Extra-intestinal coccidians

Apicomplexa

Coccidia Gregarinea

Piroplasmida Eimeriida

-Theileriidae - -Haemosporiidae - Babesiidae -Cryptosporidiidae (Plasmodium) - () (Toxoplasma)

Similarities

 Sacrocystis cruzi  Global distribution of these  Definitive host parasites  Carnivorous - canine

 Indirect life cycles -  Intermediate host

intermediate hosts  Obligatory herbivorous

 heteroxenous  bovine  Life cycle has both  Toxoplasma intestinal and tissue stages  Definitive host

 Feline  Infective stage  Intermediate host  Oocyst  Non-obligatory  Tissue cyst  Wild animals

 Domestic animals

 humans

Sarcocystosis (many species)

 Cosmopolitan distribution  Prevalence - near 100% in cattle  Meat at slaughter houses is condemned for human consumption if heavily infected  Infective:

 Oocysts

 Tissue cysts  Intermediate hosts suffer symptoms  Definitive host does not suffer pathology

 Sexual reproduction

 Merozoites invade the epithelium of small intestine and immediately form gamonts

1 Why Study Toxoplasma?

 Signficant cause of congenital birth defects

 Important opportunistic pathogen in AIDS patients

 Serious livestock pathogen

 Good apicomplexan model system

Toxoplasmosis

 Cosmopolitan distribution  Two situations can

 Seropositive prevalence produce sever disease

 15-75% (US is ~22%)  Impaired immune system  Primary infection during  Generally quite benign pregnancy disease in healthy people  Toxoplasmic encephalitis  Headache, fever, sore throat  Congenital toxoplasmosis

 Ocular involvement in rare occasions

 Zoonosis

Acquired Toxoplasmosis

• 1-4 week incubation period • acute parasitemia persists for several weeks until development of tissue cysts • often asymptomatic (>80%) • a common symptom is lymphadenopathy without fever • occasionally mononucleosis-like symptoms (fever, headache, fatigue, myalgia) • likely persists for life of patient • immunosuppression can lead to reactivation (eg., organ transplants)

2 Congenital Toxoplasmosis

 Incidence is ~ 1/1000 births  Primary infection must occur during or shortly before pregnancy  Can only occur once  Probability and severity of disease depends on when infection occurs  Early: low transmission - high severity

 Late: high transmission - low severity  Children can be asymptomatic at birth but develop symptoms later  Can result in spontaneous abortion, splenomegaly, fever, anemia, intracebral calcification, mental retardation

Congenital Toxoplasmosis

 Treatment against parasites as well as symptoms can be successful

 Despite calcification throughout the brain the 10 month old child underwent normal development

Prevalences of Outcomes 5-10% death 8-10% severe brain and eye damage 10-13% moderate-severe visual impairment 58-72% asymptomatic at birth, many developing retino-choroiditis or mental impairment

Toxoplasmic encephalitis (TE)

 25% of all seropositive AIDS patients develop severe Toxoplasmic encephalitis.  TE can be treated with pyrimethamine and sulfadrugs but not all patients tolerate side effects.  In the majority of cases this is due to reactivation of the chronic infection rather than a new infection. Tissue cyst from rat brain  Symptoms include lethargy, apathy, incoordination, dementia.

3 Toxoplasmosis treatment

 Antifolates Typical treatment involves  Pyrimethamine using both antifolates to act  pyrimidine starvation synergistically.

 DHFR inhibitor

 Sulfonamides Sulfa drugs are excreted rapidly

 Sulfadiazine (within hrs) so that repeated doses daily are required  Inhibits folic acid biosynthesis

 Other drugs Drugs tolerated well!  Clindamycin

 Translation inhibitor (prokaryotic)

 Ciprofloxacin (not approved)

 DNA gyrase inhibitor (topoisomerase)

Toxoplasma Life Cycle

Definitive Host

Unsporulated oocyst

Infective

Cysts containing bradyzoites

Tachyzoites transmitted through placenta

Intermediate Host Sporulated oocyst Infective

Toxoplasma Life cycle

Definitive Host

4 Intestinal phase in the cat

Tissue cyst Important: most healthy cats shed oocysts during an Bradyzoite acute infection, but will not shed them later.

• Cat ingests tissue cysts containing bradyzoites • Number of merogonous cycles is variable • Gametocytes develop in the small intestine but are more common in the ileum • 2-4% of gametocytes are male, each produces around 12 microgametes • Oocysts appear in the cat’s feces 3-5 days after infection with peak Oocyst production around 5 and 8 • Cats can shed oocysts for up to two weeks • Oocysts require oxygen and they sporulate in 1-5 days

Does the cat have to go if you are pregnant?

• Avoid raw or undercooked meat. (Cooking kills Toxoplasma ) • Wash raw fruits and vegetables well before eating. • Remove feces from the litter box every day, to eliminate any parasites before oocysts sporulate • Keep cats indoors to prevent hunting. Cats can become infected by eating infected rodents or birds. • Feed cats only commercial food or well-cooked meat. • Do not feed cats raw or undercooked meat. • Do not adopt cats who have lived outdoors. • Do not handle stray cats.

