Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors.

2012·1.2 MB·English

by  Bullard, Whitney

Virulence3:1,72–76;January/February2012;G2012LandesBioscience
Haptoglobin-hemoglobin receptor independent killing of African
trypanosomes by human serum and trypanosome lytic factors
Whitney Bullard,1 Rudo Kieft,1 Paul Capewell,2 Nicola J. Veitch,2 Annette Macleod2,* and Stephen L. Hajduk1,*
1DepartmentofBiochemistryandMolecularBiology;UniversityofGeorgia;Athens,GAUSA;2FacultyofVeterinaryMedicine;
WellcomeCentreforMolecularParasitology;Glasgow,UK
Thehaptoglobin-hemoglobinreceptor Introduction
(HpHbR)ofAfricantrypanosomes
plays a critical role in human innate Africantrypanosomesareeukaryoticpatho-
immunity against these parasites. gens that cause important human and
© 2012 LaLocalnized todthe felagellasr poc keBt of thie oanimsal dicseasesi. eThesenparascites ehave .
veterinary pathogen Trypanosoma brucei evolved a variety of mechanisms to escape
brucei this receptor binds Trypanosome innate and acquired immunity, including
Lytic Factor-1 (TLF-1), a subclass of the use of the variant surface glycoprotein
human high-density lipoprotein (HDL) (VSG) coatto coverthe plasmamembrane
facilitatingendocytosis,lysosomaltraffick- of the parasite providing a barrier against
ing and subsequent killing. Recently, we attack by complement.1 The VSG coat
Do not distribute.
found that group 1 Trypanosoma brucei also serves as the molecular decoy during
gambiense does not express a functional antigenic variation, presenting an ever-
HpHbR. We now show that loss of the changing target to the adaptive immune
TbbHpHbR reduces the susceptibility of system of the mammal, thus allowing the
T.b.bruceitohumanserumandTLF-1by parasites to evade antibody-mediated kill-
100- and 10,000-fold, respectively. The ing.2 The subspecies of trypanosomes
relatively high concentrations of human that infect humans face the additional
serum and TLF-1 needed to kill trypano- challenge of encountering a unique innate
somes lacking the HpHbR indicates defensemechanismmediatedbytworelated
that high affinity TbbHpHbR binding serumproteinscomplexes.Inthecirculation
Keywords: trypanosome lytic factor,
enhancesthecytotoxicity;however,inthe of humans, TLF-1 is a minor subclass
innate immunity, African sleeping
absenceofTbbHpHbR,otherreceptorsor of HDL containing apolipoprotein A-1
sickness, haptoglobin hemoglobin, high
fluid phase endocytosis are sufficient to (apoA-1), the defining protein of all
density lipoprotein, haptoglobin related
provide some level of susceptibility. HDLs, and two primate specific pro-
protein, apolipoprotein L-1
Human serum contains a second innate teins, apolipoprotein L-1 (apoL-1) and
Submitted: 05/09/11 immune factor, TLF-2, that has been haptoglobin-related protein (Hpr).3-7 In
Revised: 10/01/11 suggested to kill trypanosomes indepen- addition to these apolipoproteins, Hpr
dentlyoftheTbbHpHbR.Wefoundthat binds free hemoglobin(Hb)inthe circula-
Accepted: 10/03/11
T.b.bruceikillingbyTLF-2wasreduced tion, which is likely released from ery-
http://dx.doi.org/10.4161/viru.3.1.18295 in TbbHpHbR-deficient cells but to a throcytes during early infection.8 The
*Correspondenceto:AnnetteMacleodand lesser extent than TLF-1. This suggests heterodimericHpr/Hbcomplexisproposed
StephenL.Hajduk;Email:[email protected] that bothTLF-1 andTLF-2 canbetaken tobebifunctional,servingbothastheligand
[email protected] upviatheTbbHpHbRbutthatalternative fortheT.b.bruceiHpHbR9,10anddirectly
pathways exist for the uptake of these contributingtohighspecificactivitykilling
Addendumto:KieftR,CapewellP,TurnerCMR,
toxins. Together the findings reported bycatalyzing the peroxidation oflysosomal
VeitchNJ,MacleodA,HajdukSL.Mechanismof
here extend our previously published membrane lipids.6,7,11 The other primate
Trypanosomabruceigambiense(group1)res-
istancetohumantrypanosomelyticfactor.Proc studies and suggest that group 1 T. b. specific apolipoprotein in TLF-1, apoL-1,
NatlAcadSciUSA2010;107:16137–41; gambiense has evolved multiple mechan- is also directly involved in T. b. brucei
PMID:20805508;http://dx.doi.org/10.1073/pnas. isms to avoid killing by trypanolytic killing.5,12,13 An ion channel forming
1007074107
humanserumfactors. protein, apoL-1 undergoes conformation
72 Virulence Volume3Issue1
ARTICLEADDENDUM
changes at lysosomal pH and can integrate Materials and Methods gambiense we developed a laboratory
intomembranes.5,12,14Thecombinedaction model for TLF-1 resistance using well-
of Hpr/Hb and apoL-1 results in the TLF-1 and TLF-2 purification. Total characterized clonal cell lines of T. b.
