by 16S rRNA gene Keywords core; Francisella; Francisella victoria; lipid A; lipopolysaccharide; O-chain Correspondence E.. For a more detailed analysis of the distribution of O-linked ac
Trang 1of the fish pathogen Francisella victoria
William Kay1, Bent O Petersen2, Jens Ø Duus2, Malcolm B Perry3and Evgeny Vinogradov3
1 Department of Biochemistry and Microbiology, University of Victoria, BC, Canada
2 Carlsberg Laboratory, Copenhagen, Denmark
3 Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada
Members of the bacterial genus Francisella belong to
the Gram-negative Proteobacteria Their taxonomic
position is not completely clear, as no closely related
microorganisms have been detected Francisella
includes two species: Francisella tularensis and
Franci-sella philomiragia There are four subspecies of F
tula-rensis: tularensis, holarctica, mediasiatica, and novicida
Of all subspecies, the F tularensis subspecies tularensis
is the most infective and fatal for humans and, due to
its very low infective dose, is considered as a biological
weapon or bioterrorist agent [1] With the
introduct-ion of rapid PCR-based methods of screening of
environmental samples, potential new variants of Francisella were detected [2–4] lipopolysaccharide (LPS) of Francisella has unusually low biological activ-ity, and is considered as a potential component of antitularemia vaccines [5–8]
Recently a virulent bacterial fish pathogen was iso-lated from a moribund Tilapia (Oreochromis niloticus niloticus) Tilapia sp are warm water finfish of con-siderable commercial importance world-wide This pathogen, often and incorrectly referred to as a Rick-ettsia-like organism, was characterized and identified
as a unique Francisella sp by 16S rRNA gene
Keywords
core; Francisella; Francisella victoria; lipid A;
lipopolysaccharide; O-chain
Correspondence
E Vinogradov, Institute for Biological
Sciences, National Research Council,
100 Sussex Drive, K1A 0R6 Ottawa,
ON, Canada
Fax: +1 613 9529092
Tel: +1 613 9900832
E-mail: evguenii.vinogradov@nrc.ca
(Received 20 January 2006, revised 26 April
2006, accepted 5 May 2006)
doi:10.1111/j.1742-4658.2006.05311.x
Lipopolysaccharide (LPS) and b-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F victoria as a unique species The structure identified composes the largest oligosaccharide eluci-dated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, inclu-ding the first application of H2BC sequence to oligosaccharides The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found
in Francisella novicida The heptoseless core-lipid A region of the LPS con-tained a linear pentasaccharide fragment identical to the corresponding part of F tularensis and F novicida LPSs, differing in side-chain substitu-ents The linkage region of the O-chain also closely resembled that of other Francisella LPS preparation contained two characteristic glucans, previ-ously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual b-(1–6)-glucan with (glycerol)2 phos-phate at the reducing end
Abbreviations
BABA, b-aminobutyric acid; CHA or CHB, cysteine heart agar or broth; Fuc4N, 4-amino-4,6-dideoxygalactose; HPAEC, high-performance anion-exchange chromatography; Kdo, 3-deoxy- D -manno-octulosonic acid; LOS, lipooligosaccharide; LPS, lipopolysaccharide; LVS, live vaccine strain; P, phosphate; QuiN, 2-amino-2,6-dideoxy- D -glucose; Qui3N, 3-amino-3,6-dideoxy- D -glucose; Qui4N, 4-amino-4,6-dideoxy- D -glucose.
