1. Emerging pathogens

2. Emergence of infectious disease ~ habitat / niche BoundaryBarrier adaptation host / bacteria time homeostasis emerging infectious disease re-establish balance homeostasis bacteria & host

3. Mechanisms of host adaptation Analysis of host adaptation

4. Mechanisms of bacterial adaptation Analysis of bacterial adaptation

5. Emerging bacterial disease avoids host response aggressive pathogen stealth route overwhelm innate immune response rapid infection - no adaptive immunity frontal attack strategies engages innate immune response avoids / manipulates adaptive immunity carrier state / chronic infections

6. Development of stealth pathogens / bacteria BoundaryBarrier natural habitat homeostasis colonizes in host goal - persistance chronic infection symbiotic relationship

7. Mycobacterium tuberculosis - tuberculosis Listeria monocytogenes - listeriosis Salmonella typhi - typhoid fever Helicobacter pylori - gastric ulcers / cancer Chronic infectious agents Symbionts Rhizobium spp. - nitrogen fixing bacteria on roots / legumes Wolbachia spp. - induce sex ratio distortions in insects Mitochondria - energy production Stealth bacteria / pathogens Bacterial adaptation to host cells associated with lateral gene transfer (mobile and unstable genetic elements)

8. Establishment of chronic infections (Batut et al, Nature Reviews Microbiology, 2004) First contact with host cell Adherence / invasion Life in the environment Establishing life within the host Avoid host defenses Establishing life within the host Adapt to host environment Extending the niche Modulate host biology Extending the niche Multiply / survive in replication niche

9. Avoid host defenses : - Invade host cells rapid - Rho, Rac, Cdc42 mediated invasomes - engulf bacterial aggregates zipper - silent cell entry - Evade innate immunity lack TL4 reactive LPS - Block adaptive immunity - Evade adaptive immunity by genetic rearrangement Establish life within host: - Within phagosome arrest - phagosome acidification / fusion redirect - phagosome trafficking utilize - phagosome environment lyse phagosome - move via actin tails - Induce host cell proliferation - Inhibit host cell apoptosis - Adapt to host conditions (stomach acidity) Common strategies of stealth bacteria Manipulate host cell: - Type III secretion - Type IV secretion (J. Batut, S. Andersson, D. O’Callghan, Nature Reviews, 2004)  Proteobacteria

10. Chronic infection with Mycobacterium tuberculosis (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002) M. tuberculosis phagosome arrest

11. Development of aggressive pathogens BoundaryBarrier becomes natural habitat homeostasis colonize in new environment aggressive pathogen microorganism-rich environment (soil / water / intestine) acquire new virulence genes provide a survival advantage competes against normal flora & host defenses

12. Vibrio cholerae adhere / colonize in intestine rapid onset / severe diarrhea massive fluid loss - dehydration hypotension collapse of the circulatory system bacteria eventually wash out self-limiting disease (www.cameroon-info.net/ img/news/cholera_victim) Disease - cholera (UCLA Department of Epidemiology website) Transmission contaminated water raw seafood

13. Cholera pandemics - begin in coastal communities - link between marine environment (algal blooms) - V. cholerae survival / spread (Source: Dr. Rita Colwell, Director, National Science Foundation and Professor of Microbiology, University of Maryland) Sea surface temperature (SST) changes in Indian Ocean vs. number of cholera cases in Bangladesh. marine reservoir Vibrio cholerae

14. (www.hinduonnet.com/.../ 07/stories/0807048f.htm) Tcp - (toxin coregulated pili) - adherence origin - filamentous phage (VPI  ) cholera toxin - A-B toxin encoded on CTX phage Tcp = receptor for CTX phage Acquisition of virulence factors B 5 - subunit (G M1 ganglioside) A - subunit (ADPRT activity - Gs) Vibrio cholerae

15. 1992 - novel epidemic strain emerged in India - serotype 0139 - Bengal strain carries conjugative transposon allowed - horizontal gene transfer of DNA encoding 0139 antigen usurped El Tor until 1994 (Obtained from CDC) The global spread of cholera during the seventh pandemic (1960 - 1990’s) - El Tor biotype Emerging Vibrio cholerae disease Epidemiology remains a major problem in developing countries 155 serotypes most epidemics - serotype 01 Classical biotype - 1881-1896 & 1899-1923 pandemics El Tor biotype - ongoing cholera pandemic

