Genome-wide fitness profiling reveals adaptations required by Haemophilus in coinfection with influenza A virus in the murine lung

Bacterial coinfection represents a major cause of morbidity and mortality in epidemics of influenza A virus (IAV). The bacterium Haemophilus influenzae typically colonizes the human upper respiratory tract without causing disease, and yet in individuals infected with IAV, it can cause debilitating or lethal secondary pneumonia. Studies in murine models have detected immune components involved in susceptibility and pathology, and yet few studies have examined bacterial factors contributing to coinfection. We conducted genome-wide profiling of the H. influenzae genes that promote its fitness in a murine model of coinfection with IAV. Application of direct, high-throughput sequencing of transposon insertion sites revealed fitness phenotypes of a bank of H. influenzae mutants in viral coinfection in comparison with bacterial infection alone. One set of virulence genes was required in nonvirally infected mice but not in coinfection, consistent with a defect in anti-bacterial defenses during coinfection. Nevertheless, a core set of genes required in both in vivo conditions indicated that many bacterial countermeasures against host defenses remain critical for coinfection. The results also revealed a subset of genes required in coinfection but not in bacterial infection alone, including the iron-sulfur cluster regulator gene, iscR, which was required for oxidative stress resistance. Overexpression of the antioxidant protein Dps in the iscR mutant restored oxidative stress resistance and ability to colonize in coinfection. The results identify bacterial stress and metabolic adaptations required in an IAV coinfection model, revealing potential targets for treatment or prevention of secondary bacterial pneumonia after viral infection.The bacterium Haemophilus influenzae is a Gram-negative inhabitant of the human upper respiratory tract and a common agent in sinusitis, otitis media, lung infections in cystic fibrosis, and exacerbations of chronic obstructive pulmonary disease (COPD). In the context of prior infection by influenza A virus (IAV), H. influenzae is associated with secondary bacterial pneumonia (1). Annually, influenza and related complications cause ∼36,000 deaths, over 200,000 hospitalizations in the United States, and ∼5 million cases of severe illness worldwide (2, 3). Uncomplicated IAV infection can progress to pneumonia; however, secondary bacterial infection combined with viral infection is commonly the major cause of excess morbidity and mortality during epidemics and pandemics. For example, the 1918 influenza pandemic killed an estimated 50 million people worldwide, and the majority of deaths have been attributed to bacterial secondary infections in which Streptococcus pneumoniae, H. influenzae, and Staphylococcus spp. represent the most common isolates (1). β-Lactam antibiotics are commonly used for treatment, and yet ∼30% of H. influenzae isolates are β-lactamase–positive (4–6). Because of increasing levels of bacterial antibiotic resistance, and the continued threat of global pandemics with potential emergence of new IAV subtypes, combined IAV and bacterial infection remains a significant public health concern.In 1945, Francis and Vicente de Torregrosa demonstrated lethality of H. influenzae when introduced into the lungs of mice after infection with IAV (7). More recently, pathogenic mechanisms associated with the mouse lung model of lethal IAV coinfection with H. influenzae type b (Hib) were investigated, implicating innate immunity in disease progression (8). Coinfection did not influence viral titers and yet led to dramatically increased multiplication and persistence of bacteria. Viral enhancement of host susceptibility to bacterial infection has been examined in coinfection models with diverse bacteria, implicating modification of mucosal surfaces and dysfunctional immune responses that prevent bacterial containment including altered phagocytic capacity, defective TLR responses, and enhanced pro- and anti-inflammatory cytokine production, and decreased tolerance to tissue damage (9–14). In contrast, bacterial factors involved in coinfection have received less attention. There have been no systematic studies to identify such factors, and genes of H. influenzae involved in IAV coinfection have not been identified.We investigated the hypothesis that H. influenzae possesses genes that promote its ability to survive host defenses and exploit conditions in the lung generated by coinfection with IAV. Using a genome-scale analytical approach, we simultaneously monitored fitness of thousands of transposon mutants in the murine lung model in the presence and absence of prior IAV infection. The results reveal a core set of bacterial genes required in both models, as well as genes required uniquely in one environment but not the other. Coinfection altered bacterial requirements for known virulence genes conferring not only immune evasion properties but also those encoding regulatory factors and physiological pathways. Therefore, genome-wide analysis of the fitness of bacterial mutants serves as a probe for conditions created during bacterial/viral coinfection of murine lung and identifies bacterial adaptations that specifically promote their multiplication in this pathogenic context.