osmotic lysis of the parasite.15,16 The other serumwasobtainedfromahealthyhuman bruceithathadbeenselectedforresistance
trypanolyticserumcomplexiscalledTLF-2 donor. As previously described, two to human HDLs.23,24 We isolated TLF-1
and,whilelargelydevoidoflipidsitcontains sequential flotations on sodium bromide resistant (R) or susceptible (S) T. b. brucei
Hpr and apoA-117 and apoL-1 (this paper) gradients (ρ = 1.063 and 1.26 g/ml) lines expressing either the VSG800 or
suggesting that these complexes share a resulted in an HDL-rich fraction (TLF-1; VSG060.23 The T. b. brucei 427-800R
common origin and perhaps have a similar topthirdofthegradient)andalipoprotein- and T. b. brucei 427-060R lines showed
mechanismoftrypanosomekilling.18 deficient fraction (TLF-2; bottom third of reduced uptake of TLF-1 relative to the
The two subspecies of human sleeping the gradient).4 The TLF-1 fraction was TLF-1 susceptible parental T. b. brucei
sicknesstrypanosomeshaveevolveddistinct passed over an anti-IgM column (Sigma, 427-221ScellsandTLF-1susceptiblecells
mechanisms to survive in the human host. A9935). The unbound material was then expressing either VSG800 or VSG060. In
Trypanosomabruceirhodesienseproducesthe passed over an anti-Hpr column, washed addition,weshowedthattheexpressionof
serum resistance associated (SRA) protein withPBSE(137mMNaCl,2.7mMKCl, TbbHpHbR mRNA wasreducedapproxi-
thatbindsandinhibitsTLF-1.19-21SRA,an 10 mM Na HPO , 10 mM KH PO , mately 20-fold in resistant cells.23 These
2 4 2 4
intracellular protein largely found in endo- 3 mM EDTA) and bound protein was findings led us to examine group 1 T. b.
somes, co-localizes with TLF-1 in early eluted in 100 mM glycine (pH 2.5) and gambiense where we found that not only
endosomes and traffics to the lysosome.22 neutralized with 1 M Tris (pH 7.5). The was expression of the TbgHpHbR mRNA
©Thus, T.2b. rhod0esiense1survive2s in hu mLans aTLF-2nfractidonwasepassedsover anBanti-Hpir oreducsed buct thait meutatinons toctheegene .
largely because it is able to produce an column and washed with PBSE. Bound abolished function.23
antidote to TLF-1. While untested, it is protein was eluted in 100 mM glycine Purification and characterization of
likelythatTLF-2isinhibitedbySRAbythe (pH 2.5), neutralized with 1 M Tris TLF-1 and TLF-2. In order to determine
same mechanism since both serum com- (pH 7.5) and immediately added to an whetherloss ofTbbHpHbRwassufficient
plexes contain apoL-1. In contrast, we anti-IgM column and washed with PBSE. to provide complete protection from
recently showed that group 1 T. b. Bound protein was eluted in 100 mM human serum, TLF-1 and TLF-2 activity
Do not distribute.
