INFECTION ANI) IMMUNITY, Apr. 1994, p.

1275-1281
00 19-9567/94/$04.(0 +O
Copyright ) 1994, American Society for Microbiology

Vol. 62, No. 4

Role of YadA in Resistance of Yersinia enterocolitica to

Phagocytosis by Human Polymorphonuclear Leukocytes
BERNARD CHINA,' BAO THO N'GUYEN,2 MARC DE BRUYERE,_ AND GUY R. CORNELIS'*
Microbial Pathogenesis Unit, International Institute of Cellular and Molecuilar Pathology and Faculte de Medecine,'
and Unite Sang,2 Faculte de Medecine, Universite Catholique de Loluvain, B-1200 Brussels, Belgilum
Received 1 November 1993/Returned for modification 30 December 1993/Accepted 11 January 1994

Pathogenic Yersinia enterocolitica cells do not induce the chemiluminescence response of human polymorphonuclear leukocytes (PMNs). We tested the chemiluminescence response to Y. enterocolitica mutants affected
in the known pW-encoded factors. We did not detect any influence of the Yops in this phenomenon. By
contrast, the presence of YadA correlated with a lack of chemiluminescence. The expression of YadA at the
bacterial surface also reduced the phagocytosis by PMNs. Finally, we measured the survival of Y. enterocolitica
cells confronted with PMNs by the classical plating method and by a new luminometry assay. We observed that
YadA+ bacteria were not killed, while YadA- bacteria were killed. We conclude that the presence of YadA at
the surface of Y. enterocolitica cells prevents phagocytosis and killing by PMNs. This conclusion is in good
agreement with our recent observation that YadA protects Y. enterocolitica from opsonization by C3b.

charide (42), also play a role in this phenotype, YadA seems to

be the major determinant of resistance to complement killing,
since YadA-deficient mutants become serum sensitive (2).
When facing human PMNs, Y enterocolitica strains harboring the pYV plasmid and grown at 37C induce an unusually
low chemiluminescence (CL) response. This lack of burst
correlates with an inhibition of phagocytosis (27, 28). In the
present study, we analyzed the response of PMNs to several
well-characterized pYV mutants (2, 13, 14, 33) to identify the
pYV gene(s) involved in this inhibition.

Yersinzia enterocolitica cells are enterobacteria frequently

involved in human enterocolitis. They have a remarkable
capacity to invade the Peyer's patches and to resist the
nonspecific defense of the host, i.e., killing by complement (2)
and phagocytosis by polymorphonuclear leukocytes (PMNs)
(27) or by macrophages (39). This capacity essentially depends
on the presence of a 70-kb virulence plasmid called pYV. The
pYV plasmid specifies several thermodependent properties,
including the secretion of 11 plasmid-encoded proteins, called
Yops, and the production of at least two outer membrane
proteins, called YadA and YlpA (for reviews, see references 5
and 15). The secretion of Yops involves a machinery of a new
type encoded by (at least) loci virA (IcrA) and virC (1crC) (13,
14, 30-32, 38). A transcriptional activator encoded by gene virF
is required for the production not only of the Yops but also of
YadA and YlpA (10, 12, 14, 25).
The Yop proteins are important pathogenicity determinants
(for a review, see reference 45). For instance, YopH is a
phosphotyrosine phosphatase which confers on Yersinia
pseludotluberculosis an antiphagocytic activity against macrophages (4, 18, 39). YadA is a polymer composed of four to five
subunits with a molecular mass of 45 to 52.5 kDa (41, 43, 48)
and forming a tiny fibrillar structure at the surface of Y
enterocolitica (23, 24, 48). In the experimental oral infection of
the mouse, YadA contributes to the long-term fecal excretion
of bacteria (23). In vitro, YadA plays various roles in relation
to its surface localization. It increases the surface hydrophobicity (29) and promotes autoagglutination (41). It confers
adherence to epithelial cells (19), to ileal mucus (35), and to
the extracellular matrix by binding collagen fibers (17, 40) and
fibronectin (47). YadA also inhibits the anti-invasive effect of
interferon (6). Finally, YadA confers resistance to the bactericidal activity of human serum (2) by promoting the fixation of
factor H (9), which leads to the degradation of C3b deposited
at the bacterial surface (37). As a consequence, the formation
of the membrane attack complex is prevented (37). Although
other factors, such as the Ail protein (3, 36) and lipopolysac-

MATERIALS AND METHODS

Bacterial strains and plasmids. Y enterocolitica W22703
(nalidixic acid resistant) and W22708 (streptomycin resistant)
are two restriction mutants (Res- Mod') of the serotype 0:9
strain W227 (11). Y enterocolitica KNG22703 is a derivative of
W22703 which produces luciferase as a result of the insertion
of genes luxAB in the chromosomal gene blaA (21, 22). The
plasmids used in this study are listed in Table 1.
Bacterial growth conditions. For CL and killing experiments, Y enterocolitica was inoculated at an optical density at
600 nm (OD,(,()) of 0.1 in conical flasks containing 5 ml of brain
heart infusion (Difco, Detroit, Mich.) supplemented with 0.4%
glucose, 20 mM MgCl2, and 20 mM sodium oxalate (BHI-OX)
and containing the appropriate antibiotics. The cultures were
shaken either for 5 h at room temperature or for 2 h at room
temperature and then shifted for 3 h at 37C.
For phagocytosis experiments, bacteria were inoculated at
an GD6001 of 0.5 in a conical flask containing 5 ml of BHI-OX
supplemented with appropriate antibiotics and shaken for 3 h
at 37C. The antibiotics, purchased from Boehringer (Mannheim, Germany), were supplemented at the following final
concentrations: nalidixic acid, 35 p.g ml - '; gentamicin, 20 p.g
ml - 1; kanamycin, 50 p.g ml- 1; and tetracycline, 10 p.g ml l.
Serum. Normal human serum (NHS) was purchased from
Sigma (St. Louis, Mo.). The absence of anti-Yop antibodies
was monitored by immunoblotting as previously described (9).
Preparation of human PMNs. Human PMNs were prepared
from peripheral blood obtained from healthy individuals. A
10-mi volume of human venous blood containing heparin
(Organon Teknika, Boxtel, Holland) (5 U/ml) was mixed with

*
Corresponding author. Mailing address: Microbial Pathogenesis
Unit, University of Louvain, P.O. Box 74.49, Avenue Hippocrate 74,

B-12t)() Brussels, Belgium. Phone: 32 2 764 74 49. Fax: 32 2 764 74 98.

Electronic mail address: CornelisCa-mipa.ucl.ac.be.

1275

1276

INFEC-F. IMMUN.

CHINA ET AL.
TABLE 1. List of plasmids used in this study

Plasmid
pBC7
pBM15
pBM33
pBM79
pGBO8
pGB63
pGB910
pGC216
pGC274
pGC445
pGC559
pGC633
pGC678

pGC1152
pGC1256
pMS153
pYL4
pYVe227

Characteristic(s)

Reference(s)

pYVe227yadA::pBC9
pYVe227yopM-BM15::Tn2507
pYVe227yopQ-BM33::Tn2507
pYVe2271crVyopBD-BM79::Tn2507
pYL4yadA-GB08::Tn813-R388
pYL4-GB63::Tn813-R388
pYL4yadA-GB910::Tn8I3-R388
pGB63yscH-GC216::miniMudlac
pGB63virA-GC274::miniMudlac
pGB63yscC-GC445::miniMudlac
pGB63yopO-GC559::miniMudlac
pGB63virB-GC633::miniMudlac
pGB63virB-GC678::miniMudlac
pGB63yopH-GC1152::miniMudlac
pGB63yopE-GC1256::miniMudlac
pSelect-1; Plac-yadA
pYVe227ylpA-YL4::Tn3
Virulence plasmid of Y enterocolitica W22703

9
33
33
33
2
2
2
13
13
13
14
13
13
14
14
9
2 and 10
11

3 ml of 6% (wt/vol) dextan (Sigma) in saline, and the mixture

allowed to sediment at 37C during 30 min. The supernatant, consisting of leukocyte-rich plasma, was applied to the
upper surface of 5 ml of cold (4C) lymphocyte separation
medium (d, 1.077 0.001) (International Medical, Brussels,
Belgium) and centrifuged (1,000 x g at 4C for 15 min). The
pellet was resuspended in 10 ml of hemolysis solution (0.84%
NH4Cl 0.084%, NaHCO3, 0.043% EDTA), and the solution
was centrifuged for 7 min at 150 x g. This second pellet was
resuspended in 5 ml of Hanks balanced salt solution (HBSS)
(GIBCO, Paisley, Scotland). Cell viability, monitored by trypan
blue dye (Sigma) (final concentration, 0.2%) exclusion, was
greater than 95%. PMNs were counted with a hemocytometer
and adjusted to a final concentration of 107 cells per ml of
HBSS.
CL assay. Luminol-enhanced CL was measured with a
Pico-Lite luminometer (Packard Instrument Co., Downers
Grove, Ill.) by using a modification of the procedure described
by Lian and Pai (28). A 50-pA volume of a bacterial suspension
of luminol (5-amino-2,3in HBSS at an OD600 of 0.6, 50
dihydro-1,4-phthalazinedione; Sigma; 10-3 M), 50 of NHS,
and 100 pL of PMNs (106 cells) was mixed in a tube and placed
immediately in the luminometer cell, which was preheated at
37C. The number of counts per minute during about 20 min
was recorded (see Fig. 1).
Phagocytosis assays. We slightly modified the microscope
assay described by Lian et al. (27). Briefly, 3 x 105 PMNs in 30
of HBSS were placed in each well of a 10-well microscope
slide (Flow Laboratories, McLean, Va.) and incubated for 1 h
at 37C in 6% CO2. Bacteria from 1 ml of culture were
harvested, washed with phosphate-buffered saline (PBS) (13
mM sodium hydrogenophosphate, 2.8 mM potassium dihydrogenophosphate, 135 mM sodium chloride, [pH 7.4]), and
resuspended (OD600 of 0.6) in PBS supplemented with 10 mM
ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA) and 5 mM MgCl2. The bacterial suspension was
first incubated at 37C for 10 min with 5% (final concentration)
NHS and then washed with PBS and resuspended in 1 ml of
HBSS. This preopsonization step did not kill serum-sensitive
bacteria (survival, more than 90%). The microscope slides
containing PMNs were washed with PBS to withdraw the
nonadhering cells. A 30-pA volume of the preopsonized bacterial suspension was added to each well, and the wells were
incubated for 90 min in a 6% CO2 atmosphere. Slides were

