Molecular Microbiology (1999) 31(5), 1443±1462
The lipopolysaccharide outer core of Yersinia
enterocolitica serotype O:3 is required for virulence
and plays a role in outer membrane integrity
Mikael Skurnik,1,2* Reija Venho,1,2 JoseÂ-Antonio
Bengoechea1,2,3 and Ignacio MoriyoÂn3
1
Department of Medical Biochemistry, University of
Turku, Kiinamyllynkatu 10, 20520 Turku, Finland.
2
Centre for Biotechnology, University of Turku, Turku,
Finland.
3
Department of Microbiology, University of Navarra,
Pamplona, Spain.
Summary
Lipopolysaccharide (LPS) of Yersinia enterocolitica O:3
has an inner core linked to both the O-antigen and to an
outer core hexasaccharide that forms a branch. The
biological role of the outer core was studied using
polar and non-polar mutants of the outer core biosynthetic operon. Analysis of O-antigen- and outer corede®cient strains suggested a critical role for the outer
core in outer membrane properties relevant in resistance to antimicrobial peptides and permeability to
hydrophobic agents, and indirectly relevant in resistance to killing by normal serum. Wild-type bacteria
but not outer core mutants killed intragastrically infected mice, and the intravenous lethal dose was <104-fold
higher for outer core mutants. After intragastric infection, outer core mutants colonized Peyer's patches
and invaded mesenteric lymph nodes, spleen and
liver, and induced protective immunity against wildtype bacteria. In mice co-infected intragastrically with
an outer core mutant±wild type mixture, both strains
colonized Peyer's patches similarly during the ®rst
2 days, but the mutant was much less ef®cient in colonizing deeper organs and was cleared faster from
Peyer's patches. The results demonstrate that outer
core is required for Y. enterocolitica O:3 full virulence,
and strongly suggest that it provides resistance against
defence mechanisms (most probably those involving
bactericidal peptides).
Introduction
LPS of Yersinia enterocolitica serotype O:3 has a unique
Received 4 September, 1998; revised 22 November, 1998; accepted
25 November, 1998. *For correspondence. E-mail mikael.skurnik@
utu.®; Tel. (‡358) 2 333 7441; Fax (‡358) 2 333 7229.
Q 1999 Blackwell Science Ltd
structure in which the outer core (OC) forms a branch
(Skurnik et al., 1995). The lipid A moiety is abridged via
3-deoxy-D-manno-2-octulopyranosonic acid (Kdo) to the
inner core heptose residues onto which both the OC and
the O-antigen are bound as independent structures. The
structure of the complete core is known; the inner core is
a heptasaccharide and the OC a hexasaccharide (Radziejewska-Lebrecht et al., 1994; Shashkov et al., 1995). The
O-antigen is a homopolymer of 6-deoxy-L-altrose (6d-Alt),
in which the 6d-Alt residues are linked by a-1,2 glycosidic
bonds (Hoffman et al., 1980). The bonding of the O-antigen
to the inner core is not known. We recently reported the
characterization of the operon (Fig. 1) responsible for the
biosynthesis of the OC hexasaccharide (Skurnik et al.,
1995) and suggested that it is a relic of an ancestral heteropolymeric O-antigen operon, the function of which has been
partially replaced by the presently existing homopolymeric
O-antigen gene cluster.
As in many other smooth Gram-negative pathogens, the
O-antigen is required for full virulence of Y. enterocolitica
O:3 (Al-Hendy et al., 1992), but it has not been studied
as to whether OC plays a role in virulence. Therefore,
the relative contributions of the OC and the O-antigen to
virulence are not known. This is a new question and, as little
is known about the importance of LPS core structural variations in pathogenicity, its interest might not be limited to
our understanding of Y. enterocolitica virulence. It has not
been addressed before because in the better known Gramnegative pathogens the OC bridges the O-chain to the inner
LPS sections and therefore the lack of the O-chain is concomitant with substantial OC de®ciencies, thus preventing
an assessment of the effect that such de®ciencies could
have by themselves in virulence. In contrast, mutants lacking OC but keeping the O-chain can be produced in Y.
enterocolitica O:3. We report here that, when compared
with the WT parental strain or with strains in which the
defect was complemented by allelic exchange, such
mutants were practically non-lethal in intragastrically
(i.g.) infected mice, and showed a 104-fold decrease in
virulence in intravenously (i.v.) infected mice. Moreover,
we show that such OC-de®cient mutants were comparatively sensitive to antimicrobial peptides and hydrophobic
agents, suggesting a role of OC in outer membrane (OM)
properties linked to the resistance to innate host defence
mechanisms.
1444 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
Results
Construction of OC mutants
YeO3 (Table 1) is a fully virulent Y. enterocolitica serotype
O:3 WT patient isolate expressing complete LPS. YeO3trs11 is a polar OC gene cluster insertion mutant expressing normal O-chain and inner core (Skurnik et al., 1995).
Non-polar OC gene cluster deletion mutants YeO3-trs22
and YeO3-trs24, -trs25, -trs26 and -trs27 (Table 1) were
constructed as described in the Experimental procedures.
These same deletions were also constructed in the virulence plasmid negative (pYV ) strain, YeO3-c, to obtain
strains YeO3-c-trs22 and YeO3-c-trs24, -trs25, -trs26 and
-trs27 (Table 1). The mutant constructions were con®rmed
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1445
by Southern blotting (Fig. 1B) or by PCR (not shown). From
each of the mutants, spontaneous rough (O-antigen negative) derivatives (denoted with -R) were selected using
phage fYeO3-12, as described previously (Skurnik et al.,
1995). In the text, the relevant phenotypic characteristics
deviating from the wild-type strain are given in parentheses
after the strain name to indicate the absence of the virulence plasmid (pYV ), the serotype O:3 O-antigen (O3 )
or the outer core (OC ), i.e. YeO3-c-trs24-R (pYV O3
OC ). The truncated OC of -trs22 strains (see below) is
indicated as (OC D ).
Construction of YeO3-trs11-rev by reverse allelic
exchange
Although plasmid pRV16 carries the whole OC operon and
complements the OC defect in YeO3-trs11 in vitro (Skurnik
et al., 1995), in animal experiments, YeO3-trs11/pRV16
was not stable (data not shown). Thus, the chromosomal
defect of YeO3-trs11 was corrected by reverse allelic
exchange as described in Experimental procedures. The
resulting strain was named YeO3-trs11-rev; Southern blotting (Fig. 1B) and PCR (not shown) analyses con®rmed that
the OC gene cluster YeO3-trs11-rev was identical to that
of YeO3.
In constructing the YeO3-trs11-rev, the intermediate
merodiploid strain YeO3-trs11::pRV32 expressed complete
OC (not shown). Based on the site of the recombination
identi®ed by Southern blotting analysis (Fig. 1), presence
of promoter activity between nucleotide 2009 and the wzx
gene could be predicted, and this suggested that a weak
promoter motif upstream of the wzx gene (positions
2161±2187), identi®ed previously by sequence analysis
(Skurnik et al., 1995), is a real promoter. This conclusion
was supported by the ability of plasmid pAM200 carrying
the same 5.5 kb SphI fragment as pRV32 to complement
the OC defect in YeO3-c-trs22-R (pYV O3 OC D ) (not
shown).
LPS analysis and bacteriophage susceptibility
phenotypes of the mutants
LPS was isolated from YeO3 and mutant strains and analysed by sodium deoxycholate (DOC) polyacrylamide gel
electrophoresis (PAGE). Figure 2 shows the LPS pro®les
of the pYV ‡ strains; the pro®les of the pYV strains were
identical (not shown). This analysis demonstrates that the
OC gene cluster deletion mutant YeO3-trs24 expressed
LPS lacking the OC hexasaccharide (Fig. 2), similar to the
LPS from YeO3-trs11. The LPS patterns of YeO3-trs25,
-trs26 and -trs27 were OC (identical to that of YeO3trs24; not shown). In the LPS of YeO3-trs22, however,
there was a faint band that migrated a little faster than
the complete core band, suggesting that there may be
some WbcL activity present in the deletion mutant, perhaps
as a result of the generation of a WbcK8L8 fusion protein
(see Fig. 1A). LPS isolated from YeO3-trs11-rev was
indistinguishable from the WT LPS (Fig. 2). These analyses also proved the lack of the O-antigen smear in the
LPSs of O3 strains (the R derivatives; Fig. 2).
Bacteriophages fYeO3-12 and fR1-37 use the O-antigen and the OC as their receptors, respectively, and the
Fig. 1. Genetic organization and Southern blotting analysis of genomic DNA isolated from YeO3 OC mutants and from construction
intermediates. The YeO3 OC gene cluster contains nine genes designated wzx, wbcK-Q, and galE, ¯anked by householding genes adk,
hemH and gsk (Skurnik et al., 1995). The OC genes code for proteins responsible for the biosynthesis of the OC hexasaccharide (WbcK-Q
and GalE) and for its translocation to the periplasmic space (Wzx, the ¯ippase).
A. Restriction and genetic maps of the OC gene cluster regions of the WT and mutant strains. The sequence positions of the restriction sites
in the WT map is according to the published sequence (nucleotide sequence accession number Z47767) (Skurnik et al., 1995), the beginning
of the known sequence (0001) upstream to the adk gene is indicated. Abbreviations for the restriction enzymes: C, ClaI; N, Nsi I; Sp, SphI; X,
Xba I; Xh, Xho II. For the leftmost and rightmost ClaI sites, the approximate positions are given. The genes are drawn as open arrows below
the DNA lines. Truncated genes are indicated with a prime. The region covered by the hybridization probe (pRV3) is indicated at the top. The
vector part of the integrated pRV32 in YeO3-trs11::pRV32 is drawn as a loop with a thin line. In the YeO3-trs11, the 1.2 kb Km GenBlock
fragment substituted two Nsi I fragments; NP indicates the Nsi I/Pst I ligation sites. In YeO3-trs22, the XbaI fragment was deleted; the fusion
Xba I site is indicated by bigger characters. The mutants YeO3-trs24 to -trs27 are not drawn; the relevant Xho II restriction sites are indicated
in the YeO3 restriction map. In YeO3-trs24, the two Xho II fragments between positions 9210 and 10140 were deleted; in the YeO3-trs25
mutant, the three Xho II fragments between positions 9080 and 10140 were deleted; in the YeO3-trs26 mutant, the Xho II fragment between
positions 9575 and 10140 was deleted; and in the YeO3-trs27 mutant, the Xho II fragment between positions 9210 and 9575 was deleted.
