Kevin M. Mason Lab Projects :: The Research Institute at Nationwide Children's Hospital

Mason Lab Projects

Our projects are based on the need to understand how nontypeable Haemophilus influenzae regulates essential metabolic activities while countering immune defense strategies of the host. Projects include focusing on antimicrobial peptides, biofilms and iron regulation, filamentation, Sap and adherence and vesicles.

Antimicrobial Peptides

Antimicrobial peptides (APs) are small, positively charged molecules of the innate immune system that form pores in bacterial membranes, causing cell lysis and death.

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Detection of APs in cytoplasmic and periplasmic enriched fractions and TEM of whole cells incubated with APs suggest that NTHI resistance to these molecules depends on a complete and functional sap transporter to import AP for cytoplasmic degradation. Mutations in the sap operon and subsequent loss of function in the Sap transporter result in attenuated survival likely due to periplasmic accumulation of APs followed by rapid cell lysis. These findings indicate that targeting the sap transporter with a small molecule inhibitor may be a novel, non-antibiotic based therapeutic for treating NTHI infections such as sinusitis, exacerbations of cystic fibrosis, and otitis media.


Immunogold labeled antimicrobial peptides were detected by transmission electron microscopy. The antimicrobial peptide human beta defensin 3 (hbd3) preferentially localized to the periplasm and inner membrane of ΔsapBC permease deletion mutant. Scale bar = 200nm


Immunogold labeled antimicrobial peptides were detected by transmission electron microscopy. The antimicrobial peptide LL-37 also preferentially localized to the periplasm and inner membrane of a ΔsapBC permease deletion mutant. Scale bar = 200nm
 

In the model we demonstrate a possible explanation for the preferential localization of AP in the periplasm of the ΔsapBC permease mutant strain of NTHI. In the presence of a functional transporter SapA (periplasmic binding protein) binds APs and delivers them to the permease components (SapB and SapC) where they are translocated to the cytoplasm for degradation. APs accumulate in the periplasm of the permease mutant strain (non-functional transporter) and result in more rapid cell lysis and death.


The Sap transporter, an ABC-transporter of the PepT family –responsible for transporting small positively charged molecules—is essential for NTHI resistance to antimicrobial peptides. We hypothesize that this resistance mechanism hinges on binding and transport of APs for subsequent proteolytic degradation. To test this hypothesis we constructed a non-polar permease deletion mutant, ΔsapBC. Current studies focus on differences in AP susceptibility and transport between the wild type strain and the permease deletion strain.

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Biofilms and Iron Regulation

We have previously shown that the essential iron-containing compound heme is transported by the Sap ABC transporter.

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A mutant strain lacking the substrate binding protein SapA displays phenotypic differences in biofilm formation and an interrupted dialogue with epithelial cells. We have demonstrated that heme starvation potentiates dramatic alterations in NTHI biofilm structure and density by genetically and environmentally starving the bacteria of heme and then growing these starved cultures in media with increasing amounts of heme. Therefore, host iron sequestration may thus foster the development of unique NTHI biofilm structures that equip bacteria at infectious sites. 

This work was recently published.  View the abstract.

The SapA-deficient NTHI strain (genetically starved for heme iron) forms a phenotypically different biofilm than the parent strain when grown in rich media

By controlling heme availability (environmental starvation) we are able to promote dramatic morphological changes in biofilm structure by NTHI strain 86-028NP. Heme-starvation (panel B) promotes increased biofilm formation (90 micron thickness) compared to non-starved biofilms (40 micron thickness).


We predicted that a mutation in the SapF ATPase subunit, required for Sap transporter function, would alter biofilm phenotype. We show here that the sapF mutant biofilm is increased in thickness (approximately 4-fold) compared to the parent strain when grown in iron-free conditions for 48 hours. Intriguingly, we could measure towers of biofilm formation extending 60 microns in thickness in the sapF mutant, well above those observed in the starved parent strain. These data indicate that a loss of SapF, likely imparts an iron-starved phenotype upon the bacterial cell, and thus alters biofilm formation.

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Filamentation

We have observed that a SapA-deficient NTHI strain displayed a filamentous morphology, more prominent than filaments formed by the wild type strain, when cultured for biofilm formation on chinchilla middle ear epithelial cells.

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Filamentation by microorganisms is a survival strategy in response to environmental stressors such as oxidative stress, antimicrobial therapies, and host effectors which facilitate bacterial persistence in these stressful conditions. We hypothesize that the enhanced filamentation observed with the SapA-deficient mutant strain is due to decreased iron availability or deficiency in ability to acquire heme, stressors sufficient to induce this morphological change. An iron-restricted environment would thus induce filamentation of NTHI.

