gram-negative cause of severe diarrheal disease around 120,000 death per annum

2. Vibrio cholerae: • gram-negative • cause of severe diarrheal disease • around 120,000 death per annum • 200 known serogroups • cholera associated only with two serogroups (O1 and O139) • O1 divided in two serotypes (Inaba and Ogawa) and further in two • biotypes (classical and El Tor) • humans are the only known vertebrate host, infection by ingestion • V. cholerae is not particular ph-resistant, so infection seems to require • high dose (about a million bacteria) • small intestine is the main site of infection • idea that chemotaxis needed to find colonization niche and virulence • factor expression

3. Virulence factors • cholera toxin: • ribosylating enterotoxin • secreted AB5 subunit toxin, B unit binds to epithelia cells, A units • enter cells via endocytosis • permanent ribosylation of G proteins resulting in constitutive • cAMP production. • leads to secretion of H2O, Na+, K+, Cl-, and HCO3- into the • lumen • responsible for watery diarrhea (rice-water stool) • toxin co-regulated pilus (TCP) • required for colonization in human and animal models • pili are believed to mediate microcolony formation • gene expression is tightly regulated, no expression in extra-intestinal • growth

4. Lifecycle of pathogenic Vibrio cholerae:

5. Lifecycle of pathogenic Vibrio cholerae: • can shed ten trillion bacteria per day • these bacteria are highly motile • Shed bacteria can be ingested by other humans or settle into enviromental-reservoir • stage • Cholera is natural inhabitant of freshwater, brackish and coastal-water habitats • It can exist in a free-living form or associated with hosts like zooplankton or form • biofilms

6. Flagellar-based motility:

7. Flagellar-based motility: • Different kinds of flagellation in bacteria • Peritrichous flagella are found for example in E.coli, monotrichous flagellum in • V. Cholerae • covered by extension of the outer membrane • can achieve around 100,000 revolution per minute (sodium-motive force) • other forms of motility: twitching motility and gliding motility

8. Chemotactic in V. cholerae and other bacteria: • in flagellar motility chemotaxis is achieved by modulating direction, • speed… • best understood in E.coli

9. Chemotactic in V. cholerae and other bacteria: • in flagellar motility chemotaxis is achieved by modulating direction, • speed… • best understood in E.coli • signal reception by methyl-acccepting • chemotaxis proteins • MCP cluster at cell pole • ligand occupancy is communicated to • flagella • can respond to change of a few • molecules

10. Chemotactic in V. cholerae and other bacteria: • in flagellar motility chemotaxis is achieved by modulating direction, • speed… • best understood in E.coli • V. cholerae has many chemotaxis paralogues • organized in three operons but only operon 2 important in vitro • strains with single or combined mutations in the paralogues retain full • virulence in mouse model • speculated that operon 1 and 3 regulate flagellum-independent • motility

11. The role of chemotaxis in virulence: • motility and chemotaxis ranges from being • crucial to being dispensable • Shigella species that are non-motile but • highly infectious • invasive enteric bacteria might nor require • motility for infection (translocation through M • cells) • non-invasive pathogens (H. pylori) require • chemotaxis to stay within the mucus layer • chemotaxis inhibits V. cholera colonization • non-chemotactic mutants showed 10-fold • increased infectivity • advantage is specific to host small intestine

12. Intestine colonization by V. cholerae: • wild-type mainly colonize in the lower • half of the small intestine • bile is a possible attractant • non-chemotactic mutants are found in • the whole small intestine • less specific – greater surface area to colonize • only CCW-biased flagellar mutants show • out-competition phenotype • these mutants swim in straight runs • direction is random but the covered • distance is enough in regard to diameter • of small intestine lumen

13. Intestine colonization by V. cholerae: • wild-type colonize at the base of villi • proposed that there are antimicrobial • substances present that kill bacteria • like definsins released from Paneth cells • non-chemotactic mutants are mainly found in • the mucus layer and the luminal side of the villi • reasons for wild-type to be attracted to the • base of the villi: • signal of max. expression of cholera toxin • better protection from peristalsis • might be crucial in humans

14. Motility and V. cholerae virulence: • to determine the role of motility it must be separated from adherence • effects of the flagella • comparison of fla- and fla+mot- mutants • no differences in V. cholerae but motility itself seems to be important • in some organism motility is inhibited by virulence gene expression • - not in V. cholerae • bacteria in rice-water stool are highly motile • switched back on before exit the host • speculated that rice-water V. cholerae might be in a transiently non- • chemotactic CCW-biased state • improved infection of new human hosts

15. Thank you for your attention