genomewide characterization of host-pathogen interactions by ...

Maren Depke
Introduction
cell stimulating substances like cytokines, hormones or endotoxin, and to worsen the problem,
these substances might vary not only between the sera from different vendors but also between
batches of serum from the same manufacturer. To overcome such sources of experimental
variation, Eske et al. introduced in 2009 serum-free culture conditions for BMM. Standard cell
culture medium RPMI 1640 was supplemented with a defined mixture of proteins, hormones,
and other compounds. Differentiation of stem cells with recombinant murine GM-CSF yielded
BMM expressing macrophage markers F4/80, CD11b, CD11c, MOMA-2, and CD13 after 10 days of
cultivation, and further characteristics of BMM differentiated in serum-containing medium were
additionally retained (Eske et al. 2009).
Host-pathogen interaction experiments have revealed differences in reactions of BMM
derived from BALB/c and C57BL/6 mice when confronted with Burkholderia pseudomallei
especially after IFN-γ stimulation and at higher multiplicities of infection (MOI). Also other
infection studies uncovered differences between these two mouse strains in vivo and in vitro
(Breitbach et al. 2006, Autenrieth et al. 1994, van Erp et al. 2006). The mouse strains BALB/c and
C57BL/6 are characterized by a Th2 and Th1 centered type of immune response, respectively.
Accordingly and possibly causatively, the main type of macrophage activation differs between the
strains, with BALB/c preponderating the alternative macrophage activation pattern and C57BL/6
prevailing classical activation (Mills et al. 2000).
Against the background of genetic influences on the BMM reactions, a combined proteome
(Dinh Hoang Dang Khoa) and transcriptome (Maren Depke) study was initiated. In a first
experiment, BALB/c and C57BL/6 BMM were stimulated with IFN-γ to specify on a molecular level
the reaction to the priming signal IFN-γ as basic principle. Furthermore, the study aimed to
profile potential differences of reaction between the BMM of both mouse strains.
Model Systems for Studies of Host-Pathogen Interactions
S. aureus strain RN1HG
The genetic background of different S. aureus strains influences the reaction of the bacterium
to experimental conditions. Therefore, the strain for experimental analysis must be chosen
carefully because not all observations can be transferred from one to another S. aureus strain.
The number of strains available for studies of S. aureus has grown recently when the group of
Friedrich Götz (Department of Microbial Genetics, Eberhards-Karls University of Tübingen,
Germany) provided a rsbU + repaired RN1-derivative strain called RN1HG(001) and later tcaR + and
rsbU + tcaR + repaired RN1-derivative strains to the scientific community (Herbert et al. 2010). The
parental strain RN1 (NCTC8325) has already been widely used as model organism for diverse
studies on staphylococcal physiology. On the other hand its defect in the important regulator
rsbU was known (Kullik/Giachino 1997), which resulted in compromised conclusions from studies
addressing the regulation of virulence factors (Giachino et al. 2001). Therefore, strain RN1HG in
which the mutation in the regulatory gene rsbU has been complemented has been chosen in the
experimental setup of the studies described in this thesis.
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