Microbiology, Immunology & Molecular Genetics
Hypervirulent Human-Associated Bordetella bronchiseptica Umesh Ahuja and Jeffery F. Miller
INTRODUCTION
Model of the evolution of mammalian bordetellae
HABBs are hyperlethal
HABBs are hypercytotoxic over broad range of mammalian cells
HABB in vitro hypercytotoxicity is T3SS dependent
HABBs are better colonizers of the lungs
Secretome analysis and T3SS activity in HABBs
0
10
20
30
40
50
60
70
80
90
100
RB50 WD3 D445 Bbr77 D444 D446 D758 Bbr69 Bbr68 Bbr78 Bbr79 Bb545 Bb548 Bb599 Bb601 Bb705 Bb723 Bb782
HeLa
J774A.1
A549
0
10
20
30
40
50
60
70
80
90
100
HeLa
J774A.1
A549
FIGURE 1.: Minimum spanning tree of 3 closely related Bordetellae: A, The tree is based on the sequence of seven house keeping gene.
The sequence types (STs) sharing the highest number of single locus variants were connected first. Each circle represents a ST, the size
of which is related to the number of isolated. B, Model of the evolution of mammalian bordetellae. The bar on left indicates increasing
degree of adaptation.
Reference: Diavatopoulos, D.A., Cummings, C.A., Schouls, L.M., Brinig, M.M., Relman, D.A., and Mooi, F.R. (2005) Bordetella
pertussis, the Causative Agent of Whooping Cough, Evolved from a Distinct, Human-Associated Lineage of B. bronchiseptica. PLoS
Pathog 1: e45.
B-150
0102030405060708090
100110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Time (h)
RB50
WD3
Bbr68
D444
D445
D446
Bbr69
Bbr77
Lung
0
1
2
3
4
5
6
7
8
9
RB50 D445 RB50 D445 RB50 D445 Bbr77 Bbr77 Bbr77
ΔbscN ΔbteA
Lo
g10 C
FU
Trachea
0
1
2
3
4
5
6
7
Lo
g10 C
FU
RB50 D445 RB50 D445 RB50 D445 Bbr77 Bbr77 Bbr77
ΔbscN ΔbteA
Over all
Inflammation Perivasculitis Alveolitis Peribroncheolitis
PBS - - - -
RB50 +++ ++ ++ -/+
WD3 + - - -
RB50ΔbteA + - - -
D445 ++++++ +++++ +++++ +++++
D445ΔbscN +++ + ++ -
D445ΔbteA +++ ++ +++ +++
Bbr77 ++++++ +++++ +++++ +++++
Bbr77ΔbscN +++ + ++ ++
Bbr77ΔbteA +++ +++ +++ +++
Summary of histopathological examination of Lungs
25-
20-
15-
10-
250-
75-
50-
37-
100-
150-
A3
09
*
A3
10
*
A3
45
Bb
r69
Bb
r77
Bb
r80
*
D44
4
D44
5
D44
6
D75
8
RB
50
WD
3
RB
54
M kDa
B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent
phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Complex
IV isolates appear to have a predilection for infecting humans and they are most closely related to the progenitor from which
B. pertussis evolved. Here we report a characterization of the virulence properties of human-associated complex IV strains
of B. bronchiseptica (HABB). In in vitro cytotoxicity assays with human epithelial (HeLa), mouse monocyte/macrophage
(J774A.1) and human pneumocyte-derived (A549) cell lines, wild type HABB strains showed significantly increased
cytotoxicity in comparison to a panel of complex I strains. Some HABB isolates were remarkably cytotoxic, resulting in LDH
release levels that were 10- to 12-fold greater than the prototype complex I strain RB50. In vivo, HABB strains are
hypervirulent and rapidly kill C57/BL6 mice under conditions where RB50 establishes asymptomatic respiratory infections. In
general, mice infected with HABB succumb to rapidly progressive pneumonia, exhibiting massive peribronchiolitis,
perivasculitis and alveolitis. Following low dose inoculations, HABB strains are found in lung tissues at 10-fold (p<0.001)
greater numbers than RB50 at 3 days post infection. Two HABB strains, D445 and Bbr77, were analyzed further. Nonpolar,
in-frame deletions were introduced into bscN and bteA, which encodes for an ATPase required for T3SS activity and an
T3SS effector, respectively. For both HABB strains, the bscN/bteA mutation eliminated in vitro cytotoxicity and abrogated
hypervirulence in vivo. Preliminary analysis also indicates marked differences in the secretomes of HABB strains in
comparison to RB50. Taken together, our observations reveal a T3SS-dependent hypervirulent phenotype in HABB strains,
and justify further studies on their epidemiology and evolutionary dynamics.