B. Southern blot. Cla I-digested chromosomal DNA preparations of the indicated strains were separated by agarose gel electrophoresis, and
the DNA fragments were transferred to nylon membrane that was hybridized using 32P-labelled pRV3 DNA as a probe. Note that the Cla I site
at position 5218 is not cleaved because of overlapping dam methylase site. The hybridization positive Cla I fragments of (B) are identi®ed by
capital letters from A to H (sizes: A, 11 kb; B, 5.5 kb; C, 2.9 kb; D, 1.85 kb; E, 0.55 kb; F, 4.6 kb; G, 4.6 kb; and H, 2.2 kb). Hybridization to F
and G is seen as a double band with a stronger hybridization signal. pRV3 hybridized to three fragments (A±C) of YeO3, to four fragments
(A±E) of YeO3-trs11, and to seven fragments (A±G) of YeO3-trs11::pRV32. A second homologous recombination between the wzx8 and wzx
regions in this merodiploid strain eliminated the GenBlock and the vector sequences, and resulted in strain YeO3-trs11-rev. pRV3 hybridized
to three fragments (A±C) of three selected YeO3-trs11-rev clones. Finally, pRV3 hybridized to three fragments (A, B, H) of YeO3-trs22. The
smaller size of H re¯ects exactly the deletion of the internal 706 bp Xba I fragment from fragment C. Note that in YeO3-trs22, the Xba I deletion
generated a new fusion gene wbcK8L8, able to express a putative fusion protein of 376 amino acids with the 24 N-terminal amino acids of
WbcL replaced with the 108 N-terminal amino acids of WbcK. No direct evidence for the expression of WbcK8L8 was obtained, however, LPS
analysis and bacteriophage susceptibility (see Fig. 3) suggested a weak OC biosynthetic activity in the -trs22 mutants.
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
1446 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
Table 1. Bacterial strains, plasmids and bacteriophages used in this work.
Bacterial strains and
plasmids
Genotype or comments
References or sources
E. coli
C600
SY327 lpir
Sm10 lpir
thi thr leuB tonA lacY supE
l(lac pro ) argE (Am) rif nalA recA56 (lpir)
thi thr leuB tonA lacY supE recA::RP4-2-Tc::Mu-Km (lpir )
Appleyard (1954)
Miller and Mekalanos (1988)
Simon et al. (1983)
Y. enterocolitica
6471/76 (YeO3)
6471/76-c (YeO3-c)
YeO3-R1
YeO3-R2
YeO3-c-trs8
YeO3-c-trs8-R
YeO3-trs11
YeO3-trs11-R
YeO3-trs22
YeO3-trs22-R
YeO3-c-trs22
YeO3-c-trs22-R
YeO3-trs24
YeO3-trs24-R
YeO3-c-trs24
YeO3-c-trs24-R
YeO3-trs25
YeO3-trs25-R
YeO3-trs26
YeO3-trs26-R
YeO3-c-trs26
YeO3-c-trs26-R
YeO3-trs27
YeO3-trs27-R
YeO3-c-trs27
YeO3-c-trs27-R
Serotype O:3, patient isolate, wild type
Virulence plasmid cured derivative of 6471/76
Spontaneous rough derivative of YeO3-c
Spontaneous rough derivative of YeO3
YeO3-c, Dwzx-wbcKL::Km-GenBlock, kmR
Spontaneous rough derivative of YeO3-c-trs8, kmR
YeO3, Dwzx-wbcKL::Km-GenBlock, kmR
Spontaneous rough derivative of YeO3-trs11, kmR
YeO3 DwbcKL, deletion of a 706 bp XbaI fragment
Spontaneous rough derivative of YeO3-trs22
YeO3-c DwbcKL, deletion of a 706 bp XbaI fragment
Spontaneous rough derivative of YeO3-c-trs22
YeO3 DwbcP, 929 bp deletion of two internal Xho II fragments
Spontaneous rough derivative of YeO3-trs24
YeO3-c DwbcP, 929 bp deletion of two internal Xho II fragments
Spontaneous rough derivative of YeO3-c-trs24
YeO3 DwbcP, 1049 bp deletion of three internal Xho II fragments
Spontaneous rough derivative of YeO3-trs25
YeO3 DwbcP, deletion of an internal 566 bp Xho II fragment
Spontaneous rough derivative of YeO3-trs26
YeO3-c DwbcP, deletion of an internal 566 bp Xho II fragment
Spontaneous rough derivative of YeO3-c-trs26
YeO3 DwbcP, deletion of an internal 363 bp Xho II fragment
Spontaneous rough derivative of YeO3-trs27
YeO3-c DwbcP, deletion of an internal 363 bp Xho II fragment
Spontaneous rough derivative of YeO3-c-trs27
Skurnik (1984)
Skurnik (1984)
Al-Hendy et al. (1992)
Al-Hendy et al. (1992)
Skurnik et al. (1995)
Skurnik et al. (1995)
Skurnik et al. (1995)
Skurnik et al. (1995)
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Partial Sau 3AI fragment (nt 2009±9576) of the YeO3-c outer core operon cloned
into pBR322
The 5.5 kb Sph I fragment of pAM100 cloned into the SphI site of pTM100
Suicide vector, a derivative of pJM703.1, clmR
YeO3-c genomic library clone in pBR322, 8.2 kb insert (nt 1±8223), ampR
The YeO3-c outer core operon cloned in a 12 kb Hin dIII fragment into pTM100,
clmR
Nru I deletion derivative of pRV16, clmR
The 2.9 kb Cla I fragment (nt 2666±5562) of pRV16 cloned into the ClaI site of
pRV1, clmR
Cla I deletion derivative of pRV17 (includes nt 5563±10404), clmR
Derivative of pRV20, deletion of the 566 bp Xho II fragment, clmR
Derivative of pRV20, deletion of the 363 and 566 bp Xho II fragments, clmR
Derivative of pRV20, deletion of the 128, 363 and 566 bp XhoII fragments, clmR
Derivative of pRV20, deletion of the 363 bp Xho II fragment, clmR
Derivative of pRV19 with the 0.7 kb Xba I fragment deleted, clmR
The 1.1 kb Stu I-NruI fragment of pRV20-part.Xho II-7 cloned into pRV1, clmR
The 0.9 kb Stu I-NruI fragment of pRV20-part.Xho II-8 cloned into pRV1, clmR
The 1.5 kb Stu I-NruI fragment of pRV20-part.Xho II-1 cloned into pRV1, clmR
The 1.6 kb Stu I-NruI fragment of pRV20-part.Xho II-9 cloned into pRV1, clmR
The 5.5 kb Sph I fragment of pAM100 cloned into the SphI site of pRV1, clmR
Mobilizable vector, pACYC184-oriT of RK2, clmR
Skurnik et al. (1995)
Plasmids
pAM100
pAM200
pRV1
pRV3
pRV16
pRV17
pRV19
pRV20
pRV20-part.Xho II-1
pRV20-part.Xho II-7
pRV20-part.Xho II-8
pRV20-part.Xho II-9
pRV22
pRV24
pRV25
pRV26
pRV27
pRV32
pTM100
Bacteriophages
fYeO3±12
fR1±37
O-antigen-specific phage of Y. enterocolitica O:3
Outer core-specific phage of Y. enterocolitica O:3
Skurnik
Skurnik
Skurnik
Skurnik
et
et
et
et
al.
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al.
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Michiels and Cornelis
(1991)
Al-Hendy et al. (1991)
Skurnik et al. (1995)
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1447
Fig. 2. DOC-PAGE analysis of LPS from Y.
enterocolitica O:3 strains. The bacteria were
grown in TSB at RT. LPS samples were prepared
by hot water±phenol extraction method and, after
electrophoresis, the LPS bands (indicated on the
right) were visualized by silver staining. The lanes
are labelled by the strain names (the trs strain
names are abbreviated, all begin with YeO3-).
Note that in the gel, there was a small leakage of
sample from trs11-rev to trs11, visible at the
complete core band level. Bacteriophage
sensitivities of the strains are indicated at the
bottom. For fR1-37, sensitivity (‡) indicates that
sensitivity is dependent on growth temperature
and virulence plasmid, i.e. pYV strain grown at
378C is sensitive, whereas pYV ‡ strain grown at
378C or at RT and pYV strain grown at RT, are
resistant because of steric hindrance by abundant
YadA and/or O-antigen. YeO3-R2 is susceptible
to fR1-37 under all growth conditions.
susceptibilities of the strains to the phages were assessed
(Fig. 2). The susceptibility patterns followed the LPS patterns, and the sensitivity of the -trs22 strain to fR1-37 con®rmed the presence of at least a small amount of the phage
receptor on these bacteria.
Serum resistance
Although the most important serum resistance factors of Y.
enterocolitica are YadA (Balligand et al., 1985; Pilz et al.,
1992) (our unpublished results) and Ail (Falkow, 1991), the
Y. enterocolitica LPS O-antigen has also been associated
with serum resistance (Wachter and Brade, 1989) (our
unpublished results). To ®nd out whether the OC is involved
in serum resistance strains YeO3-trs11 (OC ), YeO3-trs22
(OC D ) and YeO3-trs24 (OC ), and the WT strain YeO3,
their pYV , O3 and pYV O3 derivatives were grown
at room temperature (RT; 22±258C) or at 378C, and
assayed for serum resistance as described in Experimental
procedures. The serum killing experiments with bacteria
expressing YadA did not indicate any direct role for OC
in serum resistance. However, in the absence of YadA,
OC seemed to potentiate the other resistance factors, in
particular Ail (not shown).
Interaction of antimicrobial peptides with the OMs of
live cells and LPS hybrids
Antimicrobial cationic peptides play a critical role in the host
defence against pathogens. These peptides share with
other bactericidal polycations the property of interacting
with anionic OM targets such as LPS core and lipid A
groups, thereby breaking the integrity of OMs (Hancock,
1984; Vaara, 1992; Nicolas and Mor, 1995). Because
pathogenic Y. enterocolitica are comparatively resistant
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
to such agents (Bengoechea et al., 1996) and this resistance relates to LPS peculiarities (Bengoechea et al.,
1998a), the role of OC was studied. To this end, bacterial
suspensions were incubated with lysozyme (a lytic enzyme
acting in the periplasm) in the absence and presence of
polymyxin B, and penetration of the probe measured as
the turbidity drop (cell lysis). Figure 3A shows that permeabilization by polymyxin B decreased in the order
YeO3-trs11-R (O3 OC ) > YeO3-trs11 (OC ) > YeO3
(WT strain). Moreover, strain YeO3-R2 (O3 OC ‡ ) yielded
results close to those of the WT strain and was more
resistant than YeO3-trs11 (O3 ‡ OC ), suggesting that
OC was more important than O-antigen in hindering the
bactericidal polycations. To con®rm this, the OM damage
caused by peptides of increasing bactericidal activity
(poly-L-ornithine, poly-L-lysine, melittin and polymyxin B;
Vaara, 1992) was measured using N-phenyl-1-naphthylamine (NPN), a hydrophobic probe whose ¯uorescence
increases in hydrophobic environments and that is ef®ciently excluded by Y. enterocolitica (Bengoechea et al.,
1996). The ¯uorescence of NPN bacterial suspensions
was low and similar (<20 RFU) for all strains tested, and
the peptides increased NPN uptake to yield RFU values
that, for each strain, correlated with the bactericidal activity
of the peptide (Fig. 3B). Moreover, the OMs of YeO3 (with
WT LPS) were less disturbed by polymyxin B and melittin
(P # 0.05) than the OMs of strains carrying defective
LPSs: YeO3-R2 (O3 ), YeO3-trs11 (OC ) and YeO3trs11-R (O3 OC ) (Fig. 3B). Furthermore, the OMs of
YeO3-R2 (O3 ) were less disturbed by melittin (P # 0.05)
than those of YeO3-trs11 (OC ) and YeO3-trs11-R (O3
OC ). Finally, YeO3 and YeO3-R2 OMs were less disturbed by poly-L-lysine and poly-L-ornithine (P # 0.05)
than those of YeO3-trs11 and YeO3-trs11-R.
