Microbiology, Immunology & Molecular Genetics

Hypervirulent Human-Associated Bordetella bronchiseptica Umesh Ahuja and Jeffery F. Miller

(uahuja@ucla.edu)

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

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RB50 WD3 D445 Bbr77 D444 D446 D758 Bbr69 Bbr68 Bbr78 Bbr79 Bb545 Bb548 Bb599 Bb601 Bb705 Bb723 Bb782

HeLa

J774A.1

A549

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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

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RB50 D445 RB50 D445 RB50 D445 Bbr77 Bbr77 Bbr77

ΔbscN ΔbteA

Lo

g10 C

FU

Trachea

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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

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250-

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50-

37-

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150-

A3

09

*

A3

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*

A3

45

Bb

r69

Bb

r77

Bb

r80

*

D44

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D44

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D44

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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

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6

7

RB50 D445

Lo

g10 C

FU

Lungs

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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

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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.

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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