Antigenic variation as adaptive process: the case of

Trypanosoma brucei

African trypanosomes infect a wide spectrumof mammalianhosts, including humans

Mechanisms of adaptation:I. Antigenic variation

A dense coat of Variant Surface Glycoprotein(VSG) covers the entiresurface of T. bruceibloodstreamforms

Bloodstreamform

Procyclic form

N-terminal VSG domains

VSG coat: protection

VSG dimers

phospholipids

- Tightly packed array of 107 molecules organized in dimers- Impenetrable to macromolecules of the host, including antibodies- Only surface loops recognizable by the host

antibody

1 2 3

Anti-1 Anti-2 Anti-3

Parasitenumber

Time

VSG coat: antigenic variation

The coat changesevery 103 to 105 cell divisions

Significance:participation in the control of the parasite burdenby attracting the lytic immune response and subsequently allowingnew antigenic variants to prolong the infection.

Genetic mechanisms of antigenicvariation in Trypanosoma brucei

7 6 5 4 8 8 3 2 1 VSG

7 6 5 4 8 8 3 2 1 VSG

VSG

VSG VSGDNA recombination(either gene conversion

or reciprocal recombination)

In situ (in)activation

~1,500 genes

~15 telomericVSG expression sites

VSG VS V G

Mechanisms of adaptation:II. Generation of adaptive proteins

Parasites must escape the defenses of their hosts, but they also need to communicate with host cells and

internalize vital host components

transferrin

antibodies

The flagellar pocket:the unique accessible

site

Surface receptors:- invariant- accessible- vitalVaccination targets??

Endocytosis in T. brucei

- only limited to 0.5% of the cell surface - highly efficient, probably due to special features (pNAL lectin?)

out

in

digestivevacuole

lysosome

Most ESAGs encode surface proteins, including a heterodimeric receptor for transferrin

and a homodimeric receptor-like adenylyl cyclase

Flagellar pocket Flagellum

plasma membrane

ATP cAMP

44444444 4444

AC

77776666 2222TF

44444444 4444

AC

?

ACAC????

plasma membrane

= VSG N-terminal domains!!!

1111

The use of different ESs, thus, the expression of different sets of ESAGs, allows a better

adaptation to a variety of different hosts:

• efficient uptake of transferrin from variousmammalian species, hence, colonization of a wide spectrumof mammals

• resistance to lysis by human serum, hence, colonization of man

10 4 8 8 3 2 1117 6 5( 9( )) VSG

10 4 8 8 3 2 1117 6 5( 9( )) VSG

The use of different BES allows a better adaptation to different hosts : efficient uptake of transferrin

The use of different BES allows a better adaptation to different hosts: resistance to lysis by human serum

T.b.rhodesiense

T.b.brucei

??

Lysis by human serumrequires endocytosis of the trypanosome lytic factor (TLF)

out

in

endocytosis

lysosome

TLF(HDL -linked)

In T. b. rhodesiense, resistance to human serumis linkedto activation of a specific VSG expression site (R-ES)

VSG

in non-human serum

VSG

R-ES

result:trypanosomessensitiveto

human serum(S clones)

VSG

in human serum

VSG

R-ES

result:trypanosomesresistant to

human serum(R clones)

Xong et al (1998) Cell 95, 839-846

The R-ES site is severely truncated and contains the Serum Resistance-Associated gene (SRA)

VSG

Expression of SRA in the R-ES appears to be a generalfeature of T. b. rhodesiense strains ;

SRA is the best available diagnostic tool of this subspecies

( )10 4 8 8 3 2 1117 6 5 9( ) VSG

B-ES

6 7 5 SRAR-ES

SRA is necessary and sufficient to confer full resistance to human serum

Xong et al (1998) Cell 95, 839-846

Trypanosoma b. b. w.t.

