RB50
ΔbtrA#1
ΔbtrA#2
RB50
ΔbtrA#1
ΔbtrA#2
time (h)
O.D
600
0 5 10 15 20 250.1
1
10
Growth rate (h)
RB50: 1.89±0.17ΔbtrA: 6.78±1.35
DC
A
B E
F
bvgΔ54RB50 ΔbtrA ΔbtrS
n=3
btrA btrS bsp22 bopN bscN bteA fhaB cyaA prn flaA bipA
relativefolddifference
0.0
0.5
1.0
1.52468
10121416
100150200250
Pellet Supernatant
Δbt
eAH
A
RB5
0HA
WD
3HA
Δbt
eAH
A
RB5
0HA
WD
3HA
Δbt
eA
RB5
0W
D3
Δbt
eA
RB5
0W
D3
BvgA
BtrA-HA
PRN
Bsp22
G HSupernatant
RB
50
Δbt
rA
Δbt
rS
25-20-
15-
37-
10-
50-
75-
100-
150-
250-
kDa
CyaA
Bsp22
Prn
FhaB
RB
50
Δbt
rA
Δbt
rS
Pellet
bvgΔ
54
bvgΔ
54
- Bsp22- BopD- BopN
BopB-
****
LDHrelease(%
max.lysis) Hela
0
20
40
60
80
100
RB50
Δbsc
NΔb
teAΔb
trA
ΔbteAΔb
trABbr7
70
20
LDHrelease(%
max.lysis) A549
40
60
80
100p <0.0001
ns
bsc locusbscP btrA btrW
btrU
btrV
alrBB1640 BB1641 BB1643
btrS
bscC
bscW
bscUbscT
bscC
bscRbscQ
fhaBcyaAprnptxbvgSbipA
btrS
bsc T3SS
BvgS
BvgA
vrgs(e.g.flagella)
FIGURE 3: btrA over expression; A, Colony morphology on Bordet-Gengou agar containing 7.5% defibrinated
blood. B, Semi-quantitative RT-PCR of indicated gene transcripts. C, qRT-PCR of indicated gene transcripts. D,
Expression and secretion of virulence gene products in pellet and culture supernatants. Coomassie brilliant blue
protein stain after SDS-PAGE of O.D. normalized pellets and supernatants of indicated strains. Lower panels,
immunoblots of the indicated virulence determinants. btrS over expression; E, Colony morphology on Bordet-
Gengou agar containing 7.5% defibrinated blood. F, Semi-quantitative RT-PCR of indicated gene transcripts. G, qRT-
PCR of indicated gene transcripts. H, Upper panel; Coomassie brilliant blue protein stain after SDS-PAGE of O.D.
normalized pellets and supernatants of indicated strain. Lower panels, immunoblots of the indicated virulence
determinants.xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
Microbiology, Immunology & Molecular Gene4cs
Host Adaptation by Bordetella species: Differential control of Type Three Secretion System Umesh Ahuja and Jeff F. Miller
ABSTRACT
Model of the Evolution of Mammalian Bordetellae
Overexpression of btrA and btrS Confer Contras4ng Phenotypes
BtrA is a T3SS Substrate That Regulates Mul4ple Virulence Genes
BtrAS Selec4vely Regulate T3SS Expression in B. pertussis
New Model of Virulence Gene Regulation
RNA-Seq Reveals Novel Regulatory Network
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. bronchiseptica isolates from diverse hosts, including humans, display potent cytotoxicity against a broad range of mammalian
cells which is dependent on BteA, a type three secretion system (T3SS) effector. In contrast, neither clinical nor laboratory isolates
of B. pertussis display T3SS-dependent phenotypes in vitro, despite the fact that bsc T3SS genes are present, intact, and nearly
identical to their B. bronchiseptica counterparts. Furthermore, bteA alleles are functionally interchangeable between these
Bordetella subspecies. An hypothesis that could explain subspecies-specific differences in T3SS activity is that the regulatory
network that controls T3SS has co-evolved with host specificity. Indeed, T3SS activity is known to be controlled at multiple levels.
