mutations in global regulatory genes pave the way for chronic … · 2012. 3. 12. · pa2894_at...
TRANSCRIPT
-
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
! " # $ %
!"
#$
%
&'()
*+%!
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
!"#$ "#" "#$ "#% "#&
!"#&
!"#$
"#"
"#$
"#%
!"#%
"#&
!"#$%&'()*'&%+),+-./0.1"2".#
'()*+,-./0.121+
324.15,-./0.121+
-'$$6
789$
3:6"
)0.;
/?7>!?$
3:6%
=@*A
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