16. WHO estimates that officially reported cases represent ~ 5-10% of actual cases worldwide (www.who.int/topics/ cholera/surveillance/) Areas reporting cholera cases in 2004Cholera, 2000-2001 Cholera Epidemiology Grey areas: countries with cholera cases Black dots: countries with imported cholera cases

17. Emergence of pathogenic Vibrio cholera BoundaryBarrier ancestral species lacked CT infection excluded by normal flora becomes natural habitat salt / fresh water homeostasis colonizes in small intestine aggressive pathogen acquired VPI  phage encoded Tcp then CTX phage encoded CT allows bacteria to flourish in intestine shed in large numbers back to natural habitat (salt / fresh water) acquired virulence factors Tcp / cholera toxin - phage 0139 serotype - conjugation

18. Emergence of pathogenic Escherichia coli

19. Not colonized Streptococcus, Lactobacillus Streptococcus, Lactobacillus, Bacteroides, Bifidobacter E. coli, Lactobacillus, Bacteroides, Bifidobacterium, Enterococcus Clostridium, 40+ species, >90% anaerobes Bacteroides, Eubacterium, Fusobacterium, Bifidobacterium, Peptostreptococcus, Ruminococcus, Enterococcus, Streptococcus, Clostridium, Enterococcus Microbial GI tract ecosystem (probiotics)

20. Not colonized Helicobacter pylori E. coli - EHEC, ETEC, Vibrio cholera C. perfringes, B. cereus, S. aureus E. coli - EHEC, EIEC, Shigella, Clostridium difficile Pathogenic bacteria of the GI tract E. coli - EPEC, Salmonella Campylobacter jejuni

21. (Dennis Kunkel Microscopy, 2001) Escherichia coli Associated disease: diarrhea / hemorrhagic colitis Transmission: contaminated food or water Bacteriology: Gram-negative rod - motile facultative anaerobe serotype - based on O, H, K antigens - LPS; H - flagellum (hauch); K - capsule (i.e. O157:H7) virotype - based on virulence factors (i.e. ETEC) (www.geocities.com/ CapeCanaveral/3504/gallery.htm)

22. Emergence of pathogenic Escherichia coli ancestral species not pathogenic not cleared by host becomes natural habitat GI tract - warm-blooded animals homeostasis exposed to GI bacteria acquired phage / plasmid encoded virulence factors from intestinal pathogenic bacteria now competes more effectively in host shed into environment transferred to other hosts virulence factors virotype dependent aggressive pathogen

23. Escherichia coli enteropathogens Enteropathogenic E. coli (EPEC) Enterotoxigenic E. coli (ETEC) Enteroaggregative E. coli (EAggEC) Enterohemorrhagic E. coli (EHEC) Enteroinvasive E. coli (EIEC)

24. EPEC - Enteropathogenic E. coli Disease: major cause of diarrhea in children (> 1 million children die / year from EPEC) associated with attaching-effacing (A/E) lesion loss of microvilli host actin-mediated pedestal formation (Knutton et al, Oxford University Press, 1998) EPEC bundle forming pili (Anantha et al, Infect. Immun., 1998) Virulence: not toxigenic / not invasive type III secretion - genes encoded - chromosomal pathogenicity island (PAI) (35 kb - LEE - locus of enterocyte effacement) adhesion - contact with host cell induces: bundle-forming pilus - (plasmid encoded)

25. EPEC - Pathogenesis Type III secretion: Apparatus - EspA, EspB, EspD (EspF) Tir- phosphorylated by host tyrosine kinase, becomes receptor for bacterial adhesin - intimin leads to intimate attachment - activation PKC (PLC  IP 3 ) results in chloride secretion - causing diarrhea - (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002) Acquisition of virulence: type III secretion - encoded on LEE PAI bundle forming pilis - plasmid encoded

26. ETEC - Enterotoxigenic E. coli Disease: childhood diarrhea in developing countries ‘traveler’s’ diarrhea’ (From: info.med.yale.edu/ library/zeiss) Virulence: toxigenic - heat labile toxin (LT) heat stable toxin (ST) (both toxins - plasmid encoded) fimbriae CFA - colonization factor antigen (plasmid encoded)

