Beyond the Axenic Culture Filtrate Secretome: BONCAT and conventional proteomics together reveal the Infection-associated secreted protein repertoire of Acinetobacter baumannii in the human lung Epithelial cell line A549

Acinetobacter baumannii (Ab) is an important opportunistic pathogen responsible for pneumonia, bloodstream infections, and sepsis. Although several virulence determinants have been characterized, the bacterial secreted protein repertoire associated with host cell infection remains incompletely understood. Conventional proteomic analyses of axenic cultures provide valuable information but cannot distinguish proteins synthesized before infection from those produced during host pathogen interactions. This study combined bioorthogonal noncanonical amino acid tagging (BONCAT) with conventional proteomics to characterize the infection associated Ab secreted protein repertoire recovered from human lung epithelial A549 cells. To achieve pathogen selective metabolic labelling, an Ab clinical isolate that had caused neonatal sepsis (Ab7) was engineered to express a methionyl tRNA synthetase variant, MetRSNLL (L10N, Y258L, H299L), capable of incorporating azidonorleucine (ANL) into newly synthesised bacterial proteins. Labelling conditions were optimized using methionine biosynthesis inhibitory amino acids, which reduced endogenous methionine availability without appreciably affecting bacterial growth. Efficient ANL incorporation was confirmed by flow cytometry, confocal microscopy, biotin streptavidin assays, and in gel fluorescence. Metabolic labelling also remained compatible with bacterial intracellular survival. Sequential labelling with azidohomoalanine (AHA) before infection and ANL during A549 infection enabled the temporal assignment of bacterial protein synthesis. Subsequent SDS PAGE fractionation and LC MS/MS identified 152 infection-associated proteins, including 23 containing experimentally detected amino acid analogue modifications. Among these, 13 were AHA only, nine were ANL only, and one contained both analogues. Notably, 117 proteins were not detected in either axenic reference proteome. The infection associated fraction exhibited enrichment of predicted signal peptide containing and outer membrane proteins, while functional annotation identified enrichment of bacterial secretion system components among infection-only proteins. These findings demonstrate the complementary strengths of BONCAT and conventional proteomics: BONCAT provides evidence of bacterial protein synthesis during infection, whereas conventional proteomics expands detection beyond proteins containing identifiable analogue modified peptides. Although extracellular recovery does not establish active secretion, the combined approach reveals a host associated bacterial protein repertoire extending beyond axenic observations. This framework supports future investigations into bacterial adaptation, host pathogen interactions, and the prioritization of candidate diagnostic biomarkers, subject to functional characterization and clinical validation. The study establishes a methodological foundation for investigating infection-associated protein synthesis and release in complex host pathogen environments.