gambiense does not bind or take up glycine(pH2.5),neutralizedwith1MTris we developed a purification protocol
TLF-1, suggesting that these cells have (pH 7.5). All protein samples were ali- exploiting physical and compositional
evolvedadifferentmechanismtoavoidthe quotedandstoredat280°C. differences in these human serum innate
cytotoxicity of TLF-1.23 The underlying Size exclusion chromatography and immune complexes (Fig.1). Freshly
basis for reduced TLF-1 uptake is 2-fold. protein gel blot analysis. Size exclusion collected human plasma was initially
First, TbgHpHpR is expressed at very low chromatography was performed on a 1 X separated by density gradient ultracentri-
levels by group 1 T. b. gambiense and PBSE equilibrated Superose 6 10/300 GL fugation to produce HDL-enriched
second, TbgHpHbR contains a number of column (GE Healthcare). Individual pro- (ρ 1.063–1.26 g/ml) and lipoprotein-
pointmutationswithinthecodingsequence tein standards were used to estimate the deficient fractions (ρ , 1.063 g/ml) that
that render the receptor non-functional.23 molecular weights of TLF-1 and TLF-2. were used as the starting materials for
The combination of mutations to the Samples of TLF-1 and TLF-2 from TLF-1 and TLF-2 purification respec-
TbgHpHbR and reduced expression abol- immuno-affinity purification (70 mg) were tively. During the purification of TLF-1
ished TLF-1 binding and uptake resulting run on the Superose 6 column at a flow small amounts of contaminating TLF-2
inresistancetoTLF-1.Thus,incontrastto rateof0.5ml/min.Fractionswerecollected were removed from the HDL-containing
T. b. rhodesiense, it appears the mechanism (0.5 ml), proteins concentrated 6-fold fraction by absorption with anti-IgM.
of group 1 T. b. gambiense resistance to with microspin S-300HR columns (GE, TLF-1 was subsequently bound to anti-
TLF-1 involves reduced uptake and avoid- 27513001) and the distribution of Hpr, Hpr resin, washed extensively at neutral
anceofthetoxin.Todatenoevidenceforan apoL-1 and IgM determined by SDS- pH to remove human HDLs lacking Hpr
inhibitory proteinwith SRA-like character- PAGE, silver staining and protein gel andelutedatlowpH.TLF-2waspurified
isticshasbeendescribedinT.b.gambiense. blotanalysis.Characterizationofantibodies from the lipoprotein-deficient serum by
In this short addendum to the Kieft et al. againstHprandapoL-1haspreviouslybeen sequential affinity chromatography with
paper23 we now show that while the described.7 Anti-IgM was purchased from anti-Hpr followed by binding and elution
TbbHpHbR enhances susceptibility to Sigma and used according to the manufac- from an anti-IgM column. The purity of
human serum, TLF-2 and TLF-1 other turer’srecommendation(Sigma,I0759). TLF-1 and TLF-2 was evaluated by size
receptors or fluid phase endocytosis also exclusion chromatography on Superose 6
contributetotrypanosomekilling. Further, Results and protein gel blot analysis with anti-
our results suggest that the resistance of Hpr, apoL-1 and IgM (Fig.1A and B).
group 1 T. b. gambiense to human serum TLF-1 resistant T. b. brucei. During the Based on size exclusion chromatography,
and TLF involves other mechanisms course of our studies on the mechanism TLF-1 and TLF-2 have estimated rela-
beyondthesimplelossofasinglereceptor. of TLF-1 resistance in group 1 T. b. tive sizes of 576 kDa and 1.6 MDa
www.landesbioscience.com Virulence 73
respectively.17 Superose 6 chromatography blots (Fig.1B; data not shown). TLF-2 amount of contaminating TLF-1 in these
of purified TLF-1 and TLF-2 revealed preparations were highly enriched in preparations to be ~10%.
