this article, we consider detachment the process by which cells in a sessile stage detach and switch to the planktonic (motile) lifestyle. The mechanism by which cells detach could include degradation of the substratum to which they are attached or cleavage of a protein or adhesin that anchors monolayers or multilayers of cells to a surface. In fully developed biofilm communities, detachment could be triggered by nutrient deprivation, accumulation of specific metabolites or toxic products, or as a consequence of external signals. The effective dispersion of motile Vibrios from a mature biofilm would require a certain degree of degradation of the biofilm matrix that holds cells together.

The genes required for the expression of flagellar motility and the biosynthesis of the biofilm exopolysaccharide and protein matrix (i.e., vps, rbm) are necessary for the efficient colonization of the small intestine. Therefore, the capacity of V. cholerae to adopt both lifestyles during infection could provide fitness in the environment of the gut. As shown in Table 1, flagellar motility could provide Vibrios with the advantage of mobility and capacity to spread along the gastrointestinal tract. On the other hand, biofilm formation could provide a mechanism of resistance to the host innate defense mechanism, facilitate a fast fecal–oral transmission route, and increase the fitness of those Vibrios that are directly shed back into the aquatic environment.

A significant amount of work on the regulation of virulence gene expression and biofilm development has been concentrated in a relatively small number of strains of serogroup O1 (classical and El Tor biotype) and O139. The focus on a reduced number of strains favors the conception of molecular models but fails to represent the broad phenotypic and genetic diversity that occurs within serogroups and biotypes. Complex phenotypes such as virulence and biofilm development integrate numerous environmental cues and can exhibit strain-specific behavior, often resulting in conflicting data. Hence, the documented regulatory connections between virulence and biofilm expression summarized above should be appreciated in the context of genetic landscapes and environment conditions that can alter the expression of mutant phenotypes. An example of genetic diversity affecting virulence and biofilm formation is quorum sensing. In this case, some O1 lineages use the quorum sensing regulator HapR, and others employ the VieA regulatory system to respond to changes in cell density [189].



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