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

<div><p>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 <i>Haemophilus</i> biofilm formation and pathogenesis. Thus, we cultured <i>Haemophilus</i> 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 <i>Haemophilus</i>, 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 <i>Haemophilus</i> 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.</p></div

Proposed model for host-mediated nutritional environment effects on NTHI pathogenesis.

<p>NTHI (dark green bacteria) asymptomatically colonizes the heme-iron sufficient nasopharynx epithelium (orange cells) (<b>A</b>). Permissive factors promote ascension up the Eustachian tube to the intact middle ear epithelium (brown cells) where NTHI are heme-iron restricted (light green bacteria) (<b>B</b>). Disease progression results in infiltration of red blood cells (red circle), immune cells (gray circle) and expansion of interstitial space (open circles) leading to potential heme-iron sources (red hexagon) for NTHI (dark green bacteria). This transition from heme-iron restricted to replete microenvironments primes NTHI to alter morphology and enhance biofilm formation, both of which promote invasion and persistence (<b>C</b>).</p

Transient restriction of heme-iron promotes biofilm tower architecture.

<p>Schematic representation of environmental heme-iron restriction. 86-028NP was cultured in the presence (+) or absence (−) of heme-iron for 24 hours generate “replete” and “restricted” populations, respectively. These populations were subcultured into medium containing heme-iron to produce continuously exposed (CE) or transiently restricted (TR) populations (<b>A</b>). The height of biofilm base or tower formed by CE or TR NTHI was measured in 20 random fields of view from a representative 48-hour biofilm (<b>B</b>). Statistical analysis was performed using a two-tailed t-test. Biofilms were visualized with live/dead stain, imaged using optical sections and reconstructed to generate a 3D view from the top (large pane) including orthogonal views of the side (top and left side) (<b>C, E</b>). Series of optical sections from an independent experiment were used to generate a surface plot to depict biofilm architecture (<b>D, F</b>). Images were processed using Image J and the height of the biofilm is indicated in each panel.</p

Robust towers arise from transiently restricted NTHI in a mixed culture.

<p>The reporter strains [86-028NP/pGM1.1 (production of GFP) and 86-028NP/pKM1.1 (production of mCherry)] were cultured for 24 hours in the presence or absence of heme-iron to generate “replete” and “restricted” populations. Restricted 86-028NP/pGM1.1 was co-cultured with equivalent numbers of replete 86-028NP/pKM1.1 for 48 hours in 2 µg heme mL⁻¹ (<b>A</b>) or 20 µg heme mL⁻¹ (<b>C</b>) in a glass chamber slide. Restricted 86-028NP/pKM1.1 was co-cultured with equivalent numbers of replete 86-028NP/pGM1.1 for 48 hours in 2 µg heme mL⁻¹ (<b>B</b>) in a glass chamber slide. Optical sections were rendered to form the top down and orthogonal views. Biofilms formed from co-culture of restricted 86-028NP/pKM1.1 with replete 86-028NP/pGM1.1 were subjected to disruption to enumerate the total numbers of each reporter strain present following 48 hours of biofilm growth (<b>D</b>). Restricted 86-028NP/pGM1.1 was co-cultured with 1000 fold more 86-028NP/pKM1.1 replete for heme-iron for 48 hours in 2 µg heme mL⁻¹ (<b>E</b>) in a glass chamber slide. Restricted 86-028NP/pKM1.1 was co-cultured with 1000 times more 86-028NP/pGM1.1 replete for heme-iron for 48 hours in 2 µg heme mL⁻¹ (<b>F</b>) in a glass chamber slide. Orthogonal views of 3D rendered image are depicted (<b>E, F</b>). Heights of the biofilm are indicated in each panel. Statistical analysis was performed using a two-tailed t-test.</p

Heme-iron status influences disease severity of NTHI.

