Sepsis is a systemic immune response responsible for considerable morbidity and mortality. Molecular modelling 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 bacterium Streptococcus pyogenes as a model system to establish a mass spectrometry based workflow for the construction of a stoichiometric surface density model between the S. pyogenes surface, the surface virulence factor M-protein, and adhered human blood plasma proteins. The workflow relies on stable isotope labelled 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 between S. pyogenes and 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 the S. pyogenes bacterial surface, revealing a dense and highly organized protein interaction network. Removal of the M-protein from S. pyogenes introduces a drastic change in the network topology, validated by electron microscopy. We propose that the stoichiometric surface density model of S. pyogenes in 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.

File Name Replicate Name Data set Sample prep Strain Replicate Concentration of Heavy KristofferS_O1504_164.raw BD_SF370_1 Density model Bacteria digest SF370 1 Roughly 1:1 to light KristofferS_O1504_165.raw BD_SF370_2 Density model Bacteria digest SF370 2 Roughly 1:1 to light KristofferS_O1504_166.raw BD_SF370_3 Density model Bacteria digest SF370 3 Roughly 1:1 to light KristofferS_O1504_167.raw BD_SF370_4 Density model Bacteria digest SF370 4 Roughly 1:1 to light KristofferS_O1504_179.raw BD_SF370_5 Density model Bacteria digest SF370 5 Roughly 1:1 to light KristofferS_O1504_180.raw BD_SF370_6 Density model Bacteria digest SF370 6 Roughly 1:1 to light KristofferS_O1504_181.raw BD_SF370_7 Density model Bacteria digest SF370 7 Roughly 1:1 to light KristofferS_O1504_182.raw BD_SF370_8 Density model Bacteria digest SF370 8 Roughly 1:1 to light KristofferS_O1504_169.raw BD_dM1_1 Density model Bacteria digest dM1 1 Roughly 1:1 to light KristofferS_O1504_170.raw BD_dM1_2 Density model Bacteria digest dM1 2 Roughly 1:1 to light KristofferS_O1504_171.raw BD_dM1_3 Density model Bacteria digest dM1 3 Roughly 1:1 to light KristofferS_O1504_172.raw BD_dM1_4 Density model Bacteria digest dM1 4 Roughly 1:1 to light KristofferS_O1504_184.raw BD_dM1_5 Density model Bacteria digest dM1 5 Roughly 1:1 to light KristofferS_O1504_185.raw BD_dM1_6 Density model Bacteria digest dM1 6 Roughly 1:1 to light KristofferS_O1504_186.raw BD_dM1_7 Density model Bacteria digest dM1 7 Roughly 1:1 to light KristofferS_O1504_187.raw BD_dM1_8 Density model Bacteria digest dM1 8 Roughly 1:1 to light KristofferS_O1504_174.raw GE_SF370_1 Density model Glycine Elution SF370 1 Roughly 1:1 to light KristofferS_O1504_175.raw GE_SF370_2 Density model Glycine Elution SF370 2 Roughly 1:1 to light KristofferS_O1504_176.raw GE_SF370_3 Density model Glycine Elution SF370 3 Roughly 1:1 to light KristofferS_O1504_177.raw GE_SF370_4 Density model Glycine Elution SF370 4 Roughly 1:1 to light KristofferS_O1504_189.raw GE_SF370_5 Density model Glycine Elution SF370 5 Roughly 1:1 to light KristofferS_O1504_190.raw GE_SF370_6 Density model Glycine Elution SF370 6 Roughly 1:1 to light KristofferS_O1504_191.raw GE_SF370_7 Density model Glycine Elution SF370 7 Roughly 1:1 to light KristofferS_O1504_192.raw GE_SF370_8 Density model Glycine Elution SF370 8 Roughly 1:1 to light KristofferS_O1611_147.raw B0e0_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 0pmol KristofferS_O1611_148.raw B1e0_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 1pmol KristofferS_O1611_149.raw B1e1_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 10pmol KristofferS_O1611_150.raw B1e2_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 100pmol KristofferS_O1611_151.raw B1e3_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 1fmol KristofferS_O1611_152.raw B1e4_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 10fmol KristofferS_O1611_153.raw B1e5_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 100fmol KristofferS_O1611_154.raw B1e6_1 DillutionSeries Spiked in peptides in dillution SF370 as background 1 1amol KristofferS_O1611_169.raw B0e0_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 0pmol KristofferS_O1611_170.raw B1e0_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 1pmol KristofferS_O1611_171.raw B1e1_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 10pmol KristofferS_O1611_172.raw B1e2_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 100pmol KristofferS_O1611_173.raw B1e3_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 1fmol KristofferS_O1611_174.raw B1e4_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 10fmol KristofferS_O1611_175.raw B1e5_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 100fmol KristofferS_O1611_176.raw B1e6_2 DillutionSeries Spiked in peptides in dillution SF370 as background 2 1amol