The above experiments strongly suggested that OC plays
1448 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
more polymyxin B (P < 0.05) than YeO3 or YeO3-R2
(O3 ) (data not shown). Finally, NPN partition into homologous or hybrid LPS aggregates (see Experimental procedures) was measured after incubation with or without
polymyxin B. Upon addition of NPN alone, there were no
signi®cant differences (P # 0.05) in the ®nal RFU values
of the YeO3, YeO3-R2 or YeO3/YeO3-R2 aggregates (all
gave <97 RFU), which were, however, signi®cantly lower
(P # 0.05) than the RFU values of YeO3-trs11 and YeO3trs11/YeO3 aggregates (151 RFU and 129 RFU respectively). It is worth noting that the difference between the
RFU values of the YeO3-trs11 and the YeO3-trs11/YeO3
aggregates was signi®cant (P # 0.05). In all cases, polymyxin B produced an increase in ¯uorescence with respect
to the values obtained without polymyxin B, but with clear
differences: the effect on YeO3 LPS aggregates (11 RFU
increase) was lower (P < 0.05) than on the other LPS aggregates (increases of 23 RFU for YeO3-R2, 32 RFU for
YeO3-trs11 and 34 RFU for YeO3-trs11/YeO3 hybrid
aggregates), and lower on YeO3-R2 than on YeO3-trs11
or on YeO3-trs11/YeO3 LPS aggregates (P < 0.05). All
these results proved that OC hinders polycation binding
to the LPS. Moreover, the differences in NPN uptake
observed with no polymyxin B suggest that the OC-de®cient LPS could create a less ef®cient barrier to hydrophobic agents in the OM and this was tested as follows.
Sensitivity of OC mutants to deoxycholate and
hydrophobic agents
Fig. 3. Effect of polycationic agents on the OM integrity of Y.
enterocolitica O:3 strains.
A. Effect of polymyxin B determined as lysozyme-induced cell lysis.
Each point represents the mean and standard deviation (covered
by the symbol) of four samples from two independently grown
batches of bacteria.
B. Effect of polymyxin B, melittin, poly-L-lysine and poly-L-ornithine
determined as NPN partition into the OM. The partition is
expressed as relative ¯uorescence units (RFU). In the absence of
any agent, the RFU value was about 20 for every strain. The
results shown represent a typical experiment (coef®cient of
variation between experiments was less than 5%). Asterisks and
open triangles indicate signi®cant differences (P < 0.05) between
the RFU obtained with YeO3 or YeO3-R2, respectively, and the
other strains studied.
a critical role in protecting against bactericidal polycations,
possibly because of an indirect role of OC in stabilizing the
OM and assisting some pYV-encoded factors or because
of reduced polycation binding by OC ‡ LPS molecules.
Three sets of experiments proved the latter hypothesis
to be the correct one. First, the lysozyme permeabilization
and ¯uorimetric experiments were repeated with the pYV
isogenic pairs of all the above strains with results identical
to those obtained with the pYV ‡ cells (not shown). Second,
the amount of polymyxin B absorbed by live bacteria was
measured, it was found that YeO3-trs11 (OC ) adsorbed
All Y. enterocolitica O:3 strains formed smooth round
colonies on Luria agar (LA), either at RT or at 378C, with
the only difference that pYV ‡ bacteria formed smaller
colonies at 378C. However, differences in colony morphology were noticed on CIN-agar in which strains YeO3trs22-R (O3 OC D ), YeO3-c-trs24-R (pYV O3 OC ),
YeO3-trs11-R (O3 OC ) and strain YeO3-c-trs22-R
(pYV O3 OC D ), in particular, formed rough irregular
colonies. This morphology was reproduced when YeO3c-trs22-R was grown on LA plates supplemented with
0.5% DOC (one of the ingredients in CIN agar), and therefore the effect of DOC was studied directly. Although all
strains were resistant to DOC-induced lysis when grown
at RT, differences became evident when grown at 378C
(Fig. 4). YeO3-trs22 (OC D ) and -trs24 (OC ) were more
sensitive than either YeO3-trs11 (OC ) or the WT bacteria, which displayed a close DOC sensitivity. The O-antigen did not seem to play a relevant role (Fig. 4) and pYV
did not affect the results (not shown).
There is indirect evidence that Y. enterocolitica uses
ef¯ux pumps to force out DOC, thereby complementing
the OM barrier (Bengoechea et al., 1998b). Thus, sensitivity to DOC (0.5%) was tested in the presence of metabolic
inhibitors that prevent ef¯ux (1 mM potassium cyanide/
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1449
Fig. 4. DOC sensitivities of Y. enterocolitica O:3 WT and OC
mutant bacteria. Cell lysis (% turbidity of the control without DOC)
after 60 min incubation in 1% DOC of bacteria grown at RT (grey
bars) or at 378C (black bars).
1 mM sodium arsenate). As expected, lysis was increased
for all the strains irrespective of the growth temperature
(data not shown), and, in addition, differences in DOC sensitivity were noted also at RT because YeO3-trs11 (OC ),
-trs22 (OC D ) and -trs24 (OC ) were more sensitive than
the WT strain. When grown at 378C, YeO3-trs22 (OC D )
and -trs24 (OC ) were most sensitive, whereas YeO3trs11 (OC ) showed a sensitivity closer to that of the
WT strain (data not shown).
Finally, sensitivity to representative hydrophobic dyes
and antibiotics was tested at 378C by the disk diffusion
method. YeO3-trs11 (OC ) was more sensitive to malachite green and novobiocin than the WT strain (inhibition
halos of 14 versus 10 mm and of 15.5 versus 0 mm respectively), and small but consistent differences were also
found with crystal violet and rifampicin (inhibition halos
of 10 versus 9 mm and of 20 versus 18 mm respectively).
For all these agents, the corresponding R strains YeO3R2 (O3 ) and YeO3-trs11-R (O3 OC ) yielded results
similar to those obtained with O-antigen-bearing strains.
Therefore, all the preceding results supported a key role
for OC in the ability of the OM to keep off hydrophobic
permeants.
Role of OC in virulence
It has been shown previously that the O-antigen plays a
role in the virulence (Al-Hendy et al., 1992). To test whether
OC is also necessary for virulence, mice were infected i.g.
with increasing doses of the YeO3 (WT), YeO3-trs11 (OC
polar) and YeO3-trs24 (OC non-polar) (Table 2, experiment 1 and 2). Altogether, YeO3 (WT) killed 15 of the 49
infected mice within 12 days, whereas YeO3-trs11(OC )
killed only 2 of 49 and YeO3-trs24 (OC ) killed 1 of 24
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
mice. As expected, YeO3-c (pYV ) did not kill any mice
even at the highest doses. In a third experiment, YeO3
killed 12 of 30 infected mice, YeO3-trs11 failed to kill any
of 29, and the highest doses of YeO3-trs22 (OC D ) and
YeO3-trs24 (OC ) killed 5 and 6 out of 30 infected mice
respectively (data not shown). These experiments were
complemented by testing the virulence of YeO3-trs11-rev
(with a repaired OC gene cluster of YeO3-trs11). This strain
showed a virulence close to that of the WT strain and killed
altogether 6 of the 25 infected mice (Table 2). Taken
together, the above results demonstrated that the OC
gene cluster is indispensable for full virulence.
To ®nd whether OC was necessary during invasion
through the gut tissues or during the later phases of infection, the bacteria were administered i.v. to bypass the mucosal immunity (Table 3). All mice infected with 0.5 ´ 102,
0.5 ´ 104 and 0.5 ´ 106 cfu of YeO3 (WT) died in the ®rst
week (thus, the LD 100 of YeO3 was < 50 cfu), and no
mice infected with YeO3-c (pYV ) showed signs of disease
(Table 3). In contrast to the results obtained in i.g. infected
mice, YeO3-trs11 (OC ) was able to kill mice, although
only at the highest dose of 0.5 ´ 106 cfu (Table 3). Mice
that received 0.5 ´ 104 cfu were clearly sick but did not
die, and mice infected with 0.5 ´ 102 cfu were seemingly
unaffected (but acquired immunity, see below). The attenuation observed (> 104-fold) suggests that OC is necessary
for the infection to progress from the infected intestinal
tissue to deeper organs.
In a preliminary experiment, the infection kinetics of
YeO3, YeO3-c (pYV ) and YeO3-trs11 (OC ) bacteria
were followed in different tissues of mice infected i.g.
with 108 bacteria. Mouse to mouse variations and the
low rates of bacterial recovery from the organs prevented
a clear assessment of possible differences in the infection
kinetics of wt bacteria and OC mutants. Thus, mice were
co-infected with a mixture of approximately equal doses
of YeO3 and YeO3-trs11 (OC ) using <109 cfu of each,
and bacterial counts in different organs were performed
(Table 4). Two days after co-infection, relatively high bacterial loads were observed in Peyer's patches, with lower
values for deeper organs. At later times, some mice began
to clear the bacteria from the tissues, whereas in others
the bacteria multiplied to very high levels which would
have ®nally caused their death. In contrast, mouse number
7 (Table 4), which was clearly moribund at 7 days after
infection and was killed, contained only low numbers of
bacteria in the organs. Of the two mice killed 9 days after
infection, mouse number 8 was in a very weak condition,
whereas mouse number 9 looked completely healthy.
This was also re¯ected in the corresponding bacterial
counts (Table 4).
The percentage of KmR colonies (YeO3-trs11) in the
recovered bacteria was determined (Table 4). Two days
after infection, YeO3-trs11 (OC ) was present in Peyer's
1450 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
Table 2. Virulence and protective immunity
development in DBA/2 mice infected i.g. with
Y. enterocolitica O:3 strains.
Primary infection
Strain
YeO3
Expt 1
Expt 2
YeO3-trs11
Expt 1
Expt 2
YeO3-trs11-rev
Expt 1
YeO3-trs24
Expt 1
YeO3-c
Expt 1
Expt 2
YeO3-R2
Expt 2
Rechallenge (YeO3, i.v.)