• Wash hands well with soap and warm water after: Gardening Yard Work Any Other Outdoor Activity Involving Contact With Soil that could be contaminated with cat feces

• There is no treatment available to prevent Toxoplasma in cats, or to prevent an infected cat from shedding the parasite in its stool.

Oocysts can survive in the environment for many months (moisture, shade, in soil or sand).

Bradyzoite stage

 Dormant, persistent state  Months or even years  Tissue cysts primarily in brain and muscle  Chronic or latent infections  Slowly dividing stage  Cysts are highly infective  Bradyzoites are resistant to low pH and digestive enzymes  Only stage that will initiate sexual reproduction  Bradyzoites are resistant to all current drugs

5 Tachyzoite stage

 Crescent shaped - 2 x 6 µm  Rapidly dividing merozoites  Acute infections  Asexual replication  Repeated rounds of merogony  Not resistant to low pH  Stage that is involved in vertical transmission to fetus

Gliding Motility

 Substrate-dependent motion that requires an actin-myosin motor

 Cytochalasins inhibit (actin destabilizer)

 Gliding is coupled to translocation of cell surface adhesins (deposit on surface)

 Differs from amoeboid movement

 Also actin based, cell deformation

 Apicomplexan gliding - no deformation

 Assists in 3 vital functions

 Migration

 Invasion

 Egress

 Movement includes:

 Circular, upright twirling, helical

Hakansson et al 1999 Toxoplasma Motility Mol Biol Cell 10: 3539

TwirlingCircular

Antibody staining of a surface antigen

6 The gliding machinery is anchored in the inner membrane complex

PM IMC MT

Gliding Translocation Model

MIC2/M2AP - hexameric complex essential role Migration

Essential role

Shed surface proteins SAG1 Proteolytic cleavage MIC1/MIC4/MIC6 MIC2/M2AP AMA1

Translocation (capping direction)

The Moving Conveyer Belt

7 The conveyor-belt model

 Motility depends of parasite actin/myosin (MyoA) The MyoA is parasite specific - different from host myosin

 Myosin is anchored into the outer IMC membrane Complex of proteins (GAP45/50, MyoA, MLC)

 Short actin filaments form and are moved towards the posterior end of the parasite by the myosin power stroke

 The short actin filaments are linked to microneme proteins by an adaptor Aldolase - moonlighting protein  Movement of actin filaments results in movement of microneme proteins

 Microneme proteins are shed at the back end (rhomboid proteases are the best candidates for this activity)

 The parasite glides over the substrate

The parasitophorous vacuole IS NOT fusing with lysosomes

 Macrophage cells were incubated with live (A/B) or heat killed (C/D) parasites

 Note that only vacuoles containing heat killed parasites show staining for a lysosomal marker protein.

 Dead parasites go in by phagocytosis, living parasites enter differently

Joiner et al. 1990, Science 249:641-6

T. gondii and cell invasion

 T. gondii does not enter the host cell by phagocytosis  Invasion results in the formation of a specialized compartment the parasitophorous vacuole  Protein secretion from several secretory organelles is involved in invasion  Parasite motility is required for invasion

8 Host Cell Invasion

 Helical gliding leads to invasion

 A constriction is apparent during invasion - “moving junction”

 Appears similar to tight junction, but molecular basis is still unknown

 Host cell entry is significantly faster than phagocytosis

 Distinction between surface antigens (generally GPI anchored) and apical invasion proteins that are retained in the secretory organelles (usually TM and soluble proteins).

Host Cell Invasion - 10-30 seconds

SAG1 surface antigen

M2AP microneme secretion

 Gliding motility (migration) is linked to virulence  Sequential release of apical complex contents

 Micronemes

 Rhoptries

 Dense granules

Host Cell Invasion

9 microneme Microneme secretion rhoptry

 Release of MIC proteins

 Adhesive proteins dense granule  Majority of these MIC proteins undergo proteolytic cleavage

 Required for motility

 Basal secretion from micronemes

 Low level regular shedding of adhesins  Ca++ regulated ↑ secretion upon cell contact

 Large repertoire of micronemal proteins still being evaluated

Lovett and Sibley 2003 J Cell Sci 116: 3009

Rhoptry secretion

 Rhoptries are secreted right at the moment of invasion

 Crucial for the formation of the PV

 Rhoptry proteins are found in the newly forming PV

 Some proteins are inserted into PVM

Dense granules

 Dense granule proteins are soluble in PV

 Modification of vacuole to prevent fusion with phagolysosomes

 Involved in establishment of a tubulovesiclular network

 Sieving pores give access to required small molecule nutrient salvage

10 Constriction is the result of the moving junction

RON4 is a rhoptry protein

Formation of the PV & moving junction

Secretion and redistribution during invasion

11 The Lytic Cycle

Timing: 24-48 hours

Egress Invasion

Replication

Egress

 Striking parallels to invasion

 Requires gliding motility

 Ca++ to activate motility

 Moving junction

 Additionally requires a mechanism that “senses” the fitness of the host cell

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