PMC

A total of 513 airway proteins were associated with S. aureus in the first 6 h of infection (see Table S1 in the supplemental material for a complete list of proteins). The proteins identified at 30 min (A) and 6 h (B) were divided into GO cellular component categories. The extracellular proteins identified at 30 min (C) and 6 h (D) were further categorized according to the corresponding GO biological processes.

Hemoglobin associates with S. aureus 30 min and 6 h postinoculation into the airway. Bacteria were recovered from the BAL fluids of mice inoculated with S. aureus, and host proteins were harvested as described in Materials and Methods. The host proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with antibodies against mouse hemoglobin. Lanes: 1, BAL fluid containing hemoglobin (control); 2, sample from 30 min postinoculation; 3, sample from 6 h postinoculation. Numbers on the left indicate molecular sizes in kilodaltons.

Laemmli buffer effectively removes airway proteins from the surfaces of S. aureus cells with minimal bacterial protein contamination. S. aureus JP1 was incubated with PBS or human BAL fluid and treated to remove the BAL proteins from the bacterial surface, and the BAL proteins were separated by SDS-PAGE and stained with SYPRO Ruby. Lanes 1, 3, 5, 7, and 9, JP1 incubated in PBS; lanes 2, 4, 6, 8, and 10, JP1 incubated in BAL fluid. BAL proteins were removed under the following conditions: lanes 1 and 2, Laemmli buffer at 100°C; lanes 3 and 4, Laemmli buffer at 37°C; lanes 5 and 6, 0.5% sodium deoxycholate at 37°C; lanes 7 and 8, 0.5% Tween 20 at 37°C; lanes 9 and 10, 0.5% Triton X-100.