Mutations in global regulatory genes pave the way for chronic infections of Pseudomonas aeruginosa

in cystic fibrosis airways

Søren Molin Department of Systems Biology Technical University of Denmark

•  CF is caused by mutations in the CFTR gene which affect chloride channels.

•  This results in decreased fluidity of mucus Impaired clearance of inhaled microbes.

Pseudomonas aeruginosa infections in Cystic Fibrosis patients

A Model System for Adaptive Evolution

Normal airway epithelia

CF airway epithelia

Thick, dehydrated mucus.

•  CF patients typically develop persistent Pseudomonas aeruginosa lung infections that lead to reduced lung function.

Niels  Høiby  

Niels  Høiby  founded  in  1972  a  unique  collec7on  of  Pseudomonas  aeruginosa  bacteria  isolated  from  CF  pa7ents  with  severe  lung  infec7ons.  This  collec7on  is  s7ll  expanding.    

’Experimental Evolutionary Biology’ at the Copenhagen Cystic Fibrosis Clinic

Høiby’s strain collection is a gold mine of resources for investigations of evolution in microbial populations

Moving from the environment to human airways

Life-style change

Phenotypes of P. aeruginosa in chronically infected CF airways

•  Loss of motility •  Loss of quorum sensing •  Reduction of virulence factors •  LPS modifications •  Antibiotic resistance •  Hypermutability •  Slow growth •  Alginate overproduction (mucoid, biofilm, population capsule)

Early infections are usually caused by environmental wild-type strains (motile, quorum sensing positive, virulent, antibiotic sensitive, fast growing, non-mucoid)

Immune evasion strategy?

Infection dynamics in chronically infected CF patients

Genotype key:

Each Color indicate specific genotypes

No color indicate unique genotypes found only once

Time

Phenotype key:

* = mucoid

Conclusions:

2 dominant clones: ‘red’ and ‘blue’

6/6 random patients have become infected with one or both

Most long-term chronic infected patients carry ‘red’ (DK1) and/or ‘blue’ (DK2). We have identified the two clones in 80 other patients.

•  6,402,658 bp

•  5884 genes

CF333 2007 genome

PADK2

1  Mbp  

2  Mbp  

3  Mbp  

4  Mbp  

5 Mbp  

6  Mbp  

Portrait of an adapted CF clone

43 isolates of the DK2 clone type have been sequenced

Phylogenetic relationships of DK2 clones isolated from cystic fibrosis patients

0

5

10

15

20

25

30

35

40

45

50

1990 1992 1994 1996 1998 2000 2002 2004 2006 2008

Tota

l SN

Ps

Year

Total SNP accumulation CF333 sub-lineage

CF333

Accumulation of SNPs in DK2 lineage

Mutation rate: 7x10-11 / base pair / generation Negative selection: dN/dS = 0.78

Transcriptomics Experimental approach

Results: An ’expression signature’ for each strain analyzed

Changes in gene expression indicate mutations in control functions

Plotting PC1 against time: Fitness?

p6 p7

1979

PC

1 re

pres

ents

40%

of t

otal

var

ianc

e

No  direct  correla7on  between  accumula7on  of  muta7ons  and  accumula7on  of  phenotypic  changes  

Could be explained by early pleiotropic mutations

Evolution playing games with sigma factors

Important mutations in early isolates

The isolate CF30-1979 which after 1979 was spread among multiple CF patients has mutations in the following regulatory genes:

Locus tag

Name

PA0763 mucA PA4462 rpoN PA1430 lasR

PA0762 algU(T) PA4777 pmrB3

PA2426 pvdS1

Locus tag

Name

PA2387 fpvI1 PA5200 amgR2 PA4102

PA0120 PA2020

1Iron uptake via pyoverdine 2Stress response and antibiotic suscept 3Colistin resistance

430ΔG

T1256C

ΔlasR

G55A

Anti-σE

N2 metabolism (σ54)

Quorum sensing

Stress (σE)

Muations in iron sigma factor genes

Heme associated iron uptake gene expression

RNA polymerase

Sigma Factor Re-Cycling

Four pleiotropic mutations

CF114-1973

CF30-1979

p6-1984

p6-2005

CF66-1973

CF43-1973

P7-2007

P7-2003

p7-1997

p7-1991 rpoN,  T1256C  

mucA  ΔG,  algU,  G55A  

Δ  lasR  

Synthetic Biology – design of a CF pathogen

•  Isogenic mutants of PAO1 carrying mutated alleles from the late blue clone were constructed by allelic replacement.