Dose
n
Deaths
Dose
n
Deaths
Survivors (%)
1.1 ´ 105
1.1 ´ 106
1.1 ´ 107
1.1 ´ 108
1.1 ´ 109
5
5
5
5
5
5, 7, 10
3, 4, 5, 11
3
3, 4, 6
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
5
2
1
4
2
4, 5, 6, 6
6, 6
20
0
100
100
100
0.8 ´ 105
0.8 ´ 106
0.8 ´ 107
0.8 ´ 108
0.8 ´ 109
4
5
5
5
5
4
4
4
5
3
5, 5, 5, 5
4, 4, 4, 4
4
4, 6
1.4 ´ 102
0.7 ´ 104
0.7 ´ 104
0.7 ´ 106
0.7 ´ 106
0
0
75
100
100
0.8 ´ 105
0.8 ´ 106
0.8 ´ 107
0.8 ´ 108
0.8 ´ 109
5
5
5
5
5
5
5
5
5
4
4, 5, 6, 6
4, 5, 6
6
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
20
40
100
100
100
1 ´ 105
1 ´ 106
1 ´ 107
1 ´ 108
1 ´ 109
5
5
5
5
5
1.4 ´ 102
0.7 ´ 104
0.7 ´ 104
0.7 ´ 106
0.7 ´ 106
4
5
5
5
5
6,
4,
5
2,
2,
0.9 ´ 105
0.9 ´ 106
0.9 ´ 107
0.9 ´ 108
0.9 ´ 109
5
5
5
5
5
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
5
5
5
2
3
4, 5, 5, 6, 6
3
6
0
80
80
100
100
1.3 ´ 105
1.3 ´ 106
1.3 ´ 107
1.3 ´ 108
1.3 ´ 109
5
5
5
5
4
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
2 ´ 102
5
5
4
5
4
5
5, 5
80
60
100
100
100
1.1 ´ 108
1.1 ´ 109
4
4
2 ´ 102
2 ´ 102
4
4
5, 5, 5
4, 5
1.5 ´ 108
1.5 ´ 109
5
5
0.7 ´ 104
0.7 ´ 106
5
5
3, 4, 4, 5, 5
2, 2, 3, 3, 3
0
0
0.5 ´ 105
0.5 ´ 106
0.5 ´ 107
0.5 ´ 108
0.5 ´ 109
5
5
5
5
5
1.4 ´ 102
1.4 ´ 102
1.4 ´ 102
0.7 ´ 104
0.7 ´ 106
5
5
5
5
4
4, 4, 6
5, 5, 5, 6, 7
5, 5, 5, 5, 6
60
0
0
100
25
12
8
25
6
5, 7, 13
4, 10
5
5
6, 6, 7
5
2, 3, 3, 3
2, 2
3, 3, 3
0
60
80
0
60
25
50
Results of two experiments (experiment 1 and 2) are shown. Sera from survivors of the rechallenge in experiment 1 were collected and analysed for specific anti-YeO3 antibodies (see text
and Figs 5 and 6).
patches in a proportion similar to that in the inoculum, but it
was clearly reduced in mesenteric lymph nodes, spleen
and liver. After 5 days, YeO3-trs11 was detected only in
Peyer's patches representing only between 17% and
4% of the bacteria, whereas YeO3 persisted in all organs.
After 7 and 9 days, only mouse number 8, which was very
sick, contained YeO3-trs11 in the Peyer's patches. These
experiments proved both that the OC-de®cient mutant
reached Peyer's patches as ef®ciently as the WT, and
that it was more rapidly cleared from all organs. Thus, it
can be postulated that OC played a role in virulence after
the invasion step.
The co-infection experiment was also performed in
mice not pretreated with desferal to exclude a differential
effect of this chelator in the elimination of YeO3-trs11.
Three mice were infected i.g. with a mixture of <1011 cfu
of each YeO3 and YeO3-trs11 (OC ) and killed 4 days
later. The proportions of YeO3-trs11 recovered were:
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1451
Table 3. Virulence and protective immunity
development in DBA/2 mice infected i.v. with
Y. enterocolitica O:3 strains.
Primary infection
Strain (route)
YeO3
Expt 1
YeO3-trs11
Expt 1
YeO3-c
Expt 1
YeO3-R2
Expt 2
Rechallenge (YeO3, i.v.)
Dose
n
Deaths
Dose
n
0.5 ´ 102
0.5 ´ 104
0.5 ´ 106
3
3
3
5, 6, 7
4, 4, 4
4, 4, 4
0.5 ´ 102
0.5 ´ 104
0.5 ´ 106
3
3
3
2 ´ 102
2 ´ 102
3
3
1.1 ´ 102
1.1 ´ 104
1.1 ´ 106
3
3
3
2 ´ 102
2 ´ 102
2 ´ 102
3
3
3
4, 4, 5
4, 4, 7
0.5 ´ 102
0.5 ´ 104
0.5 ´ 106
2
2
3
0.7 ´ 104
0.7 ´ 106
2
2
4
7
4, 4, 4
2, 3, 4
Deaths
Survivors (%)
100
100
0
0
100
50
50
Results of two experiments (experiment 1 and 2) are shown. Sera from survivors of the rechallenge in experiment 1 were collected and analysed for specific anti-YeO3 antibodies (see text
and Figs 5 and 6).
YeO3, YeO3-trs11 (OC ), YeO3-R2 (O3 ) and YeO3-c
(pYV ) using high-quality age- and sex-matched mice
[the earlier reported experiments were performed with
locally bred mice (Al-Hendy et al., 1992), and thus the
results are not directly comparable]. YeO3-R2 killed 1 of
25 mice infected i.g., whereas YeO3 and YeO3-trs11 killed
5 of 25 and 1 of 25 respectively (Table 2). When tested
i.v., YeO3-R2 yielded the same results as YeO3-trs11;
i.e. the mice were killed only by the highest dose used
(0.5 ´ 106 cfu per mouse; Table 3). These results, therefore, suggested that both O-antigen and OC are equally
important for the full virulence of Y. enterocolitica O:3 in
i.g. or i.v. infected mice.
0±5% in Peyer's patches (total bacterial counts 4.2±
6.4 ´ 108 cfu g 1 ); 2±16% in spleens (total 5.2 ´ 103 ±2.2 ´
104 cfu g 1 ); and 0% in livers (total 0.0±5.2 ´ 102 cfu g 1 ).
Although (as expected) the cfu g 1 recovered from spleens
and livers were much lower, the elimination of YeO3-trs11
correlated well with that of desferal-treated mice (Table 4).
Therefore, desferal does not directly affect OC mutants in
vivo.
Infections with rough Y. enterocolitica strain YeO3-R2
To compare the importance of the O-antigen and the OC in
virulence, experiment 2 (Table 2) was repeated with
Table 4. Bacterial counts in mouse organs at different time points after an i.g. co-infection with a bacterial mixture of 109 YeO3 and 109 YeO3trs11. Proportion of KmR colonies (YeO3-trs11) in the initial mixture was 58.5%.
Bacterial counts in organs (cfu/g tissue)a
Peyer's patches
1
Mouse no.
Day
cfu g
1
2
3
4
5
6
7
8
9
2
2
2
5
5
5
7
9
9
6.0 ´ 107
3.4 ´ 107
5.3 ´ 107
1.5 ´ 106
7.1 ´ 106
5.9 ´ 106
1.4 ´ 104
1.4 ´ 107
1.0 ´ 106
Spleen
KmR (%)
cfu g
1
40
61
62
17
9
4
0 (0/36)b
12
0
1.9 ´ 106
5.8 ´ 106
1.2 ´ 107
9.1 ´ 104
7.4 ´ 104
6.2 ´ 106
70
4.5 ´ 109
65
Lymph nodes
KmR (%)
cfu g
1
35
1
3
0
0
0
0 (0/2)
0
0 (0/1)
1.5 ´ 104
1300
3.2 ´ 104
n.g.d
2300
7.7 ´ 104
n.g.
3.1 ´ 106
n.g.
KmR (%)
17 (6/35)
n.d.c
30 (11/37)
0
0
0
Liver
cfu g
1
3.4 ´ 104
7.8 ´ 104
1.1 ´ 105
6.4 ´ 104
4.6 ´ 104
9.0 ´ 106
440
1.3 ´ 105
270
KmR (%)
14
0
0
0
0
0
0 (0/6)
0
0 (0/5)
a. Detection thresholds for the different tissues were <80 cfu g 1 for liver, <50 cfu g 1 for spleen, <800 cfu g 1 for mesenteric lymph nodes and
<400 cfu g 1 for Peyer's patches.
b. One hundred separate colonies were patched on Km plates. When fewer colonies were available, the number of KmR colonies/total patched
colonies is indicated in parentheses.
c. n.d., not determined.
d. n.g., no growth.
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
1452 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
Table 5. Bacterial counts in mouse organs at different time points after an i.g. co-infection with a bacterial mixture of 109 YeO3 and 109 YeO3-R2.
Proportion of fYeO3-12-resistant (fR ) colonies (YeO3-R2) in the initial mixture was 44%.
Bacterial counts in organs (cfu g
Peyer's patches
1
Mouse no.
Day
cfu g
1
2
3
4
5
6
7b
8
9
2
2
2
5
5
5
7
9
9
3.0 ´ 107
3.6 ´ 107
2.0 ´ 107
3.0 ´ 106
6.6 ´ 106
2.6 ´ 106
1.0 ´ 108
3.0 ´ 106
850
Spleen
%fR
cfu g
1
11
28
9
0
0
30
10
0
0 (0/1)
8.8 ´ 106
1.3 ´ 106
9.2 ´ 105
9.2 ´ 105
4.7 ´ 104
4.1 ´ 105
5.8 ´ 108
255
n.g.
1
tissue)a
Lymph nodes
%fR
cfu g
1
5
6
1
5
0
0
0
0 (0/3)c
2.6 ´ 104
n.g.d
n.g.
7000
n.g.
1800
1.0 ´ 107
n.g.
n.g.
Liver
%fR
cfu g
21 (15/71)
9.3´104
1.4 ´ 104
1.9 ´ 104
8.0 ´ 105
6500
1.6 ´ 104
8.5 ´ 108
3400
n.g.
0 (0/24)
0 (0/7)
0
1
%fR
15
8
0
0
0
2
0
0 (0/46)
a. Detection thresholds for the different tissues were <80 cfu g 1 for liver, <50 cfu g 1 for spleen, <800 cfu g 1 for mesenteric lymph nodes and
<400 cfu g 1 for Peyer's patches.
b. Mouse no. 7 died between days 6 and 7.
c. One hundred separate colonies were patched on plates containing bacteriophage fYeO3-12 (<109 per plate). When fewer colonies were available, the number of fR colonies/total patched colonies is indicated in parentheses.
d. n.g., no growth.
To pinpoint the phase of infection in which YeO3-R2
manifests the attenuation, mice were co-infected with
YeO3 and YeO3-R2 (O3 ) (Table 5). Total bacterial counts
followed very closely those seen in the YeO3 plus YeO3trs11 (OC ) co-infection experiments (Table 4). Mouse
number 7 died on the 7th day, and its organs were loaded
with bacteria (Table 5). Assessment of the percentage of
YeO3-R2 (O3 , resistant to fYeO3-12) showed its rapid
clearance from all organs (Table 5). Moreover, the percentage of YeO3-R2 in Peyer's patches after 2 days (Table 5)
was signi®cantly (P ˆ 0.01, two-tailed z-test) lower than that
of YeO3-trs11 (OC ) (Table 4). In only one mouse (number
6, Table 5), was a signi®cant number of YeO3-R2 recovered from Peyer's patches. In the dead mouse (number
7), YeO3-R2 had survived at low levels in the Peyer's
patches (Table 5). These experiments showed that the
O-antigen-de®cient mutant was more rapidly cleared from
all organs than the WT strain, and that it did not colonize
the Peyer's patches as ef®ciently as the OC mutant.
Thus, it can be postulated that the O-antigen played a
role in virulence during the invasion step.
Acquired immunity
In the experiments shown in Tables 2 and 3, many mice
infected i.g. and i.v. survived. Because OC mutants persisted in the organs, stimulation of protective immunity
was assessed by challenging the survivors of experiment
1 (Tables 2 and 3) with 200 bacteria of YeO3 ($ 5 times
the LD 100 ) administered i.v. [this route would lead to the
death of the animals (Table 3)]. As shown in Table 2
(experiment 1), the level of immunity elicited by i.g. administered YeO3-trs11 (OC ), YeO3-trs24 (OC ), YeO3 and
YeO3-trs11-rev was similar and dose dependent. YeO3-c
(pYV ), in contrast, failed to induce similar levels of protection even at the highest doses used (Table 2, experiment
1). The differences with the pYV strain were further supported by the observation that mice infected i.v. with
<50 cfu of YeO3-trs11 (OC ) developed immunity against
YeO3, whereas only the highest i.v. administered dose
(1.1 ´ 106 cfu) of YeO3-c (pYV ) elicited protection
(Table 3).