bteA and bsc apparatus genes are transcriptionally activated by an extracytoplasmic function (ECF) sigma factor, BtrS, which is
encoded by a BvgAS-activated gene. We have discovered and characterized a regulatory node in B. bronchiseptica involving
BtrA, a secreted antagonist of BtrS. RNA-seq analysis shows that BtrA negatively regulates expression of T3SS apparatus,
effector, and regulatory loci, and positively controls hundreds of genes encoding toxins, adhesins, and products of unknown
function. Deletion of btrA or over-expression of btrS leads to increased T3SS activity, while over-expression of btrA mimics the
effects of ∆btrS allele on T3SS expression and phenotypes. Antagonism is mediated by a physical interaction between BtrA and
BtrS which couples secretion to gene expression. Of particular relevance to human disease, deletion of btrA in B. pertussis
derepressess bsc T3SS gene expression and confers readily detectable BteA-dependent cytotoxicity in vitro. On the basis of
these and other results, we propose that the BtrA regulatory node determines, at least in part, subspecies-specific differences in
T3SS expression between B. bronchiseptica and B. pertussis. The implications of these results for the design of improved
vaccines against pertussis will be discussed.
A B
BtrA Physically Interacts with BtrS
FIGURE 4: A, Schematic diagram of BtrA constructs used in this study and their effect on T3SS expression, FhaB
expression and interaction with BtrS. B, Interactions between BtrA and BtrS in a yeast two-hybrid
system.XXXXXXXXXXXXXXXXXXXXXXXXXXXXXxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
FIGURE 6: A, Immunoblot analysis of normalized pellet and culture supernatant of wild type B. pertussis and its btrA isogenic null
mutant. B and C, Cytotoxicity of wild type B. pertussis and its isogenic mutant derivatives measured by LDH release. HeLa cells were
infected with indicated bacteria at different MOIs for 6h and 24h. Results are average of 3 independent experiments.
FIGURE 5: A and D, Heat map depicting the
results of cluster analysis of the log2 transformed
RPKM values. B and E, The box plots showing the
distribution of transcripts. C, Predicted regulatory
pathways. F, Log2 fold change gene expression of
known virulence-related genes compared to the
wild-type. G, Venn diagrams showing overlap of
various overexpression gene clusters (a-c) with
null-mutant gene clusters (1-4). xxxxxxxxxxxxxxxx
A B C
In view of our current findings we propose a new general model of
virulence gene regulation in Bordetella that involves intricate interaction
between BtrS and BtrA in fine-tuning the Bvg-driven response. BtrS act as
a positive regulator of T3SS locus genes while BtrA acts as an antagonist
of BtrS. The transcription of btrS is BvgAS-dependent while the activity of
BtrS is regulated by sequestration by BtrA. The transcription of btrA is
regulated by BtrS while the inhibitory intracellular levels of BtrA are
controlled by its secretion through the T3SS pore. The secretion through
the T3SS pore augurs further T3SS induction. The rate of BtrA production
and secretion through T3SS is dependent on the level of BtrS activation.
FIGURE 2: A, Genomic organization
of btrA-btrS locus.
B, T3SS-dependent secretion of BtrA.
C, Colony morphology on Bordet-
Gengou agar containing 7.5%
defibrinated blood.
D, Growth rate determination in
Stainer-Scholte medium.
E, Model of BvgAS-dependent
virulence gene regulation.
F, qRT-PCR of indicated virulence-
related genes.
G, Immunoblot analysis of indicated
virulence gene products in culture
supernatants and pellets.