Trang 2sequencing and serological cross-reactivity with other
Francisellasp., and it was subsequently named
Franci-sella victoria(unpublished results) Comparative
analy-sis of the LPS from related species is important in
order to gain an understanding of the molecular basis
of their biological properties, the host specificity and
diversity of members of this important bacterial genus,
as well as the nature of its pathogenicity The results
could also be useful for vaccine development against
fish diseases and possibly against tularemia in humans
Here we present the results of the structural analysis of
the lipopolysaccharide of the first known fish
Francisel-lasp., F victoria
Results
Silver-stained SDS⁄ PAGE of F victoria LPS,
whole-cell proteinase digests or western blot stained with
polyclonal antisera to F victoria revealed a
predomin-ant immuno-staining band of the large
lipooligosaccha-ride (LOS) component and a smaller less intense band
of the core-lipid A component (Fig 1) An additional
diffuse band of higher molecular mass components
was visible at high sample load Immunostaining using
rabbit polyclonal antisera raised to whole cells of
F tularensis live vaccine strain (LVS), F novicida or with monoclonal antibodies to LPS from F tularensis
or F novicida showed no reaction with the dominant LOS band of F victoria Cross-reactivity was observed with the low molecular weight band of F victoria (data not shown) However, staining of proteinase K-diges-ted samples of F novicida with F victoria anti-serum were negative Thus the LPS fractions of whole cells of F victoria were immunochemically distinct from the LPS fractions of the other Francisella sp Monosaccharide analysis (GC MS of alditol ace-tates) of the whole LPS revealed the presence of rhamnose, fucose, 3-amino-3,6-dideoxyglucosamine (Qui3N), quinovosamine (QuiN), 4-amino-4,6-dide-oxyhexosamine (probably a mixture of Fuc4N and Qui4N, as determined from NMR results), mannose, glucose, and glucosamine with dominant peak of glu-cose 10 times larger than that of any other compo-nent High glucose content was due to the presence of glucans in the LPS preparation GC of acetylated or trimethylsilylated (R)-2-butyl glycosides was used to determine the absolute configuration of the monosac-charides, which turned out to be l for Rha and Fuc, and d for QuiN, Qui3N, Glc, Man, and GlcN Config-urations of Fuc4N and Qui4N have not been deter-mined because of unclear results
LPS was subjected to mild acid hydrolysis, which gave water-insoluble lipid A and water-soluble prod-ucts Lipid A was purified by conventional silica gel chromatography in a CHCl3–MeOH solvent system Comparison of the1H-NMR spectra of the unfraction-ated lipid A and chromatographically fractionunfraction-ated samples indicated that the fraction eluted with 10% MeOH in CHCl3 contained the major component It was used in further studies as ‘lipid A’ Fatty acid ana-lysis of the purified lipid A showed the presence of C14 : 0, C16 : 0 (minor), C16 : 0 (3-OH), and C18 : 0 (3-OH) straight-chain acids Two-dimensional NMR spectra of this product were identical to the spectra of the lipid A from F tularensis [9] The MALDI mass spectrum of the lipid A showed one major peak at m⁄ z 1391.9, which corresponds to the [M + Na]+ ion of the structure with two GlcN, one C14 : 0, one C16 : 0 (3-OH), and two C18 : 0 (3-OH) fatty acids Smaller peaks at 1364.9 and 1419.9 indicated the presence of the variants with two methylene groups shorter or lon-ger acids, respectively A peak corresponding to the glycosyl cation of unit B was observed at 654.4 Da, corresponding to the acylation of the unit B with C14 : 0 and C18 : 0 (3-OH) acids The same results were observed for F tularensis lipid A [9]
For a more detailed analysis of the distribution of O-linked acids, the lipid A was treated with NH4OH
1 2 3
High molecular
mass components
LOS bands
Core-Lipid A band
Fig 1 Western blot of F victoria LPS products Lane 1: whole
F victoria cells treated with Proteinase K overnight at 60 C Lane
2: Non-precipitated fraction of LPS after overnight
ultracentrifuga-tion at 120 000 g Lane 3: LPS ultracentrifuge precipitate.
