(Received for publication, July 11, 1994; and in revised form, November 9, 1994)
From the
Isolation of side chain oligosaccharides from mannans of Candida albicans NIH B-792 (serotype B) and Candida
parapsilosis IFO 1396 strains has been conducted by acetolysis
under mild conditions. Structural study of these oligosaccharides by H and
C NMR and methylation analyses indicated
the presence of novel branched side chains with the following
structures in C. albicans mannan.
and
It was observed that the H-1 proton chemical shifts of the
second and the third mannose units from the reducing terminus in each
oligosaccharide are shifted upfield by substitution with an
-linked mannose unit at position 6 of the
3-O-substituted mannose unit. An agglutination inhibition
assay between factor 4 serum and cells of Candida stellatoideaIFO 1397 lacking the
-1,2-linked mannose unit, with
oligosaccharides obtained from these mannans, indicated that only the
branched oligosaccharides were active. This finding suggests that the
branched oligosaccharides correspond to the epitope of antigenic factor
4. The presence of the branched structure in other mannans was detected
by the characteristic H-1-H-2-correlated cross-peak of the
-1,2-linked mannose unit connected with the
3,6-di-O-substituted one by two-dimensional homonuclear
Hartmann-Hahn spectroscopy.
Yeasts of the genus Candida, especially of Candida albicans species are known to be pathogenic in man. Candidiasis is an opportunistic infectious disease in early childhood and in adults with predisposing conditions such as diabetes, cancer, AIDS, and treatment with immunosuppressive agents after organ transplantation(1, 2) . The antigenicity of Candida cell walls resides in the mannan. Moreover, mannan is highly soluble and can be detected in the sera of some patients with candidiasis by various techniques, including immunologic procedures(3, 4, 5, 6, 7, 8, 9) . Thus, the detection of circulating mannan is important for diagnosis of invasive candidiasis. However, the cell wall components in sera from patients with other infections, such as Mycobacterium tuberculosis(10) , Serratia marcescens(11) , and Salmonella thompson(12) may cross-react with anti-Candida mannan antibody if it is not specific to Candida species. On the other hand, there are many reports(13, 14, 15, 16, 17, 18, 19, 20) about immunomodulatory effects by the mannan or oligosaccharide of C. albicans, including the induction of suppressive effects against both B- and T-cell-mediated immune responses. Although the mechanism of the immunosuppressive effects of these components is still unknown, several reports (21, 22, 23) suggest that side chain oligomannosyl moieties participate in adherence of the C. albicans cells to mammalian cells in the initial step of Candida infection. Therefore, the fine chemical structure of cell wall mannans must be known to develop an accurate serodiagnostic procedure for candidiasis and to understand these diverse host-parasite interactions.
In 1961, Hasenclever and Mitchell (24) reported two serotypes in C. albicans strains
designated serotypes A and B. Later, Tsuchiya et al.(25) proposed the relationship between antigenic
structures of many yeasts, including seven medically important species
of Candida, based on 10 cell surface antigenic factors. In
recent years, structural analysis of cell wall mannans of C.
albicans has been developed extensively(26, 27) ,
and we have demonstrated the presence of phosphodiesterified
-1,2-linked oligomannosyl residues as a group of common epitopes
throughout the two serotype strains(28, 29) .
Furthermore, a
-1,2-linked mannose unit connected to an
-1,2-linked unit was found to correspond to a specific epitope for
serotype A strains(30, 31) . Although these two groups
of
-1,2 linkage-containing epitopes were identified as
corresponding to antigenic factors 5 and 6(32, 33) ,
the structure of factor 4 has not been determined. From the results of
agglutination assays of monoclonal anti-factor 4 antibodies with cells
of many Candida strains, it was speculated by Kagaya et
al.(34) that the antigenic factor 4 corresponds to
treebranch-like structures proposed by Suzuki et
al.(35) . However, the results of our structural studies
for C. albicans mannans provided evidence that the mannans
have a comb-like structure with an
-1,6-linked
backbone(26, 27) .
The C. albicans serotype B and Candida parapsilosis cells used in this
study have antigenic factors 1, 4, and 5 and factors 1, 13, and 13b,
respectively(25) . Therefore, the difference in structures of
the mannans of both species correlates with the presence or the absence
of antigenic factors, 4 or 13 and 13b, although the previous study by
Funayama et al.(36) could not reveal any structural
difference. This result could be attributable to the acetolysis
conditions, since these workers prepared the side chain
oligosaccharides by a conventional acetolysis procedure that cleaves
all -1,6 and
-1,2 linkages. Our recent study (37) of Saccharomyces kluyveri mannan indicated that acetolysis under
mild conditions released oligosaccharides retaining both
-1,2
linkage and part of the
-1,6 linkages of the branching mannose
unit. Therefore, we applied the mild acetolysis method to analyze
structural difference(s) between oligomannosyl side chains of the two Candida mannans. Although several reports suggest the presence
of branched side chains in the mannans of C. albicans, no
isolation of any branched oligosaccharide has been
achieved(35, 38, 39, 40) . In the
present study, we demonstrate the existence of novel branched side
chains that dominate the antigenic factor 4 specificity in the mannan
of a C. albicans serotype B strain.
