1. MICR 420 Emerging and Re-Emerging Infectious Diseases Lecture 7: C. trachomatis Dr. Nancy McQueen & Dr. Edith Porter

2. Overview C. trachomatis The genus Chlamydia Morphology Growth and metabolic characteristics Virulence factors Diseases Diagnosis Direct antigen test Culture PCR Immune response Therapy Threats: Apparently under control

3. Chlamydia Obligate gram-negative intracellular bacteria No peptidoglycan layer Major outer membrane protein accounts for 60% of all surface protein Small genome (~1.0 Mb) Depend on host molecules for replication Limited or no ATP synthesis Two forms exist Small elementary bodies (EB) Survives outside host cells Transferred to new host Similar in function to a spore  RNA:DNA = 1:1 Larger reticulate bodies (RB) Grow within cells  RNA:DNA = 3:1 Do not survive outside host Elementary Reticulate

4. Chlamydia Developmental Cycle 48h – 72h Persistent Forms EB attach to epithelial host cell Endocytosis Prevention of fusion with lysosome Development into RB RB proliferation, formation of large inclusion bodies Visible in light microscope Development into EB Rupture of host cell and release of EB

5. Chlamydia Species C. pneumoniae World wide ~ 10% of pneumonias and 5% of all bronchitis cases C. psittaci Psittacosis Zoonotic pneumonia Inhalation of droppings from infected birds Category B biological weapon C. trachomatis Various serotypes Eye infections STI

6. Inclusion conjunctivitis Transmitted to newborn's eyes during passage through the birth canal Preventive erythromycin Spread through swimming pool water Treated with tetracycline Trachoma Greatest cause of blindness worldwide Chlamydia Infection of the Eyes

7. Trachoma World wide ~ 80 (150?) million people infected and ~ 6 million blind Mostly in developing countries 3% of cause of all blindness world wide Transmitted eye-hand-eye, eye-fomite-eye, flies Infection occurs usually during childhood Chronic follicular conjunctivitis inversion of eyelashes irritation of cornea corneal ulcerations, scarring vision loss typically at age 30 – 40

8. Trachoma

9. Histopathology of Trachoma http://www.lenntech.com/images/Water%2 0Borne%20Diseases/Trachoma.jpg

10. Simplified WHO System to Assess Trachoma (from Matthew J. Burton, British Medical Bulletin, 2007) GradeDescription TFTrachomatous inflammation—Follicular: The presence of five or more follicles (>0.5 mm) in the upper tarsal conjunctiva TITrachomatous inflammation—Intense: Pronounced inflammatory thickening of the tarsal conjunctiva that obscures more than half of the deep normal vessels TSTrachomatous scarring: The presence of scarring in the tarsal conjunctiva TTTrachomatous trichiasis: At least one lash rubs on the eyeball COCorneal opacity: Easily visible corneal opacity over the pupil

11. Pathogenesis of C. trachomatis (A) Chlamydia elementary bodies (EB) translocate Tarp into an associated cell to orchestrate rearrangement of host cell actin. The C- terminal domain of Tarp directly nucleates small actin filaments followed by hostmediated signaling involving tyrosine phosphorylation (*P) cascades and ultimately Arp2/3 to mobilize actin assembly necessary for invasion. (B) Throughout development, chlamydial inclusions intercept host-derived vesicles via recruitment of Rab GTPases (Rabs 1, 4, 10, and 11) and SNARE (Vamp 3, 7, and 8) molecules through interactions with Inc proteins. (C) Established inclusions are able to interfere with NF-kB (p50 and RelA) activation through ChlaDub1-mediated prevention of IkBa ubiquitination (*Ub) or CT441-mediated degradation of RelA. This would be predicted to interfere with proinflammatory signals originating from ligand binding to TNF family (TNFR) or pattern recognition (PRR) receptors.

12. Virulence Factors of C. trachomatis (CT) Contribute to intracellular survival and proliferation In part secreted via Type III secretion apparatus Tarp (Translocated actin recruiting phosphoprotein): effects actin rearrangement and endocytic uptake of CT CT inserts proteins (Inc and others) in inclusion (endosome) membrane that prevent fusion of host lysosomes CT releases proteins (ChlaDub1, CT441) into the cytoplasma that interfere with NF  B signal transduction, a key pathway of the acute inflammatory and innate immune response.

13. Immune Response to C. trachomatis Not well understood Infection controlled by cell mediated immune response However: contributes also to the scarring Antibodies are formed Tear IgG actually enhance uptake Tear IgA appear to be protective (Matthew J. Burton, British Medical Bulletin, 2007)

14. Diagnosis of Trachoma No “gold standard” test Direct antigen test PCR ELISA to measure patient antibodies against outer membrane proteins, conserved heat shock proteins Culture inoculating specimens onto cell culture monolayers Visualization of inclusion bodies after 48 – 72 h using fluorescent labeled monoclonal antibodies against CT LPS or MOMP

15. Therapy and Prophylaxis of Trachoma Eyelid surgery Antibiotics to treat the infection Tetracycline Azithromycin Education about facial cleanliness and personal hygiene Environmental improvements WHO’s Global Alliance for the Elimination of Blinding Trachoma by 2020

16. Take Home Messages Chlamydia are obligate intracellular bacteria that have a unique life cycle alternating between infectious elementary bodies and proliferative reticulate bodies. Pathogenic factors include rearrangement of cytoskeleton promoting uptake, inhibition of fusion of lysosomes with endosomes and inhibition of NfkB activation. C. trachomatis serotypes A-C (-K) cause trachoma, a chronic conjunctivitis leading to blindness. Trachoma is diagnosed by direct antigen and nucleic acid based test and treated with tetracyline and azithromycin.

17. Resources http://www.who.int/topics/trachoma/en/ http://www.cdc.gov Textbooks Microbiology: A clinical Approach (2010) Garland Science Prescott’s Principles of Microbiology (2009) McGraw- Hill Microbiology: An Evolving Science (2009) Norton Primary literature Betts et al., Current Opinion in Microbiology, 2009, 12:81–87 Matthew J. Burton, British Medical Bulletin, 2007