Trang 3and products were analyzed by MALDI mass
spectr-ometry, as described [10] This treatment removes all
O-linked fatty acids except those acylating OH-3
of the amide-linked 3-hydroxyacyl groups F victoria
lipid A contained only one hydrolyzable acyl
substitu-ent at O-3 of residue A Indeed, the mass spectrum of
the products showed the new peak of the compound
with a mass of 1137.8, corresponding to the loss of
C16 : 0 (3-OH) acyl from O-3 of GlcN A residue
Together with the above described data, this
informa-tion can correspond only to the acyl group distribuinforma-tion
shown in Fig 2 This experiment also confirmed the
previously determined structure of F tularensis and
F novicidalipid A
Water-soluble products from the mild acid hydrolysis
of F victoria LPS were reduced with NaBH4and
separ-ated by size-exclusion chromatography It gave minor
amounts of polymeric product, which was shown to be a
starch-like glucan, and three oligosaccharide fractions
Oligosaccharides were further separated by
anion-exchange chromatography to give oligosaccharides 1
and 2 (as a mixture), 3, 4, b-glucan (see Scheme 1), and
several other products, apparently being fragments of
structure 3 Oligosaccharide 4 and b-glucan were
addi-tionally purified by high performance anion exchange
chromatography (HPAEC)
Oligosaccharides 1–4 were analyzed using
two-dimen-sional NMR spectroscopy (COSY, TOCSY, NOESY,
heteronuclear single quantum coherence (HSQC),
het-eronuclear two bond correlation (H2BC), hethet-eronuclear
multiple quantum coherence (HMQC)-TOCSY, and
heteronuclear multiple bond correlation (HMBC)) and
MS Spectra of the simplest oligosaccharide 4,
represent-ing the core part of the LPS, were completely assigned
in agreement with the proposed structure, consisting of
four mannose residues and one residue of Kdo-ol (Scheme 1, Table 1) ESI MS gave a mass of 888.9 Da, which agreed with the structure
Assignment of the spectra of the large oligosaccha-rides 1–3 presented a significant experimental challenge due to an unusually high number of nonrepeating components and consequential signal overlap even at
800 MHz (Fig 3) Additional problems arose from the presence of the oligosaccharides 1 and 2 as an unsepa-rable mixture, and partial O-acetylation of the Qui3N unit V In order to simplify spectra, oligosaccharides were O-deacetylated and spectra of native and deacyl-ated products were analyzed For the interpretation of the NMR spectra, a new method called H2BC [11–13] was used It produces spectra containing three-bond H–C–C correlations, which makes it possible to iden-tify C-2 signals starting from H-1, and C-5 starting from H-6, as well many other signals
NMR analysis of the oligosaccharide 3 showed that
it contains all components of the core oligosaccharide
4 Additionally a clearly visible nonreducing end struc-ture was present, consisting of the monosaccharide residues Y-V-U-Z-R, a core-linked sequence L-M-[K-]-J-I, and a number of glucose and fucose residues between these two fragments, with an integral intensity
of their signals being 1.5–2 times higher than that of above mentioned residues This pointed to the possible presence of loosely defined ‘repeating units’ Relative and anomeric configurations of the monosaccharides were deduced from the proton–proton coupling con-stants and chemical shifts of proton and carbon sig-nals Connections between monosaccharides were identified on the basis of NOE and HMBC correla-tions (Table 1) A very large number of the NOE cor-relations from the H-6 of the 6-deoxysugars was observed, and most of them could be rationalized within the proposed structures (Scheme 1) It should
be noted that the signals of the quinovosamine unit I were of low intensity A similar feature was observed previously in the analysis of the repeating unit-core oligosaccharides prepared from F novicida LPS [14], and probably can be explained by the restrained motion of this residue in densely packed structure The ESI MS of the oligosaccharide 3 contained triple and quadruple charged peaks corresponding to a mass
of 4381.7 Da, which corresponds to a composition Hex10dHex12HexNAc1dHexNAc3Kdool1(average mass
of 4380.1), thus involving two copies of a fragment P-W-X-[Q-]-T or X-[Q-]-T-[S-]-N (these structures are identical) The spectrum also contained smaller peaks
of oligomers of lower and higher mass, differing by hexose units (162 Da) MS-MS analysis generally con-firmed structural assignment, but provided no new data Fig 2 Structure of the F victoria lipid A.
