Epigenetic control of the ferric uptake regulator (Fur) and fumarate nitrate reductase (FNR) master regulatory proteins contributes to Haemophilus influenzae survival during lung infection

ABSTRACT DNA regulatory elements that dictate how the bacterial pathobiont Haemophilus influenzae infects and adapts to the airways of immunocompromised patients suffering from chronic obstructive pulmonary disease (COPD) are poorly understood. This is in part due to the scarcity of research integrating genetic and epigenetic perspectives to shed light on the role of distinct bacterial adaptive strategies within the human airways. In this work, global fitness profiling of H. influenzae mutants by high-throughput transposon mutant sequencing within the mouse lung identified Dam methyltransferase as an in vivo requirement for respiratory infection. Equally, single-molecule real-time sequencing methylome analyses found undermethylation of GATC motifs within putative regulatory elements and revealed the first case of phenotypic variation controlled by variable Dam methylation in H. influenzae . Moreover, RNA sequencing differential gene expression disclosed a novel regulatory network where Dam methyltransferase positively regulates the expression of the ferric uptake regulator (Fur), which in turn represses the expression of the fumarate nitrate reductase (FNR) regulator and, subsequently, of a repertoire of genes that belong to the FNR regulon and encode bacterial anaerobic defenses against, among others, reactive nitrogen species produced within the diseased airways. Our results present a multifactorial regulatory network where the interplay between the Fur and FNR master transcriptional regulators is controlled epigenetically by Dam methylation. We put forward the notion that this network regulates H. influenzae survival in diseased airway niches with high nitrosative stress where damage reduces the amount of oxygen in the lungs, as encountered in COPD. IMPORTANCE Regulatory mechanisms governing the ability of Haemophilus influenzae to survive within the human lungs remain poorly elucidated. Here, by coordinated exploitation of multiomic approaches, and using reference and clinical strains, we present evidence that the Dam methyltransferase mediates epigenetic regulatory mechanisms facilitating bacterial phenotypic diversity and flexibility, besides reversibility, to contribute to H. influenzae survival within the lungs of individuals where disease reduces the amount of oxygen, as encountered in COPD. We reveal a novel bacterial network where DNA methylation regulates the expression of and interplay between the Fur and FNR master transcriptional regulators, which act in a coordinated manner, controlling the expression of H. influenzae genes involved in bacterial defenses against the nitrosative stress encountered in the diseased lungs, and further highlight the importance of oxygen restriction within this hostile niche.