H, In vitro cytotoxicity of indicated
strains towards HeLa and A549
cells.X
wild ty
pe
bvgSΔ5
4
ΔbtrS
ΔbtrA
ΔbtrAΔbtrS
5
95
Perc
entil
e
Log 2
Tran
sfor
med
RPK
MLo
g 2 Fol
d C
hang
e (R
PKM
)
A
C
cyaCcyaAcyaBcyaDcyaEsphB1brkBpr
nfim3
ptxAptxBptxEptxDptxCptlAptlBptlCptlDptlLptlEptlFptlGptlH
fhaLbipAfhaSfimDfimCfimBfimAfhaBbvgAbvgSbvgR fim
NfimXfim2dntpagP
ΔbtrSΔbtrA ΔbtrAΔbtrS
bscFbscEbscDbcrD or
f2 orf3orf4bopNbsp22orf6bcrH1bopDbopBbcrH2bcr4bscIbscJbscK bs
cLbscNbscObscPbscQbscRbscSbscTbscUbscWbs
cC btrAbtrSbtrUbtrWbtrVbtcAbteA
-8-6-4-20246
-4-3-2-10123
B
Expr
essi
on (l
og2)
-2
0
2
4
6
8
10
12 n=89
btrS
btrA
BvgS
BvgA
Cluster 1 genes(e.g.flagella)
Expr
essi
on (l
og2)
-202468
101214 n=208
btrS
btrA
BvgS
BvgA
Cluster 2 genes(e.g.flagella)
Expr
essi
on (l
og2)
-202468
101214
n=168
btrS
btrA
BvgS
BvgA
Cluster 3 genes(e.g.fhaB/cyaA/prn)
btrS
btrA
BvgAS
Cluster 4 genes(e.g.T3SS)
n=126
wild ty
pe
bvgSΔ5
4ΔbtrS
ΔbtrA
ΔbtrAΔbtrS
Expr
essi
on (l
og2)
-202468
1012141618
Cluster 1
Cluster 2
Cluster 3
Cluster 4a
Cluster 4b
vecto
r
pbtrS
5
95
Perc
entil
e
Log 2
Tran
sfor
med
RPK
M
pbtrA
Expr
essi
on (l
og2)
Expr
essi
on (l
og2)
Expr
essi
on (l
og2)
vecto
rpbtrS
pbtrA
-202468
1012141618 n=29
-2
0
2
4
6
8
10
12n=61
-202468
101214
n=12
Cluster a
Cluster b
Cluster c
wild ty
pe
bvgSΔ5
4
ΔbtrS
ΔbtrA
ΔbtrAΔbtrS
5
95
Perc
entil
e
Log 2
Tran
sfor
med
RPK
MLo
g 2 Fol
d C
hang
e (R
PKM
)
A
C
cyaCcyaAcyaBcyaDcyaEsphB1brkBpr
nfim3
ptxAptxBptxEptxDptxCptlAptlBptlCptlDptlLptlEptlFptlGptlH
fhaLbipAfhaSfimDfimCfimBfimAfhaBbvgAbvgSbvgR fim
NfimXfim2dntpagP
ΔbtrSΔbtrA ΔbtrAΔbtrS
bscFbscEbscDbcrD or
f2 orf3orf4bopNbsp22orf6bcrH1bopDbopBbcrH2bcr4bscIbscJbscK bs
cLbscNbscObscPbscQbscRbscSbscTbscUbscWbs
cC btrAbtrSbtrUbtrWbtrVbtcAbteA
-8-6-4-20246
-4-3-2-10123
B
Expr
essi
on (l
og2)
-2
0
2
4
6
8
10
12 n=89
btrS
btrA
BvgS
BvgA
Cluster 1 genes(e.g.flagella)
Expr
essi
on (l
og2)
-202468
101214 n=208
btrS
btrA
BvgS
BvgA
Cluster 2 genes(e.g.flagella)
Expr
essi
on (l
og2)
-202468
101214
n=168
btrS
btrA
BvgS
BvgA
Cluster 3 genes(e.g.fhaB/cyaA/prn)
btrS
btrA
BvgAS
Cluster 4 genes(e.g.T3SS)
n=126
wild ty
pe
bvgSΔ5
4ΔbtrS
ΔbtrA
ΔbtrAΔbtrS
Expr
essi
on (l
og2)
-202468
1012141618
Cluster 1
Cluster 2
Cluster 3
Cluster 4a
Cluster 4b
a2 3
4abc1+2 3
4
b1+2 3
4c 3
4
D E
F
G
Supernatants Pellets
RB
50
Bp5
36
Bp5
36ΔbtrA
#1
Bp5