To assess more quantitatively the protection afforded,
in experiment 2 mice were infected with YeO3-c, YeO3,
YeO3-trs11 and YeO3-R2, and the survivors were challenged i.v. with three different doses of YeO3 (Tables 2
and 3, experiment 2). No mice initially infected i.g. with
YeO3-c (pYV ) survived a 104 or 106 cfu challenge. On
the contrary, the highest dose of i.g. administered YeO3
triggered protective immunity against the highest (106 cfu)
challenge, and initial infection with 107 but not with 106 cfu
of YeO3 gave protection against a 104 cfu challenge. A
similar degree of protection was not induced by YeO3trs11 (OC ) or YeO3-R2 (O3 ). Although lower doses
of YeO3-trs11 seemed to induce better protection than
YeO3 against a 104 cfu challenge, YeO3-trs11 did not
induce full protection against a 106 cfu challenge even at
the highest initial infection dose. In general, the protection
induced by YeO3-R2 was clearly inferior to that induced
by YeO3-trs11. Initial i.v. infection with YeO3-R2 seemed
to give better protection (Table 3).
Acquired humoral immunity
Sera from the surviving mice of experiment 1 (Tables 2
and 3) were analysed by EIA and immunoblotting. The
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1453
Fig. 5. Distribution of anti-Y. enterocolitica O:3 titres in sera of
mice that survived the i.v. rechallenge of YeO3 bacteria. Shown
are the values obtained from individual mice (empty squares) and
the mean values (horizontal bars).
distribution of the individual EIA titres (i.e. the dilution giving an absorbance of 0.3 in EIA) is shown in Fig. 5. For the
i.g. infected mice, the mean serum titres (6 standard
deviation) were: YeO3-c (pYV ), 2973 6 3424; YeO3,
540 6 507; YeO3-trs11 (OC ), 111 6 177; YeO3-trs11rev, 380 6 629; and YeO3-trs24 (OC ), 258 6 202. Accordingly, the responses induced by YeO3-trs11 were lowest
and differed from those of YeO3 (P ˆ 0.005; two-tailed
z-test), YeO3-trs11-rev (P ˆ 0.038), YeO3-trs24 (P ˆ
0.005) and YeO3-c (P ˆ 0.037). Interestingly, the three
mice infected i.g. with YeO3-c which survived the challenge
had serum titres higher than those observed for the other
four strains (P-values from 0.037 to 0.055) (Fig. 5). As
expected, the responses against YeO3 and YeO3-trs11rev did not differ from each other (P ˆ 0.13). The response
against YeO3-trs24 differed from that of YeO3 (P ˆ 0.031),
but not from that of YeO3-trs11-rev (P ˆ 0.13).
With respect to the antibody response of mice infected
i.v., the highest YeO3-c (pYV ) dose (1.1 ´ 106 ) induced
also the highest titres (735, 2900, 3570; mean 2402 6
1482). Interestingly, the six mice infected i.v. with YeO3trs11 had a mean titre of 339 6 263 (80, 115, 135, 470,
530 and 705), and this differed (P ˆ 0.012) from the
response in mice infected i.g. with the same strain.
In conclusion, the OC mutant YeO3-trs11, and to some
extent YeO3-trs24, induced signi®cantly weaker humoral
responses in i.g. infected mice than WT bacteria. YeO3-c
(pYV ), in contrast, induced the strongest responses.
When inoculated i.v., YeO3-trs11 induced a stronger
response than when inoculated i.g.
To analyse the speci®city of the antibody responses,
selected sera were tested by immunoblotting with whole
cell lysates of the different strains (Fig. 6). In general, and
no matter the infecting strain, immunoblottings revealed
a strong antibody response against protein antigens (Fig.
6A). The reactivity patterns of the sera against any single
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
strain showed only minor variability as missing bands,
mostly of low molecular weight (Fig. 6A). Although some
bands were missing from the OC mutants, this was not a
consistent ®nding and probably only re¯ected differences
in antibody titres (all sera were used at 1 : 200 dilution).
Comparison of the reacting bands of different strains
using one serum of the YeO3-infected mice revealed that
the pYV-encoded antigens were immunogenic (Fig. 6B,
®lled arrows). As expected, sera of mice infected with
YeO3-c (pYV ) were missing antibodies against pYVencoded antigens, and sera of mice infected with OC
mutants lacked antibodies against the complete core. The
chromosomally encoded proteins were recognized in the
immunoblotting with similar intensity for all the strains,
indicating that the OM protein pro®les do not vary signi®cantly between the wild-type and the mutant strains. This
was con®rmed by analysing the OM protein patterns by
SDS±PAGE (data not shown). Finally, the sera also recognized the O-antigen and the complete core (Fig. 6B, open
arrows), but not the truncated core of the OC mutants.
Discussion
OC is necessary for full virulence of Y. enterocolitica
O:3
In this work, we show that the OC of Y. enterocolitica O:3
LPS is a virulence factor. First, clear dose±response curves
showing the attenuation of OC mutants were obtained in
mice infected i.v. Second, although an accurate estimation
of the intragastric LD 50 was prevented by the fact that many
mice in different dose groups survived (Table 2), the almost
total lack of killing by the OC mutants also demonstrated
the need of the OC for full virulence by this route. Interestingly, a similar kind of `to be or not to be' phenomenon was
seen in invasion mutant studies of Y. enterocolitica O:8; in
some mice the bacterial counts in organs were low, but in
other mice the counts were high (Pepe et al., 1995). This
suggests that there exists a barrier at gut mucosal levels; it
is critical to overcome this barrier completely in order for
the bacteria to be able to proliferate in the organs and to
cause lethal infection. Finally, complementation of the OC
gene cluster defect by reversed allelic exchange restored
the virulence to the WT level, thus ful®lling Koch's molecular postulates for the identi®cation of a virulence factor
(Falkow, 1988; Gulig, 1993). To the best of our knowledge,
this represents a novel situation with respect to the importance attributed to the different LPS sections because it
demonstrates how OC structures that depart from the
classical inner core to O-chain bridge can play a role in
virulence. Moreover, if the suggestion that the OC operon
is a relic of an ancestral heteropolymeric O-antigen operon
is correct, the critical role of OC might explain why it has
not been completely replaced by the homopolymeric Oantigen gene cluster. Indeed, this interpretation is consistent
1454 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
Fig. 6. Immunoblottings of selected sera of mice that survived the i.v. rechallenge of YeO3 bacteria (see Tables 2 and 3).
A. Whole-cell lysate of YeO3-trs11 was separated with SDS±PAGE and electroblotted onto transfer membrane that was cut into strips. The
strips were incubated with 1 : 200 diluted sera from mice infected with indicated bacterial strains, and the bound speci®c antibodies were
detected by peroxidase-conjugated rabbit anti-mouse immunoglobulin antibodies. Note that the leftmost strip was misaligned (the bands
appear a few mm lower than the other strips). The serum used in the strip marked with the ®lled vertical arrow was used in 1 : 500 dilution in
immunoblotting shown in (B), in which whole cell lysates of the indicated bacterial strains were separated with SDS±PAGE and electroblotted
onto transfer membrane. Open arrows indicate the antibodies that have speci®cally reacted with the complete core present in YeO3-c and
YeO3. The typical broad band of the core can be clearly distinguished. The ®lled arrows show the virulence plasmid-speci®c bands present in
the four middle lanes, but not in the pYV YeO3-c and YeO3-R1 lanes. The asterisk indicates the distorted band in YeO3-trs22, suggesting
the presence of truncated outer core at that position. The homopolymeric O-antigen of Y. enterocolitica O:3 forms a smear in the upper half of
the gel, and speci®c antibodies reacting with it are seen as background smear in all the lanes except in the O-antigen-negative YeO3-R1 lane
in which the background is clear.
with the evidence presented here (see below) on the complementary roles of OC and O-antigen.
The co-infection experiments showed where YeO3-trs11
(OC ) failed to perform to the level of the WT strain
because they demonstrated that, although still able to
colonize the Peyer's patches as ef®ciently as the WT strain,
the mutant was comparatively unable to multiply in mesenteric lymph nodes, spleen and liver, and was cleared much
faster from these organs than from the Peyer's patches.
These results suggest that the OC-de®cient bacteria are
particularly sensitive to host defences of deeper tissues
(including phagocytosis and killing) that are successfully
overcome by the WT bacteria. It has to be stressed that
these observations cannot be due to a de®cient expression
of pYV-encoded factors in the mutants. First, the effects of
YadA expression were clearly observed in the serum
resistance experiments. Second, the immunoblots revealed
antibodies against YadA and other pYV-encoded proteins
in the sera of mice infected with OC mutants, but not of
mice infected with YeO3-c (Fig. 6A). These observations,
and the demonstration of antibodies by EIA, also suggest
that opsonizing antibodies could contribute to the elimination of the mutants from the Peyer's patches (see also
below). In vitro phagocytosis and killing experiments with
isolated mouse macrophages, polymorphonuclear granulocytes and/or monocytes should elucidate these questions.
In conclusion, all the preceding data support an important
role for OC in delaying the host-mediated killing.
The presence of an almost identical OC in many of the
so-called `environmental' or non-pathogenic Y. enterocolitica serotype strains in addition to many pathogenic Y.
enterocolitica serotype strains (Skurnik et al., 1995) (E.
ErvelaÈ and M. Skurnik, unpublished) raises the question
of the real status of the OC as a virulence factor. A virulence
factor can be de®ned as one that interacts directly with the
host defence effectors. If antimicrobial cationic peptides
are considered as defence effectors, then OC is a true virulence factor. As discussed below, we feel that OC also functions indirectly as a virulence factor by forming the pedestal
to the `true' virulence factors such as Ail and YadA.
OC mutants elicit protective immunity
The OC mutants were able to induce protective host
responses against i.v. administered WT bacteria (Tables
2 and 3). This was shown in two complementary experiments. In experiment 1 (Tables 2 and 3), all the mice
were rechallenged i.v. using a relatively small dose to ®nd
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1455
whether any protective immunity was generated at all, and
in experiment 2 (Table 2) higher doses were used to
assess protection more quantitatively. As expected, the
most potent protective responses were generated in mice
that survived the ®rst challenge with the WT bacteria
because they were able to clear <106 rechallenging bacteria. The OC mutants YeO3-trs11 and YeO3-trs24 did
not generate as strong protection, but lower rechallenging
doses were cleared ef®ciently. Interestingly, protective
responses were generated in mice infected with the 105
dose of YeO3-trs24, in contrast to YeO3- and YeO3trs11-rev-infected mice in which no protective responses
were raised by the 105 doses (Table 2). Also, the good
protection afforded by the OC mutants YeO3-trs11 and
YeO3-trs24 was in marked contrast to the low protection
elicited by YeO3-R2 or YeO3-c (Table 2). If, in future, it
is necessary to generate a live vaccine Y. enterocolitica
strain, OC mutants would make good candidates because
of the good virulence/protection ratio. The persistence of
OC mutants in Peyer's patches could be used to induce
mucosal immunity in the gut. One could also envisage
the use of OC mutants as vehicles to carry foreign antigens to intestinal mucosa.