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7

days

par

asit

emia

FCS

NHSSRA transformants

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7

days

par

asit

emia

FCS

NHS

SRA is a VSG-like glycoprotein devoid of surface loops

Models of the N-terminal domain of SRA and VSGs

SRA VSG WaTat 1.2VSG MiTat 1.2

αααα-helixA

αααα-helixB

N-term surface loops

The study of the SRA moiety necessary to confer resistance to human serumhas uncovered:• the essential role of the N-terminal αααα-helix A, which is

interactive in VSGs;• the ability of this helix to interact with apolipoprotein L-I

(antiparallel to C-terminal αααα-helix)

(serumalbumin)

Coomassie blue

control

apoL1

SRA

• ApoL1 is the trypanosome lytic factor of humanserum.

• Interactions between the N-terminal αααα-helix of SRA and the C-terminal αααα-helix of apoL1, which occurwithin the lysosome, prevent trypanolysis by humanserum.

SRA

apoL1

Vanhammeet al (2003) Nature 422, 83-87

Activity of apoL1

•The colicin-like anion-selective pore-forming domain isresponsible for lytic activity.

•The membrane-addressing domain is responsible for bothbinding to HDL and addressing to a membrane.

•The C-terminal region is not required for either activity, but isthe target for neutralisation by the trypanosome immunity proteinSRA of T. b. rhodesiense.

Pérez-Morgaet al (2005) Science 309, 469-472

endosome

flagellarPocket

lysosome

pH 5.3

apoL-I

HDL

Trafficking of apoL1 to the lysosome of T. brucei: a model

NHS/apoL1 triggers swelling of the lysosome

0 h 1 h 2 h

3 h 4 h 5 h(1 µg/ml apoL1; 33°C)

Cl-

apoL1Cl-

DIDSApoL1-driven effect

on the lysosome:a model

Pérez-Morgaet al (2005) Science 309, 469-472

Hpr

apoA-I

apoL1

Lipids

91% identity to haptoglobin(hemoglobin scavenger)

ApoL1 is associated with Haptoglobin-related protein(Hpr) on the same subset of HDL particles (HDL3);

Hpr is involved in the binding of the particlesto the trypanosome surface.

Vanhollebekeet al (2007) PNAS 104, 4118-4123

kk

Haptoglobin(r)Alexa 488

+ hemoglobin

hemoglobinAlexa 488

+ haptoglobin(r)

The haptoglobin(r)-hemoglobin complex is a ligand for T.brucei

The trypanosome receptor for Hp(r)-Hb was recently identified.

In mouse serum, this receptor appears to be responsible for the uptake of heme, which is incorporated in

hemoproteins that confer resistance of the parasite to the oxidative response of host macrophages.

In human serum, this receptor also triggers the uptake of the trypanolytic HDL particles through recognition of

the Hpr-Hb complex.

Vanhollebekeet al (2008) Science 320, 677-681

Mutual adaptations between T. bruceiand man

Mφ

ROS RNS Hp-Hb

TbHpHbRTbHpHbR

Intravascular hemolysis

CD163

Hpr

Lipids

humans

SRASRA

Human infection

apoL1Hb-

Trypanolysis

T.b.rhodesiense

HDL3

Conclusions (I)

* The telomeric VSG ESs are powerful geneticworkshops for the adaptation of the parasite:

- their high homologous recombination rate, due to both highlevel of sequence identity with other loci and high level of DNA accessibility to recombinases, allows the continuouscreation of new antigens to cope with the immune system

- their diversity allows the variation of surface receptors

- their high recombination rate leads to the generation of new adaptive proteins

Conclusions (II)

* The VSG gene seems to have been used as a major tool to construct various adaptive components (transferrin receptors, SRA, otherVSG-like proteins..??)

* Allelic exclusion is the key to adaptive variation of T. brucei

ESAG7/6 as VSG-like transferrin receptor: Didier Salmon

SRA as inhibitor of trypanolysis: Huang Van Xong, Luc Vanhamme

ApoL1 as trypanolytic factor: Luc Vanhamme, Françoise Paturiaux-Hanocq, Philippe Poelvoorde

Mechanism of trypanolytic activity of apoL1: David Pérez-Morga, Benoit Vanhollebeke

Hpr as ligand of trypanolytic HDLs : Benoit Vanhollebeke

Identification of the trypanosome Hp-Hb receptor: Benoit Vanhollebeke

Annette Pays, Patricia Tebabi, Géraldine De Muylder, Laurence Lecordier, Derek Nolan