Mason Lab

Projects

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. 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.

sapBC with hbd3
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 a ∆sapBC permease deletion mutant. Scale bar = 200nm
TEM of ∆sapBC with LL-37
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 ∆sapBC permease deletion mutant. Scale bar = 200nm
Model of AP transport in the presence of a functional or non-functional transporter
In the model we demonstrate a possible explanation for the preferential localization of AP in the periplasm of the ∆sapBC 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.

Biofilms and Iron Regulation : We have previously shown that the essential iron-containing compound heme is transported by the Sap ABC transporter. 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.

Fig-9-copy
The SapA-deficient NTHI strain (genetically starved for heme iron) forms a phenotypically different biofilm than the parent strain when grown in rich media
biofilmNSvsS
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).
SapF-biofilm
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.

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. 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.

EM-filament-on-CMEEs
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.
2-3-09-86dsapA-DIS-Starved-20ugml-heme-48hrs-lots-of-filaments
We observe extensive filamentation by the SapA-deficient strain when cultured for 48 hours in a biofilm formation assay.
86-DIS-1-1-09
Induction of filamentation of the parent NTHI strain when stressed by heme starvation.

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.

actin-polymerization-levels
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).

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.

SEM-parent-and-mutant-on-Epi
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.
SEM-parent-and-mutant-on-Epi
Wild type NTHI and the SapA-deficient strain were cultured on chinchilla middle ear epithelial (CMEE) cells in the absence of antibiotics for four days then analyzed for biofilm formation by optical sectioning. Bacteria contained a GFP-reporter vector that was constitutively expressed (green). CMEE cells were stained with Alexafluor-labeled ceramide (red).

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

Vesicles
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.
Fluorescent-Images
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 ceramide (Molecular Probes). Fluorescence micrographs were overlaid with DIC images captured in parallel.