genomewide characterization of host-pathogen interactions by ...

Maren Depke
Introduction
channel CFTR (cystic fibrosis transmembrane conductance regulator), which has been repaired in
the S9 cell line by viral transfection with the wild-type gene (American Type Culture Collection
ATCC, Manassas, VA, USA; www.atcc.org; S9 cell ATCC number CRL-2778).
In vitro infection models can be accomplished by addition of three main types of bacterial
supplement: 1) supernatant of bacterial cultures containing secreted proteins or virulence
factors, 2) PBS-washed bacterial cells which bring only their membrane-bound and intracellular
factors into the infection experiment, and 3) complete bacterial culture with both secreted
proteins from supernatant and whole bacterial cells. This last option is nearest to the in vivo
situation when the pathogen is able to influence its host with secreted factors. On the other
hand, the established bacterial culture media, which are often lysates of protein-rich raw
material (e. g. tryptic soy broth TSB), are highly artificial with reference to in vivo models or even
to eukaryotic cell culture. Therefore, a medium was developed that allows bacterial growth but
additionally has similarity to eukaryotic cell culture media (Schmidt et al. 2010). The authors
describe the use of eukaryotic cell culture medium MEM supplemented with different amino
acids, but without addition of serum. This new experimental system permits the study of hostpathogen
interactions in the context of all bacterial factors, membrane-bound and secreted, and
additionally prevents effects on bacterial physiology by prolonged handling, centrifugation, and
washing of bacteria.
Here, exponential growth phase bacterial cultures were used to infect confluent S9 cell
cultures. The strain S. aureus RN1HG has been chosen for a first insight into the molecular
reactions in this model. RN1HG is a rsbU + repaired RN1-derivative strain (Herbert et al. 2010) with
a SigB-positive phenotype.
In a combined approach of transcriptome (Maren Depke) and proteome (Melanie Gutjahr)
analysis the host reaction to infection and bacterial internalization was recorded. But not only
stood the host cell in the focus of studies, but also the bacterium. In a similar experimental setup,
internalized staphylococci were extracted from their S9 host cells and the bacterial RNA profile
was recorded using a tiling array approach (Maren Depke). Bacterial intracellular proteins were
monitored and quantified after stable isotope labeling with amino acids in cell culture, SILAC
(Sandra Scharf).
Questions and Aims of the Studies Described in this Thesis
This thesis contains results from transcriptome studies on different aspects of host-pathogen
interactions. First, liver gene expression profiles from a murine chronic stress model served to
elucidate aspects of the influence of stress on the metabolism and the immune response state of
the animals. In the experiments for this study, the in vivo model of psychological stress in a
complex mammalian host was performed without additional influences of a pathogen. Such
influence was introduced in the second study: Here, the influence of staphylococcal i. v. infection
on the host kidney gene expression was analyzed in another murine in vivo model using a wild
type S. aureus strain and its isogenic sigB mutant. Tissue expression profiling from in vivo models
has the advantage of directly recording the relevant physiological state with all its complex
interactions and influences and its vicinity to medical questions in the human. Nevertheless, it is
very difficult to distinguish the different components because the tissue samples are always a
37