•  Single, double, triple and quadruple mutants were investigated by Biolog Phenotype Microarrays and Affymetrix DNA arrays

mucA → mucA algU → mucA algU rpoN → mucA algU rpoN lasR

Sigma E/anti-sigma E

The mucoid regulators

AlgU

alg operon (alginate biosynthesis genes)

MucC

mucD mucC mucB mucA algU

MucD MucB MucA

AlgR

algR

*

*mucA mutations are most frequent causes of mucoidy. Often frame-shift mutations.

The  algU  muta7on  Expression

 int.  

No expression of algD in LB

Core binding domain DNA binding domainActivator binding domain

L419P

E507Δ

K19E

σ-70 region 4 σ-70 region 2

region 1 region 1.2 non-essential region region 2 region 3 region 4

! " # $ %

!"

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%

&'()

*+%!

R2= 0.67P < 0.0001

The algU G55A mutation

•  Partial inactivation of regulatory activity •  Reversion of mucA phenotype in aerobic conditions

•  No reversion in anaerobic conditions •  No reversion in osmotic stress conditions

Note: Conditional phenotype of the algU G55A mutation

The DK2 cells growing in the CF airways are probably mucoid in the anaerobic sectors (upper airways) and non-mucoid in the aerobic lower sectors. The AlgU mediated stress response is not lost with this particular mutation.

Sigma D and Sigma N

The  rpoN  muta7on  

assay PAO1

rpoN* ΔrpoN

Td [min] 27 32 55

Swimming +++ ++ -

Twitching +++ ++ -

PAO1            vs  

rpoN*  

Expression

 int.  

Core binding domain DNA binding domainActivator binding domain

L419P

E507Δ

K19E

σ-70 region 4 σ-70 region 2

region 1 region 1.2 non-essential region region 2 region 3 region 4

Carbon   Carbon   Nitrogen  

930 218439

904 113313

ΔrpoNrpoN*

Up-regulated genes

Down-regulated genes

Differential RpoN mediated gene expression control

-2-1

01

2

01

23

fold

-cha

nge

rela

tive

to P

AO1

genes (1-344) genes (1-216)

rpoN::GmrpoN*

rpoN::GmrpoN*

The Quadruple Mutant

up down up down

The introduction of the algT(G55A) cause a decrease in the number of overlapping genes… SD13: mucA, rpoN, lasR

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PAO1

mucA rpo

NlasRSD40SD42SD43

0.0

0.5

1.0

1.5

2.0

2.5

MIC

[µg/

ml]

PAO1

mucA rpo

NlasRSD40SD42SD43

0.0

0.5

1.0

1.5

2.0

MIC

[µg/

ml]

PAO1

mucA rpo

NlasR

SD40

SD43

0

1

2

3

4

MIC

[µg/

ml]

Tobramycin Meropenem Ceftazidime

Extensive loss of catabolic function Increased antibiotic resistance of the Q mutant

Evolutionary path in mono-clonally infected patient

All clones are mucA (ΔG430), algU (G55A) and non-mucoid

Late appearance of mucoid isolates

*Nov

*April

The late mucoid isolates are distinctly different from the non-mucoid

p720

07c.

CEL

p720

07b.

CEL

p720

07d.