In general, the OC mutants YeO3-trs24 and YeO3-trs11,
the latter in particular, induced weak humoral responses.
The WT bacteria YeO3 and YeO3-trs11-rev induced
stronger responses, but the strongest response was
clearly induced by YeO3-c (pYV ) after both i.g. and i.v.
challenge. Autenrieth et al. (1992; 1996) have presented
data suggesting that in mice protective immunity against
Y. enterocolitica is T-cell dependent. Although the fact
that no absolute correlation existed between protection
and the humoral responses suggests a role for cellular
immunity, antibodies must also play a role. Two observations support this last conclusion: (i) better protection was
elicited by the WT bacteria than by the OC mutants, and
the latter had <fourfold lower antibody titres by EIA with
no clear speci®city differences by immunoblot; and (ii)
YeO3-R2 generated a very poor protection (Table 3). It
is likely that the absence in YeO3-R2-immunized mice of
the O-antigen-speci®c antibodies, previously shown to be
protective in passive immunizations (Skurnik et al., 1996),
causes the difference. It is known that antibodies against
polysaccharide antigens are T-cell independent.
O-antigen and OC play different roles during infection
Although both O-antigen and OC were required for full virulence, these two LPS parts clearly have different roles during infection because: (i) in co-infection experiments,
YeO3-trs11 (OC ) was able to survive in Peyer's patches
during the ®rst 2 days after infection, whereas YeO3-R2
(O3 ) was rapidly eliminated; and (ii) YeO3-R2 was less
ef®cient in stimulating immunity against WT bacteria
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
than YeO3-trs11 (Table 2). These results indicate that the
O-antigen is needed during the very ®rst hours of infection,
whereas the OC is needed later. Thus, speci®c immunity
against the O-antigen would dismantle the intruding pathogen very ef®ciently during the early stages of infection.
Indeed, supporting this conclusion, monoclonal antibodies
speci®c for the O-antigen protected mice ef®ciently against
i.v. administered WT bacteria, whereas those against the
core did not (Skurnik et al., 1996). That OC and O-antigen
play different roles is also in keeping with their different
importance in modulating interactions with antimicrobial
peptides and in assisting the Ail- and YadA-mediated
serum resistance (see below).
Differences among OC mutants may also relate to
different degrees of OC de®ciency
The three OC mutants (YeO3-trs11, -trs22 and -trs24)
showed phenotypic differences that were revealed in
phage susceptibility, serum resistance, colony morphology
on DOC-containing media, and susceptibility to DOCmediated lysis. These differences may relate to the degree
of OC de®ciency. In YeO3-trs11, the mutation is polar, and
thus no unbalanced expression of the OC genes takes
place. However, LPS of the deletion mutants (YeO3-trs22
and -trs24) may carry a truncated OC and, indeed, this
was suggested for YeO3-trs22 by the LPS analyses (Fig.
2). This possibility was also supported by the serum resistance experiments in which YeO3-c-trs22-R was almost
as resistant as YeO3-R1 (not shown). It is also possible
that the mutants accumulate biosynthesis intermediates
that affect the cell envelope function, and furthermore
may activate regulatory feedback circuits. Studies are
under way to elucidate these possibilities more closely.
The higher level of humoral response induced by YeO3trs24 in comparison with YeO3-trs11 (Fig. 5) may also
re¯ect a third possibility. All the OC gene cluster genes
except for wbcP are functional and most probably
expressed in YeO3-trs24. It is possible that some of the
encoded enzymes participate in biosynthesis of another
oligosaccharide or polysaccharide species of Y. enterocolitica O:3. These would not be expressed in the polar
mutant YeO3-trs11 but would be in YeO3-trs24 and might
even have some importance during infection; this would
explain the higher level of humoral response detected by
EIA. Furthermore, if these other polysaccharides were
antigenic, then speci®c antibodies should be present in
the mouse sera from WT- and YeO3-trs24- but not in
YeO3-trs11-infected animals. Preliminary attempts to identify such antigens by immunoblotting have been unsuccessful, but its detection could be hampered by the strong
antibody response against protein antigens. A search for
these putative novel polysaccharides has been initiated
by chemical analysis.
1456 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
OC is required for OM integrity
The evidence obtained by probing the OM of live cells and
the LPS aggregates with antimicrobial peptides, the effects
of DOC on both colony morphology and cell lysis, and the
sensitivity to other hydrophobic agents prove that OC is
required for the integrity and functionality of the OM. The
presence of OC was unequivocally associated with both
a resistance to the OM-disturbing action of bactericidal
polycationic peptides and a reduced binding by both cells
and LPS aggregates. Moreover, the resistance to polycations was more marked in the O3 OC ‡ than in the
OC O3 ‡ strains. Thus, although the results with the WT
bacteria also showed that both LPS sections are relevant
in polycation resistance, it can be concluded that the role
of the OC was, in this regard, more important than that
of the O-antigen. In other enteric bacteria, the O-antigen
hinders the access of polycationic antimicrobial peptides
to inner anionic LPS targets (Peterson et al., 1986), and a
similar effect should be produced by the 6d-Alt homopolymer
that constitutes the O-antigen of Y. enterocolitica O:3.
However, in these enteric bacteria, the role of the O-antigen is less important than that played by modi®cations of
the lipid A (Helander et al., 1994; 1996; Nummila et al.,
1995), some of which are regulated by virulence genes
(Guo et al., 1997). Such modi®cations include arabinosamine substitutions in the lipid A which result in reduction
of both the negative charge and the polycation binding.
Interestingly, OC does not contain amino sugars, and so
a modulation of the overall charge of the inner LPS sections
by the OC hexasaccharide cannot be postulated. In contrast, its position as an outer branch and its size suggest
that the OC could sterically hinder bactericidal peptides
(and serum factors, see below). If so, this would represent
a novel mechanism for polycation resistance that, in addition, occurs at core level rather than at lipid A level. Consistent with the role postulated for OC in virulence, LPS
modi®cations leading to polycation resistance are thought
to be linked to virulence in other pathogenic bacteria
(Groisman, 1994; Guo et al., 1997).
The hindrance caused by the OC could be direct and/or
through interactions with other LPS molecules and OM
components. No differences in the OM protein pro®les
of the WT and OC mutants were detected, thus making
unlikely the possibility that OM protein assembly would
be affected by the LPS defect which happens in deep
rough mutants of Escherichia coli (de Cock and Tommassen, 1996). In E. coli and Salmonella typhimurium, LPS±
LPS interactions are very strong in the presence of Mg 2‡,
in all likelihood because this cation bridges the negatively
charged groups (phosphate and Kdo) of the core and lipid
A which, signi®cantly, also act as targets for polycations.
In these bacteria, such tight bridging excludes phospholipids from the outer lea¯et and, as illustrated by the
swift permeabilization caused by EDTA, it is essential for
OM integrity (Nikaido and Vaara, 1985). However, the
OMs of Y. enterocolitica are simultaneously ef®cient barriers to hydrophobic permeants and insensitive to EDTA,
and it has been suggested that this is due to some core
lipid A structural peculiarity also linked to resistance to
polycations (Bengoechea et al., 1996). Obviously, the OC
represents such structural peculiarity with respect to resistance to polycations and, by establishing interactions with
adjacent LPS molecules, could also stabilize the OM without a critical role for Mg 2‡, thereby contributing to the
resistance of hydrophobic permeants. This hypothesis is
supported by the observed increase in both the NPN
uptake by aggregates that contained OC-de®cient LPS
and the sensitivity to DOC and hydrophobic agents of
the OC mutants.
OC and O-antigen have different roles in assisting
serum resistance by Ail and YadA that may re¯ect
OM topology
The serum killing experiments con®rmed that YadA and,
to a lesser extent, Ail are the factors involved directly in
serum resistance of Y. enterocolitica O:3; no role for the
O-antigen or the OC was detected when these two proteins
were not expressed. However, both LPS sections seemed
to assist those factors, although in a different way. More
detailed analysis of the reactivities of individual complement
components with the different Y. enterocolitica O:3 mutants
is under way, and the results will be reported in a separate
communication.
In vivo role of OC
As discussed above, the OC de®ciency allows increased
binding of polymyxin B to LPS. In vivo, a similar effect
would result in increased binding of BPI, lysozyme and/
or lactoferrin to LPS (Elass-Rochardt et al., 1998) and in
inhibition of the endotoxic activity of LPS. This would result
in decreased activation of macrophages and monocytes. If
this scenario is true, then the signi®cantly lower antibody
titres present in YeO3-trs11-infected mice would be understandable because of decreased activation of immunocompetent cells. In contrast, a clearly decreased antibody
response in mice infected with the WT bacteria (YeO3)
was observed when compared with antibody responses
in mice infected with YeO3-c (pYV ). This is in concordance with the earlier ®ndings that pYV encodes immunosuppressive factors (Autenrieth et al., 1995; Nakajima et
al., 1995; Nedialkov et al., 1997). Given the importance
of the OC for virulence, the poor humoral response
induced by YeO3-trs11 may seem paradoxical. However,
the co-infection experiments elucidated this question.
The infection by YeO3-trs11 was largely restricted to the
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1457
Peyer's patches, and this may be responsible for the lower
level of stimulation of the humoral response. Indeed, mice
infected i.v. with the mutant YeO3-trs11 induced higher
levels of humoral response.
Experimental procedures
Bacterial strains and plasmids and culture conditions
Table 1 lists the bacterial strains, plasmids and bacteriophages
used and constructed in this work. For the Y. enterocolitica O:3
WT strain 6471/76, we used designation YeO3, and for its virulence plasmid-cured derivative 6471/76-c we used YeO3-c. All
the Y. enterocolitica strains used in this work are derivatives of
YeO3 and YeO3-c. Bacteria were routinely cultured in Luria
broth (LB) or tryptic soya broth (TSB) or on Luria agar (LA)
plates. Y. enterocolitica strains isolated from infected animals
were grown on Yersinia selective agar plates (CIN agar, Oxoid)
supplemented with appropriate antibiotics. E. coli strains were
grown at 378C and Y. enterocolitica strains at room temperature (228C) unless otherwise indicated. Antibiotics were added
to the growth media when appropriate at the following concentrations: kanamycin (Km), 100 mg ml 1 in agar plates and
20 mg ml 1 in broth; chloramphenicol (Clm), 20 mg ml 1; streptomycin (Sm), 25 mg ml 1; and cycloserine, 2 mg ml 1.
Nucleic acid manipulation
Routine techniques for plasmid isolation, restriction digestion,
ligation, transformation, and electroporation were used (Ausubel et al., 1987; Sambrook et al., 1989). PCR was performed
as suggested by the supplier of the thermostable DNA polymerase DynaZyme II (Finnzymes). Reaction conditions for
PCR cycles were adjusted according to the oligonucleotide
primers used and the length of the ampli®ed fragment. Mobilization of plasmids from E. coli strains to Y. enterocolitica
strains were performed as described earlier (Skurnik et al.,
1995). The suicide vector pRV1 derivatives were maintained
in E. coli SY327lpir or in Sm10lpir (Table 1). In some transformations, a rapid polyethylene glycol-mediated bacterial colony
transformation method was used (Kurien and Sco®eld, 1995).