Trang 4regarding the structure of the most obscure region
between Fuc R and Fuc L (data not shown)
After the determination of the structure of the
prod-uct 3, the full sequence of the oligosaccharides 1 and 2
was determined by NMR All signals of the components
present in the oligosaccharide 3 were found at the same
positions except for the substituted Qui3NAc residue Y
Oligosaccharide 1 contained three nonsugar
compo-nents: alanine, 3-aminobutyric acid (homoalanine or
BABA), and a novel branched amino acid designated
AA HMBC correlations allowed us to trace the BABA-acylated alanine, which was in turn linked to N-4 of terminal Fuc4N residue RR BABA had a free amino group
Component AA contained a methyl group and two other protons All proton signals were singlets and showed only NOE correlations between each other Methyl group protons gave HMBC correlations to
A
B
C
D
E
F
Scheme 1 Structures of the isolated oligosaccharide fragments of the F victoria lipopolysaccharide (LPS) (A) BABA = 3-aminobutyric acid (homoalanine) AcOH hydrolysis products (R 1 = Ac or H): full structure = 1; structure ending at PP (PP’) = 2; structure ending at Y (Y’) = 3; structure ending at F (F’) = 4 (B) O,N-deacylated LPS products: full structure = 5; structure ending at PP (PP’ in this case) = 6; structure ending at Y (Y’ in this case) = 7; structure ending at F (F’ in this case) = 8 (C) b-glucan, structures of core-lipid A backbone of different Fran-cisella LPSs: (D) F tularensis, (E) F novicid and, (F) F Victoria.
Trang 5Table 1 NMR data for oligosaccharides 1–8 Components having close chemical shift values are grouped and average data are presented for them.
Unit,
compound Nucleus H ⁄ C 1 H ⁄ C 2 H ⁄ C 3 H ⁄ C 4 H ⁄ C 5 H ⁄ C 6a H ⁄ C 7 ⁄ 6b H ⁄ C 8a ⁄ 8b
NOE from H-1
V*; 1–3 deac C 105.4 79.3 57.6 74.3 74.6 18.3
U; 1–3, 5–7 C 101.7 83.4 71.6 74.3 70.1 17.8
Z; 1–3, 5–7 C 97.8 71.4 79.0 73.0 70.1 17.9
R; 1–3, 5–7 C 101.6 68.3 75.5 69.4 68.1 16.6
P; 1–3, 5–7 C 100.8 72.8 74.0 70.6 73.8 62.0
W; 1–3, 5–7 C 101.3 75.3 70.3 82.3 69.3 16.3
X; 1–3, 5–7 C 101.1 70.0 69.8 82.5 68.8 18.0
Q; 1–3, 5–7 C 100.8 72.8 74.2 71.0 73.9 62.1
T; 1–3, 5–7 C 101.7 70.5 76.3 80.7 69.8 17.0
S; 1–3, 5–7 C 100.8 73.0 73.9 70.6 73.8 62.0
N; 1–3, 5–7 C 101.3 75.5 70.3 82.3 69.3 16.4
L; 1–3, 5–7 C 100.7 69.5 70.2 82.4 68.6 17.1
Trang 6three carbons, two of them protonated (59.4 and
66.5 p.p.m.) and one quaternary carbon at 79.0 p.p.m
Proton signals at 4.23 and 4.73 p.p.m correlated with
a quaternary carbon atom and carbonyl carbon atom
signals at 176.4 and 170.5 p.p.m These data suggested
that AA was a five-carbon dicarboxylic acid with a
methyl group at C-3 and amino or hydroxy
substitu-ents at positions 2, 3, and 4
For detailed analysis of the structure of AA, LPS
was depolymerized with anhydrous HF and Qui4NAA
was isolated using reverse-phase HPLC NMR spectra
of this monosaccharide (not shown) confirmed its
gluco configuration HMBC correlation was observed
between C-1 of AA at 170.9 p.p.m and H-4 of the
Qui4N, indicating acylation of NH2-4 of Qui4N with
one of AA carboxyl groups Spectra contained signals
