Parasitism final

kinetoplastids

broad range of free-living and parasitic protists

range of cell morphologies and is tailored to the environment parasite occupies

kinetoplast

defining feature of kinetoplastids

trypanosomatids morphologies

subgroup of kinetoplastids that are parasitic

T cruzi discovery

carolos chagas - Brazilian doctor

transfered in triatomine, or kissing bug

triatomine

bites the faces of people when the sleep

vector of T cruzi

take large blood meals and poop when they bite

T cruzi identified in posterior midgut, similar to T brucei

wattle and daub construction is ideal habitat for kissing bugs as they hide in the thatching in roof, come down to feed at night

common across all of Latin America

t cruzi in triatomine

inhabits the digestive tract

t cruzi life cycle

chronic t cruzi effects

causes range of organ defects

swelling of the lining of the esophagus and colon

peristalsis also stops making it hard to swallow and pass water

t cruzi transmission cycle

drugs for treating chagas

Nifurtimox and benznidazole are only current drugs

both prodrugs, must be activated by cells before they become toxic

T cruzi contains a bacterial-like nitroreductase, produces radicals from nitro group which is cytotoxic

explains selectivity for parasite over mammalian cells

really only work well if during acute stage of infection

nifurtimox

treats chagas

causes severe nausea and vomiting, leading to weight loss

many people don’t finish course of drug, which can facilitate development of resistance in parasite

benznidazole

treatment for chagas

better tolerated but very poor at clearing chronic infections in adults

why chagas concern in latin America

zoonotic hosts (armadillo, monkeys, cats) means T. cruzi will never be eradicated

afflicted tend to be poorest of poor, don’t seek diagnosis or treatment

deaths occur long after initial infection and are frequently not associated with disease (heart issues especially)

chagas in north america

becoming more of an issue primarily due to immigrants moving from endemic nations

insect vectors moving north

doctors never consider chagas as possible ailment

especially problematic for pregnant women

bigger problem in Europe

clearing t cruzi does not

alter progression of Chagas cardiomyopathy

manifestation of chagas cardiomyopathy

chagas diseases

95% under diagnosis rate

minimal acute infection symptoms

chronic infection can lay dormant for decades

treatment of chronic disease doesn’t undo the damage

30-40% of infected will develop heart problems

sudden death via heart attack is possible

won’t be diagnosed as due to chagas

poor people with poor access to healthcare are most at risk

t cruzi cell infection cycle

1. trypomastigote infects mammalian cell

2. differentiation into amastigotes

3. amastigotes divide

4. amastigotes lyse cell

5. amastigotes differentiate into trypomastigotes

6. amastigotes infect neighboring cells

7. trypomastigotes escape and disseminate

t cruzi cell surface coat

trans-sialidase: ~1400 genes

mucins: heavily glycosylated proteins

trans-sialidases

come in active and inactive forms

most inactive, only able to bind sugars

transfer silica acid onto t cruzi mucins

alter both parasite and host cell surfaces

trans-sialidases and immune system

can limit T cell activation

activated T cells have diminished silica acid on their cell surface, makes it easier for them to bind to other cells

aTS adds silica acid back, makes it harder for T cells to probe for infected cells

t cruzi cell infection facilitated by

disruption of host actin cytoskeleton

cytochalasin D - blocks formation of actin filments

most pathways for pathogen internalization employ phagocytosis, which requires actin filaments

disruption of host actin cytoskeleton causes redistribution of lysosomes

lysosomes

acidified compartments containing hydrolyzing enzymes

disrupted host lysosome t cruzi

disrupting host lysosomal fusion limits t cruzi infection

filling lysosomes with sucrose makes them swell and limits their ability to fuse to one another

attachment of t cruzi to host cells was not affected

t cruzi early infection

associate with lysosomes very early in invasion process

t cruzi trypomastigotes

trigger changes in host cell cytosolic calcium levels

cannot enter host cells without extracellular calcium

trigger host cell death in absence of calcium

trigger lysosome fusion with plasma membrane

lysosome exocytosis

general mechanism for healing plasma membrane damage

alternate modes of cell wounding t cruzi

cell wounding causes release of lysosomal acid sphingomyelinase

acid sphingomyelinase activity is necessary for t cruzi infection

t cruzi cell wounding

triggers rapid membrane healing pathway

wound triggers calcium influx, leads to lysosomal fusion at plasma membrane, release of ASM, production of ceramide, and rapid endocytosis

1. extracellular t cruzi trypomastigotes wound cells

2. enter cells posterior first

3. intracellular t crudi remain highly motile

protruding t cruzi

still encased in host membrane

host cell markers t cruzi

label them in progressive fashion

cells early in infection process are labeled with ceramide, but not LAMP

cells later in infection process are LAMP positive, but lack ceramide

t cruzi cell invasion mechanism

cells that are prone to damage and have elevated repair functions are likely to be preferred: muscle and heart muscle

t cruzi amastigotes

divide within the host cytosol

become amastigotes from the trypomastigotes and can be triggered to do this conversion

no parasitophorous vacuole

t infestans in arequipa

is present more in urbanized parts than rural

spreads very slowly in urban setting

does not become proficient for transmission of t cruzi within a community effectively until a certain host density is reached

developing areas in contact with environment are LESS likely to be infested, develop t cruzi infection

t cruzi in arequipa

rates of t cruzi infection reached 5% of population before insect control introduced

earliest infection only 20 years old

low levels of chagas cardiomyopathy but likely a lot more in future

prophylactic drug treatment may be good before heart issues arise

benznidiazole clearing of parasites

does not clear all parasites in mice

prolonged nifurtimox

selection identifies resistant strains of t cruzi

can select for resistant epimastigotes, but no longer mammalian-cell infectious

prolonged benznidazole

election identifies resistant strains of t cruzi

all clones show greatly diminished infection of mammalian cells

edu pulse

ethynyl deoxyuridine is incorporated into DNA

takes place of dT

ethynyl group can be selectively reacted with azides to produce covalent bonds, lots to see if DNA replication is occuring

tissue clarification

greatly enhances imaging of insect organs

fat, other non-protein components cause opacity and autofluorescence

t cruzi amastigotes in mice

become dormant in mice and tissue culture cells

lack of EdU label is not due to amastigote cell death

TUNEL: assay for apoptotic cells, only 2-3 apoptotic amastigoes observed per million parasites

dormant amastigotes t cruzi

can tolerate long drug treatment regimens

can rebound and spread throughout the host

amastigotes that survived 30 days of drug treatment are not resistant

mutations to overcome drug are not driving amastigote survival

dormancy has limits though because extended courses of high-dose BNZ clears the infection

benzoxaborole

series of these drugs have potent activity against t cruzi

targets mRNA processing

benzoxaborole AN15368 is effective at clearing t cruzi infection in non-human primate model

ancient texts t brucei

describe wasting disease that kills livestock in Africa

Egyptians used to keep livestock with game animals

purebred livestock would die of wasting disease

Zulus called disease N’gana: useless/powerless

early sleeping sickness descriptions

first descriptions came from attendants on slaving ships sailing to americas

descriptions of people in coma-like state, alive but unable to communicate

Winterbottom’s sign - swelling of the lymph nodes int he neck precede the illness

David bruce

identified infectious agent that causes N’gana

noted that wasting disease affected horses, donkeys, dogs, and cows but had not effect on local species

essentially all animals died, only some cattle survived

was convinced tsetse fly was transmitting illness

took animals into tsetse infected regions to see if they would get infected

t brucei identification

Bruce showed bite of tsetse did not transmit poison

flies that had fed on infected animals could transmit

parasite was present in every sick host, but not healthy ones

fever in animals with nagana correlated with appearance of parasites

direct blood transfer from animal transmitted disease

he did think that mosquito was just a syringe to deliver which is not true

Forde

identified trypanosomes in humans

master of steamship River Gambia was admitted with a fever, lethargy

malaria was diagnosed, but quinine did not help fever

blood smear showed trypanosomes in blood

other trypanosomes

t congolense and t vivax cause nagana but don’t infect humans

t brucei rhodiense infects humans and causes a more sever disease than t brucei gambiense

trypanosomes were isolated from cerebrospinal fluid, suggesting how the parasite might cause the coma-like stage of disease

t brucei life cycle

african trypanosomiasis progression in humans

cases of HAT

t brucei agricultural cost

livestock essential source of income for many families in sub-saharan africa

calculated cost of infection is around $2.5 billion, loss of 16% meat and 10% milk production

treating infected cattle is difficult, but suppressing tsetse fly using insecticides in effective