was

washed with ice-cold PBS, stained for 1 min with acridine

orange (0.1% [wt/vol] in PBS), counterstained for 3 min with
crystal violet (1% [wt/vol] in PBS), and washed with PBS.
Stained slides were examined under an epifluorescence microscope (Diaplan; Leitz, Wetzlar, Germany) with an oil immersion objective lens. In this assay, intracellular bacteria fluoresce
green when they are viable and red when they are nonviable
(44). Extracellular bacteria were not visible because of the
counterstain. A total of 100 PMNs were counted, and phagocytosis was expressed as the percentage of PMNs containing
intracellular bacteria (green or red).
We also measured phagocytosis by a second method based
on the flow cytometry of fluorescent bacteria (7). Bacteria were
harvested and adjusted to an OD600 of 0.6 in carbonate buffer
(0.016 M Na2CO3, 0.034 M NaHCO3 [pH 9.5]). Live bacteria
were then labeled by the addition of fluorescein isothiocyanate
(FITC; Sigma) to a final concentration of 100 ,ug ml1-' and
incubated at 37C for 30 min. The bacteria were then washed
three times with PBS. According to a plating assay, this
labeling did not affect the viability of the bacteria. It is also
known not to disturb the bacterial surface (34). Labeled
bacteria were preopsonized with 5% NHS at 37C in PBSEGTA-MgCl2 as described above. Then, 100 RI of preopsonized bacteria and 200 ,lA of PMNs suspension (107 cells ml1in HBSS) were mixed in a final volume of 1 ml. This mixture
was shaken (75 rpm) during 1 h at 37C and washed twice with
ice-cold PBS. The cells were resuspended in 1 ml of PBS, and
trypan blue (0.2% final concentration; Sigma) was added in
order to extinguish the fluorescence associated with extracellular bacteria (8). The suspension of the PMNs was then
applied to a fluorescence-activated cell sorter (FACS) (Epics
Elite; Coulter, Hialeah, Fla.). The proportion of fluorescent
PMNs was calculated and considered a measure of the percentage of phagocytosis.
Killing assay. We mixed 100 pLI of suspension of preopsonized bacteria (OD60 of 0.6 in HBSS) in a universal flask with
200 RI of a suspension of PMNs (2 x 107 cells) in a final
volume of 1 ml of HBSS. After 1 h of incubation and shaking
at 37C, 200 RI of mixture was harvested and added to 800 ,ul
of ice-cold distilled water in order to stop phagocytosis and to
lyse the PMNs. A 100-,ul volume of this dilution was plated on
tryptic soy agar (GIBCO) supplemented with appropriate
antibiotics, and the plates were incubated at 28C for 48 h. A
sample of bacteria was treated in parallel under the same
conditions but in the absence of PMNs. The ratio between the
number of CFUs counted from samples incubated with PMNs
and from samples incubated without PMNs gave the percentage of survival.
The use of strain KNG22703 allowed monitoring of survival
also by measurement of the light emitted as a result of the
luciferase activity of this strain (22, 26). The assay was performed as described by Kaniga et al. (22). A 250-pA volume of
bacterial suspension was placed in a tube containing 50 pl of
0.1% n-decanal (Sigma), and the emitted light was scored for
10 s in the luminometer. The ratio between the number of
counts per minute emitted by bacteria that have been exposed
to PMNs and by the bacteria that were not exposed to PMNs
gave the percentage of survival.
RESULTS
CL of PMNs induced by Y. enterocolitica W22703. We first
analyzed the oxidative burst induced by W22703, our wild-type
reference strain of Y. enterocolitica serotype 0:9. pYV+ and
pYV- variants of Y enterocolitica W22703 were grown in
BHI-OX either for 5 h at 25C or for 2 h at 25C and for 3 h

VOL. 62, 1994

YadA AND PMNs

120OPM (low
(100
120C
B

*ooo

1277

0)

--

SOW

Y(pYVe227*) 25 C
'YO(PVe227-) 37 C

400

-C- YO(pYVe227-) 25 C
-**- Y(pYVe227-) 37 C

200-

-*-

(31

PI x

0
0

Time (minutes)

10

Time (minutes)

12

O(PGC216) vIrC

--+- Y(PGC274)
-*-

virA

YO(pGC445) virC

-+- Y(pGBOS)(pUS153)

Y(pGC833) virD
4bpGCS7U) v/rB
0*YO(pVV*227)
I-- YW(pGC1152-9) virF

-X- O(PG8ODO)(OMS163)

-B-

YPG4O3)
---Y(PYfL4)

-4-

1--6
1

10

Time (minutes)

YOPGBO10)
1 2 3 4

lo10
a 7 8
Time (minutes)
5

12 13 14 15

FIG. 1. CL response of human PMNs to Y enterocolitica (Ye). CL is expressed in counts per minute as a function of time expressed in minutes.
Each point represents the mean of three independent experiments. CL responses induced by Y enterocolitica W22703 pYV- and pYV+ incubated
at 25C or at 37C in BHI-OX (A), by Y enterocolitica W22703 and by various yop mutants incubated at 37C in BHI-OX (B), by pleiotropic vir
mutants of Y enterocolitica W22703 incubated at 37C in BHI-OX (C), and by yadA mutants of Y enterocolitica W22708 incubated at 37C in
BHI-OX (D) are plotted. Plasmid pMS153 complements yadA4 mutants..