36ΔbtrA
#2
Bp5
36
Bp5
36ΔbtrA
#1
Bp5
36ΔbtrA
#2
mid-log stationary
RB
50
Bp5
36
Bp5
36ΔbtrA
#1
Bp5
36ΔbtrA
#2
Bp5
36
Bp5
36ΔbtrA
#1
Bp5
36ΔbtrA
#2
mid-log stationary
CyaA
Bsp22S4 subunit
Prn
FhaB
A B
C
M1
L104
T164
T134
Q19
4
BtrAFL
BtrAΔQ194-M226
BtrAΔT164-M226
BtrAΔT134-M226
BtrAΔL104-M226
M22
6
T124
S17
7
BtrAΔL101-V133
BtrAΔT134-D163
BtrAΔT164-M193
BtrAT124-S177 S81
BtrAS81-S177
+ +
+ - -
- +
+ + +
+ +
+ + +
T3S
S
FhaB
+ nd nd
- nd nd
- nd nd
- nd nd
- nd nd
BtrS
inte
ract
ion
BtrAΔT164-M226
BtrAΔT134-M226
BtrAΔL104-M226
BtrAΔL101-V133
BtrAΔT134-D163
BtrAΔT164-M193
BtrAT124-S177
BtrAS81-S177
DDO DDO X/A QDO X/A
BtrAFL
BteA644
BtrS
BtrS
BtrS
BtrS
BtrS
BtrAFL
BtrS
BteA644 BtrAFL
BtrAFL BtrS
BtrS
BtrS
BtrS
BtrS
BtrAΔQ194-M226
Bait Prey
RB50/vector
ΔbtrA /pbtrA ΔbtrA /vector
RB50/p btrA
RB50/vector
ΔbtrA/p btrA ΔbtrA /vector
RB50/p btrA
BA
RB50/vector
ΔbtrA/vector
recA
btrA
+ + + +m m m mgDNA
bopN
bsp22
btrS
fhaB
RB50/pbtrA
ΔbtrA/pbtrA
cyaA
prn
flaA
C
btrA
btrS
bsp22
bopN
cyaA
fhaB prn
flaA
relative-folddifference
0.0
0.5
1.024681012141618202224150300450
RB50/vectorRB50/pbtrA
ΔbtrA/vectorΔbtrA/pbtrA
RB50/vectorRB50/pbtrS
ΔbtrA/vectorΔbtrA/pbtrS
ΔbtrA/vector
RB50/vector
25-20-
15-
37-
10-
50-
75-100-
150-
250-
kDa
Bsp22
RB50/pbtrA
ΔbtrA/pbtrA
ΔbtrA/vector
RB50/vector
RB50/pbtrA
ΔbtrA/pbtrA
pellet supernatant
FhaB
CyaA
Prn
25-20-
15-
37-
10-
50-
75-100-
150-
250-
kDa
D
FE G H
RB50/vector
ΔbtrA /vector ΔbtrA /pbtrS
RB50/p btrS
RB50/vector
ΔbtrA /vector ΔbtrA /pbtrS
RB50/p btrS
RB50/vector
ΔbtrA/vector
recA
btrA
+ mgDNA
bopN
bsp22
btrS
fhaB
RB50/pbtrS
ΔbtrA/pbtrS
cyaA
prn
flaA
+ m + m + m
relative-folddifference
btrA
btrS
bsp22
bopN
cyaA
fhaB prn
flaA
0.0
0.4
0.815913172125150300450 Δ
btrA/vector
RB50/vector
25-20-15-
37-
10-
50-
75-100-
150-
250-
kDa
CyaA
Prn
FhaB
RB50/pbtrS
ΔbtrA/pbtrS
ΔbtrA/vector
RB50/vector
RB50/pbtrS
ΔbtrA/pbtrS
pellet supernatant
Bsp22
25-20-15-
37-
10-
50-
75-100-
150-
250-
kDa
BvgS
BvgA
[Adenylate cyclase/CyaA]
CyaE
CyaB
BteA
[Pertussis toxin] (Bp only)
Capsule
[Pertac4n]
[FHA] TCT
Type I CyaD
[Type III]
Vag8, BrkA Tcf
Pep4doglycan layer
OM
IM
FimC usher
FimD
Fim2 & 3
FimB
Type IV
vrg
“Phospho-‐relay”
bvgA bvgS bvgR
+ +
FhaB FhaC
Adapted from Locht et. al. Current Opinion in Microbiology 2001, 4:82–89
B A
F G H
A
B C D E
California NanoSystems Ins4tute