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. 2018 Sep 12;3(5):e00286-18.
doi: 10.1128/mSphere.00286-18.

Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

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Free PMC article

Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

Rachael L Hardison et al. mSphere. .
Free PMC article

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Abstract

Nutrient limitation restricts bacterial growth in privileged sites such as the middle ear. Transient heme-iron restriction of nontypeable Haemophilus influenzae (NTHI), the major causative agent of chronic and recurrent otitis media (OM), promotes new and diverse phenotypes that can influence planktonic, biofilm, and intracellular lifestyles of NTHI. However, the bacterial responses to nutrient restriction that impact intracellular fate and survival of NTHI are unknown. In this work, we provide evidence for the role of transient heme-iron restriction in promoting the formation of intracellular bacterial communities (IBCs) of NTHI both in vitro and in vivo in a preclinical model of OM. We show that transient heme-iron restriction of NTHI results in significantly increased invasion and intracellular populations that escape or evade the endolysosomal pathway for increased intracellular survival. In contrast, NTHI continuously exposed to heme-iron traffics through the endolysosomal pathway for degradation. The use of pharmacological inhibitors revealed that prior heme-iron status does not appear to influence NTHI internalization through endocytic pathways. However, inhibition of macropinocytosis altered the intracellular fate of transiently restricted NTHI for degradation in the endolysosomal pathway. Furthermore, prevention of macropinocytosis significantly reduced the number of IBCs in cultured middle ear epithelial cells, providing evidence for the feasibility of this approach to reduce OM persistence. These results reveal that microenvironmental cues can influence the intracellular fate of NTHI, leading to new mechanisms for survival during disease progression.IMPORTANCE Otitis media is the most common bacterial infection in childhood. Current therapies are limited in the prevention of chronic or recurrent otitis media which leads to increased antibiotic exposure and represents a significant socioeconomic burden. In this study, we delineate the effect of nutritional limitation on the intracellular trafficking pathways used by nontypeable Haemophilus influenzae (NTHI). Moreover, transient limitation of heme-iron led to the development of intracellular bacterial communities that are known to contribute to persistence and recurrence in other diseases. New approaches for therapeutic interventions that reduce the production of intracellular bacterial communities and promote trafficking through the endolysosomal pathway were revealed through the use of pharmacological inhibition of macropinocytosis. This work demonstrates the importance of an intracellular niche for NTHI and provides new approaches for intervention for acute, chronic, and recurring episodes of otitis media.

Keywords: Haemophilus; NTHI; endolysosomal pathway; host cell invasion; intracellular bacterial community; macropinocytosis; nutritional immunity.

