trans

CYCLIC NUCLEOTIDES 261
Other enzymic components of the
trypanosomatid cAMP-signaling
pathways
Few attempts have been made so far to functionally
characterize other components of the
trypanosomatid cAMP signal transduction
pathways. This should change rapidly in the
near future, stimulated by the rapid progress
of the trypanosomatid Genome Projects. For
example, at the time of writing, sequences corresponding
to putative catalytic (L. major and
T. brucei) and regulatory (L. major) subunits of
PKA have been deposited in the database. Two
types of T. brucei cAMP-specific phosphodiesterase
have also been identified. One, which
belongs to the class 1 phosphodiesterases,
was isolated after complementation of yeast
mutants. TbPDE1 is encoded by a single-copy
gene and is expressed constitutively. The second
type corresponds to the class 2 phosphodiesterases,
and is encoded by a family of at least
five genes, some of which are clustered. The
activity of the expressed product of one of these
genes (TbPDE2A) was reduced by some, but not
all, of a group of phosphodiesterase inhibitors.
Interestingly, the active inhibitors trequinsin,
dipyridamole, sildenafil citrate (Viagra) and
ethaverine also block the proliferation of bloodstream
parasites, suggesting that this phosphodiesterase
activity could be essential.
Possible roles for cAMP signaling
in trypanosomatids
Given the abundance of AC genes in the
T. brucei genome, it would seem apparent that
cAMP-signaling is of major regulatory importance
during the parasite life cycle. However,
surprisingly little is known about the role(s) of
this signaling system, the nature of the external
activators, and why T. brucei requires such
an extensive repertoire of genes. Most of the
available evidence suggests a role in differentiation.
When bloodstream T. brucei are triggered to
differentiate to the procyclic form by the addition
of citrate/cis-aconitate and reduction of
the ambient temperature to 27°C, two phases
of transient AC activation can be detected. The
first occurs after 6–10 h, and is immediately
followed by the release of the surface VSGs.
This pulse of AC activity also precedes the first
cell division, and the loss of the surface AC
isoforms encoded by the ESAG4 genes. The
second pulse of activity, after 20–40 h, coincides
with the beginning of cell proliferation.
Treatment of bloodstream forms of T.brucei to
induce shedding of the VSG (low pH and trypsin
digestion) also leads to activation of AC. In addition,
activation of AC and release of VSG can
be induced by specific protein kinase C (PKC)
inhibitors, suggesting a possible inhibitory
role for this regulatory kinase. Despite these
findings, it appears that the simultaneous
release of VSG and the activation of the cAMP
pathway in response to cellular stress occur
independently and are not mechanistically
linked. Further evidence for the antagonistic
action of PKC and the cAMP signal transduction
pathway has come from a study of disaggregation
of T. brucei bloodstream forms
following exposure to anti-VSG antibodies or
immune serum. Disaggregation is a regulated
PKC-dependent process that can be inhibited
by increased levels of cAMP.
Compelling evidence of a role for cAMP
signaling in differentiation has come from
studies on the development of the non-dividing
stumpy forms of the parasite, that are preadapted
for transmission to the tsetse fly.
Stumpy cell induction is a response to cell density,
and appears to be mediated by the release
of a low molecular weight ‘stumpy induction
factor’ (SIF). SIF causes cell-cycle arrest in the
G 1 /G 0 phase of the slender bloodstream forms
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA