Leishmaniasis - 4 (8/9) Flashcards

Question 1

Q

Leishmaniasis is also known as

Answer

A

orient boils
Baghdad boils
kala azar
black fever
sandfly disease
DumDum fever
espundia

Question 2

Q

Leishmaniasis distribution

Answer

A

88 countries
- tropical and sub-tropical countries
2 million new cases each year
12 million people infected / 52,000 deaths in 2010
350 million people at risk (will increase with global warming)
- flies that spread the disease moving up the Italian peninsula
- moving north as the earth warms up
spectrum of diseases ranging from mild to severe
due to militariy in Iraq etc. have gotten the disease in non-endemic sites
- military gone into the area, go back home, bringing parasite with them
- insect vector found in parts of the US
- secondary foci - esp Southern US (eg Texas)

Question 3

Q

Leishmaniasis

transmission

Answer

A

parasite transmitted by phlebotomine sand flies
- Phlebotomus in Africa, Asia, and Europe
- Lutzomyia in Americas
adult sandflies ~ 2mm in length
adult sandflies characterized by hairy body and wings

Question 4

Q

Leishmaniasis

transmission

Phlebotomus spp

Answer

A

desert/semi-arid ecosystems
some breed in peridomestic situations and enter human habitations
flies transmitting in the old world
some breed/propagate in areas surrounding houses, occassionally can transmit vector within human habitation
moving into towns more and more, becoming more urban

Question 5

Q

Leishmaniasis

transmission

Lutzomyia spp

Answer

A

forest dwelling
- new world, associated with forests
disease transmission associated with humans living or working near forests

Question 6

Q

Leishmaniasis

transmission

sandflies fly via

Answer

A

“hopping”

short bursts of flight/few seconds of rest
short flight range

Question 7

Q

Leishmaniasis

transmission

sandflies: …. spread the disease

Answer

A

only females spread the diseae - males play no role in disease transmisson

male flies don’t feed on any mammals - feed on phloem of plants
only females feed on blood = only vectors for disease transmission

Question 8

Q

Leishmaniasis

transmission

sandflies feed

Answer

A

most actively at twilight/night

Question 9

Q

Leishmaniasis

transmission

sandflies are

Answer

A

pool feeders

saw-like
short mouthparts
drink blood/lymph fluid from wound

Question 10

Q

Leishmaniasis

transmission

parasite not in

Answer

A

insect salivary gland

parasite found anterior portion of gut and pharynx
transmitted to humans not from fly saliva in formation of wound BUT spread by vomit of the insect
- parasite regurgitated by insect vector

Question 11

Q

Leishmaniasis

transmission

zoonotic or anthroponotic

Answer

A

zoonotic = animal to human
anthroponotic = human to human
different transmission cycles in different parts of the world (higher human density in some regions)
generally in areas with sparse human populations = zoonotic transmission
many humans = anthroponotic
different flies have different preferences for what they feed on

Question 12

Q

Leishmaniasis

transmission

zoonotic

Answer

A

in Mediterranean and Latin America
small rodents and canines (and humans)
- rodents and canines are reservoirs in these regions

Question 13

Q

Leishmaniasis

transmission

anthroponotic

Answer

A

Indian subcontinent
more people packed in so tend to have human-human transmission
can still go through zoonotic cycle but in this region more associated with urban environment
other methods of human to human transmissions:
- blood transfusion
- organ/bone marrow transplants
- congenital
- drug usage (needle sharing)

Question 14

Q

Leishmaniasis

causative agent

Answer

A

21 different Leishmania species can infect humans
- L. donovani
- L. infantum
- L. braziliensis
- L. major
some species are synonymous
- L. infantum = L. chagasi (Old World vs New world)
  - old world anmes don’t tend to name location
morphologically indistinguishable
- hard to definitively diagnose because identical
- hard to treat
molecularly distinct
- PCR
- isoenzyme analysis
- monoclonal antibodies

Question 15

Q

Leishmaniasis

major forms of the parasite

(list)

Answer

A

promastigote
- non-infectious promastigotes
- infectious metacycic promastigotes
amastigote

Question 16

Q

Leishmaniasis

major forms of the parasite

promastigote

Answer

A

15-20 μm in length
flagellated
motile
common morphology in insect host
- 2 types of promastigote form, both arise in insect vector
- flagellum pocket - anterior of nucleus
- flagellum - not attached to cell body like in epimastigote or trypomastigote
  - sticks straight out from body
- kinetoplast - located in front of nucleus, near anterior end of cell body

promastigote not in T. brucei or T. cruzi

Question 17

Q

Leishmaniasis

causative agent

2 types of promastigote

Answer

A

non-infectious promastigotes

insect gut
able to divide by binary fission

infectious metacyclic promastigotes

attach/invade neutrophils/macrophages
non-dividing (mechanism from going insect to mammalian host)
inside the gut the non-infectious promastigote becomes infectious metacyclic promastigote
metacyclic - form of the parasite that goes insect to human
- generally true for most

Question 18

Q

Leishmaniasis

major form of the parasite

amastigote

Answer

A

2-4 μm in diameter
stumpy flagellated
non-motile
intracellular form
- in phagolysosome of immune cells
divides by binary fission
- replicates within neutrophils and macrophages
  - in macrophage usually within vesicular compartment - membrane around them derived from host
  - can be transmitted back into the insect vector or in new neutrophils/macrophages
many (500+) parasites in 1 host cell
infectious - can invade macrophages
- transmissable from humans to humans
flagellum pocket - anterior of nucleus
flagellum - very short (doesn’t normally extend beyond the cell body), projecting only slightly beyond flagellum pocket
kinetoplast - located in front of the nucleus, near anterior endo f cell body

Question 19

Q

Leishmaniasis
life cycle

(picture)

Question 20

Q

Leishmaniasis

life cycle 1

picture

Question 21

Q

Leishmaniasis

life cycle 1

Answer

A

sand fly takes a blood meal
metacyclic promastigotes regurgitated into victim’s bloodstream (pool of fluid it’s feeding from from bite)
- regurgitation caused by the parasite…
infected sandflies have dysfunctional stomodeal valve at foregut/midgut
- valve that acts as boundary between foregut and midgut of insect vector’s digestive tract
junction → damage caused by parasite
- parasite in midgut, damages the valve between the 2
valve can’t close → blood moves back into mouthpiece and wound
when the insect vector sucks up blood/lymph that goes down the digestive tract, when it stops at the valve it goes back up, washing the parasite into the wound
reflux reaction → expulsion of parasite from anterior of gut into bite wound
exacerbated as parasite secretes promastigote secretory gel → physical barrier to fly feeding
- gel acts as bung - promotes insect vector to suck and doesn’t feel as though it’s feeding
- not much blood/lymph getting past this gel
  - getting more food than it thinkgs, so continues feeding
  - greater chance parasite going back into pool that insect vector is feeding on
secreted gel (and insect saliva) may potentiate fly infectivity
- gel and saliva contain elements that our immune systems will respond to
  - immune system responds to physical damage from cut, also gel and insect saliva → magnifies attraction at that site
caused b promastigote’s secreted gel plus physical wound etc. = causes neutrophils to go to an area
- the parasite invasdes the nuetrophils
  - always through neutrophil first, aters expression of neutrophil genes
  - does this to promote itself

Question 22

Q

Leishmaniasis

life cycle 1.5

Answer

A

metacyclic promastigote phagocytized by host neutrophils
infectious form taken up by neutrophil
use neutrophil to get into macrophages - 2 hypotheses
- both hypotheses lead to amastigote forms in macrophages
  - amastigotes multiply in macrophages
  - macrophages rupture, releasing amastigotes into bloodstream to continue cycle (infect new macrophages)
  - makes a long-lasting infection

Question 23

Q

Leishmaniasis

life cycle 1.5

trojan horse hypothesis

Answer

A

trojan horse hypothesis (in L. major)

infected neutrophils undergo apoptosis
neutrophil apoptotic markers onto surface of neutrophils
apoptotic cells phagocytosed by macrophages
metacyclic promastigote cargo delivered into macrophages
- in addition to taking up dying neutrophil also takes up metacyclic promastigotes
once within the macrophage differentiates into amastigotes

Question 24

Q

Leishmaniasis

life cycle 1.5

trojan horse hypothesis

(picture)

Question 25

Q

Leishmaniasis

life cycle 1.5

hypothesis 2

Answer

A

neutrophil becomes in fected
metacyclic promastigote changes to amastigote which multiplies
causes neutrophil to burst
amastigotes into blood/lymph taken up by neutrophils
from one cell type (doesn’t pack but only amplifies a few times) and neutrophil lyses to release amastigotes which are then taken up

Question 26

Q

Leishmaniasis

life cycle 1.5

hypothesis 2

(picture)

Question 27

Q

Leishmaniasis

life cycle 2

picture

Question 28

Q

Leishmaniasis

life cycle 2

Answer

A

back into the insect host
sandfly takes a blood meal from an infected mammal, ingests macrophages with infected amastigotes
macrophages rupture in insect vector gut
- into midgut macrophage is digested and ruptures
- releasing parasite amastigotes into the intracellular environment/lumen

Question 29

Q

Leishmaniasis

life cycle 3

picture

Question 30

Q

Leishmaniasis

life cycle 3

Answer

A

in the midgut the amastigotes transform into non-infectious nondividing promastigotes
- from decrease in temperature, increase in pH
initally parasite is surrounded by mucus layer from the insect itself
- parasite is trapped in this meshwork (peritrophic matrix)
  - peritrophic matrix = chitin and protein mesh secreted by the midgut epithelium that encloses the blood being digested
various enzymes produced by insect and parasite will degrade away the matrix and release the noninfectious promastigote into the lumen
- host and parasite chitinase
- after a few days the parasite escapes from peritrophic matrix
released parasite move toward anterior of midgut
- attach to microvilli of insect epithelial cells via lipophosphoglycans (LPG) to prevent excretion in feces
- the noninfectious promastigote then fires LPG that sticks to the epithelial layer of the gut wall
  - prevents the parasite being excreted

Question 31

Q

Leishmaniasis

life cycle 4

picture

Question 32

Q

Leishmaniasis

life cycle 4

Answer

A

the promastigotes divide in the anterior midgut
produce promastigote secretory gel
some promastigotes form metacyclic promastigotes (infective)
detach from midgut wall (LPG changes)

Question 33

Q

Leishmaniasis

cell invasion

(general)

Answer

A

Leishmania species are obligate intracellular parasites in the mammalian host
infects cells with microbicidal capability - macrophages
how do the parasites:
- avoid instant killing?
- replicate in a hostile environment?
- establish chronic infections?
have to overcome immune insults that our body will mount against infection
may use immune systems to gain entry to mammalian cells

Question 34

Q

Innate immune mechanisms

Answer

A

complement-mediated lysis
lysosomal pH
lysosomal enzymes
oxidative stress

Question 35

Q

Innate immune mechanisms

complement-mediated lysis

picture

Question 36

Q

Innate immune mechanisms

complement-mediated lysis

Answer

A

/alternative activation pathway
binding of early complement components to organism is followed by assembly of membrane attack complex (C5-C9)
use this to get into the cell - so we can exploit this
Leishmania parasites exploit this cascade to get into the parasite
can overcome this and utilize it at the same time

Question 37

Q

Innate immune mechanisms

lysosomal pH

picture

Question 38

Q

Innate immune mechanisms

lysosomal pH

Answer

A

phagosomal compartment is acified upon fusion with lysosomes to pH5
microbes in a phagosome (a vacuole formed around a particle)
once within the macrophage or neutrophil, must overcome the cellular mechanisms of those particular cell lines to destroy infectious organisms
in neutrophil/macrophage vacular compartment becomes acidified
- this is usually sufficient to damage membrane of infectious agent
- downshift in pH
- parasites use this to recognize the switch required in the life cycle
these pockets are targeted by degradative enzymes
- parasite resistant to all of these

Question 39

Q

Innate immune mechanisms

lysosomal enzymes

Answer

A

lysosomes contain an array of digestive enzymes including:
- proteases
- nucleases
- lipases
pockets (phagosomes) targeted by degradative enzymes
- parasite resistant to all of these

Question 40

Q

Innate immune mechanisms

oxidative stress

Answer

A

mechanisms by which our cells generate oxidative stress

NADPH oxidase (Phox) complex on phagosomal membrane generates superoxide
- leads to production of hydrogen peroxide and hypochlorite
- breaks down to stuff iwth antimicrobial activities
reactions with transition metals (eg iron) lead to the formation of hydroxyl radicals
nitric oxide synthase generates nitric oxide which combines with superoxide to form peroxynitrite
- this vacuole is targeted for something that makes nitric oxide with antimicrobial expression
  - these are amplified when come with superoxide to make something very damaging

parasite must overcome all of these systems

Question 41

Q

The players of Leishmania infection

infective

Answer

A

metacyclic promastigotes

Question 42

Q

The players of Leishmania infection

host target cells

Answer

A

neutrophils, then macrophages

Question 43

Q

The players of Leishmania infection

mode of entry

Answer

A

passive, opsonized phagocytosis
doesn’t require energy
opsonized - something that macrophages/neutrophils find attractive

Question 44

Q

The players of Leishmania infection

host receptors

Answer

A

CR1
CR3

(complement receptors)

Question 45

Q

The players of Leishmania infection

parasite molecules

Answer

A

gp63 protease
- zinc protease
lipophosphoglycan (LPG)

Question 46

Q

The players of Leishmania infection

site of replication

Answer

A

phagolysosome vacuole

Question 47

Q

Infection involves

(2 ~steps)

Answer

A

complement evasion
host cell invasion

Question 48

Q

Complement evasion

parasite surface

Answer

A

parasite surface changes during differentiation

replicative promastigote (non-infective)

short LPG
low level of GP64

metacyclic promastigote (infective)

long LPG
high level of GP63

Question 49

Q

Complement evasion

LPG

Answer

A

lipophosphoglycan
sugar - fat phospho sugar mix
coat all over the cell surface
3-5 million copies per cell
contains
- oligosaccharide cap
  - toward external environment
- phosphoglycan region = trunk, can have branches
  - galactose-mannose-phosphate
  - side chains
  - galactosyl in replicative proastigotes
- arabinosyl then caps then galactosyl in infective promastigotes
galactosyl side chain binds to galectin
- binds parasite to wall of insect gut
capping prevents binding to galectin, facilitating detachment from wall of midgut
replicative promstigote with short LPG side chains always have galacto side chain that can bind to galectin on epithelial layer of insect vector (galactose interacts with galectin, lets parasites stick to wall)
into infectious metacyclic form - galactose is capped (sugar on the end) by arabinosyl that’s sufficient to inhibit the interaction of LPG with the galectin, releasing metacyclic off the epithelial layer

Question 50

Q

LPG picture

Question 51

Q

Answer

A

LPG

pink = inside cell
green dots = phosphoglycan
- can have disc branches

Question 52

Q

LPG picture - detailed

Question 53

Q

Complement evasion

LPG - continued

difference between noninfectious promatigotes and

non-replicative infectious forms

Answer

A

all linked by GPI anchor
- way of packing molecules very close together on the surface
apart from arabinosyl and galactose side chains, main difference is size
- noninfectious promastigotes have short galactose mannose phosphate repeat sequences
- significantly longer in non-replicative infectious forms
- LPGs longer in infectious forms - more repeats

Question 54

Q

Answer

A

blue is more sugars

Question 55

Q

Cutaneous leishmaniasis

pathology

Answer

A

most common manifestation - 10 million cases
doesn’t usually progress beyond this
known as:
- orient/bay sore
- uta
- Baghdad/Dehli/Kandahar/Lahore boil
- city boil
L. tropica in cities
L. major in rural
starts as raised, generally painless red lesion at site of bite
can ulcerate/break the skin
small spot to progressively larger lesion, eventually immune system will kick in and wipe it out

Question 56

Q

Cutaneous Leishmaniasis

ulcer classified as wet or dry (crust formation)

Answer

A

wet ulcer = L. major
- remains open
dry ulcer = L. tropica
no pus formation unless get bacterial infection
ulcer self heals
- causes scarring
- eg L. major
- in about 6 months
- usually treatment not provided because will heal on oyur own
  - drugs saved for more severe form of the disease
- if face bitten will be given the drug to speed up healing
once healed, individuals immune for life
- once moutned appropriate immune response will be immune to that particular species
in some individuals diffuse lesions develop
- wet ulcer may disappear, reappear on different parts of the body later in time

Question 57

Q

Diffusive Leishmaniasis

pathology

Answer

A

2 types

diffuse cutaneous leishmaniasis (DCL)
mucocutaneous leishmaniasis (MCL)

Question 58

Q

Diffusive Leishmaniasis

diffuse cutaneous leishmaniasis (DCL)

Answer

A

metastasizes over the whole body
non-ulcerated, scaly lesions (nodular)
generally associated with anergy (lack of immune response)
amastigotes abundant in lesions
- packed with macrophages and amastigotes
eg L. aethiopica, L. mexicana
can appear anywhere on the body

Question 59

Q

Diffusive Leishmaniasis

mucocutaneous leishmaniasis (MCL)

Answer

A

primarily L. braziliensis (3-5% of cases move from cutaneous to this kind of form)
- most prevalent in Latin America
metastasizes mucocutaneous junctoins
- mouth/nose/soft palate or anus/genitalia
can occur weeks/years after first degree infection
tissue destroyed → severe disfigurement
seconary bacterial infections common
may affect eg breathing, prone to secondary infections

Question 60

Q

Visceral Leishmaniasis

pathology

Answer

A

starts as cutaneous lesion to visceral form
known as
- Kala-azar
- DumDum fever
most serious form
200-500,000 cases/50,000 deaths per year
typically caused by
- L. donovani (India/Pakistan)
- L. infantum (Mediterranean) = L. chagasi (Latin America)
high fatality rate
darkening of the skin
others highly disfiguring or may lead to death through secondary infection, but are not lethal itself

Question 61

Q

Visceral Leishmaniasis

symptoms

Answer

A

generalized infection of the reticuloendothelial system
involves liver, spleen, bone marrow, lymph nodes
characterized by
- fever (sudden or gradually rising)
- splenomegaly (enlargement of spleen)
- hepatomegaly (enlargement of the liver)
- enlarged lymph nodes/tonsils (→ anemia, leucopenia, thrombocytopenia)
disease progression leads to malaise, tiredness, lassitude, weaness
patient exhibits wasting syndrome despite good appetite
- muscle wastage - musculature doesn’t develop to the same degree, remains extremely thin
secondary infections are common
kills by organ failure
- affects organs associated with eg clearing blood and helping immune system = prone to secondar infections
  - pneumonia
  - tuberculosis
  - dysentery
50,000 deaths directly attributed to Leishmania
- but if have visceral Leishmaniasis will die of secondary infection and won’t be attributed to Leishmania (which weakens the immune system)

Question 62

Q

Leishmaniasis

disease severity depends on

Answer

A

immune status of the individual

Question 63

Q

Th1-type response

Answer

A

cellular immune response triggered - no/low antibody formation
recover from infection, are immune (cutaneous)

Question 64

Q

Th2-type response

Answer

A

humoral immune response triggered, antibody formation occurs
succumb to infection (visceral)
Th2 by mistake = develops to more serious forms
antibodies → succumb, more severe forms
some of the more dangerous and highly infectious strains appear to be able to switch host’s immune response from something that will clear the parasitemia to something that will enhance the parasitemia
- some strong L. donovani strains force host to switch from Th1 to Th2 response (mechanisms unknown)

Answer 54

A

epidemiological history
symptoms
definitive diagnosis - detection of parasites, seriology

Answer 55

A

dermal and visceral both

suspected
geographical presence of parasite
history of sandfly bite

Answer 56

A

chronic, painless lesion (CL)
mucocutaneous lesion (MCL)
scaly lesions (DCL)

Answer 57

A

prolonged fever
splenomegaly
hepatomegaly
anemia
leucopenia
thrombocytopenia

Answer 58

A

amastigotes in scraping

CL - incision of lesion
dermal scraping examined by Giemsa staining/microscopy

amastigotes in aspirates

CL - injectio saline in lesion
examine aspirate following giemsa staining/microscopy

in vitro culturing of promastigotes from aspirates

CL - injection saline in lesion
transfer to tissue culture medium
monitor for promastigote growth (~1 week)
amastigote parasites break out in tissue culture of macrophage and spontaneously generate the promastigote form (grow out the form that grows in insect vector, but takes time)

inoculation of aspirates in model organism

CL - injection saline in lesion
transfer to model organism (hamster)
monitor for clinical pathology (2-3 months)

Answer 59

A

amastigotes in bone marrow, aspirates, or biopsy

CL- injection saline in lesion
examine aspirate following Giemsa staining/microscopy

in vitro culturing of promastigotes from aspirates

CL - injection saline in lesion
transfer to tissue culture medium
monitor for promastigote growth (~1 week)

Answer 60

A

Leishmanin skin test

looks at person’s response to fixed parasites (dead) or parasitse in extracts
intradermal injection of promastigotes
- extracts or whole, killed
monitor for local dealyed hypersensitivity (local redness)
development of erythema if leish +ve
- if some time in the past the person was infected - parsite may be wiped out but still be responding as if the parasite was there

Answer 61

A

serological test (direct agglutination, K39 dipstick)

direct agglutination

fixed coloured parasites + patient serum
- = anti-Leishmania antibodies → agglutinate fixed coloured parasites = blue/purple
- (react with colored parasites to agglutinate them)

K39 dipstick

patient serum applied to immunochromatographic dipstick
dipstick contains antigen to K39, abundant amastigote surface antigen
- K39 is a 39 amino acid repeat sequence
K39 antigen on strip, let patient’s serum migrate
if immune response will bind to K39 antigen and change color
patient serum reacts to K39 if contains anti-Leishmania antibodies
in figure lower band = control, 2 bands appear = infection

Answer 62

A

drugs based on antimony - can draw comparisons with the arsenic-based drug for HAT

sodium stibogluconate (Pentostam) → GSK

meglumine antimoniate (Glucantime) → Sanofi S.A

pro-drug
administered in inactive state (pentavalent)
activated (to trivalent state) only by amastigote
introduced in 1945
daily intravenous infusion (Pentostam) or intramuscular injections (Glucantime)
20mg/kg/day fro 10 days (CL)
20mg/kg/day for 30 days (VL/DCL/MCL)

Answer 63

A

not all Leishmania species are the same

those that cause the more severe form are more susceptible to these pentavalent antimonials
L. donovani >*
L. braziliensis* >
L. mexicana* >
L. major*

VL species > DCL/MCL species > CL species

Answer 64

A

high level of toxicity

fatigue, nausea, muscle/joint pain, changes in ECG, cardiotoxicity, hepatic/renal dysfunction, shock, and (rarely) sudden death
haevy metal = heavy metal poisining
similar problems to melarsoprol

don’t always work

may fail to clear parasitemia
resistance in field
- esp in India

Answer 65

A

activation can occur inside/outside cell
both Sb^V and Sb^III can be taken up by cell
- prodrug = Sb^V
- drug = Sb^III
conversion from prodrug (pentavalent) to drug (trivalent) can only occur if amastigotes present
don’t know where conversion occurs
- surface of amastigote before drug goes into the cell
- within the cell itself
- could be both
if activated outside know pentavalent can be taken in, as can trivalent

Answer 66

A

Sb^IIItaken up by AQP (aquaglyceroporin)
- same with melarsoprol
- resistance toward antimonials have been associated with reduced uptake of trivalent form by AQP
Sb^V - don’t know what’s used for uptake

Answer 67

A

conversion outside pentavalent → trivalent
- don’t know what mediates it
Sb^v→ Sb^III inside by reductases
- ACR2 - arsenite reductase
- TDR1 - thiol-dependent reductase

Answer 68

A

trivalent form binds conjugate agents - especially thiols
trivalent form binds trypanothione, inhibits trypanothione-dependent enzymes
- eg trypanothione reductase
- which has major implications how these parasites:
  - respond to oxidative stress
  - synthesize DNA
- heavy metal works the same as melarsoprol - sticks to thiols, messes up downstream pathways

Answer 69

A

1980s antimonials resistance observed
today 50-65% of VL cases in Bihar
antimonials obsolete in India
why?
- unrestricted availability
- incorrect doses
- inconsistent quality
  - cut with other substances
  - may not be up to required therapeutic doses
mechanism of resistance in the field is unknown
lab approaches used to dissect pathways
- resistance by parasites can be readily generated in the lab by growin cells on sub-lethal levels of the drug

Answer 70

A

Sb^R Leishmania amplified specific genetic loci from genome → episome
- amplify part of genome, throw it out to generate a plasmid
- amplify up to give high copy number
- very plastic genome
sequence/mapping/biochemical studies shown that episome contains ABC transporter
- localized to unknown organelle that’s very close to the flagella pocket
in mammals ABC transporters involved in
- multidrug resistance
- heavy metal resistance
Leishmania ABC transporter designated as MPRA (multipdrug resistance protein A)
previously shown to function as metal efflux pump that recognizes metals conjugated to thiols
- MRPA pumps metal-thiol conjugates into this organelle
- trypanothione conjugates the antimonial, pumped via this mechanism into the uknown organelle
overexpression in parasites causes resistance
- higher level of MRPA leads to resistance to antimonials
- can knock gene out - not essential to parasite genome, makes parasite more sensitive to the drug

Answer 71

A

MRPA localized to unknown organelle near flagella pocket

biochemical/functional analysis shows that MRPA

transports metals conjugated to thiols
over-expression causes resistance to antimonials
null mutants are sensitive to antimonials

Answer 72

A

resistance to antimonials is via sequestratoin of trypanothione-drug conjugate

Answer 73

A

pentavalent to trivalent form
trivalent form interacts with trypanothil (TS) to form metal-thiol conjugate to hit the target enzymes (trypanothione-dependent enzymes)

Answer 74

A

in resistant cell lines

get diversion away from the target
because you amplify up MRPA gene - increases the amount of transporter such that the metal-thil conjugate isn’t getting to the target but is pumped out into the unknown organelle
rate-limiting step is amount of TS2 (trypanothione)
so is the level of trypanothione affected in resistant cell lines?

Answer 75

A

resistant parasites have elevated levels of trypanothione
- Halmeur et al., 2002
amplification of trypanothione synthesis genes
- Grondin et al., 1997
- Haimeur et al., 1999
inhibitors of trypanothione synthesis (buthionine sulfoxice/eflornithine) can reverse resistance
- Grondin et al., 1997
- Haimeur et al., 1999
high levels of trypanothione, enzymes in trypanothine synthesis amplified
make more TS2 to bind out the drug
use inhibitors that block synthesis of trypanothione to make cells sensitive to antimonial
as well as upregulating transporter, upregulating trypanothione biosynthesis pathway to bind out lots of antimonial that can be cleared out of the cell into this organelle

Answer 76

A

sequestration of trypanothione-drug conjugate

AND

increased trypanothione synthesis

Answer 77

A

amphotericin B
miltefosine
paromomycin

Answer 78

A

polyene antibiotic
originally developed as antifungal
effective against antimonial resistant VL
infusion, severe toxic side effects
lipid formulation (AmBisome) have reduced toxicity
- mycels = lipid globules into which you can insert drug = reduced toxicity
amphotericin B binds to 24-substituted sterols (eg ergosterol) disrupting osmotic integrity of the membrane
- binds sterols in parasite cell membrane = punches holes at point of sterol to make parasite cell leaky

Answer 79

A

phosphocholine analogue
developed in 1980-90s as anticancer drug, abandoned due to toxicity problems
activity against Leishmania in vitro and in vivo (1987-1996)
daily, oral administration for 28 days
clinical trials for VL in Bihar State, India, >95% cure, including Sb(V) resistant cases
- Registered in India & launched in 2003
registered for VL/CL in Colombia, 90% cure: clinical trials in Brazil
BUT it is teratogenic (causes malformations in embryos), cannot be used by women of child-bearing age
mode of action – uncertain, possibly by inhibiting phospholipid formation, signal transduction or calcium homeostasis

Answer 80

A

aminoglycosides antibiotic (related to kanamycin, neomycin)
developed as antibacterial, used to treat Entamoeba and Cryptosporidium infections
anti-leishmania activity reported in 1960s
daily, oral administration for 20 days
phase 2 trials for VL in India/Kenya, ~90% cure
phase 3 trials ongoing/pharmaceutical manufacturer secured
mode of action – uncertain. Possible effects in mitochondrion?

Answer 81

A

vector control

insecticides and repellants
screens and bednets
difficult to carry out due to:
- cost
- re-emergence after cessation of spraying programs
- diversity of epidemiology (rural vs urban)

control of reservoir species

canine vaccines
culling
Mediterranean basin and Latin America
try to control flies
requires money and political will = problem