Abstract
Haemophilus influenzae is a major cause of community-acquired pneumonia. While studied extensively in various laboratory models, less is known about the cell function while inside the human lung. We present the first analysis of the global gene expression of H. influenzae while the bacteria are in the lung during pneumonia (in vivo conditions) and contrast it with bacterial isolates that have been cultured under standard laboratory conditions (in vitro conditions). Patients with pneumonia were recruited from emergency departments and intensive care units during 2018-2020 (n = 102). Lower respiratory samples were collected for bacterial culture and RNA extraction. Patient samples with H. influenzae (n = 8) and colonies from bacterial cultures (n = 6) underwent RNA sequencing. The reads were then pseudo-aligned to core and pan genomes created from 15 reference strains. While bacteria cultured in vitro clustered tightly by principal component analysis of core genome (n = 1067) gene expression, bacteria in the patient samples had more diverse transcriptomic signatures and did not group with their lab-cultured counterparts. In total, 328 core genes were significantly differentially expressed between in vitro and in vivo conditions. The most highly upregulated genes in vivo included tbpA and fbpA, which are involved in the acquisition of iron from transferrin, and the stress response gene msrAB. The biosynthesis of nucleotides/purines and molybdopterin-scavenging processes were also significantly enriched in vivo. In contrast, major metabolic pathways and iron-sequestering genes were downregulated under this condition. In conclusion, extensive transcriptomic differences were found between bacteria while in the human lung and bacteria that were cultured in vitro. IMPORTANCE The human-specific pathogen Haemophilus influenzae is generally not well suited for studying in animal models, and most laboratory models are unlikely to approximate the diverse environments encountered by bacteria in the human airways accurately. Thus, we have examined the global gene expression of H. influenzae during pneumonia. Extensive differences in the global gene expression profiles were found in H. influenzae while in the human lung compared to bacteria that were grown in the laboratory. In contrast, the gene expression profiles of isolates collected from different patients were found to cluster together when grown under the same laboratory conditions. Interesting observations were made of how H. influenzae acquires and uses iron and molybdate, endures oxidative stress, and regulates central metabolism while in the lung. Our results indicate important processes during infection and can guide future research on genes and pathways that are relevant in the pathogenesis of H. influenzae pneumonia.