ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

TgMyoA: a candidate motor to power gliding
motility
C. Hettmann, A. Geiter , F, Delbac
T. gondii myosin A (TgMyoA) is constitutively
expressed and localizes beneath the plasma membrane
of the parasite. Thus, TgMyoA is in an
ideal position to transmit mechanical energy into
forward motion propelling parasites into host cells.
We mapped the membrane localization determinant
within the short carboxy-terminal tail of TgMyoA,
and site-directed mutagenesis revealed two essential
arginine residues. The closest homologue in P. falciparum,
PfMyoA is transcribed specifically in the
invasive blood stage form and shows the same subcellular
localization in merozoites. The nature of the
receptor at the plasma membrane remains to be determined.
TgMyoD and TgMyoE are similar to TgMyoA, but
predominantly transcribed in the dormant bradyzoite
stage. We have abrogated the expression of TgMyoD
by double homologous recombination in the tachyzoites
of the virulent RH strain without any apparent
detectable phenotype. We need now to reproduce this
knockout in a persistent strain capable to differentiate
into bradyzoites, in order to analyze the phenotypic
consequences of the absence of TgMyoD in the
appropriate stage.
Biochemical characterization of TgMyoA
A. Herm, in collaboration with M. Geeves (Kent
UK), D. Manstein (MPI, Heidelberg) and E. Meyhöfer
(Hannover)
To power gliding motility, TgMyoA must be able to
track along actin filaments upon ATP hydrolysis at
a speed of 1-3 µm/s. We have determined the biochemical
and physical properties of histidine tagged-
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TgMyoA purified from culture recombinant parasites.
Transient kinetic analysis of TgMyoA was
obtained by stopped-flow and flash photolysis methods
and revealed strong similarities to typical fast
myosins, like rabbit skeletal myosin. The sliding
velocity of fluorescently labeled actin filaments on
TgMyoA attached to nitrocellulose-coated glass was
5-6 µm/s. A step size of 5 to 6 nm was determined by
single molecule assay monitoring filament displacement
using optical tweezers. TgMyoA shows all the
characteristics of a fast myosin of the class II but
considering the significant divergence in the converter
domain and the quasi absence of neck, it is
unclear at the moment how such a motor can generate
a power stroke.
TgMyoB/C: One gene, two tails, and two localizations.
F. Delbac, C. Hettmann, A. Sänger
TgMyoB and TgMyoC are alternative-spliced variants
of the same gene, which produce two myosins
exhibiting different tails. TgMyoB distributes evenly
in the cytosol and is essentially soluble. In contrast,
TgMyoC localizes at the posterior pole of the parasites
describing a ring structure reminiscent of the
termination of the inner membrane complex (IMC).
TgMyoC protein fractionates with membranes and
partitions in the detergent-insoluble fraction. Inhibitors
of myosin ATPase and actin polymerization disrupt
the orderly turnover of mother cell organelles
during daughter cell formation. The circular distribution
of TgMyoC suggests a possible role of this
myosin in the assembly and elongation of the IMCmicrotubule
complex during parasite division. Moreover
the overexpression of TgMyoB slows the rate of
replication and leads to the formation of large resid-
ual bodies, providing an additional indication of the
involvement of TgMyoB/C in cell division.
Figure 1. Immunolocalization of Myosin C (in red) and
MIC6 (in green).
II. Micronemal proteins of several apicomplexan
parasites are sharing common
structural and functional features
The rapid and efficient nature of invasion by Apicomplexa
relies on a sequence of events that are
tightly controlled in time and space. Micronemes are
involved in the trafficking and sequestration of binding
ligands for host cell receptors. These organelles
ensure the appropriate release of ligands in high concentration,
at the tip of the parasite and upon response
to external stimuli which senses contact with the
host cells. To accommodate for the broad host range
specificity of T. gondii, we postulated that adhesion
should involve the recognition of ubiquitous surfaceexposed
host molecules or, alternatively, the presence
of various parasite attachment molecules able
to recognize different host cell receptors. We identified
and characterized a soluble protein MIC4, as
well as a novel family of transmembrane microne-
mal proteins in T. gondii. These proteins are released
at the time of invasion and share domains of homology
with proteins described in other members of the
phylum Apicomplexa, supporting the hypothesis of a
common molecular mechanism for host recognition
attachment and invasion. They contain a combination
of adhesive motifs including thrombospondin, integrin,
apple and EGF-like domains. Invasion is successfully
achieved when the newly formed parasitophorous
vacuole is sealed and at this point, the
tight interaction between host cell receptors and parasite
ligands must be disrupted. The proteolytic processing
of micronemal proteins that occurs on the
parasite surface could fulfil such a function.
TgMIC4 carries six apple domains and binds
to host cells
S. Brecht, U. Jäkle
TgMIC4 carries six apple domains, a signal peptide
at the amino terminus and no apparent membranespanning
domain. Apple domains are found in plasma
coagulation factors, kallikrein and factor XI and are
involved in highly specific protein-protein interactions.
MIC4 is expressed in all invasive stages of the
parasites, secreted at the time of invasion and proteolytically
processed twice onto the parasite surface,
after secretion by the micronemes. MIC4 binds efficiently
to host cells and the adhesive properties map
within the carboxy-terminal apple domain of the protein.
Preliminary experiments showed that galactose
inhibits specifically binding to host cells and we are
currently characterizing further the lectin properties
of MIC4 and attempting to identify the host cell
receptor(s) by expressing heterologously the apple
domain in P. pastoris.
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