FlgO is an outer membrane protein that localizes throughout the membrane and not at the flagellar pole. Although FlgO and FlgP do not specifically localize to the flagellum, they are required for flagellar stability. Due to the nature of these motility defects, we established that the flagellum is not sufficient for adherence; rather, motility is the essential factor required for attachment and thus colonization by V. cholerae O1 of the classical biotype. This study reveals a novel mechanism for which the outer membrane proteins FlgO and FlgP function in motility to mediate flagellar stability and influence attachment and colonization.The causative agent of the infectious diarrheal disease cholera is the bacterium Vibrio cholerae. This enteric pathogen naturally inhabits an aquatic environment and is motile by the action of a single polar flagellum. The mechanism of pathogenesis by which V. cholerae causes disease is a stepwise and cyclic process. First, contaminated food or water is ingested; then, the bacteria pass through the stomach. After withstanding that acidic environment, the bacteria enter the lumen of the intestine and attach to the intestinal epithelium of the human host. Once the bacteria establish an initial attachment, the bacteria begin to express the toxin-coregulated pilus, which facilitates bacterium-bacterium interactions and the formation of microcolonies. Subsequently, an enterotoxin, cholera toxin, is expressed and secreted by the bacteria, which through a cascade of events leads to profuse, watery diarrhea. Eventually, the bacteria are shed from the host and are dispersed back into the environment, thus renewing the cycle.Although the later stages of the disease, which include the production of toxin-coregulated pilus and cholera toxin, are well characterized, little is known about the initial stages of pathogenesis. The attachment factor GbpA has been shown to bind chitin, an abundant substance found in the environment that is a polymer of N-acetylglucosamine (GlcNAc) carbohydrate subunits (17). GbpA is also involved in the initial attachment of V. cholerae to epithelial cells, which have exposed GlcNAc moieties on their surfaces. In the absence of gbpA, only a 50% reduction in attachment is observed, suggesting that there may be additional factors that are involved in initiating attachment to the intestinal epithelium.Colonization requires a functional flagellum; therefore, motility is a crucial virulence factor of V. cholerae (9, 12). The flagellum functions to propel the cell through its environment. In the host, the flagellum functions as a means to gain access to the microvilli of the epithelial cells by facilitating the penetration of the intestinal mucus layer, an action that is important for colonization (1,4,14). The flagellum is composed of a base, a hook, and a filament. The flagellar apparatus is assembled in an ordered cascade of events. The genes necessary for motility and chemotaxis are found distributed among six regions on the large chromosome of V. cholerae (regions I t...

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Vibrio cholerae relies on two main virulence factors, the toxin coregulated pilus (TCP) and cholera toxin, to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protects against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here we report the de novo x-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function.

As an alternative approach toward the development of a cholera vaccine, the potential of peptide mimics of Vibrio cholerae lipopolysaccharide (LPS) to elicit cross-reactive immune responses against LPS was investigated. Two closely related protective monoclonal antibodies, S-20-4 and A-20-6, which are specific for Ogawa O-antigen (O-specific polysaccharide; O-SP) of V. cholerae O1, were used as the target antibodies (Abs) to pan phage display libraries under different elution conditions. Six phage clones identified from S-20-4 panning showed significant binding to both S-20-4 and A-20-6. Thus, it is likely that these phage-displayed peptides mimic an important conformational epitope of Ogawa antigens and are not simply functionally recognized by S-20-4. Each of the six phage clones that could bind to both monoclonal antibodies also competed with LPS for binding to S-20-4, suggesting that the peptides bind close to the paratope of the Ab. In order to predict how these peptide mimics interact with S-20-4 compared with its carbohydrate counterpart, one peptide mimic, 4P-8, which is one of the highest affinity binders and shares motifs with several other peptide mimics, was selected for further studies using computer modeling methods and site-directed mutagenesis. These studies suggest that 4P-8 is recognized as a hairpin structure that mimics some O-SP interactions with S-20-4 and also makes unique ligand interactions with S-20-4. In addition, 4P-8-KLH was able to elicit anti-LPS Abs in mice, but the immune response was not vibriocidal or protective. However, boosting with 4P-8-KLH after immunizing with LPS prolonged the LPS-reactive IgG and IgM Ab responses as well as vibriocidal titers and provided a much greater degree of protection than priming with LPS alone.Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae, which is prevalent in many developing countries. According to the World Health Organization, an estimated 120,000 deaths occur from cholera worldwide each year. Although the establishment of clean water and food, adequate personal hygiene, and sanitation are the long term solutions for cholera control, in the short term, sufficient improvements in these areas are difficult to achieve in most cholera-endemic areas. Meanwhile, there is an urgent need for improved vaccines as an additional public health tool for cholera prevention (1). Since the affected population is very poor, development of an inexpensive vaccine is most desirable.Currently available vaccines for cholera are based on killed, whole cell, or live attenuated formulations. The killed, whole cell (WC and WCrBS) vaccines provide only partial short term protection. The live, attenuated strains, such as CVD 103-HgR, have greatly improved efficacy in North American volunteers but have not been proven efficacious in field trials (1, 2). However, field trials of the live attenuated strain Peru-15 have proven to be safe, immunogenic, and efficacious (3). An approach that could serve as an alternative or be used to augment e...

The licensed oral, live-attenuated bacterial vaccine for typhoid fever, Salmonella enterica serovar Typhi strain Ty21a, has also been utilized as a vaccine delivery platform for expression of diverse foreign antigens that stimulate protection against shigellosis, anthrax, plague, or human papilloma virus. However, Ty21a is acid-labile and, for effective oral immunization, stomach acidity has to be either neutralized with buffer or by-passed with Ty21a in an enteric-coated capsule (ECC). Several studies have shown that efficacy is reduced when Ty21a is administered in an ECC versus as a buffered liquid formulation, the former limiting exposure to GI tract lymphoid tissues. However, the ECC was selected as a more practical delivery format for both packaging/shipping and vaccine administration ease. We have sought to increase Ty21a acid-resistance to allow for removal from the ECC and immune enhancement. To improve Ty21a acid-resistance, glutamate-dependent acid resistance genes (GAD; responsible for Shigella spp. survival at very low pH) were cloned on a multi-copy plasmid (pGad) under a controllable arabinose-inducible promoter. pGad enhanced acid survival of Ty21a by 5 logs after 3 hours at pH 2.5, when cells were pre-grown in arabinose and under conditions that promote an acid-tolerance response (ATR). For genetically 100% stable expression, we inserted the gad genes into the Ty21a chromosome, using a method that allowed for subsequent removal of a selectable antibiotic resistance marker. Further, both bacterial growth curves and survival assays in cultured human monocytes/macrophages suggest that neither the genetic methods employed nor the resulting acid-resistance conferred by expression of the Gad proteins in Ty21a had any effect on the existing attenuation of this vaccine strain.