Extra-intestinal coccidians
Apicomplexa
Coccidia Gregarinea
Piroplasmida Eimeriida Haemosporida
-Theileriidae -Eimeriidae -Haemosporiidae - Babesiidae -Cryptosporidiidae (Plasmodium) -Sarcocystidae (Sarcocystis) (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
12