Rhoptry proteins modify the function of the host cell nucleus

1. Rhoptry proteins modify the function of the host cell nucleus • Several rhoptry proteins are injected into the host cell cytoplasm during invasion • They accumulate in the host cell nucleus • Interestingly, many of them are enzymes capable of changing the phosphorylation state of proteins (kinases & phosphatases) • Their precise function remains to be determined but it appears that they modulate gene expression in the host cell and that their activity is required for rapid growth and the ability to cause disease (virulence – one of our seminars will feature the activity of one of these proteins)

2. Rhoptry proteins have multiple functions in vacuole formation & host manipulation Bradley & Sibley, Current Opinion in Microbiology 10: 582-587

3. Invasion depends on sequential protein secretion from three organelles • Dense granules (DG): secretion of dense granules occurs after the vacuole is fully formed and continues throughout the intracellular growth of the parasites • Dense granules are secreted from the basal end of the parasites • Dense granule proteins likely play a role in modification of the vacuole into an environment supportive of parasite growth

4. Dense granule proteins are secreted into the PV • Certain dense granule proteins are soluble in the lumen of the PV others integrate into the membrane • These proteins are probably involved in modifying the vacuole (will do a paper on host cell modification by dense granule proteins in seminar section)

5. The PV is highly modified to suite the parasite’s needs • Tubular network increases surface (dense granule) • Sieving pores give access to small nutrient molecules in the host cell cytoplasm (probably dense granule) • Specific host cell organelles are recruited close to the PV membrane (rhoptry)

6. Dense granules are involved in establishing the intravacuolar network

7. the parasitophorous vacuole contains a sieving pore

8. Host cell mitochondria and ER are recruited to the PVM

9. Apicomplexan invasion • Active, parasite driven process • Depends on parasite actin/myosin motility (conveyor belt model) • Involves secretion of micronemes (attachment, motility), rhoptries (PV & MJ formation) and dense granules (makes PV into a suitable home) • Sets up a parasitophorous vacuole which initially is derived from the host cell cell-membrane • A moving junction is formed which screens out host membrane proteins from the PV, the PV is fusion incompetent and the parasite protected

10. Theileria -- cell transfomation by intracellular parasites

11. Devastating outbreaks follow the big cattle drives • In the 1860s and 1870s Texas longhorns were driven in huge numbers to the railheads in Kansas (from there they went by train to the slaughterhouses of Chicago and other big nothern cities) • Farmers in Kansas and Missouri were plagued by outbreaks of “Texas” fever in their herds which they linked to the cattle drives • Several standoffs ensued as local farmers tried to block drives • Theobald Smith and Frederick Kilbourne show (1889-1893) that the disease is caused by a protozoan parasite transmitted by ticks (first disease shown to be transmitted by an arthropode)

12. Border Cowboys patrol the U.S. Mexican border for ticks • Boophilus ticks and with them the Texas tick fever have been eradicated from the Southern U.S. but they are still present in Central America • USDA employs 60 cowboys which patrol the Southern border to find and check stray-cattle for ticks to prevent the reintroduction • See short New York Times feature on border cowboys posted on the class web site Eddie Dillard, left, and Jack Gilpin are tick riders (NYT 7/03)

13. Theileria • Infects mainly ruminants (cattle, goats, sheep, deer) • Several different species causing both pathogenic and benign disease • Infection in wild animals is mostly asymptomatic cattle: disease Cape buffalo: reservoir

14. Distribution of theileriosis red = T. annulata 250 million cattle at risk orange = T. parva 50 million cattle at risk grey = T. buffeli/orientalis/sergenti relatively benign

15. Life cycle of Theileria spec. • Infects mainly ruminants (cattle, goats, sheep, deer, horse) • Several different species causing both pathogenic and benign disease

16. Two stages are found in the bovine host: Koch’s bodies and piroplasms

17. Theileria (sporozoite) invasion differs from Toxoplasma invasion

18. Sporozoites do not enter the host cell apical end first

19. Theileria sporozoites do not form a moving junction during invasion

20. Theileria escapes the vacuole into the cytoplasm • Escape occurs <15 min after invasion • No vacuolar acidification is observed • Escape coincides with discharge of rhoptries and micronemes

21. Host cell microtubles are found in close association with the parasite

22. Theileria invasion • Zipper mechanism of entry into lymphocyte • Escape from vacuole into cytoplas coincides with rhopthry & microneme discharge • Parasites free in the cytoplasm associate with host MT Animated version