LD
H r
ele
ase (
% m
ax.
lysis
)
A B
A
B
LD
H r
ele
ase (
% m
ax.
lysis
)
Complex-IV strains Complex-I strains MOI 50:1
t=3h
MOI 1:50
J774A.1
HeLa
MOI 1:50
t=3h
FIGURE 2. Survival of C57BL/6 mice
following inoculation with various
B. bronchiseptica strains. C57BL/6 mice
(n=4) were inoculated intranasally with 40 µl
of PBS containing 4-5× 105 CFU of indicated
strains. XXXXXXXX.
FIGURE 5: Respiratory tract colonization by various B. bronchiseptica strains. C57BL/6 mice (n=10-12) were inoculated intranasally
with 40 µl of PBS containing 4-5 × 105 CFU of indicated strains and the numbers of bacteria present in the trachea (A) and lungs (B)
were determined at the day 3 post-inoculation. *, P < 0.01.
FIGURE 3. A, Comparative analysis of
cytotoxicity induced by various B. bronchiseptica
strains. Cytotoxicity of Bordetella is measured
by LDH release. Indicated mammalian cells
were infected with bacteria at an MOI of 50:1 for
3 h. Results are average of 4-6 independent
experiments. B, Time course of LDH release
from J774A.1 cells upon infection with indicated
strains.
Trachea
0
1
2
3
4
5
6
7
RB50 D445
Lo
g10 C
FU
Lungs
0
1
2
3
4
5
6
7
8
RB50 D445
Lo
g10 C
FU
P<0.0001
FIGURE 6. Respiratory tract colonization by
B. bronchiseptica strains RB50 (red circles) and D445 (blue
circles). C57BL/6 mice (n=8) were inoculated intranasally
with 40 µl of PBS containing 2.5-3 × 105 CFU of indicated
strains and the numbers of bacteria present in the trachea
(A) and lungs (B) were determined at the day 3 post-
inoculation. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
A B Infection with low dose
Histology
FIGURE 7: Wild-type C57Bl/6 mice were intranasally inoculated
with 4-5 × 105 CFU of indicated strain in a 40 µl volume. The
mice were sacrificed at 3 day post inoculation and the lungs
were perfused and fixed in 10% formaldehyde. Lung sections (4
μm) were cut and stained with haemolysin and eosin.
LD
H r
ele
ase (
% m
ax.
lysis
)
A3
09
*
A3
10
*
A3
45
Bb
r69
Bb
r77
Bb
r80
*
D44
4
D44
5
D44
6
D75
8
RB
50
WD
3
RB
54
Bsp22
Bsp22
Complex-IV strains
A B
FIGURE 8: Comparison of secreted proteins in minimal media by different HABB strains.
A, supernatent samples (after normalization) were TCA precipitated, separated by 4-
15% gradient SDS-polyacrylamide gel and Comassie stained. B, Western blot analysis
of pellets (upper panel) and supernatent (lower panel) from the indicated strains with
anti-Bsp22 antibody, a conserved type III needle component of Bordetellae. Strains
marked with “*” were always found in minus- (avirulent) phase. XXXXXXXX
XXXXXXXXXXXXXXXX
Pellets
Supernatents
FIGURE 4. Comparative analysis of
cytotoxicity induced by various B.
bronchiseptica strains and their
respective type III secretion defective
(ΔbscN), a type III effector deficient
(ΔbteA) and corresponding BteA
complemented strain (pbteA).XXXXX
Cytotoxicity of Bordetella is measured
by LDH release. Indicated mammalian
cells were infected with bacteria at an
MOI of 50:1 for 3 h. Results are
average of 3 independent experiments.
0
20
40
60
80
100
120
Perc
en
tag
e s
urv
ival
Days
RB50
D445
Bbr77
D444
D446
Bbr69
Secreted proteins from the various HABB strains clearly differ both quantitative and qualitative. Currently we are in process of
identifying the unique proteins from each strain by mass spectrometery. XXXXXXXX XXXXXXXXXXXXXXXX
P<0.0001
P<0.0001
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