In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075

The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.

lysate. This protein band was only apparent in the SDS protein extracts for AB5075 cells, and was not detected in the soluble lysates (Fig. 4A, B, D). Full blot image is shown in Supplementary Fig. 17.

LC-MS ReACT analysis
A real-time informatics strategy (ReACT), previously described 1 , was used to improve the throughput of cross-linked peptide analysis. In MS 1 scan events, ions of charge states ≥ 4+ were identified using FTICR mass analyzer with 50,000 resolving power.

Generating the stage 1 protein database with BDP-NHP cross-linkers in AB5075
The purified proteins from the cross-linked AB5075 cells, which was in the buffer of 50 mM Only fully tryptic peptide sequences were considered and up to 2 missed cleavages were allowed. For QE plus data, the same database and Comet parameter settings were used as Velos-FT data, except for that fragment bin tolerance 0.1 and fragment bin offset 0 were used for QE plus data. The false discovery rate for the peptide identification was determined with the target-decoy approach, and was controlled at 1%.
A total of 1,741 proteins were identified with two or more unique peptides. The list of the proteins is provided in Supplementary Data 3.

Anti-HA co-IP with the BDP-NHP cross-linked AB5075-pMMB-oxa23 cells
Since membrane protein structures are highly dependent upon the presence of the lipid membrane environment, it only stands to reason that cell lysis and extraction to remove proteins from this lipid environment causes membrane protein structural perturbations and loss of relevant binding partners in a way not equaled with soluble protein complexes. Thus, the relative success rate of co-IP of soluble protein interactions is indisputably higher compared to complexes that involve membrane proteins. One way to improve the Co-IP success rate for membrane proteins is to add a cross-linking step before the Co- NaCl, 0.05% Triton X-100] to SDS concentration less than 0.1%. Anti-HA agarose was added to the mixture and were incubated at room temperature for five hours. Anti-HA agarose was spun down with 3,000 × g for 3 min at 4 °C, and was washed four times with the cross-linking IP buffer. Proteins were eluted with 2 × Laemmli buffer (Bio-Rad) and boiling at 95 °C for 5 min.

SDS-PAGE and immunoblot analysis
Proteins were analyzed with the Bio-Rad Mini-PROTEAN system, and were transferred to Streptavidin IRdye (680 or 800 channels) (LI-COR Bioscience) was used in a 1:20,000 dilution ratio with PBST, and was imaged with LI-COR Odyssey.

Colony forming units (CFU) analysis
A. baumannii cells that underwent BDP-NHP or DMSO treatments were pelleted with 3, 000 × g centrifugation at 4 °C for 15 min. Cell pellets were resuspended with the sterile PBS solution (pH 7.4), and were divided into two halves. One half of the cells were serially diluted with PBS, and 5 µl of the cells at 10 5 and 10 6 dilutions were spotted onto LB agar plates. The CFU observed from the two dilutions were averaged, and was converted to CFU/ml based on the starting volume and the plated volume.
The other half of the cells were lysed with SDS. The extracted proteins were separated with SDS-PAGE, and were analyzed with Infrared dye-labeled Streptavidins (LI-COR Bioscience) or with Coomassie protein stain (Bio-Rad).  Fig. 10) was based on measurements of two biological replicates (i.e. two independent protein purification events). Three technical replicates were analyzed for each biological replicate.

RNA extraction and quantitative polymerase chain reaction (qPCR)
AB5075 wild type or mutant cells were cryoground using Mixer Mill MM 400 (Retsch, Haan, Germany). Total RNA was extracted from the frozen cell powder with RNeasy Mini Kit (Qiagen, Hilden, Germany) following QiaGen protocol, including the on-column DNase digestion step using Ambion Dnase. RNA concentration was measured with Nanodrop N1000 (ThermoFisher Scientific). The cDNA was synthesized with ImProm-II ReverseTranscription System kit (Promega) using the random hexamer primers, with the RNA template amount of 800 ng in each reaction.
The qPCR primers include a primer set for 16S RNA 20

Parallel reaction monitoring (PRM) assays for Oxa-23 proteins
PRM analysis for label-free peptide quantitation was performed with a Q Exactive Plus system coupled to a Thermo EASY-nLC 1000. Peptides were separated with LC separation gradients 2% -10% acetonitrile for 1 min, and 10% -30% acetonitrile for 89 min, at flow rate of 300 nL/mim, using a 3 cm (IVAFALNMEMR, 241-251) could be detected in both the wild type and mutant forms, and were used to quantify the relative abundance of Oxa-23 proteins.
The PRM data was analyzed with Skyline (version 3.5.0) 23 as previously described 24 . The log2 of peptide ratios were averaged to yield the protein-level ratios.