somewhat dispersed distributions consist- particles containing Hpr, apoL-1 and Susceptibility ofT.b.bruceitohuman
ent with particle heterogeneity but there IgM; however, these preparations also serum, TLF-1 and TLF-2. Our previous
was minimal overlap of the TLF-1 and contained small amounts of IgM deficient studies compared the short-term killing of
TLF-2 absorbance peaks at 280 nM complexeswithanelutiontime(~28min) trypanosomes to TLF-1.23 Here we have
(Fig.1A).Proteingelblotanalysisrevealed from the Superose 6 column consistent re-examined the susceptibility of these
no contaminating TLF-2 in our purified with TLF-1. Based on the distribution of T.b.bruceilinesusingalong-termgrowth
TLF-1 preparations based on the lack of the Hpr dimer and IgM across the size assay (Fig.2). Consistent with previous
anti-IgM reactive material on protein gel exclusion fractions we estimate the studies,theparentalT.b.brucei427-221S,
© 2012 Landes Bioscience.
Do not distribute.
Figure1.Characterizationofpurified
TLF-1andTLF-2.(A)Superose6size
exclusionchromatographyofTLF-1
andTLF-2.Absorbanceprofiles
(280nM)ofTLF-1andTLF-2,super-
imposedonindividuallyranmarker
proteins[1,thyroglobulin(660kDa);
2,apoferritin(480kDa);3,conalbu-
min(67kDa);4,ovalbumin(45kDa)].
(B)AnalysisofindividualSuperose6
columnfractionsofTLF-1andTLF-2
separatedonnon-denaturing12%
SDS-PAGEandsilverstained(top
panel).Hpr,apoL1andIgMwere
detectedbyproteingelblot.NA,not
analyzed.
74 Virulence Volume3Issue1
T. b. brucei 427-800S, and T. b. brucei TbbHpHbR-independent mechanisms of the TbbHpHbR. To test this possibility,
427-060S were highly susceptible to TLF- TLF-1 uptake play a significant role in wetreatedTLF-1resistantandsusceptible
1 with a calculated LG of 0.8–6 ng/ml. trypanosome killing. T. b. brucei lines with human serum
50
T. b. brucei 427-800R and T. b. brucei Based on our studies with both the (Fig.2B). We found that T. b. brucei
427-060R were . 10,000-fold more group 1 T. b. gambiense and the TLF-1 427-800R and T. b. brucei 427-060R were
resistant to TLF-1 than wild-type T. b. resistant T. b. brucei lines we predicted approximately 100-fold more resistant to
brucei, suggesting the TbbHbHbR is that loss of a functional TbgHpHbR human serum killing than either T. b.
important in TLF-1 susceptibility. played a critical role in human infection brucei 427-800S or T. b. brucei 427-060S
However, concentrations of . 10 mg/ml by African trypanosomes.23 The dramatic (Fig.2B). Based on these results, we con-
overcametheTbbHpHbRdeficiencylead- reductioninsusceptibilitytoTLF-1inthe clude that loss of TbbHpHbR expression
ingtoreducedsurvival(Fig.2A).Sincethe TbbHpHbR-deficient cell lines supports contributes to the overall resistance of
concentration of TLF-1 needed to kill this prediction (Fig.2A). However, the these cells to human serum; however the
T. b. brucei 427-800R and T. b. brucei possibility remained that human serum level of resistance is much less than the
427-060R is similar to that found in contained additional innate immune fac- high level of resistance seen for TLF-1
human serum it is likely that tors,suchasTLF-2,thatmightnotrequire (10,000-fold). A possible interpretation of
these findings is that other human serum
components, such as TLF-2, are less
dependent on TbbHpHbR binding than
TLF-1.
© 2012 Landes BioItshas bceen piropeosednthat TcLF-2ecan .
bind to T. b. brucei in the absence of
the TbbHpHbR.18 We tested whether
highly purified TLF-2 was able to kill
T. b. brucei 427-800R and T. b. brucei
427-060R (Fig.2C). Similar to our find-
ings with complete human serum, these
Do not distribute.
TbbHpHbR-deficient cells were more
resistant to TLF-2 relative to the wild-
type TbbHpHbR-expressing cell lines.