23. The Theileria paradox • Although Theileria replicates in lymphocytes these cells seem to proliferate enormously in infected animals (most of these proliferating lymphocytes are infected) -- this is in contrast to other infections like malaria or babesiosis where parasite replication is associated with the decline of the host cell population causing anemia • Also, the sporozoite (injected by the tick) appears to be the only stage capable of invading lymphocytes • How can the parasite spread to new lymphocytes? • The trick: Theileria hijacks and exploits two key features of the lymphocyte’s cell biology: cell division and growth control

24. Divide & conquer

25. Divide & conquer • Parasites do not egress from (and in the process destroy) their host cells and infect new lymphocytes but proliferate along with them • The tight association of parasites with host cell microtubules ensures that they are segregated by the host cell mitotic spindle between the two daughter cells • A recently divided infected lymphocyte (the arrow indicates the cleavage furrow at which cytokinesis occurred. Blue (DNA), red (host cell centrioles), green (parasite surface membrane), HN (host nucleus)

26. Theileriosis is a lympho-proliferative disease • Lymphocytes are usually arrested and only expand upon antigen presentation • If parasite replication requires host cell replication the parasite has to somehow promote proliferation of its host cells • Indeed theileriosis is a lympho-proliferative disease • Swelling and proliferation of the lymph node draining the bite site is the first sign of disease

27. Pathology is mainly due to lymphoproliferation • Lymphocytes proliferate heavily invading multiple organs causing disease similar to a lymphoma (cancer of lymphocytes) • (Top) Infiltration of kidney by Theileria parva infected lymphocytes • (Bottom) Abdominal ulcers due to transformed lymphocytes • Death is in most cases due to infiltration of the lung resulting in lung edema (the abnormal build up of fluid within the lung)

28. Theileria infected cells show characteristics of transformation • Theileria infection seems to share many of the features seen in the transformation of normal cells into cancer cells • Uncontrolled growth • Loss of differentiation • Immortalization (infected cells taken into culture will grow indefinitely) • Growth in the absence of external growth factors • Enhanced ability to migrate and to infiltrate organs • Enhanced resistance to apoptosis

29. Transformation by Theileria is completely reversible • Theileria infection of transformed cells can be cured with BW720c • Cells cease to proliferate and apoptose • Optimal serum conditions and IL-2 treatment (diamonds) prolong life of cells

30. How does Theileria interfere with lymphocyte growth and cause cancer?

31. NF-kB -- a major regulator of lymphocyte growth • NFkB (nuclear factor, p50 & p65) is an important and very well studied transcription factor (a protein that interacts with the promoter of genes and stimulates gene expression) • It is a major player in the stimulation and clonal expansion of lymphcytes (among other functions) • NFkB is bound by IkB (its inhibitor) which sequesters it in the cytoplasm and keeps it inactive • Phosphorylation followed by ubiquitinylation and degradation of IkB leads to import into the nucleus and transcriptional activity • Theileria interferes with this pathway by causing the destruction of IkB

32. Theileria induces the degradation of IkBa and b • NFkB is constitutively activated in transformed cells and dependent on the continuos presence of parasites • Theileria seems to induce constitutive degradation of IkB a and b

33. The IKK complex regulates IkB turnover • IkB is tagged for destruction by phosphorylation through the IKK complex • In normal lymphocytes this provides a way to relay the reception of signals from the surface of the cell to gene expression (e.g. stimulate clonal expansion) • Theileria hijacks and activates the IKK signaling complex independent of the usually required external stimulation

34. Hijacking and activation of IKK transforms infected cells • Theileria parasites (green) interact with and activate IKK (red) of their host lymphocytes • IKK tags IkB for destruction • NfKb free of its inhibitor enters the nucleus and cells start dividing rapidly

36. summary • Theileria sporozoites invade using a zippering mechanism • The PV is lysed upon rhoptry secretion and the parasites resides in the cytoplasm and associates with the host cell’s microtubuli & centrosomes • When the host cell divides the parasite divides and segregates alongside using the host cell’s mitotic machinery • Theileria schizonts transform their hosts lyphocytes (induce uncontrolled ‘cancer-like’ growth) • Transformation is parasite dependent and reversible • Parasites interfere with NFkB growth control by activating the IKK signalling pathway