of one N-acetyl group acylating N-4 of the AA The ESI mass spectrum of Qui4NAA showed the molecular mass of 361.3 Da, 18 units less than expected for the linear structure of the AA, which pointed to the lac-tam formation MS⁄ MS experiments led to the obser-vation of a signal at m⁄ z 199.2, corresponding to a cyclic AA component The cyclic structure of the AA was confirmed by the observation of the weak C-5: H-2 HMBC correlation There was no data for the determination of the configuration of chiral atoms Taken together, these experimental data agreed with the structure (Fig 4)
As only one Qui4N (residue PP) was present in the oligosaccharides 1 and 2, isolated Qui4NAA repre-sented residue PP monosaccharide Close values of NMR shifts for AA in the oligosaccharides and in the
Table 1 (Continued).
Unit,
compound Nucleus H ⁄ C 1 H ⁄ C 2 H ⁄ C 3 H ⁄ C 4 H ⁄ C 5 H ⁄ C 6a H ⁄ C 7 ⁄ 6b H ⁄ C 8a ⁄ 8b
NOE from H-1
F; 1–3, 5–7 C 101.3 76.2 71.6 78.6 76.5 62.0
GlcNol H 3.65 ⁄ 3.78 3.59 3.73 3.93 3.88 4.12 3.74
Trang 7HF-released product indicated that its structure was
not modified during HF treatment
A number of oligosaccharide fractions not presented
in Scheme 1 were isolated after acetic acid hydrolysis,
which had no components of the core, and contained
mostly fragments of the oligosaccharide chain from unit
L to Y or from J to Y, including side chains Their
NMR spectra contained many minor signals, mostly of
a-glucose As PAGE of the LPS showed the presence of
high molecular mass chains, it seems reasonable to
believe that these oligosaccharides formed the polymeric
chain beyond units Y or RR, and for some reason were
cleaved off in both acidic and alkaline conditions
Deacylation of the LPS with 4 m KOH in the
pres-ence of NaBH4 with subsequent fractionation by
gel-chromatography on Sephadex G50 gave four
frac-tions As in the case of acetic acid hydrolysis, the
product eluted with the void volume turned out to be starch-like material A second (major) fraction contained large oligosaccharides 5–7 A third fraction contained b-glucan with aglycon, modified due to alka-line conditions; it was not further analyzed The lowest molecular mass component, eluted near the salt peak, contained mostly b-glucan and core oligosaccharide 8, purified further by HPAEC Minor fractions from HPAEC contained the variants of structure 8 with partly degraded lipid A glucosamine due to alkaline conditions of deacylation Products 5–7 were found impossible to separate in conditions used, and they were analyzed in the mixture
Oligosaccharide 8 was analyzed by NMR Complete assignment of two-dimensional NMR spectra led to the identification of two a-Kdo residues, one b-GlcN, one glucosaminitol, and four mannose residues The b-configuration of the Man F followed from the observa-tion of intraresidual strong NOEs between H-1 and H-3, H-5, and also from the low field position of the C-5 signal at 78.6 p.p.m Characteristic NOE between H-3 of the Kdo C and H-6 of the Kdo D residues indi-cated the attachment of Kdo D in a-configuration to O-4 of Kdo C All glycosidic linkages were identified
on the basis of transglycosidic NOE and HMBC
[p.p.m.]
Fig 3 The 800 MHz1H-NMR spectrum of the mixture of
oligosac-charides 1 and 2 is shown.