cattle function as important reservoir for t brucei rhodiense

drugs for HAT

trypan blue - effective for killing lab animals but turns them blue

1917: bayer 205 identified, colorless, very trypanocidial, still in use today for early-stage T. b. rhodiense infections. suramin

arsenicals - arsenic containing

ataxy: developed in 1960s, somewhat effective but blinds 2% of patients

melarsoprol: no blinding, effective against brain-stage HAT. causes severe encephalitis in 5-10%, kills 1-5%, damages veins when administered. still in use

nifurtimox: nitro prodrug, used to treat chagas

elfornithine: inhibitor of ornithine decarboxylase, blocks synthesis of polyamides in cells. originally chemotherapeutic. low toxicity, very effective

elfornithine

nearly lost as HAT treatment

rights to drug were sold several times

failed as chemotherapeutic, eventually sold for $750 per treatment, but eventually sold as a hair removal product

Doctors Without Borders and trypanosome researchers pushed BMS to provide free eflornithine for HAT

NECT treatment

for trypanosomiasis

combined elfonithine and nifurtimox

fexindazole

important treatment for HAT

orally bioavailable, 10-day course

low toxicity, as effective as NECT and doesn’t cross blood brain barrier

could significantly improve treatment course

still requires hospitalization because you need to make sure patient doesn’t have parasites in CSF. lumbar puncture

acoziborole

improved treatment for HAT

targets CPSF3- part of polyadenylation pathway

same target as compound for treating t cruzi

orally available, crosses blood-brain barrier, single-does, can use prophylactically, no need to monitor patients

3 mutations in CPSF3 can increase resistance

tsetse fly

gives live birth

feed “milk” to maggot, produces one at a time. needs additional blood meal to produce another offspring, takes about 9 days

susceptible to climate change and populations are rapidly declining

agglutination test

rapid screening for t brucei

can be performed with pinprick of blood, can screen entire towns

positive card tests are referred for microscopy tests to identify parasites in blood

teams can be deployed to towns via motorcycle or small boat

tsetse nets

tiny target nets effectively capture the flies

net deployment leads to 80% decline in tsetse population, 3-fold decline in disease prevalence. in control area, disease prevalence increased by 10-fold

t brucei extracellular

exclusively extracellular parasite

in blood, organs, and lymphatic system during early stages, then crosses blood-brain barrier in second stage illness

parasite is constantly exposed to host immune system

designed to swim in blood, motility is optimized for crowded environments and high viscosity. cells actually swim faster in blood than in media

t brucei parasitemia

shows cycles of high and low

waves tended to correlate with increased symptoms: fever, exhaustion

t brucei bloodstream form

have thick cell surface

variant surface glycoprotein (VSG) comprises most of the bloodstream form cell surface

VSG

variant surface glycoprotein

more than 10^7 copies of protein per cell

99% of cell surface covered

GPI anchored, not integral membrane protein

varying oligomerization states

~1600 copies of VSG gene in trypanosome genome

forms tight array on cell surface that minimizes the exposure of antigens to the host immune system

new VSG structures suggest wider diversity than previously thought and may have additional function beyond immune evasion

antigenic variation

t brucei can escape immune response

cells expressing one VSG grow to high density, but immune system recognizes cells and produces antibodies, almost all parasites cleared by antibody

low number of parasites “switch” to a different VSG gene, antibodies no longer function allowing them to survive

expresses only a single VSG variant at a time and maximizes the repertoire of VSGs in genome

switching is rare, successful immune response clears out all parasites with current VSG

antigenic variation mechanism

vsg in nucleus

other telomeric VSGs held inactive in nuclear periphery

active expression site body labeled with Pol I antibody

VSG switching can occur by switching to new VSG expression site

15 known sites in T brucei

VSG switching in genome

can occur via homologous recombination at VSG

DNA break at VSG allows replacement of VSG gene

either VSG at other inactive site can replace VSG or other VSGs

t brucei has 11 primary chromosomes, inactive VSGs are maintained at “subtelomeric” (near telomeres) sites

minichromosomes contain hundreds of VSGs and function as repositories for “other” VSGs

stumpy initiation factor

controls parasite number and transmission in T brucei

SIF-short oligopeptides generated by proteases secreted by t brucei

stumpy form - does not divide, predisposed to survive within tsetse fly

ratio of slender to stumpy t brucei

dependent on levels of parasitemia

t brucei subspecies hosts

congolense, brucei bruxei, vivas cannot cause infections in humans

brucei gambiense and brucei rhodiense can infect humans.

