Trypanosoma brucei brucei: endocytic recycling is important for mouse infectivity.
Endocytosis in the African trypanosome, Trypanosoma brucei, is intimately involved in maintaining homeostasis of the cell surface proteome, morphology of the flagellar pocket and has recently been demonstrated as a bona fide drug target. RNAi-mediated knockdown of many factors required for endocytic transport, including several small GTPases, the major coat protein clathrin and a clathrin-associated receptor, epsinR, results in rapid cell death in vitro. Rapid loss of viability in vitro precludes meaningful investigation by RNAi of the roles of trypanosome endocytosis in vivo. Here we have sought to address this issue using strategies designed to produce milder effects on the endocytic system than complete functional ablation. We created a trypanosome clathrin heavy chain hemizygote and several lines expressing mutant forms of Rab5 and Rab11, described previously. All are viable in in vitro culture, with negligible impact to proliferative rates or cell cycle. Clathrin hemizygotes express clathrin heavy chain at ∼50% of wild type levels, but despite this demonstrate no defect to growth in mice, while none of the Rab5 mutants affected proliferation in vivo, despite clear evidence for effects on endocytosis. By contrast we find that expressing a dominantly active Rab11 mutant led to compromised growth in mice. These data indicate that trypanosomes likely tolerate the effects of partly decreased clathrin expression and alterations in early endocytosis, but are more sensitive to alterations in the recycling arm of the pathway.
Department of Pathology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QP, UK.
Senthil Kumar A
SK
Natesan
Alana
A
Black
Keith R
KR
Matthews
Jeremy C
JC
Mottram
Mark C
MC
Field
D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D004705:Endocytosis / Q000502:physiology*; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D051379:Mice; D008813:Mice, Inbred ICR; D018512:Parasitemia / Q000469:parasitology; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D014353:Trypanosomiasis, African / Q000276:immunology / Q000469:parasitology*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D033942:Adaptor Proteins, Vesicular Transport; D033922:Clathrin Heavy Chains; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C000595657:EpsinR protein, mouse
Eukaryot Cell. 2005 May;4(5):971-80
15879531
Cell. 2007 Nov 2;131(3):505-15
17981118
Nature. 2010 Apr 1;464(7289):728-32
20360736
J Cell Sci. 2006 Mar 15;119(Pt 6):1105-17
16507595
Mol Biochem Parasitol. 2004 Nov;138(1):67-77
15500917
BMC Res Notes. 2010 Mar 05;3:63
20205710
Biochem J. 2003 Sep 1;374(Pt 2):443-51
12744719
J Biol Chem. 2004 Oct 22;279(43):45047-56
15284229
Microsc Microanal. 2004 Oct;10(5):621-36
15525435
Eukaryot Cell. 2007 Nov;6(11):2029-37
17905918
Mol Biol Cell. 2003 May;14(5):2029-40
12802073
J Biol Chem. 2004 Nov 12;279(46):48426-33
15326171
Acta Trop. 1979 Sep;36(3):289-92
43092
J Biol Chem. 2008 Oct 24;283(43):28934-43
18701454
EMBO J. 2003 Oct 1;22(19):4991-5002
14517238
BMC Genomics. 2008 Jun 23;9:298
18573209
Traffic. 2009 Jul;10(7):894-911
19416477
Mol Biochem Parasitol. 1993 Jun;59(2):327-9
8341329
EMBO J. 2004 Feb 25;23(4):780-9
14963487
Nat Rev Microbiol. 2009 Nov;7(11):775-86
19806154
Parasitol Today. 1994 Feb;10(2):80-4
15275508
Traffic. 2008 Sep;9(10):1681-97
18657071
J Cell Sci. 1979 Jun;37:287-302
383734