The Haemophilus influenzae in vivo gene expression reveals major clues about bacterial central metabolism, acquisition of trace elements, and other essential pathways during infection of the human lung

Haemophilus influenzae is a major cause of community and hospital acquired pneumonia. While extensively studied in various laboratory models, less is known about how this species persists and causes infection inside the human lung. We present the first study on the H. influenzae in vivo transcriptome during pneumonia, and contrast this with isolates cultured in vitro under standard laboratory conditions. Patients with pneumonia were recruited from emergency departments and intensive care units in a Swedish referral hospital during 2018-2020 (n=102). Duplicates of lower respiratory samples were collected for bacterial culture and RNA-extraction. Patient samples with H. influenzae (n=18) from which bacterial mRNA of adequate quantity and quality could be extracted (n=8) underwent RNA-sequencing, along with duplicates of lab-cultured counterparts (n=7). The transcripts were aligned to core and pan genomes created from 15 reference strains. While in vitro bacteria clustered tightly in principal component analyses of core genome (n=1067) expression, the in vivo samples displayed diverse transcriptomic signatures and did not group with their lab-grown counterparts. In total, 328 core genes were significantly differentially expressed between in vitro and in vivo conditions. The most upregulated genes in vivo included the transferrin-acquisition genes tbp1 and fbpA and the reductase gene msrAB involved in stress response pathways. Biosynthesis of nucleotides/purines, response-to-heat systems, and molybdopterin-scavenging processes were also significantly upregulated in vivo. Major metabolic pathways and iron-sequestering processes were downregulated in vivo. In conclusion, extensive transcriptomic differences were found between bacteria collected in the human lung during pneumonia and isogenic bacteria cultured in vitro.