genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
Kidney Gene Expression Pattern in an in vivo Infection Model
of 70 % ethanol, air dried for approximately 2 min and solved over night at 4°C in DNA
Rehydration Solution (Promega, 10 mM Tris/HCl pH 7.4, 1 mM EDTA pH 8.0; 50 µl / pellet). Both
aliquots of each sample were combined on the following day, the concentration was determined
photometrically (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE, USA), and the
quality was checked by electrophoresis in 0.8 % agarose-TBE gels.
Molecular estimation of infection rate with qPCR
The DNA from infected tissue consists of a mix of host and pathogen DNA, which contains
almost constant levels of host DNA and a varying proportion of bacterial DNA that very much
depends on the infection rate. Therefore, quantifying the ratio of pathogen to host DNA allows
an estimation of the infection rate, which can also be calibrated with the aid of artificially mixed
standard samples.
The highly conserved staphylococcal genes nuc (coding for thermonuclease) and dapA
(encoding dihydrodipicolinate synthase) were quantified in reference to the mouse cytoskeleton
gene Actb using 20x TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA;
Actb: inventoried assay Mm00607939_s1; nuc: custom assay, forward primer
GATCCAACAGTATATAGTGCAACTTCAACT, reverse primer ACCGTATCACCATCAATCGCTTT, reporter
AACCTGCGACATTAATT; dapA: custom assay, forward primer CTTTGAAGCGATTGCAGATGCT,
reverse primer TGGTTCAATTGTCATGTTCGTTCTTG, reporter ACGACTGGTAATTTC; each reporter’s
5’ end is labeled with FAM; a non-fluorescent quencher (NFQ) and a Minor Groove Binder (MGB)
molecule is linked to the 3’ end).
The real-time PCR was performed in triplicates with 20 mM Tris/HCl pH 7.4, 50 mM KCl, 3 mM
MgCl 2 , 4 % glycerol, 0.2 mM dNTP mix (dATP, dCTP, dGTP, dTTP, 0.2 mM each), 1 % ROX reference
dye (Invitrogen, Karlsruhe, Germany) and 0.35 U Platinum Taq DNA Polymerase (Invitrogen) using
20 ng of DNA as template in each reaction. The so-called “universal settings” for Applied
Biosystems’ TaqMan Assays were applied: 2 min at 50°C, 10 min at 95°C followed by 40 cycles of
15 s at 95°C and 1 min 60°C.
A calibration curve with DNA prepared from a mixture of non-infected kidney tissue and
in vitro cultivated staphylococcal cells was recorded in analogous reactions. Each standard
contained a defined number of cfu per 10 mg tissue ranging from 1.32E+05 cfu/10 mg to
3.31E+08 cfu/10 mg (Fig. M.2.1).
A
B
8.0
6.0
4.0
2.0
mean
Linear (mean)
y = -3.3265x + 23.246
R² = 0.9946
8.0
6.0
4.0
2.0
mean
Linear (mean)
y = -3.2883x + 22.211
R² = 0.9956
ΔCt
0.0
ΔCt 0.0
-2.0
-2.0
-4.0
-4.0
-6.0
-6.0
-8.0
5.0 6.0 7.0 8.0 9.0
log ( cfu / 10 mg tissue)
-8.0
5.0 6.0 7.0 8.0 9.0
log ( cfu / 10 mg tissue)
Fig. M.2.1: Calibration curves for molecular estimation of infection rate using the staphylococcal genes dapA (A) and nuc (B).
Mean values of ΔCt (ΔCt = Ct bacterial gene – Ct Actb) from n = 2 or n = 3 independent standard samples and the range (minimum to
maximum) of these independent measurements are displayed.
The Ct values of bacterial genes and Actb were used to calculate ΔCt
(ΔCt = Ct bacterial gene − Ct Actb ). This value negatively correlates to the log-transformed values of
cfu/10 mg and can be described by a linear equation. The use of this linear correlation as
calibration curve allows calculation the infection rate of the unknown experimental samples.
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