ABSTRACTHaemophilus influenzaeis a human-adapted bacterial pathogen that causes airway infections. Bacterial and host elements associated with the fitness ofH. influenzaewithin the host lung are not well understood. Here, we exploited the strength ofin vivo-omic analyses to study host-microbe interactions during infection. We usedin vivotranscriptome sequencing (RNA-seq) for genome-wide profiling of both host and bacterial gene expression during mouse lung infection. Profiling of murine lung gene expression upon infection showed upregulation of lung inflammatory response and ribosomal organization genes, and downregulation of cell adhesion and cytoskeleton genes. Transcriptomic analysis of bacteria recovered from bronchoalveolar lavage fluid samples from infected mice showed a significant metabolic rewiring during infection, which was highly different from that obtained upon bacterialin vitrogrowth in an artificial sputum medium suitable forH. influenzae.In vivoRNA-seq revealed upregulation of bacterialde novopurine biosynthesis, genes involved in non-aromatic amino acid biosynthesis, and part of the natural competence machinery. In contrast, the expression of genes involved in fatty acid and cell wall synthesis and lipooligosaccharide decoration was downregulated. Correlations between upregulated gene expression and mutant attenuationin vivowere established, as observed uponpurHgene inactivation leading to purine auxotrophy. Likewise, the purine analogs 6-thioguanine and 6-mercaptopurine reducedH. influenzaeviability in a dose-dependent manner. These data expand our understanding ofH. influenzaerequirements during infection. In particular,H. influenzaeexploits purine nucleotide synthesis as a fitness determinant, raising the possibility of purine synthesis as an anti-H. influenzaetarget.IMPORTANCEIn vivo-omic strategies offer great opportunities for increased understanding of host-pathogen interplay and for identification of therapeutic targets. Here, using transcriptome sequencing, we profiled host and pathogen gene expression duringH. influenzaeinfection within the murine airways. Lung pro-inflammatory gene expression reprogramming was observed. Moreover, we uncovered bacterial metabolic requirements during infection. In particular, we identified purine synthesis as a key player, highlighting thatH. influenzaemay face restrictions in purine nucleotide availability within the host airways. Therefore, blocking this biosynthetic process may have therapeutic potential, as supported by the observed inhibitory effect of 6-thioguanine and 6-mercaptopurine onH. influenzaegrowth. Together, we present key outcomes and challenges for implementingin vivo-omics in bacterial airway pathogenesis. Our findings provide metabolic insights intoH. influenzaeinfection biology, raising the possibility of purine synthesis as an anti-H. influenzaetarget and of purine analog repurposing as an antimicrobial strategy against this pathogen.