Viral infections facilitate bacterial trafficking to the lower respiratory tract resulting in bacterial viral co infections. Bacterial dissemination to the lower respiratory tract is enhanced by influenza A virus induced epithelial cell damage and dysregulation of immune responses. Epithelial cells act as the first line of defense and detect pathogens by a high variety of pattern recognition receptors. The post translational modification ubiquitin is involved in almost every cellular process. Moreover, ubiquitination contributes to the regulation of host immune responses, influenza A virus uncoating and transport within host cells. We applied proteomics with a special focus on ubiquitination to assess the impact of single bacterial and viral as well as bacterial viral co-infections on bronchial epithelial cells. We used Tandem Ubiquitin Binding Entities to enrich polyubiquitinated proteins and assess changes in the ubiquitinome. Infecting 16HBE cells with Streptococcus pyogenes led to an increased abundance of proteins related to mitochondrial translation and energy metabolism in proteome and ubiquitinome. In contrast, influenza A virus infection mainly altered the ubiquitinome. Co-infections had no additional impact on protein abundances or affected pathways. Changes in protein abundance and enriched pathways were assigned to imprints of both infecting pathogens.

LC-MS/MS was applied to uncover peptide compositions and quantities. Therefore, an EASY nLC 1200 (Thermo Fischer) was coupled to a QExactive mass spectrometer (Thermo Fisher). Peptides were loaded onto in-house packed fused silica columns of 20 cm length and an inner diameter of 75 µm, filled with Dr. Maisch ReproSil Pur 120 C18-AQ 1.9 µm (Dr. Maisch). Peptides were eluted using a non linear binary gradient of 166 min from 2% to 99% solvent B (0.1% (v/v) acetic acid in acetonitrile) in solvent A (0.1% (v/v) acetic acid). For PRM measurements of selected proteins precursor ions were isolated with an isolation width of 1.4 Th and fragmented by higher-energy collisional dissociation (HCD) at a normalized collision energy of 27. Fragment ion spectra were recorded with a resolution of 70,000 at 200 m/z. Selected precursor ions were time scheduled.

Proteins were digested on micro S Traps (Protifi) following the vendor’s protocol with slight modifications. Briefly, lysates containing 50 µg protein were transferred into new tubes and the volume was adjusted to 50 µl using freshly prepared lysis buffer. SDS and TEAB have been added to final concentrations of 5% (w/v) and 50 mM, respectively. Disulfide bonds were reduced by adding TCEP (5 mM final concentration; 45 min; 65 °C). Thiol groups were alkylated by adding iodoacetamide (IAA; 10 mM final concentration; 15 min; RT; in the dark). Phosphoric acid was added to the samples to a final concertation of 1.2%. Afterwards, 500 µl S Trap binding buffer (90% methanol, 100 mM TEAB) was added to the acidified samples. The protein colloid solution was subsequently loaded onto the S-Trap micro columns by centrifugation (4,000×g). Trapped proteins were washed three times with 165 µl S Trap binding buffer. Proteins were digested by adding 25 µl digestion buffer (50 mM TEAB) containing 2 µg trypsin (Promega) followed by an incubation for 3 hours at 47 °C. Peptides were subsequently eluted with 40 µl 50 mM TEAB; 0.1% (v/v) acetic acid; 60% (v/v) ACN in 0.1% (v/v) acetic acid. All eluted fractions of a sample were pooled and dried in a vacuum concentrator. Dry peptides were stored at -80 °C. Peptides were fractionated using Pierce Micro-Spin Columns (Thermo Fisher) packed with 15 mg of Dr. Maisch Reprosil-Gold 300 C18, 5 µm (Dr. Maisch) particles. Columns were washed three times with 300 µl acetonitrile and equilibrated two times with 300 µl 0.1% (v/v) TFA by centrifugation (5,000×g; 2 min). Dried peptides were reconstituted in 300 µl 0.1% (v/v) TFA and loaded onto the spin column by centrifugation (4 min; 3,000×g). Peptides were subsequently eluted with 5%; 7.5%; 10%; 12.5%; 15%; 17.5%; 20% and 60% (v/v) acetonitrile in 0.1% (v/v) triethylamine, 300 µl each (4 min; 3,000×g). Fractions were concatenated using the scheme: 5% and 15%; 7.5% and 17.5%; 10% and 20%; 12.5% and 60%. Concatenated fractions were dried by vacuum centrifugation and resolved with 20 µl 0.1% (v/v) acetic acid containing iRT peptides (Biognosys). 5 µl of each fraction were pooled for validation of the label free quantification by parallel reaction monitoring (PRM).