Acetolysis under
conventional conditions was carried out as described by Kocourek and
Ballou (42) using a 10/10/1 (v/v) mixture of
(CHCO)
O, CH
COOH, and
H
SO
, and the resultant solution was kept at 40
°C for 12 h.
Figure 1:
Elution patterns of oligosaccharides
obtained from C. parapsilosis (A and B) and C. albicans (C and D) mannans by acetolysis. A and C, acetolysis was performed with
(CHCO)
O, CH
COOH,
H
SO
(10:10:1, v/v) at 40 °C for 12 h
(conventional conditions). B and D, acetolysis was
performed with (CH
CO)
O, CH
COOH,
H
SO
(100:100:1, v/v) at 40 °C for 36 h
(mild conditions). M, M
, M
, M
,
M
, M
, and M
indicate mannose,
mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose,
and mannoheptaose, respectively. Vo, void
volume.
Figure 2:
The
anomeric region of the H NMR spectra of oligosaccharides
obtained from C. parapsilosis (A) and C. albicans (B) mannans by acetolysis under the mild conditions.
Spectra were recorded using a JEOL JNM-GSX 400 spectrometer in
D
O solution at 45 °C using acetone as the standard
(2.217 ppm). M
-M
are designated as in the
legend to Fig. 1.
Figure 3:
C NMR spectra of
oligosaccharides obtained from fraction A. DEPT 135
C NMR
spectra of AM
(A), AM
(B),
AM
(C), AM
(D), and
AM
-H (E), and HMBC of AM
-H (F) are shown. Spectra were recorded using a JEOL JNM-GSX 400
spectrometer in D
O solution at 45 °C using
CD
OD as the standard (49.00 ppm). Negative signals on DEPT
135 spectra correspond to C-6 carbon. The dashed lines among
those spectra indicate downfield shift of C-6 signals by substitution
with another mannose unit or by reduction on the reducing terminal
mannose unit.
Figure 4:
Partial two-dimensional HOHAHA analysis of
mannooligosaccharides PM, PM
, AM
,
and AM
, obtained by mild acetolysis and the reduction
products PM
-H, PM
-H, AM
-H, and
AM
-H. The cross-peaks for all ring protons, H-2 to H-6, are
indicated by a vertical line correlated with the H-1 signal of
each mannose unit. The dashed line between the cross-peaks of
parent and reduced oligosaccharides indicates a shift of the H-1 signal
caused by the reduction by
NaBH
.
To identify the H-1
proton of the second mannose unit from the reducing terminal unit,
Man-B, AM and AM
were reduced with
NaBH
. The H-1 and H-2 signals of Man-B were easily assigned
based on their upfield shift,
=
0.06 and 0.1
ppm, respectively(37) . Namely, the signals at 5.24 and 5.27
ppm of AM
and AM
were assigned to the H-1
proton of Man-B. These results indicate that the upfield shift of the
H-1 signal of Man-C from 5.28 to 5.22 ppm,
= 0.06
ppm, by the attachment of an
-1,6-linked mannose unit to Man-D is
larger than that of Man-B from 5.27 to 5.24 ppm,
=
0.03 ppm. The shift value of the H-1 signal of Man-C is similar to that
observed on the branched oligosaccharide from S. kluyveri mannan,
= 0.05 ppm (37) . Therefore, we
propose that the chemical structures of the branched oligosaccharides
in the isomer mixtures, AM
and AM
, are as
follows (Structures 1 and 2, respectively).
Figure 5:
Sequential connectivities of mannose units
of AM-H (A) and AM
-H (B). The right side of the diagonal shows COSY, and the left
side of the diagonal shows rotating frame NOE spectroscopy. Primed
letters indicate interresidue H-1-H-2` or H-1-H-3` NOE
cross-peaks, and unprimed uppercase and lowercase letters indicate the H-1-H-2 and the H-2-H-3-correlated cross-peaks,
respectively, caused by J-coupling; e.g.B indicates the H-1-H-2-correlated cross-peak of the second mannose
unit, Man-B, and B` indicates the interresidue NOE cross-peak
between H-2 of Man-B and H-1 of an adjacent mannose unit, Man-C. By
this procedure, H-1 and H-2 signals were sequentially assigned from the
H-1 of the Man-B, B-B`-C-C`-D-d-D`-E for AM
-H and
B-B`-C-C`-D-d-D`-E-E`-F for
AM
-H.
Figure 6:
Partial two-dimensional HOHAHA spectra of
fractions P and A and other mannans of related yeasts. H-C COSY of
fractions P and A are also shown. Cross-peaks 1 and 2 indicate -1,6-linked backbone and
-1,6-linked branching
mannose units, respectively. Cross-peak 3 indicates an
-1,2-linked mannose unit substituted by a
3,6-di-O-substituted mannose unit.
The present comparative study between the structures of the
mannans of C. parapsilosis and C. albicans serotype B
strains clearly demonstrates the existence of branched side chains in
the latter mannan. Now we propose the entire chemical structures of the
mannans of C. parapsilosis IFO 1396 and C. albicans NIH B-792 strains based on the results of the present and recent
studies (29, 52) as shown in Fig. 7. There was
a report (35) on the presence of a treebranch-like structure in
the mannan of C. albicans serotype A strain. Based on our
structural studies of C. albicans mannan, we find no evidence
for such a structure. The branched structure is predominant in the
mannan of the C. albicans serotype B strain, but small amounts
occur in that of the C. albicans serotype A strain. Therefore,
the structures recognized by monoclonal antibodies specific for
antigenic factor 4 proposed by Kagaya et al.(34) were
also speculative. Funayama et al.(36) reported that
antigenic factors 13 and 13b correspond to a mannohexaose moiety with a
linear structure,
Man1
2Man
1
3Man
1
2Man
1
2Man
1
2Man. Therefore, it is apparent that the antigenic factor 4
corresponding to the branched mannoheptaose moiety would be degraded to
structures corresponding to antigenic factor 13b by conventional
acetolysis(29, 30, 36, 58) . The
above results suggest that the C. parapsilosis IFO 1396 strain
cannot synthesize branched side chains and express antigenic factors 13
and 13b but not factor 4. However, in the cells of the C. albicans serotype B strain, these side chains are branched by the addition
of
-1,6-linked mannose units to make the epitope corresponding to
antigenic factor 4 instead of 13b. Therefore, we can say that the
relationship between the structures of antigenic factors 13b and 4 is
the same as that seen in blood groups H and A or B.
Figure 7:
Possible structure of C.
parapsilosis IFO 1396 and C. albicans NIH B-792 strain
mannans. M denotes a D-mannopyranose unit. The side
chain sequence is not specified. -1,6-Linked mannose units in
brackets indicate partial absence of this
unit.
Factor 4 serum
is prepared by absorption of anti-C. albicans serotype A
strain whole-cell serum with C. parapsilosis cells(25) . Because C. albicans serotype A and C. parapsilosis cells have epitopes corresponding to antigenic
factors 1, 4, 5, and 6 and factors 1, 13, and 13b, respectively, the
factor 4 serum contains antibodies against antigenic factors 5 and 6 as
well as factor 4(32) . Therefore, some Candida species
react strongly with factor 4 serum, despite a low density of the real
antigenic factor 4 in the mannans. For example, the C. albicans serotype A strain mannan seems to contain only a small amount of
branched side chain as judged from the intensity of cross-peak 3 in the
two-dimensional HOHAHA spectrum. However, because C. albicans serotype A strain mannans contain -1,2-linked mannose units
connected to an
-1,2-linked mannose to give antigenic factor 6,
strong cross-reactions with factor 4 serum can be observed. As
additional evidence, Kobayashi et al.(59) and Okawa et al.(60) observed a significant decrease in the
reactivity of C. albicans serotype A strain cells to factor 4
serum in addition to the disappearance of reactivities to factor 5 and
6 sera when the cells were cultivated at low pH or at high temperature.
This result suggests that the presence of two types of
-1,2-linkage-containing side chains, corresponding to antigenic
factors 5 and 6, are responsible for the strong reactivity of the C. albicans serotype A strain cells to factor 4 serum.
The
upfield shift of the H-1 proton of an -1,2-linked mannose unit by
a steric effect, found in branched mannooligosaccharides obtained from
the mannan of S. kluyveri(37) , was also observed in
the branched oligosaccharides of C. albicans mannan. In the
case of the latter oligosaccharides, the
-1,6-linked branching
mannose unit affects the H-1 proton chemical shifts of both the third
mannose unit and the second one from the reducing terminal. Therefore,
the three-dimensional structure of these branched oligosaccharides in
aqueous solution would be of interest.
A strong cross-peak 3 in two-dimensional HOHAHA spectra of the mannans of C. guilliermondii and C. stellatoidea suggests that these mannans contain significant amounts of branched side chains. The structural analysis of the mannans of these strains is in progress.