International Journal of Biological Macromolecules 42 (2008) 93–102
The opportunistic fungal pathogen Scedosporium prolificans:
Carbohydrate epitopes of its glycoproteins
E. Barreto-Bergter
a
, G.L. Sassaki
b
, R. Wagner
b
, L.M. Souza
b
,
M.V.A.R. Souza
a
, M.R. Pinto
a,c
, M.I.D. da Silva
a
, P.A.J. Gorin
b,∗
a
Instituto de Microbiologia, C.C.S., Universidade Federal do Rio de Janeiro, 21944-590 Rio de Janeiro, RJ, Brazil
b
Departamento de Bioqu´ ımica e Biologia Molecular, Universidade Federal do Paran´ a, 81531-990 Curitiba, PR, Brazil
c
Departamento de Microbiologia, I.C.B., Universidade de S˜ ao Paulo, 05508-000 S˜ ao Paulo, SP, Brazil
Received 21 September 2007; accepted 26 September 2007
Available online 29 September 2007
Abstract
Isolated from the mycelium of Scedosporium prolificans were complex glycoproteins (RMP-Sp), with three structurally related components
(HPSEC). RMP-Sp contained 35% protein and 62% carbohydrate with Rha, Ara, Man, Gal, Glc, and GlcNH
2
in a 18:1:24:8:6:5 molar ratio.
Methylation analysis showed mainly nonreducing end- of Galp (13%), nonreducing end- (9%), 2-O- (13%), and 3-O-subst. Rhap (7%), nonreducing
end- (11%), 2-O- (10%), 3-O- (14%), and 2,6-di-O-subst. Manp units (13%). Mild reductive -elimination of RMP-Sp gave -l-Rhap-(1 → 2)--
l-Rhap-(1 → 3)--l-Rhap-(1 → 3)--d-Manp-(1 → 2)-d-Man-ol, with Man-ol substituted at O-6 with -d-Galp units, a related pentasaccharide
lacking -d-Galp units, and -d-Galp-(1 → 6)-[-d-Manp-(1 → 2)]-d-Man-ol in a 16:3:1 w/w ratio. Traces of Man-ol and Rha-ol were detected.
ESI-MS showed HexHex-ol and Hex
3–6
Hex-ol components. Three rhamnosyl units were peeled off successively from the penta- and hexasaccharide
by ESI-MS-MS. The carbohydrate epitopes of RMP-Sp differ from those of the glycoprotein of Pseudallescheria boydii, a related opportunistic
pathogen.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Scedosporium prolificans; Scedosporiosis; Mycelium; Rhamnomannogalactoprotein; Carbohydrate epitopes
1. Introduction
Scedosporium prolificans is a fungus occurring worldwide in
soil and plant residues. It is an opportunistic pathogen, causing
localized human infections restricted to bone and soft tissues
in immunocompetent patients, and is deeply invasive with dis-
seminated infections in immunocompromized patients. It is
related to Pseudallescheria boydii, the teleomorph state (per-
fect form) of Scedosporium apiospermum. These fungi cause
up to 10% of invasive fungal infections in predisposed patients
[1]. The mycelium of P. boydii contained a complex mixture
of glycoproteins (RMP-Pb), isolated on hot aqueous extraction,
followed by Cetavlon precipitation. Some of its carbohydrates
were identified [2], one of these was a branched hexasac-
charide epitope, liberated on reductive, alkaline -elimination
∗
Corresponding author. Tel.: +55 41 3361 1670; fax: +55 41 3266 2042.
E-mail address: cesarat@ufpr.br (P.A.J. Gorin).
of O-linked glycoprotein [3]. We now describe a structural
investigation on a complex mixture of carbohydrate epitopes
of rhamnogalactomannoprotein (RMP-Sp), similarly prepared
from S. prolificans. These could serve as a basis for eventual
immunological comparison of its epitopes with those of P. boydii
and other opportunistic fungal pathogens.
2. Materials and methods
2.1. Microorganism and growth conditions
A culture of S. prolificans was kindly supplied by Dr. J.
Guarro, Unitat de Microbiologia, Facultat de Medicina e Insti-
tut d’Estudis Avanc ¸ats, R´ eus, Spain. It was maintained on (g/L):
Difco peptone, 10; Difco yeast extract, 5; Difco agar, 20; glu-
cose, 40. Cells were grown on Sabouraud solid slants and
inoculated into Erlenmeyer flasks (500 mL) containing culture
medium (200 mL) and incubated at 25
◦
C for 7 days with shak-
ing. Cultures were then transferred to the same medium (3 L)
0141-8130/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2007.09.015