trans

234 TRYPANOSOMATID CARBOHYDRATES
repeating Gal-Man-phosphate units. Unlike the
situation in L. major, however, in L. donovani
terminally exposed capping sugars are lost,
which appears to be due not to a downregulation
of their expression, but to a conformational
change affecting their availability for
binding. Such changes in LPG structure have
profound implications on function, and suggest
important points of regulation of the glycosyltransferases
involved in LPG biosynthesis.
These structural changes are believed to
obscure epitopes that promote adherence to
the midgut and allow passage of the parasites
to the mouthparts of the sand fly. Genetic
results obtained with LPG-deficient mutants
strongly support the conclusions that LPG is
not essential for survival in the early blood-fed
midgut but, along with other secreted phosphoglycan-containing
glycoconjugates, can protect
promastigotes from the digestive enzymes
in the gut. Furthermore, the LPG-deficient
mutants provided additional evidence that
LPG is required to mediate midgut attachment
and to maintain infection in the fly during
excretion of the digested blood meal.
LPG expression is not only developmentally
regulated within the promastigote during metacyclogenesis,
but also during promastigote–
amastigote conversions. The number of LPG
molecules expressed by the intracellular
amastigotes is substantially lower than in the
promastigote. Promastigotes express about
1–5 10 6 copies per cell, while amastigotes
express about 100 or 1000 copies per cell. The
amastigote LPG from L. major is biochemically
and antigenically distinct from promastigote
LPG. The glycan core and PI anchor structures
are conserved among promastigotes and
amastigotes with slight variations in the level
of glucosylation. The amastigote LPG contains
an average of 36 repeat units, 70% of which
are unsubstituted, with the rest containing
glucopyranose and galactopyranose, but not
arabinose. Also, side chains of up to 11 Gal
residues have been identified, compared to the
typical 1–3 Gal residues in the LPG side chains of
promastigotes. The cap structure is predominately
Gal(1,4)Man(1-) whereas the major
promastigote LPG cap is Man(1,2)Man(1).
No functions have yet been attributed to the
amastigote version of LPG.
LPG has been implicated in a number of
functions within the mammalian host. Within
the bloodstream, LPG prevents complementmediated
lysis by preventing insertion of the
C5b-9 membrane attack complex into the promastigote
membrane. In addition, LPG serves
as a ligand for receptor-mediated endocytosis
by the macrophage via complement receptors
as well as the mannose receptor. Inside the
macrophage, LPG inhibits PKC and the microbicidal
oxidative burst as well as inhibiting
phagosome–endosome fusion. These in vitro
data suggest that LPG is a virulence factor;
however, the molecular tools to unequivocally
substantiate this claim have been lacking.
Furthermore, there are complications with the
structural relatedness of LPG with other glycoconjugates,
such as the PPGs, which present
difficulties in assigning the proper structure–
function relationships. Recent advances in the
knockout of genes involved in phosphoglycan
biosynthesis and the isolation of the corresponding
genes have now permitted the direct
testing of LPG as a virulence factor in accord
with Koch’s postulates. Surprisingly, while LPG
is clearly a virulence factor in Leishmania–sand
fly interactions, two independent approaches
have resulted in apparently conflicting conclusions
in mice. In L. major, use of a knockout
line that lacks an LPG biosynthetic gene
(LPG1, a putative galactofuranosyltransferase
gene), resulted in a dramatic reduction in
virulence. Importantly, the re-introduction of
LPG1 restored virulence. In contrast, similar
approaches using two distinct L. mexicana
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA