Targeted Proteomics and Absolute Protein Quantification for the Construction of a Stoichiometric Host-Pathogen Surface Density Model.
Sepsis is a systemic immune response responsible for considerable morbidity and mortality. Molecular modeling of host-pathogen interactions in the disease state represents a promising strategy to define molecular events of importance for the transition from superficial to invasive infectious diseases. Here we used the Gram-positive bacteriumStreptococcus pyogenesas a model system to establish a mass spectrometry based workflow for the construction of a stoichiometric surface density model between theS. pyogenessurface, the surface virulence factor M-protein, and adhered human blood plasma proteins. The workflow relies on stable isotope labeled reference peptides and selected reaction monitoring mass spectrometry analysis of a wild-type strain and an M-protein deficient mutant strain, to generate absolutely quantified protein stoichiometry ratios betweenS. pyogenesand interacting plasma proteins. The stoichiometry ratios in combination with a novel targeted mass spectrometry method to measure cell numbers enabled the construction of a stoichiometric surface density model using protein structures available from the protein data bank. The model outlines the topology and density of the host-pathogen protein interaction network on theS. pyogenesbacterial surface, revealing a dense and highly organized protein interaction network. Removal of the M-protein fromS. pyogenesintroduces a drastic change in the network topology, validated by electron microscopy. We propose that the stoichiometric surface density model ofS. pyogenesin human blood plasma represents a scalable framework that can continuously be refined with the emergence of new results. Future integration of new results will improve the understanding of protein-protein interactions and their importance for bacterial virulence. Furthermore, we anticipate that the general properties of the developed workflow will facilitate the production of stoichiometric surface density models for other types of host-pathogen interactions.
From the ‡Department of Immunotechnology, Faculty of Engineering, Lund University, Sweden.|§Division of Infection Medicine, Department of Clinical Sciences, Lund University, Sweden.
Kristoffer
K
Sjöholm
§Division of Infection Medicine, Department of Clinical Sciences, Lund University, Sweden.
Ola
O
Kilsgård
§Division of Infection Medicine, Department of Clinical Sciences, Lund University, Sweden.
Johan
J
Teleman
§Division of Infection Medicine, Department of Clinical Sciences, Lund University, Sweden.
Lotta
L
Happonen
¶S3IT, University of Zurich, Zurich, Switzerland.
Lars
L
Malmström
§Division of Infection Medicine, Department of Clinical Sciences, Lund University, Sweden; johan.malmstrom@med.lu.se.
Johan
J
Malmström
D000942:Antigens, Bacterial / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D013290:Streptococcal Infections / Q000382:microbiology*; D013297:Streptococcus pyogenes / Q000378:metabolism / Q000502:physiology*
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001798:Blood Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; C014689:streptococcal M protein