t brucei HDL

component of these particles in blood lyses certain trypanosome sub-species

particles filled with lipids and cholesterol

same VDL/HDLs that are tested for cholesterol levels

APOL-1 component of lipid particles

ApoL-1

effectively kills certain trypanosome sub-species

lysis appears to occur by swelling of the endosome/lysosome

similar appearance and kinetics compared to lysis with human serum

ApoL1-mediated lysis mechanism

blocks ApoL1-mediated lysis in gHAT

TgsGP: T. b. gambiense-specific glycoprotein

stiffens membranes, thought to limit ApoL1 insertion into membrane

t brucei transmitted by bites

remain in nearby tissue

parasite associates with many cell types associated with skin

cells appear to be dividing and viable

abundant in skin after tsetse bite

• alive and outside the vasculature

present in skin 12 days after infection in injection

t brucei eradication complications

skin resevoirs

people carrying skin infection had no symptoms of HAT

skin infection could have minimal/different symptoms

t brucei important resevoirs

fat and skin

fat resident ones stay primarily slender

RNA-seq

allows specific mRNAs and their expression levels to be determined

captures poly-A mRNAs selectively using oligo-dT

conversion of mRNAs into cDNA using reverse transcriptase

next-gen sequencing to identify sequence of mRNAs

abundance of specific sequences reflects the amount of mRNA in cells

t brucei motility

essential for evading host acquired immune response

antibody internalization is dependent on parasite motility

t brucei antibody isotypes

display different internalization rates

hydrodynamic flow forces can drag antibody-VSG complexes towards cell posteior

larger antibody, more drag experiences so explains different rates of internalization for IgG, IgM, and Fab fragments

suggests that parasite motility is an important part of antibody clearance from cell surfaces

trypanosome swimming behavior

function of viscosity

higher viscosity gives higher motility

they can navigate crowded environments well

testing crowded environments

PDMS pillar arrays

biocompatible plastic

speed is affected by pillar spacing

tuned to size of erythrocytest

trypanosome cellular waveforms

affect their swimming speed in pillar arrays

zebra fish as host

t carassi can infect

attach to surfaces and erythrocytes

have all three modes of motility

sandfly

causes skin lesions

start small, but frequently ulcerate and develops “crateriform” borders

can take years to heal, leaving disfiguring scars

can also cause the formation of diffuse lesions and scarring

can cause erosion of mucous membranes

kala-azar

swelling of liver and spleen, weight loss, anemia, but no cyclical fevers or malaria present in blood smear

high degree of mortality noted

patients can resolve severe dermatitis after resolution

caused by leishmania

sir William boog leishman

identified parasites in spleens of malaria-like patients

identified small, ovoid cells in sample

noted “micronucleus” similar to what had previously been seen in t brucei

thought it was t brucei that had died and shriveled up

leishmania life cycle

ancient leishmania

conservation of life cycle stages and presence in sandfly argues that digenetic lifestyle is an ancient adaptation

presence in Asia and South America raises questions about dispersal of leishmania

Gondwana theory might give insight, ancestral leishmania developed there and two populations isolated during dissolution of supercontinent. developed separately afterwards

leishmania treatments

many different species, each of which has different sensitivities to drugs

some parasite may cause different diseases and we don’t know why

resistance is arising

leishmania cell morphology

leishmania infection cycle

sandfly neutrophils

rapidly recruited to sandfly bite

neutrophils efficiently capture leishmania at sandfly bite site

internalized leishmania survive neutrophil lysis and can be recaptured

leishmania and neutrophils

depletion of neutrophils decreases number of leishmania in bitten ears

may serve as initial host cells. can be phagocytose by other immune cells, such as macrophages

neutrophils produce

NETs upon exposure to various pathogens

leishmania and NETs

leishmania LPG (lipophosphoglycan) blocks cell death caused by NETs

NET production is independent of LPG

leishmania in neutrophil

leishmania inside “loose” phagosome are killed

ones in “tight” phagosome tend to survive

recruitment of ER components to vacuolar membrane may block acidification and limit presence of lysosomal components

infected neutrophils are phagocytose by macrophages

neutrophils may facilitate leishmania infection of macrophages which are preferred host

leishmania and opsonization

opsonization facilitates uptake into immune cells