Table 2 NMR data for b-glucan A¢ is nonreducing end residue, A – repeating, A¢ – linked to Gro.
Fig 4 Structure of the isolated 4-amino-4,6-dideoxy glucose, N-ac-ylated with the amino acid AA (unit PP-AA in the oligosaccharides).
Trang 8correlations The following NOEs were observed in the
product 8: B1A6, C3D6, E1C5, E1C7, F1E4, F1E6,
G1F2, G1F3, H1E6, which corresponds to the
struc-ture presented on Scheme 1
Analysis of the mixture of oligosaccharides 5–7 by
NMR (Fig 5, Table 1) confirmed the sugar backbone
structure determined from the analysis of the products
1–3 with all nonsugar components of 1–3 absent in
5–7 Compound 5 had the most complete structure;
in the oligosaccharide 6 terminal Fuc4N was missing;
in 7, the nonreducing sequence
b-Fuc4N-3-b-Qui4N-4-b-Glc- was missing (Scheme 1)
The structure of b-glucan (Scheme 1) was studied by
NMR (Table 2), MS and chemical analysis
Monosac-charide analysis showed the presence of glucose and glycerol NMR data indicated that short b-(1–6)-linked glucose oligomers have an aglycon, consisting of two glycerol residues, linked by a phosphodiester bond (31P-NMR signal at 1.08 p.p.m.) Methylation analysis
of b-glucan revealed the presence of terminal and 4-substituted glucopyranose Positive-mode MALDI mass spectrum of the b-glucan (Fig 6) contained a ser-ies of peaks that could be attributed to ions [M + Na]+and [M +2Na)1]+, with maximum con-tent of oligomer Glc9, which gave disodium peak at
m⁄ z 1749.2 The same b-glucans were found previously
in LPS preparations from F victoria and F tularensis [9,15]
4.5 4.0 3.5
3.0
K2
K4
K3
S2 Q2
Q4 P4 P4
P2
P5
Q3
U3+U1:Z3
U4
U2 K1:J3 Q1:T3
S1:N2
Q1:T4 P1:W2
E2
E1:C5
E3
X2 X4
X1:T4 X3
X1:T3
N3
W1:X5
T3 T2 W2 G2
G1:F2
G3
W1:X4
J2 J3 J4
Z2
Z1:R3 Z1:R4 Z3
L2
R2 R3 R4
T1:W4 L3
H2
H3 H1:E6
V1:U2 F2
F1:E4 I1:F4 F3
F5
V2
Y'2
Y'5
W35 T35 T45
J45 J35
W23 N23 X5:T3
X35
R35 R45
W5:X2
M3 V5
I3
B2
V3,4 RR2 RR4
Y'3
Y3 I2
I4 Y4
B3,5
M2 KK2
PP2 PP'3 PP'2
PP4 PP'4
KK3
W34 N34 Y1:V2
RR3
PP1:KK4
KK6
PP3 Y2
Fig 5 Overlap of COSY (green), TOCSY (magenta) and NOESY (black) spectra of the mixture of oligosaccharides 5–7, showing correlations from anomeric protons Intensity is set to high level for clarity, but some important correlations became invisible.
