Chlamydiae Obligate intracellular pathogens. Obligate intracellular pathogens. Acute and/or persistent infections. Acute and/or persistent infections. C. trachomatis – mucosal surfaces: C. trachomatis – mucosal surfaces: Ocular infections - trachoma Genital infections - pelvic inflammation, infertility Reactive arthritis C. pneumoniae C. pneumoniaePneumoniaAtherosclerosis

Chlamydia Life Cycle Elementary body (EB) Elementary body (EB) -metabolically inactive -highly infective stage Reticulate body (RB) Reticulate body (RB) -metabolically active -intracellular growth stage Persistent body (PB) Persistent body (PB) -life cycle pause between EB and RB stages -stable association with host cell

Apoptosis vs Necrosis Apoptosis – programmed cell death. Apoptosis – programmed cell death. -eliminate and phagocytose cells in an orderly fashion. -phosphatidylserine (PS) receptor on phagocytes increases anti-inflammatory cytokines TGF-beta and IL-10. -needed for embryogenesis, immune system maintenance. Necrosis – non-programmed cell death. Necrosis – non-programmed cell death. -cellular debris is a ‘danger signal’ in the cell, so inflammatory response follows DSR interaction with phagocytic cells.

Apoptosis vs Necrosis

How might apoptosis help pathogens? Facilitating pathogen propagation Facilitating pathogen propagation -pathogens within apoptotic cells can be taken up by other phagocytic cells without the pathogen having to navigate the extracellular environment. Avoiding inflammatory responses Avoiding inflammatory responses -apoptosis can release anti-inflammatory cytokines that down regulate the immune response.

Pathogen manipulation of apoptosis Viruses – often inhibit apoptosis Viruses – often inhibit apoptosis - Oncogenic viruses destroys p53 surveillance system - Inhibit extrinsic and intrinsic apoptosis pathways Protozoa – often inhibit apoptosis Protozoa – often inhibit apoptosis - Toxoplasma, Trypanosomes, Cryptosporidium - Heat shock proteins, NF-KB Bacteria – often induce apoptosis Bacteria – often induce apoptosis - Helicobacter, Shigella, Salmonella - Toxins, protein synthesis inhibitors, TTSS

Mechanisms of Apoptosis Extrinsic pathway – Receptor mediated Extrinsic pathway – Receptor mediated - FasL-death receptor interactions - Initiator caspases – caspases 8, 9 - Effector caspases – caspases 3, 6, 7 Intrinsic pathway – Intracellular origin Intrinsic pathway – Intracellular origin - Caspase activation or intracellular stress signals - Mitochondrial release of cytochrome c - Apoptosome formation

Extrinsic pathway Type I cells – activate initiator caspase 8, then effector caspase 3, then apoptosis commenses. Type I cells – activate initiator caspase 8, then effector caspase 3, then apoptosis commenses. Type II cells – require mitochondrial amplification. BAX, BAK stop being inhibited by BCL-2, BCL-X and cause mitochondrial release of cytochrome c, then apoptosome forms, activates caspase 3, and commenses apoptosis (DNA fragmentation, nuclear condensation, membrane blebbing, etc.) Type II cells – require mitochondrial amplification. BAX, BAK stop being inhibited by BCL-2, BCL-X and cause mitochondrial release of cytochrome c, then apoptosome forms, activates caspase 3, and commenses apoptosis (DNA fragmentation, nuclear condensation, membrane blebbing, etc.)

Chlamydial apoptosis manipulation When to inhibit apoptosis? When to inhibit apoptosis? When to induce apoptosis? When to induce apoptosis?

Chlamydial apoptosis manipulation When to inhibit apoptosis? When to inhibit apoptosis? - for chronic or persistent infections - when intracellular growth stages dominate When to induce apoptosis? When to induce apoptosis? - for acute infections - when infectious Elementary Body stages dominate

How to manipulate apoptosis? ‘Chlamydia protein associating with death domains’ = CADD, is an oxidoreductase, so accumulation of reactive oxygen species could lead to necrosis, while interactions with Fas could inhibit apoptosis. ‘Chlamydia protein associating with death domains’ = CADD, is an oxidoreductase, so accumulation of reactive oxygen species could lead to necrosis, while interactions with Fas could inhibit apoptosis. Chlamydia interferes with mitochondrial apoptosis signals, perhaps by secreting Bcl-2 anti- apoptotic proteins or inactivating pro-apoptotic proteins. Type III Secretion Systems available. Chlamydia interferes with mitochondrial apoptosis signals, perhaps by secreting Bcl-2 anti- apoptotic proteins or inactivating pro-apoptotic proteins. Type III Secretion Systems available.

How to induce apoptosis? Caspase-independent apoptosis occurs. Caspase-independent apoptosis occurs. Necrosis occurs in some cases… by design or accident? Necrosis occurs in some cases… by design or accident? Cell type specific interactions. Cell type specific interactions. Over-expression of BAX, BAK cause cell death. Over-expression of BAX, BAK cause cell death. - BAX deficient cells and mice had fewer Chlamydial organisms, so perhaps BAX-induced apoptosis is important for propagation of the infection. Increased mitochondrial metabolism and oxidative stress observed in infected cells. Increased mitochondrial metabolism and oxidative stress observed in infected cells.

How to inhibit apoptosis? Inhibit cytochrome c release from mitochondria. Inhibit cytochrome c release from mitochondria. MEK/ERK – MAPK signalling pathways. MEK/ERK – MAPK signalling pathways. NF-KB – as with MEK/ERK, upregulate transcription of anti-apoptotic genes. NF-KB – as with MEK/ERK, upregulate transcription of anti-apoptotic genes. IAP – upregulate Inhibitors of Apoptosis Proteins. IAP – upregulate Inhibitors of Apoptosis Proteins.

Future Work No methods exist for genetic transfer (yet), so much is unknown about virulence and pathogenesis! Is Chlamydiae-induced apoptosis associated with acute disease while Chlamydiae-inhibited apoptosis is associated with chronic disease?

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