at 37C. They were subsequently engaged in a CL test (1), with

PMNs in a ratio of 50 bacteria per PMN in the presence of

20% NHS. The photons emitted in the presence of luminol
were counted in a luminometer. Bacteria grown at 25C
induced a strong CL response, irrespective of the presence
or absence of the pYV plasmid (Fig. 1A). By contrast, the
CL response induced by bacteria grown at 37C was different according to the presence or the absence of the pYV
plasmid: Y enterocolitica W22703 pYV- induced a significant
CL response, while the pYVW bacteria induced a five- to
sevenfold-lower CL response, indicating a lack of oxidative
burst.
These results are in agreement with those obtained previously (28, 46) with a Y. enterocolitica strain of serotype 0:3 and
confirmed that the lack of induction of CL by wild-type Y.
enterocolitica correlates with the expression of pYV plasmidencoded functions at 37C.
Role of the Yops in CL inhibition. Since there was a
coincidence between the inhibition of the oxidative burst and
the secretion of Yops, both occurring at 37C and in the
absence of Ca2", we monitored the CL response induced by
mutants affected in the production of one or several individual
Yops. We tested Y. enterocolitica W22703(pGC1256), mutated
in yopE; W22703(pGC1152), affected in yopH; W22703
(pGC559), deficient in yopO and yopP; W22703(pBM15),

mutated in yopM; W22703(pBM33), deficient in yopQ; and

W22703(pBM79), affected inyopB,yopD, and 1crV. All of these
yop mutants induced the same level of CL as the wild pYV+
strain (Fig. 1B), indicating that none of these Yops was
responsible for the inhibition of stimulation of PMNs. Because
we could not test mutants that were deficient in YopN and
YopR (15), we also tested the pleiotropic virA, virB, virC, and
virF mutants. Only the virF mutant induced a significant CL
response (Fig. 1C). Since the virA, virB, and virC mutants do
not secrete any of the Yops but nevertheless inhibit the CL
response, we inferred that neither a particular Yop nor a
combination of Yops was involved in the reduction of the
oxidative burst.
Role of YadA in CL inhibition. We then focused on the
YadA protein, because it is produced by virA, virB, and virC
mutants but not by virF mutants. We tested the yadA ylpA
double mutants W22708(pGB08) and W22708(pGB910), as
well as the parental ylpA mutant W22708(pYL4) (2, 11). We
also tested W22703(pBC7), a yad4 mutant constructed by
recombinational integration of a suicide plasmid (9). The yadA4
mutant and yadA ylpA double mutants induced a strong CL
response, while the ylpA mutant behaved like the wild type
(Fig. 1D), indicating that YadA was responsible for the
inhibition of the oxidative burst.
To confirm the role of YadA, we complemented the three

1278

INFEC'F. IMMUN.

CHINA ET AL.

1pm

1pm

FIG. 2. Light microscopy analysis of phagocytosis of Y enterocolitica by PMNs. (A) Y enterocolitica(pYVe227) incubated at 37C in BHI-OX
in contact with PMNs; (B) fluorescence examination of cells in panel A; (C) Y enterocolitica(pBC7) incubated at 37C in BHI-OX in contact with
PMNs; (D) fluorescence examination of cells in panel C. The arrows indicate individual bacteria; the green fluorescence of the nucleus (n) of an
individual PMN is shown. Magnification, x 1,250.

yadA mutants with plasmid pMS153, which contains an amplified yadA4 gene cloned under the control of the lac promoter
(9). As expected, strains carrying pMS153 produced YadA
(data not shown) and no longer induced a CL response (Fig.
1D). YadA, thus, is involved in the CL inhibition phenomenon.
YadA and phagocytosis. To determine whether the inhibition of oxidative burst was due to a reduction of phagocytosis,
as already suggested by Lian et al. (27), we first used the
microscopic method described by these authors. Preopsonized
Y enterocolitica W22703 pYV+, W22703 pYV-, W22703
(pBC7), and W22703(pBC7)(pMS153) were added to microscope slide wells containing adherent PMNs, and the wells
were incubated for 90 min at 37C. The slides were then
stained with acridine orange and counterstained with crystal
violet. Acridine orange diffuses in PMNs and accumulates in
acidic cellular compartments such as phagolysosomes (16). It
intercalates in DNA in the same way as ethidium bromide
and stains bacteria and the nuclei of PMNs (Fig. 2). Crystal
violet does not enter PMNs, but it stains the bacterial wall
and quenches the fluorescence of extracellular bacteria. We
examined 100 PMNs and counted those containing bacteria.
As shown in Table 2, we observed a clear correlation between
the presence of YadA and the reduction of phagocytosis.
This method was quite difficult to apply, because the distinc-

tion between intracellular bacteria and acidic granules stained

by acridine orange was sometimes difficult. To confirm our
result, we thus turned to another approach, based on the use of
fluorescent bacteria and flow cytometry. We labeled live
bacteria with FITC and checked that the labeling did not affect
their viability (data not shown). The FITC-labeled bacteria
were then opsonized and mixed with PMNs (ratio, 50/1) for 1
h at 37C. The mixture of PMNs and bacteria was then
counterstained with trypan blue to quench the fluorescence of
TABLE 2. Microscope study of phagocytosis of Y enterocolitica
by human PMNsa
Y enterocolitica strain

W22703(pYVe227)
W22703

W22703(pBC7)
W22703(pBC7)(pMS153)

Presence
YadA

+
+

% Phagocytosis
%Paoyoi

20
91
75
35

4
8
12
11

a
PMN monolayers were incubated for 90 min with preopsonized bacteria.
Percentages of PMNs having engulfed bacteria and standard deviations for three
independent experiments are given. According to a hierarchical F test, the
difference between the YadA+ and the YadA- bacteria is significant (P < 0.01).