Trang 9The LPS of F victoria contains two variants: a
rough-type structure consisting of the core and lipid A, and a
much larger structure with a nonrepetitive
oligosaccha-ride linked to the core SDS⁄ PAGE of the proteinase
K-treated whole cells or of the purified LPS appears to
reflect this composition with low molecular weight
bands, probably representing the lipid A and core
oligosaccharide regions, and the more abundant,
higher molecular weight bands, probably representing
lipooligosaccharide conjugated to the polysaccharide
component None of these LPS components were
cross-reactive with LPS of the other Francisella sp.,
with the exception of some nonreciprocal
cross-reactiv-ity with F novicida, perhaps due to the similarcross-reactiv-ity of
their core-region oligosaccharides These results serve
to emphasize the uniqueness of the F victoria
oligosac-charide and to confirm it as a unique Francisella sp
Lipid A had the same structure as determined earlier
for F tularensis [9,16] and F novicida [14,15] LPS with
a characteristic nonphosphorylated free reducing end
Another variant of the lipid A, which seems to be not
substituted with core and has a phosphorylated
redu-cing end, and which has been found in F tularensis
and F novicida, was not detected in F victoria The
inner core of the LPS of F victoria resembles the
core of F tularensis and F novicida in the presence of
oligosaccharide fragment b-Man-4-a-Man-5-a-Kdo
(marked in bold font together with lipid A backbone,
Scheme 1), but it has an additional side-chain Kdo
residue and different branching substituents The
poly-saccharide part is linked to the core via b-N-acetylqui-novosamine or its close relative b-N,N-diacetyl-bacillosamine in all Francisella LPSs (Scheme 1) Oligosaccharide 1 was found to consist of 33 monosaccharide residues and some nonsugar compo-nents, which to the best of our knowledge is the lar-gest complex carbohydrate structure elucidated to date The oligosaccharide had no repeating units in the usual sense, although it contained two copies of the same pentasaccharide fragment Its analysis required application of all available NMR methods, and it is still matter of good luck that signals were spread sufficiently to allow interpretation of the spec-tra Assignment relied strongly on the combined usage of several heteronuclear 1H–13C correlated experiment including the new experiment H2BC, as described recently [13]
LPS preparation from F victoria contained two pol-ymers of glucose, a starch-like polymeric material and short b-1–6-glucan, found previously in F tularensis and F novicida [9,15] These components seem to be characteristic for Francisella species Overall the simi-larity of structural elements of LPS and other compo-nents clearly shows that newly discovered F victoria is indeed a new species of Francisella genus
There are several unresolved questions concerning Francisella LPS biological activity Thus the role of the ‘starch’-like material and other glucans, coex-tracted with LPS, is not clear Polymers such as these are conserved microbial structures called
‘pathogen-associated molecular patterns’, which are ligands for pattern recognition receptors expressed Fig 6 MALDI mass spectrum of the b-glucan, isolated from acetic acid hydrolysis products.
Trang 10on various immune cells as part of the innate
immu-nity recognition system [17,18] b-1,3⁄ 1,6-Glucans are
cell wall components of various bacteria, fungi, and
plants, which effect the immune response of various
vertebrates species, including fish [19] However,
these b-glucan polymers are known to have a
schizo-phrenic activity; at low concentrations they have
been shown to be immuno-stimulatory, whereas at
higher concentrations they can be immuno-inhibitory
[20,21] and seem to modulate the release of potent
cytokines induced by LPS Possibly the starch-like
polymer and glucans coextracted here with F victoria
LPS are immuno-modulatory ancillary polymers A
structural understanding of specific polymers, such as
those shown here, may shed light on how Francisella
sp so effectively evades or suppresses the host
immune response and why, after so many years,
there are still no efficacious vaccines available
Experimental procedures
Growth of F victoria
