Dual proteomics of infected macrophages reveals bacterial and host players involved in the Francisella dissemination

Facultative intracellular bacteria adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. We combine cell sorting to enrich infected host cells and in depth proteomics analysis to observe bacterial and host players involved in the Francisella intracellular life cycle and dissemination.

Macrophages were infected with GFP-expressing bacteria for 10 hours, then sorted using the FACS Sony MA900 and compared to non-infected cells (five biological replicates). Cells were then lysed and digested using S-Trap spin column. Tryptic peptides were analysed on nanoElute HPLC - timsTOF Pro MS (Bruker Daltonics) using PASEF DDA method over the m/z range of 100 to 1700 Th with ion mobility values from 0.75 to 1.25 V·s/cm² (1/k0). The total cycle time was 1.17 seconds with 10 PASEF MS/MS scans per cycle. Ramp time was set to 100 ms, with target intensity and intensity threshold set to 15,000 and 500, respectively. The MS files were processed with the MaxQuant v2.0.1.0. Gene Ontology Over Representation Analysis (GO ORA) was performed on the signifcant up- and downregulated proteins using ClusterProfler R package (v4.6.2). Bacterial multiplication was monitored in the fully automated microscope Incucyte® S3 (Essen BioScience).

Over 900 bacterial proteins were identified in infected macrophages after a 10 hour infection, representing more than 50% of Francisella estimated proteome. Biotin biosynthesis-related proteins were found upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, most of the proteins encoded by the Francisella pathogenicity island were downregulated, supporting the importance of the Francisella Type VI Secretion System during the early phase of infection but not during cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. GO ORA highlighted cell-cell contact and actin polymerization as the main deregulated pathways in macrophage. Using flow cytometry and live cell imaging, we demonstrated that cell-to-cell contacts and actin polymerization-dependent processes are essential for dissemination and we illustrated the merocytophagy as one of the mechanisms of dissemination.

These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in a variety of host-pathogen models.