Global changes in Staphylococcus aureus gene
expression during human prosthetic joint infection
1
Xu ,
1
Maltesen ,
1,2
Larsen ,
2
Schønheyder ,
Yijuan
Raluca Georgiana
Lone Heimann
Henrik Carl
Jeppe Lund
1
1
1,3
1
3
Nielsen , Per Halkjær Nielsen , Trine Rolighed Thomsen , Kåre Lehmann Nielsen , The PRIS Study Group
1Department
of Biotechnology, Chemistry, and Environmental Engineering, Aalborg University, Denmark. 2Department of Clinical Microbiology, Aalborg Hospital, Aalborg
University Hospital, Denmark. 3The Danish Technological Institute, Life Science Division, Denmark.
Conclusions
Introduction
Staphylococcus aureus is one of the leading
causes of community- and hospital-acquired
infections worldwide. It can cause acute
infections and adapt to a biofilm mode of
growth and thereby cause persistent and
recurrent infections, particularly in devicerelated infections. Little is known about
regulation of gene activity of S. aureus during
actual human infection.
Here we characterize the metabolome using
NMR, and the transcriptome using RNA-seq, of
S. aureus infected joint fluid derived from an
acute human prosthetic joint infection, and
compare them with the genome, transcriptome
and metabolome of an isolate obtained from
the same sample grown in vitro (LB medium).
• S. aureus sustained on a versatile human-cell-based diet consisting of amino acids,
glycans and nucleosides in the hypoxic joint fluid during human prosthetic joint
infection.
• Many, but not all, of the known virulence factor genes were upregulated in situ.
Results
• S. aureus monoinfection was determined by culture, 16S amplicon sequencing and FISH.
• 436 Protein-coding genes (17% of total) were differentially expressed (322 upregulated and 114
downregulated in situ).
• 131 Known or proposed virulence factors in the genome: 47 upregulated and 9 downregulated.
Particularly γ-hemolysins, a few superantigen-like proteins, adhesins and immune evasion
molecules as well as SaeRS and VraSR two-component systems were overexpressed in situ.
Deoxyadenosine
Deoxyguanosine
Phosphate
DeoD
DeoD
Adenine
Aim
Deoxyuridine
Phosphate
Phosphate
Guanine
Thymidine
Phosphate
DeoA
Uracil
DeoA
Thymine
Deoxyribose-1-phosphate
β-D-glucose-6-phosphate
GLYCOLYSIS I
Spontaneous
PURINE/PYRIMIDINE
DEOXYRIBONUCLEOSIDES
DEGRADATION
2-Deoxy-α-D-ribose 1-phosphate
D-fructose-6-phosphate
Phosphate
DeoB
fbp
To characterize the in situ virulence gene
expression and metabolism of S. aureus using
RNA-seq and NMR metabolite analysis.
H2O
2-Deoxy-D-ribose-5-phosphate
Fructose-1,6-bisphosphate
DeoC
Acetaldehyde
NAD+
Coenzyme A
NADH
H+
D-glyceraldehyde-3-phophate
AldA
ADH
Phosphate
NAD+
H+
NADH
Acetyl-CoA
Dihydroxyacetone phosphate
gapA
Tagatose-1,6-bisphosphate
LacD
ADP
H+
ATP
1,3-Bisphospho-D-glycerate
LACTOSE AND GALACTOSE
DEGRADATION I
LacC
Tagatose-6-phosphate
Methods
3-Phospho-D-glycerate
NAD+
Coenzyme A
H+
NADH
gpmA
2-Phospho-D-glycerate
Lactose
6-phosphate
LacA
LacB
b-D-glucose
H2O
D-galactose
6-phosphate
L-SERINE
DEGRADATION
Identification
Lactose 6-phosphate
LacG
Phosphoenolpyruvate
H+
ammonia
L-serine
L-arginine
ARGININE
DEIMINASE
PATHWAY
NAD+
H2O
Phosphate
H+
L-ornithine
pflB
ald
L-alanine
argI
argF
Carbamoyl-phosphate
ADP
H+
Ammonia
ATP
yqeA
arcC
CO2
FISH
nanA
H+ NAD+
NADH
Acetyl-CoA
aldA
ADH
Spontaneous
Acetaldehyde
butA
Diacetyl
CO2
ADH
adhP
NADH
H+
PYRUVATE TO
ETHANOL
FERMENTATION I
Ethanol
(S)-acetoin
NAD+
(S)-ACETOIN BIOSYNTHESIS
N-ACETYLNEURAMINATE
DEGRADATION
ORNITHINE
SAUREUSv1_20053 DEGRADATION I
L-proline
An oxidized electron
acceptor
H+
A reduced electron acceptor
SAUREUSv1_30035 PROLINE
DEGRADATION
(S)-1-pyrroline-5-carboxylate
2 H2O
NAD+
H+
NADH
16S amplicon
sequencing
Coenzyme A
NAD+
NADH
H+
An oxidized electron
acceptor
A reduced electron
+
H
acceptor
N-acetyl-β-D-mannosamine
L-ornithine
Ammonium
formate
(S)-2-acetolactate
N-acetylneuraminate
L-arginine
Coenzyme A
Pyruvate
NADH
Ammonia
2 H+
ALANINE
DEGRADATION IV
arcA
L-citrulline
Genome
sequencing and
annotation
Culture
tdcB
H2O
Ammonia
H+
L-histidine
HISTIDINE DEGRADATION I
Ammonia hutH
H+
rocA
Urocanate
H2O
L-glutamate
H+
hutU
4-Imidazolone-5-propionate
Gene expression
H2O
H+
hutU
Formate
H2 O
N-forminino-L-glutamate
Fig. 1 Overexpressed metabolic pathways in the infection. The pathway names are according to the MetaCyc database. Each
pathway is assigned with a specific color and the upregulated enzymes in each pathway are indicated.
Isolate
(OD600~0.5)
12
Synovial
fluid
AAU
Bioinformatics
RNA-seq
A) Amino acids
B) Carbohydrates
10
(EdgeR)
3
Metabolite analysis
Isolate
Concentration (mM)
Concentration (mM)
8
6
4
2,5
2
1,5
1
0,5
0
(OD600~0.5)
2
0
Synovial
fluid
NMR measurement
LB (OD600=0)
C) Fermentation products
D) Nucleosides
40,5
This paper was prepared within the framework of the
‘Prosthetic-Related Infection and Pain’ (PRIS) - Innovation
project. http://www.joint-prosthesis-infection-pain.dk The
study was supported by a grant for the PRIS Innovations
Consortium from The Danish Council for
Technology and Innovation (no. 09–052174).
trt@teknologisk.dk
39
Concentration (mM)
Acknowledgement
39,5
3
2,5
2
Concentration (mM)
40
1,2
Joint fluid
1
0,8
0,6
0,4
1,5
1
LB (OD600=0.5)
0,2
0,5
0
0
www.cmc.aau.dk
Fig. 2 Concentration of metabolites
determined by NMR analysis.
LB(OD600=0) (blue) and joint fluid
(green) were analyzed in technical
triplicates while LB (OD600=0.5) (red)
was done in biological replicates. The
detection limit of NMR is 2 μM.