<p>Middle ear pressure was measured by tympanometry (mean ± S.D.) on each day following transbullar introduction of heme-iron replete (black squares) or restricted (gray circles) NTHI (<b>A</b>). Histopathology was assessed by examination of hematoxylin and eosin stained thin sections of naïve (<b>B</b>) or infected middle ear bullae 7 days following introduction of heme-iron replete (<b>C, D</b>) or restricted (<b>E, F</b>) NTHI. Scale bar = 25 µm (<b>B</b>), 50 µm (<b>F</b>), 100 µm (<b>C, D, E</b>). Abbreviations = Bone (B), mucosa (M), biofilm (Bio). Arrows denote capillaries.</p

Heme-iron restriction provides a selective advantage during experimental otitis media in a mixed infection model.

<p>Restricted 86-028NP/pKM1.1 was mixed with 10 fold more 86-028NP/pGM1.1 replete for heme-iron for inoculation into the chinchilla middle ear. On days one and four post-inoculation, the total bacterial burden was enumerated from middle ear mucosal homogenates (<b>A</b>). The fluorescence emission spectrum was used to determine the origin of each colony. Statistical analysis was performed using a two-tailed t-test. Infected middle ear inferior bullae were analyzed by hematoxylin and eosin staining of thin sections with biofilm (Bio) as well as mucosa (M) and bone (B) highlighted (<b>B</b>). 86-028NP that was restricted (<b>D</b>) or replete (<b>C</b>) for heme-iron was individually inoculated into the chinchilla middle ears. Four days post inoculation, infected middle ear bullae were fixed and processed for histological examination. Scale bar = 50 µm.</p

Transient restriction promotes intracellular bacterial community formation.

<p>86-028NP that was restricted (<b>A, B</b>) for heme-iron was individually inoculated into the chinchilla middle ears. Four days post inoculation, infected middle ear bullae was fixed and processed for histological examination. Scale bar = 10 µm. 86-028NP/pGM1.1 was cultured for 24 hours in the presence or absence of heme-iron to generate “replete” and “restricted” populations (CE or TR). Nutritionally conditioned NTHI were co-cultured with chinchilla middle ear epithelial cells for 48 hours. Epithelial cell surface was labeled with wheat germ agglutinin conjugated to Alexa-Fluor 594 (red). Biofilms were visualized by GFP fluorescence (green), imaged using optical sections and reconstructed to generate a 3D view from the top (large pane) including orthogonal views of the side (top and left side) (<b>C, D</b>). Optical sections that depict additional representative images of the localization of NTHI based upon nutritional conditioning (<b>E, F</b>). Scale bar = 10 µm.</p

Lace-like architecture and towers arise from SulA-mediated filamentous morphotypes.

<p>Restricted or replete 86-028NP were cultured in medium containing 2 µg heme mL⁻¹ for 48 hours on glass chamber slides and visualized by fluorescent microscopy using a live/dead stain (<b>A, B</b>). Restricted 86-028NP/pGM1.1 was grown in the presence 2 µg heme mL⁻¹ for 6 hours and subjected to centrifugation through a 5 µm pore filter to enrich for bacillary and filamentous subpopulations. Enriched bacillary (<b>C</b>) and filamentous (<b>D</b>) populations were used to initiate biofilm formation on glass chamber slides in the presence of 2 µg heme mL⁻¹ for 48 hours. Scale bar = 10 µm. Restricted 86-028NP/pGM1.1) (<b>E</b>) and restricted <i>sulA</i> (86-028NP Δ<i>sulA</i>/pGM1.1) (<b>F</b>) were used to initiate biofilm formation for 48 hours. The height of biofilm base or tower formed by CE or TR NTHI was measured in 20 random fields of view from a representative 48-hour biofilm (<b>G</b>). Statistical analysis was performed using a two-tailed t-test. 86-028NP/pGZRS39A and 86-028NP Δ<i>sulA</i>/pSPEC1 were restricted of heme-iron for 24 hours. Both strains were normalized for viability and equivalent numbers of each strain were co-inoculated into the middle ears of chinchillas. Effusions were collected on day 4, 7 and 10 days following inoculation and plated on selective media (<b>H</b>). Dashed line indicated limit of detection. Statistical analysis was performed using a Mann-Whitney U-test.</p

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM