Sand Fly Cutaneous Leishmaniasis L. tropica

Muco-cutaneous VisceralPKDLDetection

L. brazillensis &L. mexicana

L. Donovani (Kala-azar)& L.infantum

Detection of parasite from splenic aspiration or from bone marrow puncture.

Treatment:Pentavalent antimonial like sodium stibogluconate, meglumine:

Very toxic sometimes with life-threatening adverse side effect like cardiac failure & acute pancreatitisAmphotericin B:

Costly also very toxic causes cardiotoxicity and nephrotoxicity, require hospitalization.Miltefosine:

Originally anticancer drug used in VL, toxic and very costly.

No vaccine and appropriate diagnostic procedure are available against leishmaniasis.

50,000 death/year500,000 new cases/year

Hemoglobin endocytosis in Leishmania:A novel target

Leishmania lack a complete heme biosynthetic pathway and must acquire heme from external sources for their survival.

How they acquire heme?

Is it from host hemoglobin?

46-kDa protein located in the flagellar pocket of Leishmaniainvolved in hemoglobin endocytosis

dc

ba

Hb-gold

Purification and characterization of hemoglobin receptor from Leishmania promastigotes

•Membrane proteins from Leishmania promastigotes were solubilized in octyl β-glucoside and ~46 kDa protein was enriched by 50% ammonium sulfate precipitation. Pellet resuspended in low salt buffer containing Triton-X100 was loaded onto SP-Sepharose column. Protein was eluted along a linear gradient of salt concentration. The ~46 kDa receptor protein eluted in fractions corresponding to salt concentration of about 140-190 mM. Hb specifically binds with purified protein.

46kDa

45kDa31kDa21kDa

1 2 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

bFp

a Merge

Nu

1 2 3

Localization of the 46-kDa protein in Leishmania1. GXXXK 9. MXHEMVEGXEGR2. VVXQK 10. MXPSFVYTSDAPK3. FVXPK 11. MMSENAPDDXEKR4. QNXQR 12. TQGXATVAVDGSVYEK5. TPSFKR 13. DGSGVGAAMX - - - K6. VNNXXSHXAXR 14. GNAVTPXNMECaGNFDSK7. ASAVTKDPAVSAR 15. - - - GMXAAD - - -8. XVSGMYXSVXSR 16. MMSENAPDDXEK17. SAXVGDATDXFDFXAQSVKBlast search indicated that some of the peptides could partially alligned with full length hexokinasefrom different organisms.

Time (min)0 5 10 15 20

Hex

okin

ase

activ

ity (O

.D.)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

GSTPHbRHis-HbRHbR-HisGST-HbR

Time (min)0 5 10 15 20

Hex

okin

ase

activ

ity (O

.D.)

0.0

0.1

0.2

0.3

0.4

0.5GST-HbRHis-HbRHbR-His

Time (min)0 5 10 15 20

Hex

okin

ase

activ

ity (O

.D.)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

pXGpXG-HbR

pXG-HbR pXGpXG-HbR pXG

Hemoglobin receptor in Leishmaniais a hexokinase

Role of clathrin and dynamin in hemoglobin endocytosis in Leishmania

MergeClathrin

Hb

K Nu

Hb

NuKClathrin

Clathrin

ClathrinClathrin

Marker Lysate

220kDa

97kDa

66kDa

45kDa

Ld-CHC MergeHb (5 min)

Hb (15 min)

Hb (30 min)

Hb

Hb

Hb

Ld-CHC

Ld-CHC

Ld-CHC

Role of clathrin in hemoglobin trafficking in Leishmania

How hemoglobin is transported to the lysosomess in Leishmania

Intracellular localization of Rab proteins

Late endosome

Lysosome

Early endosome

Recycling endosome

NucleusER

Rab5a,b,c

Rab27

Rab7

Rab6a.b

Trans-Golgi

Cis-Golgi

Rab1

Rab9

Rab24

Rab1,2

Rab3a,b,c,dRab8Rab26Rab37

Rab10Rab12Rab13Rab30Rab33bRab36

Rab11Rab17 Rab25

Rab4Rab15Rab18Rab20Rab22

How hemoglobin is transported to the late compartment to generate heme

after intracellular degradation

Cloning and expression of endocytic Rab GTPasesfrom Leishmania

Fractionation of early compartment from Leishmania containing endocytic probes

Rab7:WTRab5: WT

Localization of Rab5 and Rab7in Leishmania

PhaseLTR MergeRab7-GFP

Rab5-GFP LTR Phase MergeRab5-GFP

Rab7-GFP

Lysosomes

Lysosomes

Merge

Nu

Rab7:WT

Nu

Rab7:Q66L

Rab7:T21N Nu

LdRab7:WTNu

K

LdRab7:Q66L

Nu

K

LdRab7:T21N

Nu

K

Characterization & localization of different mutants of Rab7

in Leishmania

Rab7: C4 GSTRab7:Q66L Rab7:T21NRab7:WT

GTP BindingLd

Rab7

:Q66

L

MRa

b7:W

TLD

Rab7

:WT

LdRa

b7:Δ

C4

LdRa

b7:T

21N

GTP Hydrolysis

GDP

GTP

CaaxxCCCxC

F T DxxG ExSAnKxDGxxxxGKs

N-terminus GNB-Domain C-terminalExtension

C-terminus

PM1 G1 PM2 PM3 G2 G3

X= any residuea = aliphatics = Ser or Thr

Main sequence elements and very conserved residues of ras-like proteins

SytoHbK

Nu Hb

20 min10 min 60 min

Hb

Rab7:WTRab7:WT

Hb

Rab7:Q66L Rab7:Q66L

Hb

HbRab7:T21N

Rab7:Q66L

Hb

Rab7: WT

Hb

Regulation of hemoglobin trafficking by Rab7 in Leishmania

Time (Hrs)0 1 2 3 4

Hb

degr

aded

(ng)

0

200

400

600

800

1000

1200

ControlRab7:WTRab7:Q66LRab7:T21N

Time (Hrs)0 1 2 3 4

Hb

upta

ke (n

g)

0

100

200

300

400

500

600

Control Rab7:WTRab7:Q66LRab7:T21N

Uptake and degradation of 125I-Hb By Rab7 overexpressed Leishmania

Rab7 in GTP form target the hemoglobin to the late lysosomalcompartment for degradation.

Time (Hrs)

0 5 10 15 20 25 30 35

3 H-T

hym

idin

e in

corp

orat

ion

(C.P

.M.)

0

5000

10000

15000

20000

25000

30000

Control Rab7:WTRab7:T21NRab7:T21N + Hemin

Role of hemoglobin degradation on the growth of Leishmania

Inhibition of hemoglobin trafficking to the lysosomes in the cells over-expressing Rab7 negative mutant (T21N) compromised the growth of the Leishmaniademonstrating that Rab7 mediated hemoglobin degradation to generate intracellular heme is necessary for The gorwthof Leishmania.

Hemoglobin endocytosis in Leishmania

Presence of specific Hb receptor in the flagellar pocket.J. Biol. Chem. (1999) 274:2758

Bound Hb is internalized into early endosomal compartment by Rab5 regulated process.EMBO J. (2003) 22:5712

Subsequently,Hb is targeted to the lysosomes where it is degraded to generate intracellular heme.PNAS, USA. (2008) 105:3980.

HbR

Hb

Hb binds with its receptor which is a surface localized hexokinase.J. Biol. Chem. (2005) 280:5884

Mechanism of Phagocytosis

Konark MukherjeeShehla HashimShadab SiddiqueParashuramanMalabika BhattacharyaNamrata OhjaRicha Madan

Intracellular traffickingIn Leishmania

Shantanu SenguptaRuchi TandonJelaj TripathiSudha Bala SinghVikramNitin PatelGanga KrishnamurthySenthil KumarShruti AgarwalRuchir RastugirSurbhi BhalSonali BoseDeepika Gupta

Site specific drugDelivery

Vikram PrasadS. SrividyaSaubiya Siddique

Funded by:Department of Biotechnology,Department of Science & Technology,Indian Council of Medical Research,Council of Scientific & Industrial Research.Indo-French Center for Promotion of Advanced Research

I have shown you how endocytosis and intracellular trafficking is regulated in a unicellular protozoan pathogen through hemoglobinendocytosis in Leishmania. This receptor system appears to be a very significant process to acquire heme from the intracellular degradation of internalized hemoglobin by Leishmania. Inhibition of hemoglobin uptake or its intracellular degradation is found to be detrimental to the cells suggesting that HbR could be a novel therapeutic taget for Leishmaniasis.