CEL

mun

ov07

a.C

EL

mun

ov07

b.C

EL

mun

ov07

c.C

EL

mua

pr07

c.C

EL

mua

pr07

a.C

EL

mua

pr07

b.C

EL

PA0427_oprM_at

PA1756_cysH_at

PA2729_at

PA2726_at

PA1199_at

PA5465_at

PA3925_at

PA0537_at

PA3222_at

PA2712_at

PA5364_at

PA0294_at

PA0629_at

PA0638_at

PA0635_at

PA0639_at

PA2687_pfeS_at

PA0097_at

PA0260_at

PA3191_at

PA0079_at

PA3401_at

PA1087_flgL_at

PA1396_at

PA1845_at

PA0261_at

PA4267_rpsG_at

PA2792_at

PA3294_s_at

PA0262_at

PA1450_at

PA0096_at

PA5136_at

PA5138_at

PA5025_metY_at

PA3059_at

PA0073_at

PA1326_ilvA2_at

PA1015_at

PA1086_flgK_at

PA3061_at

PA2728_at

PA3021_at

PA0064_at

PA0637_at

PA2894_at

PA0360_at

PA1395_at

PA0088_at

PA0076_at

PA3729_at

PA0095_at

PA5089_at

PA0855_at

PA5273_at

PA0098_at

ig_6125795_6125079_at

PA0632_at

PA1844_at

PA0070_at

PA3064_at

PA4801_at

PA1639_at

PA0072_at

PA0170_at

PA0172_at

PA0505_at

PA0091_at

PA0094_at

PA4512_at

PA0126_at

PA0089_at

PA1511_at

PA1493_cysP_at

PA5085_at

PA2624_idh_at

PA1654_at

PA4740_pnp_at

PA0316_serA_at

PA3977_hemL_at

PA2536_at

PA0101_at

PA2537_at

PA0046_at

PA0045_at

Pae_tRNA_Thr_s_at

PA0248_at

PA3731_at

PA3732_at

PA1527_at

PA0418_at

PA3850_at

PA4856_at

PA0092_at

PA2774_at

PA3716_at

PA0075_at

PA0074_ppkA_at

PA0087_at

PA4800_at

PA0024_hemF_at

PA0081_at

PA0570_at

PA2540_at

PA0171_at

PA3730_at

PA0077_at

PA3679_at

PA4516_at

PA2685_at

PA2684_at

PA4317_at

PA5441_at

PA1069_at

PA0047_at

PA2539_at

PA0071_at

PA0080_at

PA0078_at

PA0563_at

PA0082_at

PA0083_at

PA3485_r_at

PA0084_at

PA0090_at

PA0086_at

PA0085_at

PA3363_amiR_at

PA3622_rpoS_at

PA2114_at

PA2113_at

PA2109_at

PA2116_at

PA2410_at

PA2405_at

PA2404_at

PA2478_at

PA2749_endA_at

PA0156_at

PA0314_at

PA1741_at

PA2750_at

PA1727_at

PA5446_i_at

PA4378_inaA_at

PA2174_at

PA4573_at

ig_4889111_4888194_at

PA4377_at

PA4345_at

PA4338_at

PA3366_amiE_at

PA1948_rbsC_at

PA4204_at

PA4575_at

PA4413_ftsW_at

PA3023_at

PA0290_at

PA0059_osmC_at

PA3762_at

PA2528_at

PA5422_at

PA1944_at

PA1641_at

PA2963_at

PA0853_at

PA5261_algR_at

PA3177_at

PA2045_at

PA0060_at

PA3031_at

PA3289_at

PA4390_at

PA2778_at

PA2443_sdaA_at

PA0134_at

PA3986_at

PA1048_at

PA5182_at

PA1995_i_at

PA2954_at

PA2414_at

PA3460_at

PA2754_at

PA2146_i_at

PA1114_at

PA5108_at

PA3572_at

PA2172_at

PA2176_at

PA5463_at

PA2708_at

PA0943_at

PA2190_at

PA0990_at

PA3687_ppc_at

PA5060_phaF_at

PA2177_at

PA4876_osmE_at

PA2883_at

PA3461_at

PA3547_algL_at

PA3459_at

PA2562_at

PA3548_algI_at

PA3541_at

PA3549_algJ_at

PA3546_algX_at

PA3544_algE_at

PA3543_algK_at

PA3545_algG_at

PA3540_algD_at

PA3550_algF_at

PA3542_at

PA3551_algA_at

PA0869_pbpG_at

PA3238_at

PA1189_at

PA5452_wbpW_at

PA4829_lpd3_at

PA0803_at

PA4552_pilW_at

PA2787_cpg2_at

PA1889_at

PA3902_at

PA4750_folP_at

PA1296_at

PA0104_at

PA0462_at

PA1471_at

PA5423_at

PA3945_at

PA0762_algU_at

PA1518_at

PA4983_at

PA5497_at

PA1474_at

PA5209_at

PA0857_bolA_at