F victoriawas initially isolated as the predominant
Gram-negative pathogen from the kidney of a moribund,
appar-ently wild Tilapia sp., Oreochromis niloticus F victoria
grew slowly at <30C on cysteine heart agar or broth
(CHA or CHB)
Stock cultures were held at)80 C in 25% glycerol and
cultured at 28C on CHA Three colonies were inoculated
into 0.2 mL of CHB, grown at 28C and sequentially
scaled up to 5 mL and 100 mL of CHB, which was then
used to inoculate a 25-L CHB fermentation broth
Fermen-tation was carried out in CHB at 28C in a Chemap
fer-mentor fitted with Chemap’s Fundafom foam breaker and
a bottom-driven, three-tier, flat-bladed impeller system
Aeration was initially adjusted to 25 LÆmin)1 The control
systems provide proportional control of impeller speed,
air-flow, and temperature; pH and aeration were under control
Growth was monitored at A600 until the culture grew to
4 A600(48 h) Data from the fermentor was logged using
a PC and GenesisTMprocess control software pH was
trolled by the addition of 5% H2SO4 For harvest and
con-centration a high capacity Millipore PelliconTM tangential
flow filtration system was used and cells were finally
pellet-ed by centrifugation at 10 000 g
PAGE of F victoria LPS
LPS samples (1 mgÆmL)1) were boiled in SDS sample
buf-fer for 10 min and 50 lL used for SDS⁄ PAGE according
to the Laemmli method as modified SDS gels were washed
for 15 min in dH2O and chemically stained for LPS with
Gelcode (Pierce, Rockford, IL, USA) For western blotting,
whole cells were grown in CHB at 28C overnight, harves-ted, washed once with NaCl⁄ Pi, resuspended in SDS⁄ PAGE sample buffer and 50 lL samples digested with
5 lL Proteinase K (1 mgÆmL)1) for 2 h at 60C and boiled for 10 min to stop the digestion For western blotting, the prospective antigens were electrophoretically transferred to nitrocellulose membranes for 1 h at 50 mAÆgel)1(Bio-Rad, Hercules, CA, USA) These transblots were blocked using 5% w⁄ v skimmed milk ⁄ NaCl ⁄ Pi⁄ Tween-20 and reacted for
1 h at room temperature with a 1 : 3000 dilution of rabbit polyclonal antisera (anti-F tularensis LVS; anti-F novicida; anti-F victoria) or mouse mAb (anti-F tularensis LVS LPS; anti-F novicida LPS) in NaCl⁄ Pi)0.5% Tween-20 The membranes were then washed and further reacted with a
1 : 4000 dilution of second antibody, goat antirabbit IgG conjugated to alkaline phosphatase (Caltag Laboratories, Burlingame, CA, USA) and developed for 4 h at room tem-perature with 5-bromo-chloro-3-indolyl phosphate and 4-nitro blue tetrazolium chloride Francisella sp and LPS-spe-cific antisera were kindly provided by F Nano, University
of Victoria, BC, Canada
For large-scale LPS preparations F victoria cells (300 g wet mass) were extracted by stirring with 50% aqueous phenol (500 mL, 70C, 15 min) The cooled extract was diluted by equal amounts of water, dialyzed against tap water until phenol-free and lyophilized The respective resi-dues were resuspended in 50 mL 0.02 m sodium acetate,
pH 7.0, and treated sequentially with RNase, DNase and proteinase K (37C, 2 h each) Enzyme-treated samples were subjected to ultracentrifugation (120 000 g, 12 h,
4C) and the precipitated gels were dissolved in water and lyophilized to yield 350 mg of LPS
Mild acid hydrolysis
Aqueous phase LPS (100 mg) was hydrolyzed with 2% acetic acid (6 mL) at 100C for 4 h and, following removal
of precipitated lipid A by centrifugation at 30 000 g, the concentrated water soluble products were fractionated by Sephadex G-50 chromatography to yield for fractions I–IV Fractions II–IV were further separated by anion exchange chromatography on Hitrap Q column (5 mL, Amersham, Piscataway, NJ, USA) in a 3 mLÆmin)1 gradient of water (first 20 min) to 1 m NaCl over 1 h with UV detection at
220 nm and sugar detection by charring aliquots from each fraction on silica gel TLC plates after dipping in 2% H2SO4
in MeOH Thus acidic oligosaccharides 1–3 were isolated from fraction II, b-glucan from fraction III, and oligosac-charide 4 from fraction IV Neutral oligosacoligosac-charides eluted with water were not numbered
O,N-Deacylation of the LPS
LPS (80 mg) was dissolved in 4 m KOH (4 mL) containing NaBH4(50 mg), kept overnight at 100C, and neutralized