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

Abstract

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.

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment

“…We showed that Arp/2/3 plays important roles in NTHI invasion into NHBE cells. We have previously shown that the NTHI responses to nutritional immunity promote intracellular invasion and survival within host cells (113). We therefore suggest that therapeutic modulation of actin remodeling to prevent invasion will increase the vulnerability of NTHI to the host immune responses and antimicrobial therapies.…”

Therapeutic Transcutaneous Immunization with a Band-Aid Vaccine Resolves Experimental Otitis Media

“…2A). Each middle ear was individually assessed for the relative quantities of NTHI within MEF and mucosal biofilms, as is standard for this experimental model (18,19,25,26,(31)(32)(33)(34)(35)(36)(37)(38)(39). Four days after middle ear challenge with NTHI, all middle ears exhibited robust experimental OM, with high concentrations of NTHI within both MEF and mucosal biofilms ( Fig.…”

“…The adherent middle ear mucosal biofilms were also collected and individually homogenized in sterile saline solution. It is well established in chinchilla models of OM that each middle ear should be considered independently (18,19,25,26,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40); therefore, MEF and biofilm homogenates collected from the left and right middle ears were individually plated on chocolate agar to semiquantitatively assess the relative numbers of NTHI in the planktonic and adherent populations. MEF samples were then clarified by centrifugation, and supernatants were stored at Ϫ80°C until determination of immunoglobulin profiles as described below.…”

Host-to-host variation of ecological interactions in polymicrobial infections

“…The bacterial species also depend on the host for extracting essential nutrients such as metals for their growth. For example, the Gram-negative NTHI and Gram-positive Sp require iron extracted from the serum generated by the host during inflammation(16, 17). Therefore, it is plausible that variations of these factors across hosts would lead to differences in infection kinetics between hosts.…”