Thus, reduced expression of TbbHpHbR
expression caused a reduced susceptibility
of TLF-2 killing suggesting that TLF-2
can bind to the TbbHpHbR. However,
the toxic concentration of TLF-2 is
.10-fold less than TLF-1, indicating that
TbbHpHbR-independent mechanisms
may play a greater role in TLF-2 binding,
uptake and killing.
Discussion
In the studies presented here human
serum, TLF-1 and TLF-2 susceptibility
was examined in isogenic lines of T. b.
bruceidifferinginTbbHpHbRexpression.
Cells deficient in TbbHpHbR expression
were 10,000-fold more resistant to TLF-1
Figure2.Invitroactivityofhumanserum,TLF-1andTLF-2.TLF-1resistant(R)andsusceptible(S) relative to wild-type susceptible cells.
clonalcelllinesofbloodstreamformT.b.bruceiLister427expressingVSG221,800and060were However, at concentrations of TLF-1
preparedaspreviouslydescribed.24,25Thepercentagesurvivingcellswasdetermined,usingphase typically found in serum (.10 mg/ml),
contrastmicroscopy,14hfollowingtheadditionofTLF-1,TLF-2orcompletehumanserumto
both resistant and susceptible cell lines
exponentiallygrowingculturesat37°C.(A)TLF-1susceptibilityofT.b.brucei427-221S(black),T.b.
brucei427-800S(blue),T.b.brucei427-800R(red),T.b.brucei427-060S(yellow)and427-060R(green). were killed. Human serum killing was
(B)Normalhumanserum(NHS)susceptibilityofT.b.brucei427-221S(black),T.b.brucei427-800S also reduced approximately 100-fold in
(blue),T.b.brucei427-800R(red),T.b.brucei427-060S(yellow)and427-060R(green).(C)TLF-2 cell lines expressing reduced levels of
susceptibilityofT.b.brucei427-221S(black),T.b.brucei427-800S(blue),T.b.brucei427-800R(red),
TbbHpHbR. However, significant kill-
T.b.brucei427-060S(yellow)and427-060R(green).
ing was still observed at human serum
www.landesbioscience.com Virulence 75
concentrations above 100 mg/ml. Since results have been used subsequently to TbbHpHbRthanisTLF-110,17.Finally,we
the only difference in the susceptible and argue that TLF-2 does not bind the propose that group 1 T. b. gambiense have
resistancecelllinesisthelevelsofexpression TbbHpHbR.10Itispossiblethatourresults evolved multiple mechanisms, including
ofTbbHpHbR,itseemslikelythathuman are influenced by the small amount of but not limited to the loss of a functional
serum contains a second trypanolytic contaminating TLF-1 in our TLF-2 pre- HpHbR, to avoid the cytotoxicity of the
activity that interacts with T. b. brucei parations. Clearly, a detailed characteriza- trypanosomelyticfactors.Wearecurrently
independentlyoftheTbbHpHbR.Basedon tion of the TLF-2 binding, uptake and exploringthesemechanisms.
this interpretation of the human serum cellularlocationisneeded.
killing results we decided to investigate Our results are consistent with previous Acknowledgments
whether TLF-2 killing of T. b. brucei was findings indicating the importance of the WethankcurrentmembersoftheHajduk
independent on the level of expression of TbbHpHbR in TLF-1 killing but also Laboratory for comments and suggestions
TbbHpHbR.WefoundthatTLF-2killing suggest that other mechanisms of TLF-1 on TLF and its mechanism of killing
was reduced 500–1000-fold in cell lines binding and uptake may contribute to trypanosomes. The work on TLF in the
withreducedlevelsofTbbHpHbR,suggest- trypanosome killing. The most likely path- Hajduk lab is supported by NIH
ing that TLF-2 also binds TbbHpHbR. ways for TbbHpHbR-independent uptake (AI039033). C.M.R.T. and A.M.L. thank
These results are in apparent contrast to ofTLF-1iseitherbyfluidphaseendocytosis the Wellcome Trust for financial support.
previous studies on TLF-2 showing that or the trypanosome lipoprotein scavenger A.M.L. is a Wellcome Trust Research
TLF-2 killing was not inhibited by the receptor18,25. The findings presented here CareerDevelopmentFellow.P.C.isaUK
addition of haptoglobin, an inhibitor of further support the findings of others that Biotechnology and Biological Sciences
©HpHb bi2ndingt0otheT1bbHpH2bR.17 ThLese aTLF-2nkillindg is leess despend entBon thie oReseasrch Ccounciil reesearchnstudecnt. e.
References 11. BishopJR,ShimamuraM,HajdukSL.Insightintothe 19. De Greef C, Hamers R. The serum resistance-
mechanism of trypanosome lytic factor-1 killing of associated(SRA)geneofTrypanosomabruceirhodesiense
1. PaysE.Thevariantsurfaceglycoproteinasatoolfor
Trypanosoma brucei brucei.. Mol Biochem Parasitol encodesavariantsurfaceglycoprotein-likeprotein.Mol
adaptation in African trypanosomes. Microbes Infect
2001;118:33-40;PMID:11704271;http://dx.doi.org/ BiochemParasitol1994;68:277-84;PMID:7739673;
2006; 8:930-7; PMID:16480910; http://dx.doi.org/
10.1016/S0166-6851(01)00361-9 http://dx.doi.org/10.1016/0166-6851(94)90172-4
10.1016/j.micinf.2005.10.002
12. Pérez-MorgaD,VanhollebekeB,Paturiaux-HanocqF, 20. Turner CMR, McLellan S, Lindergard LAG,
2. TaylorJE,RudenkoG.Switchingtrypanosomecoats:
what’sinthewardrobe.TDrendsGeonet2006 ;22n:614- oNotlanD P,LdinsL,HoimblesF,etalt.ApolripoproiteinbL-I uBisonti L,eTait A,.MacLeod A. Human infectivity
promotes trypanosome lysis by forming pores in trait in Trypanosoma brucei: stability, heritability and
20; PMID:16908087; http://dx.doi.org/10.1016/j.tig.
lysosomal membranes. Science 2005; 309:469-72; relation to sra expression. Parasitology 2004; 129:
2006.08.003
PMID:16020735; http://dx.doi.org/10.1126/science. 445-54;PMID:15521633;http://dx.doi.org/10.1017/
3. RifkinMR.Identificationofthetrypanocidalfactorin 1114566 S0031182004005906
normal human serum: high-density lipoprotein. Proc
13. Molina-Portela MP, Samanovic M, Raper J. Distinct 21. XongHV,VanhammeL,ChamekhM,ChimfwembeCE,
NatlAcadSciUSA1978;75:3450-4;PMID:210461;
rolesofapolipoproteincomponentswithinthetrypano- VanDenAbbeeleJ,PaysA,etal.AVSGexpressionsite-
http://dx.doi.org/10.1073/pnas.75.7.3450
somelyticfactorcomplexrevealedinanoveltransgenic associated gene confers resistance to human serum in
4. HajdukSL,MooreDR,VasudevacharyaJ,SiqueiraH, mousemodel.JExpMed2008;205:1721-8;PMID: Trypanosomarhodesiense.Cell1998;95:839-46;PMID:
TorriAF,TytlerEM,etal.LysisofTrypanosomabrucei 18606856;http://dx.doi.org/10.1084/jem.20071463 9865701; http://dx.doi.org/10.1016/S0092-8674(00)
byatoxicsubspeciesofhumanhighdensitylipopro- 14. Molina-PortelaMP,LugliEB,Recio-PintoE,RaperJ. 81706-7
tein.JBiolChem1989;264:5210-7;PMID:2494183
Trypanosome lytic factor, a subclass of high-density 22. StephensNA,HajdukSL.EndosomalLocalizationof
5. Vanhamme L, Paturiaux-Hanocq F, Poelvoorde P, lipoprotein,formscation-selectiveporesinmembranes. the serum resistance-associated protein in African
NolanDP, LinsL,VanDen Abbeele J,etal. Apoli- Mol Biochem Parasitol 2005; 144:218-26; PMID: trypanosomes confers human infectivity. Eukaryot
poproteinL-Iisthetrypanosomelyticfactorofhuman 16202458; http://dx.doi.org/10.1016/j.molbiopara. Cell 2011; 10:1023-33; PMID:21705681; http://dx.