VOL. 62, 1994

YadA AND PMNs

1279

LC0~

a7

-4~
~
~
~
~
~ ~

~~~~~~~~~~~~~~~-i

FLUORESCENCE iNTENSiTY
FIG. 3. Flow cytometry analysis of phagocytosis. The bacteria were labeled with FITC before contact with PMNs. The fluorescence of
extracellular bacteria was extinguished by staining with trypan blue. Only PMNs were analyzed by the cytometer. The intensity of fluorescence is
given as a function of the relative number of PMNs. Y enterocolitica W22703(pYVe227) (A), W22703 (B), W22703(pBC7) (C), and
W22703(pBC7)(pMS153) (D) are shown. The surface under the curve gives an estimate of the number of fluorescent PMNs on 5,000 counted
PMNs. The numbers in the upper right-hand corners are the percentages of fluorescent PMNs for each bacterial strain.

extracellular bacteria and was analyzed in the FACS. The

FACS monitored all of the PMNs and calculated the percentage of cells that were fluorescent as a result of engulfment of
bacteria. The experiment was carried out with Y enterocolitica
W22703(pYVe227), W22703 pYV-, W22703(pBC7), and
W22703(pBC7)(pMS153). As shown in Fig. 3, bacteria expressing YadA were less phagocytosed than the bacteria
that did not express YadA. This second approach thus confirmed our microscopic observations. There was a correlation
between the lack of oxidative burst and the lack of phagocytosis.
YadA and killing. The CL experiments did not resolve the
fate of the bacteria ingested by PMNs. We thus measured the
killing of Y enterocolitica. For this assay, we used our strain
KNG22703, a derivative of W22703 which produces luciferase
as a result of the insertion of genes luxAB in the chromosomal
blaA gene (21). Preopsonized bacteria were suspended either
in HBSS containing PMNs or in HBSS without PMNs. Bacteria and PMNs were mixed in a ratio of 50 bacteria per PMN.
After 60 min of incubation, the PMNs were lysed with ice-cold
distilled water, and bacteria were plated on tryptic soy agar
supplemented with nalidixic acid for strain KNG22703, with
nalidixic acid and gentamicin for strain KNG22703(pBC7), and
with nalidixic acid, gentamicin, and tetracycline for strain
KNG22703(pBC7)(pMS153). As shown in Table 3, pYV+
bacteria were not killed by PMNs, while most of the pYVbacteria did not survive. The pYV+ yadA mutant was also
killed; however, it survived when the mutation was complemented by the introduction of a cloned yadA gene.
Since KNG22703 emits photons in the presence of n-decanal
and since there is a linear relation between the number of
counts per minute emitted and the number of CFUs (22), we
also measured the survival rate by luminometry as described

by Kaniga et al. (22). The experiment was realized with

Y enterocolitica KNG22703(pYVe227), KNG22703 pYV-,
KNG22703(pBC7), and KNG22703(pBC7)(pMS153). For
each culture, the number of surviving bacteria was estimated
both by plating and by luminometry. The results obtained were
in very good agreement with those obtained by plating, indicating that the activation of PMNs did not interfere with the
light emission resulting from luciferase activity (Table 3). We
conclude from these experiments that there is a correlation
between the expression of YadA on the cell surface, the
inhibition of the CL response of the PMNs, the reduction of
phagocytosis by PMNs, and the survival against killing by
PMNs.
TABLE 3. Killing of Y enterocolitica by human PMNsa
Y enterocolitica strain

KNG22703(pYVe227)
KNG22703

KNG22703(pBC7)
KNG22703(pBC7)(pMS153)

Presence
of % Survival % Luminescence
YadA
+
+

112
15
16
95

18
7
5
22

96
28
32
92

11
5
7
9

a Preopsonized bacteria were incubated in HBSS or in HBSS containing PMNs

for 60 min at 37C. After lysis of PMNs, the numbers of viable bacteria were
determined by plating (percent survival) or by bioluminescence (percent luminescence). The values given are the percentages of CFU (percent survival) or of
counts per minute (percent luminescence) obtained for bacterial suspensions
incubated with PMNs, compared with those for control bacterial suspensions
incubated in HBSS. According to a analysis of variance 2 analysis, the two
methods were not significantly different. Orthogonal-contrast analysis of results
obtained by the two methods showed that KNG22703(pYe227) was significantly
different from KNG22703(pBC7) (P < 0.01) but that KNG22703(pYVe227) was
not significantly different from KNG22703(pBC7)(pMS153) and that KNG22703
was not significantly different from KNG22703(pBC7).

1280

CHINA ET AL.

DISCUSSION

Lian and Pai (28), showed that Y enterocolitica cells harboring the virulence plasmid and grown at 37C do not induce the
oxidative burst of PMNs. They showed that a protein present in
