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  

       (uahuja@ucla.edu)    

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