27. ETEC - pathogenesis ST- heat-stable toxin guanilyn hormone-like) - stimulates guanylate cyclase retains toxicity - 100 o C for 30 min processed 18-19 amino acid peptide / stabilized by disulfide bonds ST1a ST1b LT- heat-labile toxin cholera-like (AB 5 ) toxin B-subunit receptor - G M1 ganglioside A-subunit enzyme - ADP-ribosyltransferase ADP-ribosylates / activates G s stimulates - adenylate cyclase secondary effects - stimulates prostaglandins, leukotrienes, cytokines AB 5 -toxin

28. Acquisition of virulence: LT / ST - plasmid encoded fimbriae CFA - plasmid encoded ETEC - pathogenesis

29. Disease: childhood diarrhea in developing countries - linked to growth retardation mucous diarrhea - can persist for > 14 days persistent colonization - forms mucous biofilm EAggEC - Enteroaggregative E. coli (J. Nataro, T. Steiner, R. Guerrant Emerging Infectious Diseases, 1998) EAEC adherence to HEp-2 cells Virulence: Adhesion / mucous biofilm related AAF/I - (aggregative adherence fimbriae) characteristic - adherence to epithelial cells as aggregates - plasmid encoded EAST1 - heat stable (ST) enterotoxin encoded on 100 kD plasmid Acquisition of virulence: - AAF/I and EAST1 - plasmid encoded

30. Disease: watery followed by bloody diarrhea intense abdominal pain O157:H7 - most important serotype hemorrhagic colitis (HC) severe cases - hemolytic uremic syndrome (HUS) edema, hemorrhage, acute kidney failure hemolytic anemia, thrombocytopenia (5-10% mortality rate) EHEC - Enterohemorrhagic E. coli (STEC) Virulence: Toxigenic / non invasive Shiga-like toxin (Stx1, Stx2-vero toxins) bacteriophage encoded EAST1 - heat stable toxin LEE - pathogenicity island - A/E lesion (www.yamagiku.co.jp/pathology/case/case006) Bacteria attached to crypt epithelium in region of active inflammation (HE, high power) Colon biopsy patient suffering from O157:H7 infected patient shows focal inflammation in lamina propria (HE, low power)

31. Shiga-like (Stx) AB 5 - toxin L enzyme activity N-glycosidase A-subunit B-subunit receptor binding Gb 3 (glycolipid globotriaosylceramide) Receptor - Gb 3 (not on enterocytes - high expression on kidney cells) Enzyme activity - N-glycosidase - cleaves adenine from residue 4324 of 28S rRNA - inhibits protein synthesis - retrograde transfer to cytosol - ERAD (ER associated protein degradation) (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002) Acquisition of virulence: Stx - phage encoded T3S - encoded on LEE PAI EAST 1 - plasmid encoded EHEC - Pathogenesis

32. EIEC - Enteroinvasive E. coli Disease: inflammatory diarrhea - closely related to Shigellosis (infectious dose 2-3 logs higher than Shigella) E. coli - biochemical characterization Shigella - genotype / phenotype characterization Virulence: Invasive disease invasion genes - located on 140 MDa plasmid encodes type III secretion system - mxi / spa loci does not produce Shiga toxin EIEC invasion (Prasadarao et al, Infect. Immun. 1999) Virulence relates to : - hybrid - Shigella - E. coli organism - type III secretory process - affecting invasion plasmid encoded

33. Summary emerging Escherichia coli pathogens normal flora E. coli - host GI homeostasis ETEC plasmid - LT / ST LT - CT-like EAggEC plasmid - AAF/1 fimbriae EHEC LEE PAI - T3S phage - Stx Stx - Shiga-like toxin EIEC Shigella-like plasmid - T3S Shigella aggressive GI pathogen EPEC LEE PAI - T3S plasmid - bfp Salmonella Aggressive GI pathogen 100 million years

34. Concepts - emerging pathogens How new bacterial diseases emerge Strategies used by stealth / chronic pathogens Origin / strategies used by aggressive pathogens