outer membrane fragment preparations was responsible for
this property, and they hypothesized that this protein could be
the outer membrane protein now known as YadA. Tertti et al.
(46) confirmed the lack of burst, and they hypothesized that it
was due to Yop proteins. Moreover, they showed that there
was a correlation between the lack of oxidative burst and the
reduction of opsonization by complement molecule C3b.
In this study, we tested several well-characterized pYV
mutants for their abilities to prevent the oxidative burst of
PMNs. The yadA mutants did not prevent the oxidative burst,
and this effect of the mutation could be complemented by a
cloned yadA gene supplied in trans. Thus, YadA is involved in
this phenomenon, as was suggested by Lian et al. (27). We then
investigated phagocytosis of Y enterocolitica and observed that
YadA+ Y enterocolitica cells were less phagocytosed than the
YadA- variant. According to acridine orange staining, the
intracellular YadA - bacteria that were phagocytosed were
localized in an acidic compartment, an acidified phagosome or
a phagolysosome. This acid compartment seemed to be lethal
for the bacteria. This conclusion was confirmed by the killing
experiments, which showed that the bacteria harboring YadA
survive in the presence of PMNs. In conclusion, YadAbacteria are phagocytosed and killed, while YadA+ bacteria
resist phagocytosis and killing. We do not know the fate of the
few YadA+ bacteria that are phagocytosed. The fact that
YadA inhibits phagocytosis by PMNs could be related to the
capacity of YadA to reduce the opsonization by C3b molecules
(9). We hypothesize that the presence of YadA prevents the
opsonization by C3b molecules, which leads to a reduction of
opsonophagocytosis by PMNs as well as to resistance to the
bactericidal activity of complement. Y. enterocolitica cells also
resist phagocytosis by macrophages, the other professional
phagocytes (39). This second resistance involves YopH (39), a
phosphotyrosine-phosphatase dephosphorylating macrophage
protein (4). We do not see any contradiction between our
observation that the inhibition of opsonophagocytosis by
PMNs is mediated by YadA and the known role of YopH in
the inhibition of phagocytosis by macrophages in the absence
of serum opsonins. Indeed, the experiments of phagocytosis by
macrophages were carried out in the absence of autologous
complement. The systems are thus different. We wondered
whether YopH would also prevent phagocytosis by PMNs in
the absence of serum. However, in the absence of serum, we
could not detect any significant CL response to pYV- Y
enterocolitica, and most pYV- Y enterocolitica remained extracellular (lOa). Hence, in contrast to macrophages, PMNs
require opsonization of the target, to carry out phagocytosis. It
is thus not too surprising that Y enterocolitica developed
different protection strategies to face different situations. Since
opsonization is required, preventing opsonization seems to be
a first-choice strategy to protect from PMNs. This strategy is
classical in bacterial pathogenesis. Among others, it is exemplified by group A Streptococcus pyogenes. The M protein of
group A S. pyogenes prevents phagocytosis by a reduction of
complement opsonization (20). We would like to point out the
functional similarity between protein M and YadA. Like YadA
(9), protein M was indeed reported to enhance the fixation of
factor H (20). It is difficult to say whether this functional
parallelism results from a similar structure, because the structure of YadA remains unclear.

INFEc-r. IMMUN.

ACKNOWLEDGMENTS
We thank C. Debande for drawing the figures and Ralf Schulte for
a critical reading of the manuscript.
B.C. was the recipient of a fellowship from the Belgian Institut pour
l'Encouragement de la Recherche Scientifique dans l'Industrie et
l'Agriculture. This work was supported by grants from the Belgian
Fonds National de la Recherche Scientifique Medicale (convention
3.4514.93) and by the Belgian Program on Interuniversity Poles of
Attraction initiated by the Belgian State Prime Minister's Office
(Science Policy Programming).
REFERENCES
1. Allen, R. C., and L. D. Loose. 1992. Phagocytic activation of a
luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages. Biochem. Biophys. Res. Commun. 69:245252.
2. Balligand, G., Y. Laroche, and G. Cornelis. 1985. Genetic analysis
of virulence plasmid from serogroup 9 Yersinia enterocolitica
strain: role of outer membrane protein P1 in resistance to human
serum and autoagglutination. Infect. Immun. 48:782-786.
3. Bliska, J., and S. Falkow. 1992. Bacterial resistance to complement
killing mediated by the Ail protein of Yersinia enterocolitica. Proc.