Construction of OC mutants
The construction of wbcK-L deletion mutant was started from
plasmid pRV19 from which a 706 bp XbaI fragment was
deleted, and the resulting plasmid was named pRV22 (Table
1). pRV22 was transformed into E. coli Sm10lpir from where
it was introduced by mobilization into YeO3 and YeO3-c and
in which the plasmid integrated by homologous recombination into the chromosome. The bacteria with an appropriate
second crossing over were enriched by cycloserine as described earlier (Skurnik et al., 1995). DNA from the resulting
Clms transconjugants was analysed by PCR using primers
from both sides of the XbaI fragment, and transconjugants
with the desired deletion were selected and named as
YeO3-trs22 and YeO3-c-trs22. Southern blotting was performed to con®rm the deletion of the XbaI fragment from
the OC operon (see Fig. 1).
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
The construction of wbcP deletion mutants was started
from plasmid pRV17 (Table 1). Plasmid pRV20 was ®rst constructed by deleting from pRV17 the ClaI fragments carrying
the 58 end of the OC operon until sequence position 5565;
note that in pRV17 there was one ClaI site upstream of the
hemH gene in the vector pTM100. Plasmid pRV20 was partially digested with XhoII, ligated and transformed into E.
coli C600. This resulted in plasmid constructs which lacked
different parts of the wbcP gene (Table 1). The StuI±NruI fragments from the plasmids were puri®ed and cloned into the
EcoRV site of the suicide vector pRV1. The new plasmids
were named pRV24±pRV27, and they had 929, 1049, 566
and 363 bp deletions in the wbcP gene respectively (Table
1). From E. coli Sm10lpir , these plasmids were mobilized into
YeO3 and YeO3-c. The transconjugants were subjected to
the cycloserine enrichment, ClmS transconjugants were selected and the deletions were con®rmed by PCR using primers
from both sides of the XhoII fragments (not shown). The
resulting wbcP deletion derivatives were named YeO3-trs24
to -trs27 and YeO3-c-trs24 to -trs27 (Table 1).
Construction of YeO3-trs11-rev by reverse allelic
exchange
The construction of the suicide plasmid for reversing the sitedirected mutation in YeO3-trs11 was started by cloning from
pAM100 the 5.5 kb SphI fragment, which contains the nucleotide sequence of the OC operon between positions 2009 and
7362 and a short piece of pBR322, into suicide vector pRV1,
and the resulting plasmid was named pRV32. From E. coli
SY327lpir , pRV32 was transformed using the polyethylene
glycol colony transformation method (Kurien and Sco®eld,
1995) into E. coli Sm10lpir , which then mobilized pRV32 into
YeO3-trs11. A transconjugant having pRV32 integrated by
homologous recombination (YeO3-trs11::pRV32, Fig. 1) was
subjected to cycloserine enrichment to select a derivative
that had deleted, by a second homologous recombination
event, the Km GenBlock and the vector sequences from the
genome. Chromosomal DNA from three of the resulting KmS
ClmS clones was analysed by Southern hybridization (Fig.
1B) to verify the proper insertion of the WT OC gene cluster
sequence back into the genome of YeO3-trs11. One clone
with the right hybridization pattern was selected and named
as YeO3-trs-11-rev.
Isolation and analysis of LPS
LPS samples were prepared by the small-scale hot phenol±
water extraction method from 3 ml of the different Y. enterocolitica bacteria grown overnight in 5 ml of TSB at RT
(Zhang and Skurnik, 1994), analysed in DOC-PAGE and the
LPS bands were visualized by silver staining as described
earlier (Skurnik et al., 1995). Alternatively, SDS±PAGE and
silver staining for LPS were performed as described previously (Tsai and Frasch, 1982). For large-scale extractions
of LPS from YeO3, YeO3-trs11 and YeO3-R2, bacteria
were grown in TSB in 2 l ¯asks (800 ml per ¯ask) in an orbital
shaker (200 r.p.m.) for 24 h at 378C. The LPSs were obtained
with the phenol±water method as previously described
(Westphal and Jann, 1965). To purify the LPSs, the crude
1458 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
preparations were dispersed (10 mg ml 1 ) in 0.8% (w/v) NaCl/
0.05% (w/v) NaN3 /0.1 M tris-HCl (pH 7), digested ®rst with
nucleases and then with proteinase K as described previously
(Ames et al., 1974). The puri®ed LPSs were sedimented by
ultracentrifugation (6 h, 100 000 ´ g ), and freeze dried. The
Kdo content was determined by the thiobarbituric acid
method (DõÂaz-Aparicio et al., 1993) for each LPS; being
2.72% for YeO3 LPS, 4.17% for YeO3-R2 LPS and 3.70%
for YeO3-trs11 LPS. The protein content of each LPS was
determined by the modi®ed Lowry method (Markwell et al.,
1978) and was less than 1%.
Serum resistance experiments
Human sera containing no speci®c antibodies for Y. enterocolitica serotype O:3 were obtained from eight healthy persons,
pooled, divided into small aliquots, and stored at 708C until
use. Before use, sera were thawed on ice, and were never
used refrozen. Complement was inactivated by incubating
the sera at 568C for 30 min
For the serum bactericidal tests, bacteria were grown overnight in 5 ml of MedECa (Skurnik, 1985) at RT or 378C. The
cell density in the overnight cultures was about 3±6 ´ 108
bacteria ml 1. For the tests, bacteria were diluted into PBS,
as indicated. Serum resistance was assayed using the direct
plating method, modi®ed from the one described by Olling
(1977). From the overnight culture of bacteria, 1 : 10 000±
1 : 50 000 dilutions were prepared so that 10 ml of the dilution
would contain about 300±600 bacteria. From the dilutions,
10 ml samples were mixed with 20 ml of normal serum or
inactivated serum giving a ®nal serum concentration of
66.7%. The mixtures were then incubated at 378C for 30 or
120 min, after which 50 ml of brain±heart infusion broth (BHI)
was added to each mixture to stop the complement function.
The tubes were kept on ice until the contents of each tube
were plated on LA plates to determine the viable bacterial
numbers in the samples. The experiments were carried out
in duplicate and repeated several times. The serum bactericidal effect was calculated as survival percentage, taking the
bacterial counts obtained with bacteria incubated in inactivated
serum as 100%.
Fluorimetry
NPN (Ferosa) is a hydrophobic ¯uorescent probe whose
¯uorescence (quantum yield) increases in hydrophobic
environments (i.e. membranes and LPS moiety), and that is
ef®ciently excluded by Y. enterocolitica (Loh et al., 1984; MartõÂnez de Tejada and MoriyoÂn, 1993; MartõÂnez de Tejada and
MoriyoÂn, 1995; Bengoechea et al., 1996; Freer et al., 1996).
Viable cells. Fluorimetric assays were carried out as
previously described (MartõÂnez de Tejada and MoriyoÂn,
1995; Bengoechea et al., 1996) with minor modi®cations.
The bacterial strains were stored in 10% skimmed milk
powder in water at 808C, and, for the experiments carried
out with the pYV ‡ isogenic pairs, inoculi were taken directly
from the frozen seeds to minimize the loss of pYV. All were
grown in TSB in sidearm ¯asks on an orbital shaker
(250 r.p.m.) at 378C, and growth was monitored by measuring the OD 540 . Cells were harvested (5000 ´ g, 20 min, 58C)
in the exponential phase of growth, resuspended in 1 mM
KCN/2 mM HEPES, pH 7.2, at an OD 600 of 0.5 and
transferred immediately to 1 cm ¯uorimetric cuvettes. One
minute before addition of NPN, the polycationic agents were
added at the following ®nal concentrations: polymyxin B,
3 mg ml 1; poly-L-lysine, 40 mg ml 1; poly-L-ornithine, 40 mg
ml 1; and melittin, 10 mg ml 1. Polymyxin B (8000 units mg 1;
molecular weight 1385), poly-L-lysine (molecular weight
7000±10 000), poly-L-ornithine (molecular weight 12 000±
22 000) and melittin were all purchased from Sigma. After
stabilization of the mixture, NPN (500 mM in acetone) was
added at a ®nal concentration of 10 mM, and the ¯uorescence
measured in relative ¯uorescence units (RFU) at 378C in a LS50 ¯uorimeter (Perkin-Elmer) set as follows: excitation
350 nm; emission 420 nm; slit width 2.5 nm (quenching
under these experimental conditions was not observed).
For each strain or isogenic pair, two independently grown
batches of bacteria were used in the above-described
¯uorescence measurements, and each measurement was
repeated twice.
LPS aggregates. In preliminary experiments, it was found
that LPS of some mutant strains did not produce stable
suspensions suitable for ¯uorimetric measurements. To
circumvent this problem, LPS aggregates and hybrid
aggregates were prepared as described previously (Rudbach
et al., 1976). Brie¯y, 2 mg of LPS (for hybrid aggregates,
1 mg of each type of LPS) was suspended in 2 ml of doubledistilled water, and the suspension was sonicated for 30 s at
maximum frequency. After this, 2 ml of 2% DOC in 0.1 M trisHCl (pH 8.5) was added and the mixture was incubated for
15 min at room temperature. The formed aggregates were
precipitated with six volumes of cold ethanol at 208C for
18 h, centrifuged at 10 000 ´ g, washed with ethanol twice
and resuspended in water. The aggregates were extensively
dialysed against double-distilled water, and ®nally adjusted to
a concentration of 400 mg ml 1.
These aggregates were tested as follows. Suspensions
containing 400 mg ml 1 were prepared in distilled water, and
1.5 ml transferred to 1 cm ¯uorimetric cuvettes. NPN was
added at a ®nal concentration of 10 mM, and ¯uorescence
was monitored as described above. The interaction of polymyxin B with the LPS aggregates was tested by adding the
polycation to a ®nal concentration of 10 mg ml 1 before NPN.
For all LPS aggregates, three measurements were performed.
Quenching under these experimental conditions was not
observed.
Permeability to lysozyme
Bacteria were grown as described above for ¯uorimetry and
the cells were resuspended in 2 mM HEPES (pH 7.1) at an
OD 500 of 0.8, and lysozyme (3 mg ml 1 ) and polymyxin B
(9 mg ml 1 ) were added. The suspensions were incubated at
378C, and cell lysis was monitored by the decrease in the
OD 500 . Lysozyme or polymyxin B alone did not produce any
OD decrease under these experimental conditions. Results
were expressed as percentages of the optical density of controls incubated in the absence of lysozyme and polymyxin B.
All experiments were run with quadruplicate samples from two
independently grown batches of cells.
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
Y. enterocolitica O:3 lipopolysaccharide outer core 1459
Binding of polymyxin B by viable cells
The assay described by Freer et al. (1996) was carried out with
minor modi®cations. Brie¯y, cells grown as described above for
¯uorimetry were resuspended in 2 mM HEPES, pH 7.5, at
<1.25 ´ 1010 cfu ml 1, and 100 ml aliquots were mixed with
12 ml containing different amounts of polymyxin B. After 5 min
incubation at 378C, the cells with the bound polymyxin B
were sedimented (12 000 ´ g, 10 min), the supernatant centrifuged two more times under the same conditions, and the
unbound polymyxin B measured in a bioassay. To this end,
3-mm-diameter wells were punched on standard Petri dishes
containing 10 ml of 1% (w/v) glucose/1% (w/v) peptone/0.8%
(w/v) yeast extract/1% (w/v) agarose (type II-A, medium electroendosmosis; Sigma Chemical) previously inoculated with
E. coli O111 (6.1 ´ 105 cfu ml 1 of molten medium equilibrated
at 408C). The wells were ®lled with 5 ml of the supernatants,
and the plates were incubated overnight at 378C in a wet
chamber. The diameter of zones of growth inhibition around
wells were measured to quantify polymyxin B antimicrobial
activity, and the results expressed in units (10 units ˆ 1 mm).
Sensitivity to hydrophobic agents
Sensitivities to crystal violet, malachite green, rifampicin and
novobiocin (all purchased from Sigma) were assessed on
Mueller-Hinton agar using the disk diffusion test (Ames et al.,
1974). The disks (concentration disks, é 6.5 mm, Difco Laboratories) were prepared by loading per disk 40 mg crystal violet,
40 mg malachite green, 50 mg novobiocin, or 30 mg rifampicin
dissolved in 20 ml distilled water. The disks were dried overnight at 378C. Fresh, exponentially growing bacteria grown
as described for ¯uorimetry were resuspended in saline to a
®nal concentration of 108 cfu ml 1, a lawn was prepared on
the agar plates with a sterile swab, and the antibiotic-loaded
disks were placed on the lawns. After incubation for 18 h at
378C, the diameters of the inhibition halos (in mm) were
recorded. All experiments were performed with duplicate
samples from two independently grown batches of bacteria,
with similar results.
DOC sensitivity experiments were performed as previously
described (Bengoechea et al., 1998b) with minor modi®cations.
Exponentially growing bacteria were resuspended in 2 mM
HEPES (pH 7.2) to an OD 450 of 0.21, and 2 ml was transferred
to two tubes: one containing 200 ml of 10% DOC in 2 mM
HEPES, and the other, the control tube, 200 ml of 2 mM
HEPES. Thus, the ®nal DOC concentration was <1%. The
tubes were incubated for 60 min at the growth temperature
of the bacteria, and then the OD 450 of the suspensions was
measured. The DOC sensitivity of the bacteria was seen as
cellular lysis, which was measured as decrease in OD 450 ,
and the results were expressed as percentage turbidity of
the DOC tube from the control tube without DOC. When the
effect of metabolic inhibitors potassium cyanide and sodium
arsenate (®nal concentration of 1 mM) on DOC sensitivity
was assessed, the ®nal concentration of DOC was 0.5%. All
experiments were performed in duplicate with two independently grown batches of bacteria.
Animal experiments
The animal work described in this article has been approved
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1443±1462
by the ethical committee of the University of Turku and it has a
formal authorization of the Province of Western Finland's government (decision no. 11735TT). Animal experiments were
performed on three separate occasions (165 mice in experiment 1, 110 in experiment 2, and 132 in experiment 3). Inbred
female DBA/2, 6- to 8-week-old mice, tested to be free of the
microbial pathogens, were purchased from Bomholtgarden.
The mice were maintained under ®lter tops, and kept after
receipt from the breeder for 2 weeks before the onset of the
experiments to adjust the mice to the housing conditions. Virulence experiments in mice using Y. enterocolitica serotype O:3
strains require i.p. injection of the iron-chelating compound
desferal (desferroxamine) (Autenrieth et al., 1994; RobinsBrowne and Prpic, 1985), a treatment without which the
bacteria do not kill the mice. Thus, mice were injected intraperitoneally (i.p.) with 10 mg of desferal (Ciba Geigy) in
100 ml of sterile water 1 day before administration of the test
bacteria (Robins-Browne and Prpic, 1985).
The bacteria for animal experiments were grown in 200 ml
of LB, supplemented with appropriate antibiotics when necessary, in 2 l Erlenmeyer ¯asks under aeration for 16±18 h at RT.
The bacteria were pelleted and resuspended into 10 ml of
phosphate-buffered saline, pH 7.4 (PBS). Three 1 ml portions
of the suspension were centrifuged in preweighed Eppendorftype tubes at 14 000 r.p.m. for 10 min. The supernatant was
carefully but completely removed, and the wet weight of the
bacterial pellet was determined from the three tubes. One
hundred milligrams of bacterial pellet contains about 1011 bacteria, and the mean of the three determinations was used to
adjust the bacterial suspension to 1010 bacteria ml 1. From
this suspension, serial 10-fold dilutions were prepared, and
100 ml of appropriate dilutions were used for animal inoculations. In addition, to determine exact bacterial counts, samples
from dilutions estimated to contain 103, 102 or 10 bacteria per
100 ml were plated on LA plates containing antibiotics when
appropriate.
Mice that were inoculated i.g. were kept without solid food
for 4±12 h before bacterial challenge. The bacterial suspension (100 ml) was administered i.g. directly to the stomach of
the mice using a 20 gauge stainless-steel ball-tipped catheter.
For i.v. inoculations, the bacterial suspensions were injected
into the tail veins of the mice using a 30 gauge needle and a
light table to warm the tail and help visualization of the vein.
In virulence experiments, the mice were followed daily for
35 days and the deaths were recorded. For infection kinetics
experiments, mice were inoculated i.g. with a single bacterial
dose of <108 bacteria per mouse and killed 6, 48 and 168 h
after the injection of the bacteria. Peyer's patches, mesentery,
spleen, and liver were aseptically removed, weighed and
homogenized into 0.5, 2, 2 and 5 ml, respectively, of PBS.
Presence of Y. enterocolitica was quanti®ed by determining
the bacterial numbers by plating serially diluted samples on
CIN plates supplemented with appropriate antibiotics when
necessary.
For co-infection experiments, nine mice were inoculated i.g.
with a bacterial mixture containing <109 YeO3 and 109 YeO3trs11 bacteria. The mixture was serially diluted and appropriate dilutions were plated on LA plates to determine exact
bacterial counts. Two hundred colonies were patched on Kmcontaining plates to determine the percentage of YeO3-trs11
bacteria in the inoculation mixture. Three mice at a time
1460 M. Skurnik, R. Venho, J.-A. Bengoechea and I. MoriyoÂn
were killed 2, 5 and 9 days after infection, and their spleens,
livers, mesenteric lymph nodes and Peyer's patches were
prepared as above and homogenized into 1, 3, 1 and 0.5 ml,
respectively, of sterile PBS. Y. enterocolitica from the homogenates and serial dilutions thereof were recovered on CIN
plates without antibiotics. Percentage of YeO3-trs11 was
determined from the recovered colonies by patching 100 or
all the recovered colonies (if less than 100) onto Km-containing
plates. Another co-infection experiment was performed with
strains YeO3 and YeO3-R2. The experiment was carried out
exactly as described above, except that percentage of
YeO3-R2 was determined by patching the colonies on LA
plates saturated with bacteriophage fYeO3-12. YeO3-R2 is
resistant to this phage whereas YeO3 is susceptible, allowing
the identi®cation of the YeO3-R2 colonies among the YeO3
colonies.
To monitor the development of protective immunity after i.g.
infections, the mice that survived the 35 days after the primary
i.g. or i.v. challenge with different strains were given 10 mg
of desferal as described above, and the following day were
challenged with 200 YeO3 bacteria i.v. in experiment 1 and
with different doses of YeO3 in experiment 2. These mice
were followed daily for 15 days and the deaths were recorded.
The mice that survived the secondary i.v. challenge in experiment 1 were killed, blood samples were recovered and sera
separated from the blood after clotting. The sera were stored frozen at 208C until tested for speci®c antibodies against YeO3.
Enzyme immunoassay (EIA)
The wells of a 96 well microtitre plate (Nunc) were coated with a
100 ml volume of antigen in PBS overnight at RT. An SDS
extract of whole bacteria (0.5 mg of protein ml 1 ), prepared as
described earlier (Granfors et al., 1989), was used as antigen.
After washing three times with PBS, the wells were blocked by
100 ml of 5% skimmed milk powder in PBS (SM-PBS) for 2 h at
RT, and then washed again three times with PBS. The mouse
sera were diluted in SM-PBS to obtain dilutions 1 : 240, 1 : 960,
1 : 3840 and 1 : 15360 (the sera giving no or very low absorbances at 1 : 240 dilution were retested using 1 : 30 and 1 : 120
dilutions), and 75 ml of the dilutions were incubated in the
wells for 1.5 h at RT and washed four times with PBS. Then,
80 ml of rabbit horseradish peroxidase-conjugated immunoglobulin against mouse immunoglobulins (P260, Dako) diluted
1 : 1000 in SM-PBS was added and incubated for 1 h at RT.
The plates were washed four times with PBS and 80 ml of substrate solution [3 mg ml 1 of 1,2-phenylenediamine and 0.02%
H 2O2 in citrate buffer (per litre: 4.97 g of citric acid.H 2O and
9.9 g of Na2HPO4 .2H 2O)] was added and incubated for
30 min at RT. The reactions were terminated by adding
130 ml of 1 M HCl to the wells. Optical absorbances were
measured at 492 nm with a Labsystems Multiskan MCC/340
photometer. Each sample was analysed in duplicate, and for
each serum the test was repeated at least twice. For negative
controls, wells with similarly diluted normal mouse serum and
wells without any mouse serum were included. The monoclonal antibody A6, speci®c for the Y. enterocolitica O:3 O- antigen
(Al-Hendy et al., 1991), was used as a positive control.
The mean absorbance values of matched normal mouse
serum dilutions (<0.05±0.13) were deducted from the test
serum absorbances, and the resulting values were plotted
against the dilutions. From the plots, a dilution giving an
absorbance value of 0.3 was estimated and the reciprocal
of that dilution was used to indicate the speci®c antibody
titre in the serum.
Immunoblotting
Bacteria were grown overnight at 378C in 5 ml of MedECa,
and whole cell lysates were prepared from the bacterial pellet
by resuspending the bacteria in 100 ml of Laemmli sample buffer and heating the mixture at 958C for 10 min before loading
onto the gel. The lysates (<2±3 ml per lane) were separated
in SDS±PAGE using the Miniprotean II device (Bio-Rad) with
12% acrylamide separating gels and 4% stacking gels. The
separated samples were transferred to nitrocellulose membrane (BAS 83, Schleicher and Schuell) using the Trans-Blot
Semi-Dry Transfer Cell (Bio-Rad), according to the manufacturer's instructions. The membrane was blocked overnight at
RT in SM-PBS. After rinsing the ®lter with PBS, it was cut into
strips or processed as a whole membrane and incubated with
diluted mouse sera (1 : 200 or 1 : 500 in SM-PBS) overnight at
RT. The membranes were washed 4´ 10 min in PBS, and then
incubated with 1 : 2000 P260 for 1±4 h at RT. After four washes
as above, the bound peroxidase was detected using the ECL
Western blotting kit (RPN 2106, Amersham International),
according to the manufacturer's instructions.
Acknowledgements
This work was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, the Centre for International Mobility, the Turku University Foundation and from
the Plan de InvestigacioÂn de la Universidad de Navarra.
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