successful liver stage development, confirmed that
49c does not affect merozoite development (Fig. 4,
B and C). An in vivo time-course experiment was
conducted with mice infected with luciferase-expressing P. berghei sporozoites and either treated
twice with 100 mg/kg 49c or left untreated (Fig. 4D).
The livers of drug-treated and control mice were
comparably infected at 44 hours postinfection,
as revealed by bioluminescence imaging. Control mice exhibited the typical disappearance of
signal from the liver after 55 hours and the concomitant appearance of signal in blood after
65 hours (Fig. 4E). The liver load was prolonged
in the presence of 49c, likely as a result of impaired egress, and blood-stage development was
strongly delayed, as analyzed by flow cytometry
in the blood of infected animals (Fig. 4F). Treatment with 49c had a strong effect on the establishment of blood-stage parasites, although, at the
administered doses, a complete block was not
Curative and preventive strategies for malaria
treatment should ideally target three malarial
life-cycle stages: exoerythrocytic forms, the asexual blood stages, and the transmission stages.
Here we show that the pleiotropic plasmepsin
inhibitor 49c inhibits malarial PMIX and PMX,
resulting in a block in blood-stage parasite egress
and invasion as well as hepatic-stage egress and
transmission. Taken together, PMIX and PMX
qualify as very promising dual targets toward
REFERENCES AND NOTES
1. B. E. Sleebs et al., J. Med. Chem. 57, 7644–7662 (2014).
2. J. A. Boddey et al., Nature 463, 627–631 (2010).
3. I. Russo et al., Nature 463, 632–636 (2010).
4. C. R. Collins, F. Hackett, J. Atid, M. S. Y. Tan, M. J. Blackman,
PLOS Pathog. 13, e1006453 (2017).
5. G. E. Weiss, B. S. Crabb, P. R. Gilson, Trends Parasitol. 32,
6. R. Recacha et al., Acta Crystallogr. F Struct. Biol. Commun. 71,
7. C. L. Ciana et al., Bioorg. Med. Chem. Lett. 23, 658–662
8. W. A. Guiguemde et al., Nature 465, 311–315 (2010).
9. L. M. Sanz et al., PLOS ONE 7, e30949 (2012).
10. S. Glushakova, J. Mazar, M. F. Hohmann-Marriott, E. Hama,
J. Zimmerberg, Cell. Microbiol. 11, 95–105 (2009).
11. S. Das et al., Cell Host Microbe 18, 433–444 (2015).
12. M. Sajid, C. Withers-Martinez, M. J. Blackman, J. Biol. Chem.
275, 631–641 (2000).
13. S. Yeoh et al., Cell 131, 1072–1083 (2007).
14. T. Chu, K. Lingelbach, J. M. Przyborski, PLOS ONE 6, e18396
15. S. Glushakova et al., Curr. Biol. 20, 1117–1121 (2010).
16. M. J. Boyle et al., Proc. Natl. Acad. Sci. U.S.A. 107,
17. M. H. Lamarque et al., Nat. Commun. 5, 4098 (2014).
18. A. Yap et al., Cell. Microbiol. 16, 642–656 (2014).
19. J. Healer, T. Triglia, A. N. Hodder, A. W. Gemmill, A. F. Cowman,
Infect. Immun. 73, 2444–2451 (2005).
20. R. Banerjee, S. E. Francis, D. E. Goldberg, Mol. Biochem.
Parasitol. 129, 157–165 (2003).
21. C. Pfander et al., Nat. Methods 8, 1078–1082 (2011).
22. C. R. Collins et al., Mol. Microbiol. 88, 687–701 (2013).
23. N. Philip, A. P. Waters, Cell Host Microbe 18, 122–131
24. Z. Bozdech et al., PLOS Biol. 1, e5 (2003).
25. A. Mbengue, N. Audiger, E. Vialla, J.-F. Dubremetz,
C. Braun-Breton, Mol. Microbiol. 88, 425–442 (2013).
26. D. L. Baldi, R. Good, M. T. Duraisingh, B. S. Crabb,
A. F. Cowman, Infect. Immun. 70, 5236–5245 (2002).
27. A. H. O’Keeffe, J. L. Green, M. Grainger, A. A. Holder,
Mol. Biochem. Parasitol. 140, 61–68 (2005).
28. R. F. Howard, D. L. Narum, M. Blackman, J. Thurman,
Mol. Biochem. Parasitol. 92, 111–122 (1998).
29. D. Soldati, A. Lassen, J.-F. Dubremetz, J. C. Boothroyd,
Mol. Biochem. Parasitol. 96, 37–48 (1998).
30. C. Crosnier et al., Nature 480, 534–537 (2011).
31. J. C. Volz et al., Cell Host Microbe 20, 60–71 (2016).
32. F. Galaway et al., Nat. Commun. 8, 14333 (2017).
33. S. A. Howell, C. Withers-Martinez, C. H. M. Kocken,
A. W. Thomas, M. J. Blackman, J. Biol. Chem. 276,
34. T. Kariu, T. Ishino, K. Yano, Y. Chinzei, M. Yuda, Mol. Microbiol.
59, 1369–1379 (2006).
35. C. Suarez, K. Volkmann, A. R. Gomes, O. Billker, M. J. Blackman,
PLOS Pathog. 9, e1003811 (2013).
36. L. Tawk et al., J. Biol. Chem. 288, 33336–33346 (2013).
37. O. Silvie et al., J. Biol. Chem. 279, 9490–9496 (2004).
38. A. Sturm et al., Science 313, 1287–1290 (2006).
39. M. A. Child, C. Epp, H. Bujard, M. J. Blackman, Mol. Microbiol.
78, 187–202 (2010).
40. R. Stallmach et al., Mol. Microbiol. 96, 368–387 (2015).
We are grateful to C. Boss (Actelion Pharmaceuticals Ltd.)
and S. Wittlin for providing us with the initial 49c stock
and for their help with the chemical synthesis. We thank
V. Polonais for her exploratory work on the project and
G. Wright, M. Lebrun, D. Gaur, and A. Cowman for the gift of
numerous antibodies. We are grateful to O. Billker for the
gift of compound 2 and F. Hackett, J. B. Marq, and J. Xu for their
technical assistance. We thank the Biocenter Oulu Mass
Spectrometry Core Facility for their services. We would like
to thank the PlasmoGEM (Plasmodium genetic modification
project) team (Wellcome Trust Sanger Institute) for providing
the PlasmoGEM vectors. We would like to thank the Netherlands
Cancer Institute Protein Facility for provision of the ligation-
independent cloning vector, which was acquired by material
transfer agreement and the Nederlandse Organisatie voor
Wetenschappelijk Onderzoek (NWO) for financial support to the
facility (grant 175.010.2007.012). This work was funded by
Carigest SA (D.S.-F.), the Swiss National Foundation (grants
156825 to P.P., 310030B_166678 to D.S.-F., 310030_159519 to
V.H., and BSSGI0_155852 to M.B.), SystemsX.ch (grant
51TRPO_151032 to V.H. and D.S.-F.), and the Academy of
Finland (grants 257537 and 292718 to I.K.). Bo.M. is funded by
the European Research Council under the European Union’s
Horizon 2020 Research and Innovation program under grant
agreement no. 695596. Funding to M.J.B. was from the Francis
Crick Institute ( www.crick.ac.uk/) and Wellcome Institutional
Strategic Support Fund (ISSF2) funding to the London School of
Hygiene & Tropical Medicine. All the data required to
understand and interpret the paper are available in the main
text and the supplementary materials.
Materials and Methods
Figs. S1 to S8
Movies S1 and S2
11 March 2017; accepted 18 September 2017
Ne-Fatty acylation of Rho GTPases
by a MARTX toxin effector
Yan Zhou,1 Chunfeng Huang,1 Li Yin,1 Muyang Wan,1 Xiaofei Wang,1 Lin Li,2
Yanhua Liu,3 Zhao Wang,1 Panhan Fu,1 Ni Zhang,1 She Chen,2 Xiaoyun Liu,3
Feng Shao,2 Yongqun Zhu1†
The multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are a family of
large toxins that are extensively distributed in bacterial pathogens. MARTX toxins are
autocatalytically cleaved to multiple effector domains, which are released into host cells
to modulate the host signaling pathways. The Rho guanosine triphosphatase (GTPase)
inactivation domain (RID), a conserved effector domain of MARTX toxins, is implicated in
cell rounding by disrupting the host actin cytoskeleton. We found that the RID is an Ne-fatty
acyltransferase that covalently modifies the lysine residues in the C-terminal polybasic
region of Rho GTPases. The resulting fatty acylation inhibited Rho GTPases and disrupted
Rho GTPase–mediated signaling in the host. Thus, RID can mediate the lysine Ne-fatty
acylation of mammalian proteins and represents a family of toxins that harbor N-fatty
acyltransferase activities in bacterial pathogens.
MARTX toxins are critical virulence factors of many bacterial pathogens (1). The se- creted MARTX toxins insert into the host cell plasma membrane, where they can be autocatalytically cleaved by their conserved inositol hexakisphosphate–activating cysteine protease domain to release multiple effector
domains into the cytosol of host cells (2) (fig. S1).
The Rho guanosine triphosphatase (GTPase) inactivation domain (RID) was implicated in cell
rounding by inducing a substantial decrease in the
cellular levels of active G TP-bound Rho G TPases
and disrupting the host actin cytoskeleton through
an unknown mechanism (3).
The mutations Cys3022 → Ser (C3022S) and
His2782 → Ala (H2782A) of RID of the Vibrio
cholerae MARTX toxin (RIDvc) abolished its
effect on the actin cytoskeleton (4) (Fig. 1A),
which indicates that RIDvc may be an enzyme
with the catalytic residues Cys3022 and His2782.
We isolated proteins that associated with recombinant maltose-binding protein (MBP)–fused
RIDvc C3022S in bovine brain cell extract. A
1Life Sciences Institute and Innovation Center for Cell
Signaling Network, Zhejiang University, Hangzhou, Zhejiang
310058, China. 2National Institute of Biological Sciences,
Beijing 102206, China. 3College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, China.
*These authors contributed equally to this work. †Corresponding
author. Email: firstname.lastname@example.org