Natl. Acad. Sci. USA 89:3561-3565.
4. Bliska, J. B., C. Clemens, J. E. Dixon, and S. Falkow. 1992. The
Yersinia tyrosine phosphatase: specificity of a bacterial virulence
determinant for phosphoproteins in J774A.1 macrophage. J. Exp.
Med. 176:1625-1630.
5. Brubaker, R. R. 1991. Factors promoting acute and chronic
diseases caused by Yersiniae. Clin. Microbiol. Rev. 4:309-324.
6. Bukholm, G., G. Kapperud, and M. Skurnik 1990. Genetic
evidence that the yopA gene-encoded Yersinia outer membrane
protein Yopl mediates inhibition of anti-invasive effect of interferon. Infect. Immun. 58:2245-2251.
7. Cantinieaux, B., C. Hariga, P. Courtoy, J. Hulpin, and P. Fondu.
1989. Staphylococcus aureus phagocytosis. A new cytofluorometric
method using FITC and paraformaldehyde. J. Immunol. Methods
121:203-208.
8. Chateau, M.-T., H. Rabesandratana, and R. Caravano. 1991.
Suspended mouse peritoneal macrophages preparation and properties. J. Immunol. Methods 143:103-109.
9. China, B., M.-P. Sory, B. T. N'Guyen, M. De Bruyere, and G. R.
Cornelis. 1993. Role of the YadA protein in the prevention of
opsonization of Yersinia enterocolitica by C3b molecules. Infect.
Immun. 61:3129-3136.
10. China, B., T. Michiels, and G. R. Cornelis. 1990. The pYV plasmid
of Yersinia encodes a lipoprotein, YpA, related to TraT. Mol.
Microbiol. 4:1585-1593.
10a.China, B., B. T. N'Guyen, M. De Bruyere, and Guy R. Cornelis.
Unpublished data.
11. Cornelis, G., and C. Colson. 1975. Restriction of DNA in Yersinia
enterocolitica detected by recipient ability for a derepressed R
factor from Escherichia coli. J. Gen. Microbiol. 87:285-291.
12. Cornelis, G., C. Sluiters, C. Lambert de Rouvroit, and T. Michiels.
1989. Homology between VirF, the transcriptional activator of the
Yersinia virulence regulon, and AraC, the Escherichia coli arabinose operon regulator. J. Bacteriol. 171:254-262.
13. Cornelis, G., M.-P. Sory, Y. Laroche, and I. Derclaye. 1986.
Genetic analysis of the plasmid region controlling virulence in
Yersinia enterocolitica 0:9 by mini-Mu insertions and lac gene
fusions. Microb. Pathog. 1:349-359.
14. Cornelis, G., J.-C. Vanooteghem, and C. Sluiters. 1987. Transcription of the yop regulon from Y enterocolitica requires trans acting
pYV and chromosomal genes. Microb. Pathog. 2:367-379.
15. Cornelis, G. R. 1992. Yersinia, finely tuned pathogens, p. 231-266.
In C. E. Hormaeche, C. W. Penn, and C. J. Smyth (ed.), Molecular
biology of bacterial infection: current status and future perspectives. Society for general microbiology symposium, vol. 49, Cambridge University Press, Cambridge.
16. D'Arcy Hart, P., and M. R. Young. 1975. Interference with normal
phagosome-lysosome fusion in macrophages, using ingested yeast
cells and suramin. Nature (London) 256:47-49.
17. Emody, L, J. Heesemann, H. Wolf-Watz, M. Skurnik, G. Kapperud, P. O'Toole, and T. Wadstrom. 1989. Binding to collagen by

VOL. 62, 1994

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.
29.
30.
31.

32.
33.

Yersinia enterocolitica and Yersinia pseudotuberculosis: evidence for

yopA-mediated and chromosomally encoded mechanisms. J. Bacteriol. 171:6674-6679.
Guan, K., and J. E. Dixon. 1990. Protein tyrosine phosphatase
activity of an essential virulence determinant in Yersinia. Science
249:553-556.
Heesemann, J., and L. Griiter. 1987. Genetic evidence that the
outer membrane protein YOP1 of Yersinia enterocolitica mediates
adherence and phagocytosis resistance to human epithelial cells.
FEMS Microbiol. Lett. 40:37-41.
Horstmann, R. D., H. J. Sievertsen, J. Knobloch, and V. A.
Fischetti. 1988. Antiphagocytic activity of streptococcal M protein:
selective binding of complement control protein factor H. Proc.
Natl. Acad. Sci. USA 85:1657-1661.
Kaniga, K., I. Delor, and G. R. Cornelis. 1991. A wide-host-range
suicide vector for improving reverse genetics in gram-negative
bacteria: inactivation of blaA gene of Yersinia enterocolitica. Gene
109:137-141.
Kaniga, K., M.-P. Sory, I. Delor, C. Saegerman, J. N. Limet, and
G. R. Cornelis. 1992. Monitoring of Yersinia enterocolitica in
murine and bovine feces on the basis of chromosomally integrated
luxAB marker gene. Appl. Environ. Microbiol. 58:1024-1026.
Kapperud, G., E. Namork, M. Skurnik, and T. Nesbakken. 1987.
Plasmid-mediated surface fibrillae of Yersinia pseudotuberculosis
and Yersinia enterocolitica: relationship to the outer membrane
YOP1 and possible importance for pathogenesis. Infect. Immun.
55:2247-2254.
Lachica, R. V., D. L. Zink, and W. R. Ferris. 1984. Association of
fibril structure formation with cell surface properties of Yersinia
enterocolitica. Infect. Immun. 46:272-275.
