Haemophilus Responses to Nutritional Immunity: Epigenetic and Morphological Contribution to Biofilm Architecture, Invasion, Persistence and Disease Severity

In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites.
Author Summary Clinical management of upper and lower respiratory tract diseases caused by nontypeable Haemophilus influenzae (NTHI) is a significant socioeconomic burden. Therapies targeting the pathogenic lifestyle of NTHI remain non-existent due to a lack of understanding of host microenvironmental cues and bacterial responses that dictate NTHI persistence. Iron availability influences bacterial virulence traits and biofilm formation; yet, host sequestration of iron serves to restrict bacterial growth. We predicted that fluctuations in availability of iron-containing compounds, typically associated with infection, would impact NTHI pathogenesis. We demonstrated that transient restriction of heme-iron triggered an epigenetic developmental program that enhanced NTHI biofilm architecture, directly influenced by induced morphological changes in bacterial length. Heme-iron restricted bacteria were primed for survival in the mammalian middle ear, due in part to an observed reduction in host inflammation coinciding with a striking reduction in host mucosal epithelial damage, compared to that observed in response to heme-iron replete NTHI. Moreover, transiently restricted NTHI were more invasive of epithelial cells resulting in formation of intracellular bacterial communities. Our findings significantly advance our understanding of how host immune pressure and nutrient availability influence pathogenic behaviors that impact disease severity.