Time-resolved quantitative proteome profiling of host-pathogen interactions: the response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells.
Staphylococcus aureus is a versatile gram-positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse-chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on-membrane digestion, and high-sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof-of-principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.
Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.
Frank
F
Schmidt
Sandra S
SS
Scharf
Petra
P
Hildebrandt
Marc
M
Burian
Jörg
J
Bernhardt
Vishnu
V
Dhople
Julia
J
Kalinka
Melanie
M
Gutjahr
Elke
E
Hammer
Uwe
U
Völker
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001980:Bronchi / Q000166:cytology*; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000191:economics / Q000379:methods*; D013203:Staphylococcal Infections / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000502:physiology*; D013997:Time Factors
D001426:Bacterial Proteins; D020543:Proteome