serum.Nature2003;422:83-7;PMID:12621437;http:// 2005.08.018 doi.org/10.1128/EC.05112-11
dx.doi.org/10.1038/nature01461
15. Rifkin MR. Trypanosoma brucei: Biochemical and 23. KieftR,CapewellP,TurnerCM,VeitchNJ,MacLeodA,
6. SmithAB,EskoJD,HajdukSL.Killing oftrypano- morphologicalstudiesofcytotoxicitycausedbynormal HajdukS.MechanismofTrypanosomabruceigambiense
somesbyhumanhaptoglobinrelatedprotein.Science human serum. Exp Parasitol 1984; 58:81-93; PMID: (group1)resistancetohumantrypanosomelyticfactor.
1995; 268:284-6; PMID:7716520; http://dx.doi.org/ 6745390; http://dx.doi.org/10.1016/0014-4894(84) ProcNatlAcadSciUSA2010;107:16137-41;PMID:
10.1126/science.7716520 90023-7 20805508;http://dx.doi.org/10.1073/pnas.1007074107
7. ShiflettAM,BishopJR,PahwaA,HajdukSL.Human 16. Vanhollebeke B, Lecordier L, Perez-Morga D, 24. FaulknerSD,OliMW,KieftR,CotlinL,WidenerJ,
highdensitylipoproteinsareplatformsfortheassembly Amiguet-Vercher A, Pays E. Human serum lyses Shiflett A, et al. In vitro generation of human high
ofmulti-componentinnateimmunecomplexes.JBiol Trypanosomabruceibytriggeringuncontrolledswelling densitylipoproteinresistantTrypanosomabruceibrucei.
Chem2005;280:32578-85;PMID:16046400;http:// oftheparasitelysosome.JEukaryotMicrobiol2007; Eukaryot Cell 2006; 5:1276-86; PMID:16896212;
dx.doi.org/10.1074/jbc.M503510200 54:448-51; PMID:17910690; http://dx.doi.org/10. http://dx.doi.org/10.1128/EC.00116-06
8. WidenerJ,NielsenMJ,ShiflettA,MoestrupS,HajdukS. 1111/j.1550-7408.2007.00285.x 25. Green HP, Pilar Molina Portela M, St. Jean EM,
Hemoglobinisaco-factorofhumantrypanosomelytic 17. RaperJ,FungR,GhisoJ,NussenzweigV,TomlinsonS. LugliEB,RaperJ.EvidenceforaTrypanosomabrucei
factor.PLoSPathog2007;3:1250-61;PMID:17845074; Characterization of a novel trypanosome lytic factor lipoprotein scavenger receptor. J Biol Chem 2003;
http://dx.doi.org/10.1371/journal.ppat.0030129 in human serum. Infect Immun 1999; 67:1910-6; 278:422-7; PMID:12401813; http://dx.doi.org/10.
9. Drain J, Bishop R, Hajduk SL. Haptoglobin-related PMID:10085035 1074/jbc.M207215200
protein mediates trypanosome lytic factor binding to 18. Vanhollebeke B, Pays E. The trypanolytic factor of
trypanosomes.JBiolChem2001;276:30254-60;PMID: humanserum;manywaystoentertheparasite,asingle
11352898;http://dx.doi.org/10.1074/jbc.M010198200 waytokill.MolMicrobiol2010;76:806-14;PMID:
10. VanhollebekeB,DeMuylderG,NielsenMJ,PaysA, 20398209; http://dx.doi.org/10.1111/j.1365-2958.
Tebabi P, Dieu M, et al. A haptoglobin-hemoglobin 2010.07156.x
receptorconveysinnateimmunitytoTrypanosomabrucei
in humans. Science 2008; 320:677-81; PMID:
18451305;http://dx.doi.org/10.1126/science.1156296
76 Virulence Volume3Issue1