PA0555_fda_at

PA0967_ruvB_at

PA1243_at

PA0833_at

PA1354_at

PA1356_at

PA3740_at

PA1118_at

PA5329_at

PA0148_at

PA1349_at

PA3239_at

ig_2240302_2239267_at

PA1605_at

PA0764_mucB_at

PA1408_at

PA0738_at

PA0737_at

PA1880_at

PA5473_at

PA4972_at

PA5488_at

PA0372_at

PA0369_at

PA5077_mdoH_at

PA5489_dsbA_at

PA2988_at

PA2987_at

PA3612_at

PA0854_fumC2_at

PA2071_fusA2_at

PA1946_rbsB_at

PA5323_argB_at

PA2746_at

PA3598_at

PA4788_at

PA2145_at

PA4183_at

PA5212_i_at

PA3451_at

PA2168_at

PA2485_at

PA2433_at

PA1784_at

PA3819_at

ig_2893827_2894451_at

PA1745_at

PA2779_at

PA5424_at

PA3385_at

PA0061_at

PA2884_at

PA2167_at

PA1404_at

PA0062_at

PA2777_at

PA5183_at

PA2569_at

PA2171_at

PA5526_at

PA2173_at

PA0490_at

PA2023_galU_at

PA0309_at

PA3952_at

PA5322_algC_at

−2 −1 0 1 2Row Z−Score

Color Key

Heat map of gene expression in isogenic variants of DK2 from 2007 samples

2007-NM 2007-MNov 2007-MApr

Highly similar gene expression in two mucoid isolates

Mutations in mucoid isolates Gene  ID Gene  name Protein mutaAon

P707Apr mucoid

PA2164 probable  glycosyl  hydrolase G1735T;E579_

PA4415 mraY phospho-‐N-‐acetylmuramoyl-‐pentapepAde-‐transferase

G781A;A261T      

PA0576 rpoD sigma  factor  RpoD 1517-‐1519deltaAAG

No  blast Miscellaneous

P707Nov mucoid

PA4776 pmrA two-‐component  regulator  system  response  regulator  PmrA

T14C;L5P          

PA4418 WsI penicillin-‐binding  protein  3 G524A;R175H       PA2630 conserved  hypotheAcal  

protein     C172A;R58S        

PA0436 ?   probable  transcripAonal  regulator

C426T;V142V,  silent

PA1802 clpX ATP-‐dependent  Clp  protease  ATP-‐binding  subunit  ClpX

C381T;T127T,  silent    

PA0762 algU Sigma  factor  AlgU G55A;  E19K          Reversion  to  wt

Mutations in two different sigma factors result in highly similar phenotypes

Sigma D and Sigma N

The  rpoD  muta7on  

Core binding domain DNA binding domainAcivator binding domain

L419P

E507Δ

K19E

σ-70 region 4 σ-70 region 2

region 1 region 1.2 non-essential region region 2 region 3 region 4

RpoD  (σ70)  

Region 3 is involved in binding to the core RNA polymerase, but may be also in promoter binding

Eσ70 Eσ22

Mucoid

Non-mucoid

Mucoid

modified σ factor

The late mucoid phenotype represents a novel evolutionary leap

p6 p7

1979

PC

1 re

pres

ents

40%

of t

otal

var

ianc

e

Mucoid

Conclusions

•  Successful transmissible and competitive lineages •  Constant low mutation rate over 200,000 generations •  Frequent hypermutator lineages •  Genetic drift more than adaptive evolution •  Early fast increase in fitness followed by constancy •  Pleiotropic mutations result in adaptive peaking •  Key mutations in sigma factors •  Conversion of opportunistic pathogen to optimized

primary CF pathogen •  ….until a novelty mutation created a new jump in

fitness

Acknowledgements

•  Lei Yang •  Martin Holm Rau •  Lars Jelsbak •  Rasmus Marvig •  Søren Damkiær •  Morten Sommer •  Chris Workman •  Helle K. Johansen •  Niels Høiby