Host-Microbe Protein Interactions during Bacterial Infection

Abstract

Interspecies protein-protein interactions are essential mediators of infection. While bacterial proteins required for host cell invasion and infection can be identified through bacterial mutant library screens, information about host target proteins and interspecies complex structures has been more difficult to acquire. Using an unbiased chemical crosslinking/mass spectrometry approach, we identified interspecies protein-protein interactions in human lung epithelial cells infected with Acinetobacter baumannii. These efforts resulted in identification of 3,076 crosslinked peptide pairs and 46 interspecies protein-protein interactions. Most notably, the key A. baumannii virulence factor, OmpA, was identified as crosslinked to host proteins involved in desmosomes, specialized structures that mediate host cell-to-cell adhesion. Co-immunoprecipitation and transposon mutant experiments were used to verify these interactions and demonstrate relevance for host cell invasion and acute murine lung infection. These results shed new light on A. baumannii-host protein interactions and their structural features, and the presented approach is generally applicable to other systems.

Figure 1. Identification of protein interactions from infected lung epithelial cells

(a) H292 cells were infected with Ab5075, crosslinked using BDP-NHP. Digested peptides were enriched and analyzed by LC-MS/MS. (b) PPI map of human (blue) and bacterial (green) proteins. Interspecies crosslinks are highlighted in red. (c) Total number of PPIs within the dataset, their breakdown by species (Human-human, Ab5075-Ab5075, or Ab5075-Human), and the relative matched interactions from orthogonal data (i.e. previously observed in PrePPI[35]/IntAct[36] or intraprotein PPIs within a single protein).

Figure 2. Intraprotein interactions mapped to known and predicted protein structures

Crosslinked sites identified in the large-scale proteomic infection experiment were mapped to Phyre2 predicted structures [83] (green lysine sites, light grey model) or known crystal structures (magenta lysine sites, dark grey model) for the bacterial proteins Ab57_2983 and Oxa23 and the human protein plakoglobin. For plakoglobin, while the central domain has been crystalized (PDB: 3IFQ), the N- and C- terminal portions have not been crystalized, yet a site within the C-terminus was identified in an intraprotein crosslinked relationship and shown within the predicted structural model of this region.

Figure 3. Depth of interspecies and OmpA specific intercellular interactions

(a) Force-directed network of the interspecies protein interactions identified between A. baumannii and human proteins. Insets depict how multiple site-to-site crosslink interactions underlie each PPI. Interspecies crosslinks are red. (b) Site-to-site interactions for all proteins (bacterial and human) interacting with OmpA in the cell infection model. Nodes are individual lysine sites identified in crosslinked relationships between human (blue nodes) and bacterial (green nodes) proteins. Interspecies links are red.

Figure 4. OmpA-related virulence in Ab5075

(a) Ab5075 invasion assays comparing WT Ab5075, Ab5075 with transposon disruption of OmpA (tn-ompA), and WT Ab5075 treated with control IgG serum, α-OmpA serum, or purified α-OmpA antibodies. Mean +/− SEM. (b) Murine intratracheal infection with WT Ab5075 (grey) or tn-ompa (black, log-rank p-value = 0.025).

Figure 5. Functional significance of uncharacterized interspecies interactors and OmpA interactors

(a) Space filling models for crystal structures of OmpA (green, PDB: 4G4Y) and DSP (blue, PDB: 1LM5). Identified sites of crosslinking are highlighted in orange. Sites crosslinked between DSP and OmpA are highlighted in magenta. (b) Immunoprecipitation of DSP from Ab5075 cells, H292 cells alone or Ab5075-infected H292 cells (with and without crosslinker). (c) Ab5075 invasion assays comparing WT Ab5075 to Ab5075 with transposon disruption mutants of GUNK1, GUNK2, GUNK3 (Ab57_1108, Ab57_2521, and Ab57_2983). Mean +/− SEM. Inset: crosslinked site interactions between GUNK proteins and OmpA from Ab5075 crosslinked alone (nodes: non-redundant crosslinked lysines; edges: crosslinked relationships).

Figure 6. Ab5075 host-pathogen interactions

(a) Confocal micrographs of Ab5075-infected H292 cells stained with primary antibodies against desmoplakin (green) and OmpA (red). DNA was stained with Hoechst 33342 (blue). Confocal images and overlaid images from replicate analyses at a minimal thickness of 1.1μm, scale bars represent 10μm. (b) The 46 host-pathogen interactions identified in this study (green bar) compared to the number of interspecies interactions identified for other bacterial species (HPIDB 2.0 database) [60]. If substrains were present in the database, the strain with the highest number of interactions was shown. Due to scale, HPIDB 2.0 host pathogen interactions for Yersinia pestis (n=4018), Bacillus anthracis (n=3061), and Francisella tularensis subsp. tularensis SCHU S4 (n=1346) were not shown.