The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4-to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.

Cholera epidemics caused by Vibrio cholerae El Tor O1 strains are typified by a large number of asymptomatic carriers who excrete vibrios but do not develop diarrhea. This carriage state was important for the spread of the seventh cholera pandemic as the bacterium was mobilized geographically, allowing the global dispersion of this less virulent strain. Virulence factors associated with the development of the carriage state have not been previously identified. We have developed an animal model of cholera in adult C57BL/6 mice wherein V. cholerae colonizes the mucus layer and forms microcolonies in the crypts of the distal small bowel. Colonization occurred 1 to 3 h after oral inoculation and peaked at 10 to 12 h, when bacterial loads exceeded the inoculum by 10-to 200-fold, indicating bacterial growth within the small intestine. After a clearance phase, the number of bacteria within the small intestine, but not those in the cecum or colon, stabilized and persisted for at least 72 h. The ability of V. cholerae to prevent clearance and establish this prolonged colonization was associated with the accessory toxins hemolysin, the multifunctional autoprocessing RTX toxin, and hemagglutinin/protease and did not require cholera toxin or toxin-coregulated pili. The defect in colonization attributed to the loss of the accessory toxins may be extracellularly complemented by inoculation of the defective strain with an isogenic colonization-proficient V. cholerae strain. This work thus demonstrates that secreted accessory toxins modify the host environment to enable prolonged colonization of the small intestine in the absence of overt disease symptoms and thereby contribute to disease dissemination via asymptomatic carriers.

Vibrio cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this colonization were investigated. Successful colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX), and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. cholerae encounters barriers to infection including an acidic environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause cholera disease. This adult mouse model of intestinal V. cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.

Although the presence of phosphorylcholine (PC) in Trichinella spiralis is well established, the precise structure of the PC-bearing molecules is not known. In this paper, we report structural studies of N-glycans released from T.spiralis affinity-purified antigens by peptide N-glycosidase F. Three classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and eight N-acetylhexosamine residues. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine.

Following intranasal inoculation, Vibrio cholerae KFV101 (⌬ctxAB ⌬hapA ⌬hlyA ⌬rtxA) colonizes and stimulates tumor necrosis factor alpha and interleukin 1␤ (IL-1␤) in mice, similar to what occurs with isogenic strain P4 (⌬ctxAB), but is less virulent and stimulates reduced levels of IL-6, demonstrating a role for accessory toxins in pathogenesis. Morbidity is enhanced in C3H/HeJ mice, indicating that Toll-like receptor 4 is important for infection containment.Vibrio cholerae is a gram-negative pathogen that induces diarrhea dependent upon the action of the cyclic AMP-stimulating cholera toxin (CT) (11). During cholera disease, patients in the acute phase do not have extensive tissue damage such as might be observed with shigellosis (21, 22). However, low levels of infiltration of neutrophils, mast cells, and eosinophils in infection and the presence of mediators of inflammatory cell regulation on the second day of cholera disease indicate that V. cholerae stimulates a proinflammatory response early during infection (21). This inflammatory response possibly becomes excessive in the absence of genes for CT since volunteers given CT-deficient strains have significantly increased titers of lactoferrin and CXCL8, indicative of more severely inflammatory disease typified by symptoms that include vomiting, fever, mild diarrhea, and cramping (25). These symptoms are also typical of nonepidemic strains of V. cholerae that do not produce CT (4,7,10,13,16,17). This increase in the inflammatory nature of V. cholerae infection could be due to the absence of the immunomodulatory activity of the B subunit of CT that blocks the secretion of proinflammatory cytokines by macrophages, dendritic cells, and epithelial cells in response to bacterial lipopolysaccharide (LPS) (24) due to downregulation of mitogen-activated protein kinase pathways (6).A major problem with this residual disease of non-CT-producing V. cholerae is that these strains are frequently sufficiently virulent to make them unsafe for use as live attenuated vaccines (2,14,26,27). It has been proposed that the actions of accessory toxins of V. cholerae are responsible for this increased inflammation and contribute to the reactogenicity of live attenuated vaccine strains (24). Recently, we developed a murine model that is sensitive to accessory toxins and can be used to investigate early events in the initiation of the host response to non-CT-producing V. cholerae. Although adult mice are resistant to intestinal colonization by V. cholerae (12), we found that acute inflammation develops in the lung tissue after intranasal inoculation with V. cholerae, providing a model for the assessment of the toxic action on inflammation of a mucosal epithelial layer (8). This model is based on experiments using Shigella flexneri dependent on the premise that the bronchial tree is composed of a mucosal lining with relevant characteristics similar to those of the intestine, including simple cubodial-to-columnar epithelial cells, lymphoid aggregates, bronchiole-associated lymphoid ti...