NTHi alter their morphology when co-cultured on epithelial cells. The NTHI parent (left) and sapA-deficient strain (right) initiate biofilm formation on chinchilla middle ear epithelial cells, typically with a filamentous morphology in close proximity to the epithelial cell.

We observe extensive filamentation by the SapA-deficient strain when cultured for 48 hours in a biofilm formation assay.

Induction of filamentation of the parent NTHI strain when stressed by heme starvation.

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Sap and Adherence

Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterium that colonizes the human nasopharynx. Alteration of bacterial factors important for commensalism, which currently remain unknown, can result in bacterial pathogenicity, i.e. infection of the middle ear (Otitis Media). Changes in adherence or bacterial metabolism appear to disrupt commensal colonization and thus alter the host’s response to NTHI.

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Our laboratory has previously shown that the sapA transporter-deficient NTHI strain (an iron-starved and antimicrobial peptide (AP) susceptible phenotype) induces actin polymerization and membrane ruffling of bacteria-infected chinchilla middle ear epithelial cells (CMEE), in contrast to the unremarkable response observed when these cells are cultured in the presence of the parent strain. These data suggest that a functional Sap transporter is critical for NTHI commensal behavior. The Mason lab seeks to further understand this shift from commensal to pathogenic behavior as it specifically relates to bacterial metabolism, adherence and immune resistance mechanisms. We are currently creating adherence deletion mutants in the sap A-deficient background to determine which adhesins contribute to this enhanced cellular response. Our studies will expand our understanding of the complex, yet essential, biological processes of iron metabolism, AP resistance and adherence to host epithelium.

Actin polymerization coincides with NTHI adherence. Chinchilla nasopharyngeal epithelial (CNPE) cells were inoculated with 86-028NP sapA::kan containing a constitutively expressed GFP-reporter vector (Green) and incubated for four days. CNPE cells were labeled with Alexafluor-labeled ceramide (Red) and actin polymerization is observed by phaloidin labeling (Purple).

The parent strain (top) or its sapA mutant (bottom), each expressing GFP, were grown to mid-log phase, used to inoculate normal human bronchial epithelial cells (NHBE), and monitored for biofilm formation 2 days post-inoculation by epifluorescence microscopy. After fixation, epithelial cells were surface labeled with Alexa594 wheat-germ agglutinin (WGA), permeabilized and actin polymerization was monitored by Alexa350-Phalloidin labeling. Fluorescent micrographs were captured at 100x magnification. The mutant (bottom) displayed a unique phenotype depicted by increased membrane ruffling and actin polymerization compared to that caused by the parent strain (top). These observations suggest that this is perhaps a compensatory phenotype due to the lack of SapA-mediated homeostatic events.

NTHI parent (top panel) and sapA-deficient strain (bottom panel) were cultured on differentiated chinchilla nasopharyngeal epithelial (CNPE) cells at an air-liquid interface, for 3 days, fixed and bacterial-host cell interaction was monitored by scanning electron microscopy. The parent strain formed a biofilm on the cell surface. In contrast, loss of a functional Sap transporter caused an altered host cell response to colonization as depicted by increased membrane ruffling, actin polymerization and ultimate cell lysis.

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Vesicles

We are interested in studying Haemophilus outer membrane release to determine selective packaging of outer membrane and periplasmic proteins and whether these particles impart an immunogenic response when cultured with epithelial cells or immune cells.

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We hypothesize that OMV release is important for Haemophilus biofilm formation, resistance to innate immune components such as antimicrobial peptides, and pathogenesis in vivo.

This work was recently published.  View the abstract.

Haemophilus produces outer membrane vesicles (OMVs). Bacteria were grown overnight, whole cells removed by centrifugation and supernatants were filtered. Vesicles were pelleted by high speed centrifugation and loaded on top of an Optiprep gradient for purification. Purified vesicles were floated on copper grids and stained with uranyl acetate for electron microscopy.

Outer Membrane Vesicles adhere to host cells. Purified vesicles (1 ug) were conjugated to Alexa Fluor-488 (Invitrogen) and applied to Detroit 562 cells for 24 hours. Cells were fixed and plasma membranes were visualized using wheat germ agglutinin (Molecular Probes).

OM vesicles are proteo- liposomes consisting of Outer Membrane (OM) phospholipids and LPS, a subset of OM proteins, and periplasmic (luminal) proteins. Proteins and lipids of the Inner Membrane (IM) and cytosolic content are excluded from OM vesicles. Vesicles are likely to bud at sites where the links between the peptidoglycan and OM are infrequent, absent, or broken. (LPS) Lipopolysaccharide; (Pp) periplasm; (OM) outer membrane; (PG) peptidoglycan; (IM) inner membrane; (Cyt) cytosol. Model of vesicle biogenesis modified from Kesty and Kuehn, 2005.

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