Lambert de Rouvroit, C., C. Sluiters, and G. R. Cornelis. 1992.
Role of the transcriptional activator, VirF, and temperature in the
expression of the pYV plasmid genes of Yersinia enterocolitica.
Mol. Microbiol. 6:395-409.
Legocki, R. P., M. Legocki, T. 0. Baldwin, and A. A. Szalay. 1986.
Bioluminescence in soybean root nodules: demonstration of a
general approach to assay gene expression in vivo by using
bacterial luciferase. Proc. Natl. Acad. Sci. USA 83:9080-9084.
Lian, C.-J., W. S. Hwang, and C. H. Pai. 1987. Plasmid-mediated
resistance to phagocytosis in Yersinia enterocolitica. Infect. Immun.
55:1176-1183.
Lian, C.-J., and C. H. Pai. 1985. Inhibition of human neutrophil
chemiluminescence by plasmid-mediated outer membrane proteins of Yersinia enterocolitica. Infect. Immun. 49:145-151.
Martinez, R. J. 1989. Thermoregulation-dependent expression of
Yersinia enterocolitica protein 1 imparts serum resistance to Escherichia coli K-12. J. Bacteriol. 171:3732-3739.
Michiels, T., and G. R. Cornelis. 1991. Secretion of hybrid proteins
by the Yersinia Yop export system. J. Bacteriol. 173:1677-1685.
Michiels, T., J.-C. Vanooteghem, C. Lambert de Rouvroit, B.
China, A. Gustin, P. Boudry, and G. R. Cornelis. 1991. Analysis of
virC, an operon involved in the secretion of Yop proteins by
Yersinia enterocolitica. J. Bacteriol. 173:4994-5009.
Michiels, T., P. Wattiau, R. Brasseur, J.-M. Ruysschaert, and
G. R. Cornelis. 1990. Secretion of Yop proteins by Yersiniae.
Infect. Immun. 58:2840-2849.
Mulder, B., T. Michiels, M. Simonet, M.-P. Sory, and G. R.

YadA AND PMNs

34.

35.

36.
37.

38.
39.

40.

41.
42.
43.
44.

45.

1281

Cornelis. 1989. Identification of additional virulence determinants

on the pYV plasmid of Yersinia enterocolitica W227. Infect.
Immun. 57:2534-2541.
Mundi, H., B. Bjorksten, C. Svanborg, L. Ohman, and C. Dahlgren. 1991. Extracellular release of reactive oxygen species from
human neutrophils upon interaction with Escherichia coli strains
causing renal scarring. Infect. Immun. 59:4168-4172.
Paeregaard, A., F. Espersen, 0. M. Jensen, and M. Skurnik. 1991.
Interactions between Yersinia enterocolitica and rabbit ileal mucus:
growth, adhesion, penetration, and subsequent changes in surface
hydrophobicity and ability to adhere to ileal brush border membrane vesicles. Infect. Immun. 59:253-260.
Pierson, D. E., and S. Falkow. 1993. The ail gene of Yersinia
enterocolitica has a role in the ability of the organism to survive
serum killing. Infect. Immun. 61:1846-1852.
Pilz, D., T. Vocke, J. Heesemann, and V. Brade. 1992. Mechanism
of YadA-mediated serum resistance of Yersinia enterocolitica
serotype 03. Infect. Immun. 60:189-195.
Piano, G. V., S. S. Barve, and S. C. Straley. 1991. LcrD, a
membrane-bound regulator of the Yersinia pestis low-calcium
response. J. Bacteriol. 173:7293-7303.
Rosqvist, R., I. Bolin, and H. Wolf-Watz. 1988. Inhibition of
phagocytosis in Yersinia pseudotuberculosis: a virulence plasmidencoded ability involving the Yop2b protein. Infect. Immun.
56:2139-2143.
Schulze-Koops, H., H. Burkhardt, J. Heesemann, K. von der
Mark, and F. Emmrich. 1992. Plasmid-encoded outer membrane
protein YadA mediates specific binding of enteropathogenic
Yersiniae to various types of collagen. Infect. Immun. 60:21532159.
Skurnik, M., L. Bolin, H. Heikkinen, S. Piha, and H. Wolf-Watz.
1984. Virulence plasmid-associated agglutination in Yersinia spp.
J. Bacteriol. 158:1033-1036.
Skurnik, M., and P. Toivanen. 1993. Yersinia enterocolitica lipopolysaccharide: genetics and virulence. Trends Microbiol. 1:148152.
Skurnik, M., and H. Wolf-Watz. 1989. Analysis of the yopA gene
encoding the Yopl virulence determinants of Yersinia spp. Mol.
Microbiol. 3:517-529.
Smith, D. L., and F. Rommel. 1977. A rapid micro method for the
simultaneous determination of phagocytic-microbicidal activity of
human peripheral blood leukocytes in vivo. J. Immunol. Methods
17:241-247.
Straley, S. C., E. Skrzypek, G. V. Plano, and J. B. Bliska. 1993.
Yops of Yersinia spp. pathogenic for humans. Infect. Immun.

61:3105-3110.
46. Tertti, R., E. Eerola, 0. P. Lehtonon, T. H. Stahlberg, M. Vinder,
and A. Toivanen. 1987. Virulence-plasmid is associated with the
inhibition of opsonization in Yersinia enterocolitica and Yersinia
pseudotuberculosis. Clin. Exp. Immunol. 68:266-274.
47. Tertti, R., M. Skurnik, T. Vartio, and P. Kuusela. 1992. Adhesion
protein YadA of Yersinia species mediates binding of bacteria to
fibronectin. Infect. Immun. 60:3021-3024.
48. Zaleska, M., K. Lounatmaa, M. Nurminen, E. Wahlstrom, and
P. H. Makela. 1985. A novel virulence associated cell surface
structure composed of 47-kd protein subunits in Yersinia enterocolitica. EMBO J. 4:1013-1018.