Figures

FIG 1
FIG 1
Transient heme-iron restriction of NTHI promotes intracellular bacterial community formation in a preclinical model of otitis media. (A) Schematic representation of environmental heme-iron conditioning of NTHI strain 86-028NP and inoculation of chinchilla middle ears. Two days postinoculation, middle ears were excised, fixed, and paraffin embedded and thin sections from sham-treated, transiently restricted, or continuously exposed infected ears were processed for microscopy. TR, transiently restricted NTHI; CE, continuously exposed NTHI. (B) Four-nanometer thin sections of sham-treated middle ear bullae were stained with hematoxylin and eosin (left panel) or processed for immunofluorescence microscopy and stained with wheat germ agglutinin and 4′,6-diamidino-2-phenylindole (right panel). The lumen, mucosa, and bone are denoted for anatomical orientation. (C) Immunofluorescence micrograph gallery depicting the surface staining of transiently restricted NTHI by anti-OMP labeling (green), staining of the host cell membrane by wheat germ agglutinin (red), and staining of host and bacterial DNA by Hoechst stain (blue). (D) Representative images of transiently restricted NTHI IBCs within middle ear mucosal epithelium, including a three-dimensional rendering of a series of optical sections (left panel) to depict the orthogonal views of IBCs filling the entirety of the epithelial cell and visualized using the fluorophores depicted in panel C. Multiple IBCs were observed in each thin section. (E) A no-primary-antibody control of a thin section of a middle ear infected with transiently restricted NTHI. The thin section is sequential to those in panel D and depicts the specificity of NTHI labeling. (F) Representative image of middle ears infected with continuously exposed NTHI depicts the absence of IBCs in these thin sections. Bar, 10 µm for all images. (G) Quantification of IBCs in thin sections of ears infected with transiently restricted or continuously exposed NTHI. Three sequential thin sections were counted for three ears from each cohort. Significance was determined using a two-tailed Student t test.
FIG 2
FIG 2
Transiently restricted NTHI invades and survives within human epithelial cells in intracellular bacterial communities. (A) Normal human bronchial epithelial (NHBE) cells were cocultured with either transiently restricted (TR) or continuously exposed (CE) NTHI for 1 h, and total bacterial association (no gentamicin) or intracellular bacteria only (+ gentamicin) were quantified. Results are depicted as the percentage of inoculum that remained viable with or without gentamicin treatment. Statistical significance was determined using a two-tailed Student t test of the means from duplicate wells from each of three biological replicates. Error bars indicate standard errors of the means. ns, not significant. (B) The invasion index of transiently restricted (TR) and continuously exposed (CE) NTHI defined as the number of viable intracellular bacteria when normalized for total association. Statistical significance was determined using a two-tailed Student t test on the mean from technical duplicates from each of three biological replicates. Error bars represent standard errors of the means. (C) Intracellular survival of transiently restricted (TR) and continuously exposed (CE) NTHI following infection of NHBE cells for 2 or 24 h. Results are depicted as the percentage of inoculum that remained viable following gentamicin treatment. Statistical significance was determined using a two-tailed Student t test on the mean from duplicates from each of eight biological replicates for the 2-h time point and five biological replicates for the 24-h time point. Error bars represent standard errors of the means. (D to G) NTHI strain 86-028NP(pGM1.1) expressing green fluorescent protein was transiently restricted (TR) or continuously exposed (CE) to heme-iron and then cocultured with NHBE cells for 4 (D and E) or 24 (F and G) hours. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red), and bacteria were visualized by GFP fluorescence (green). Bar, 10 µm. Images depict the formation of early IBCs by TR NTHI as early as 4 h postinoculation (D) that progress to mature IBCs at 24 h (F). CE NTHI localizes in circular compartments (E, inset) and does not form IBCs 24 h postinoculation (G).
FIG 3
FIG 3
Transient heme-iron conditioning of NTHI alters trafficking to early endosomes. (A) NHBE cells were cocultured with either transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) for 4 h. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 350 (blue), and bacteria were visualized by GFP fluorescence (green). Early endosomes were labeled with rabbit antibody to early endosomal antigen 1 (EEA1) protein and visualized with donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Representative images are shown for each condition (TR or CE) with individual and merged fluorescence images shown for depiction of colocalization. Colocalization of bacteria with EEA1 is observed as either yellow (merged) or red EEA1 label closely surrounding clusters of green NTHI bacteria. Bar, 10 µm. (B) Additional images representative of those depicted in panel A. (C) The number of colocalization events per cell was quantified by visual assessment of 200 individually infected cells. Statistical significance was determined by Mann-Whitney U test, and error bars represent standard errors of the means. (D and E) Transmission electron microscopy of NHBE cells cocultured with TR or CE conditioned NTHI for 4 h and subsequently immunolabeled to detect bacterial association with EEA1. Early endosomes were labeled with rabbit antibody to EEA1 and visualized with anti-rabbit IgG antibody conjugated to an 18-nm colloidal gold particle. EEA1-containing vesicles devoid of bacteria (labeled B) are indicated by yellow arrows, while red arrows indicate EEA1-containing vesicles associated with bacteria. Bar, 500 nm.
FIG 4
FIG 4
Transient heme-iron restriction of NTHI alters lysosomal trafficking and biogenesis. (A and B) NHBE cells were cocultured with transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) and assessed for colocalization of NTHI with LAMP1 by immunofluorescence at 24 h postinoculation. Cell membranes were visualized with wheat germ agglutinin conjugated to Alexa Fluor 350 (blue), and NTHI was visualized by GFP fluorescence (green). LAMP1 bar, 10 µm. (C and D) Colocalization of TR and CE NTHI with LAMP1 was assessed by TEM. LAMP1 was labeled with rabbit anti-LAMP1 and detected with anti-rabbit IgG conjugated to 18-nm colloidal gold. LAMP1-positive vesicles devoid of bacteria (labeled B) are indicated by yellow arrows, and LAMP1-positive vesicles associated with bacteria are indicated with red arrows. Bar, 500 nm. (E to G) LAMP1 staining patterns depict differences in lysosomal biogenesis in NHBE cells infected with TR NTHI (F) or CE NTHI (G) at 5 h postinoculation. Uninfected cells are included for comparison (E). LAMP1 was visualized using rat anti-LAMP1 and detected with anti-rat IgG conjugated to Alexa Fluor 594 (red). TR NTHI infection promotes a diffuse LAMP1 staining pattern while CE NTHI infection promotes a punctate and often perinuclear staining pattern in NHBE cells. Uninfected NHBE cells display a combination of both diffuse and punctate staining. Bar, 10 µm. (H) LAMP1 staining patterns were quantified by visual counting of 100 individual NHBE cells from each condition (infected with either TR or CE NTHI). Statistical significance was determined using a two-tailed Student t test.
FIG 5
FIG 5
Pharmacological inhibition of endocytosis pathways reveals that nutritionally conditioned NTHI is internalized into cells through multiple mechanisms. (A) Representative images depicting inhibition of uptake of known fluorescent cargo conjugates (Alexa Fluor 488, green) into NHBE cells in the presence of each pharmacological inhibitor [CPZ, chlorpromazine; MβCD, methyl-β-cyclodextrin; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride]. The optimal concentration of cytochalasin D (CytoD) was determined by staining F-actin with phalloidin conjugated to Alexa Fluor 350. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red). Bar, 10 µm. (B) Fluorescence microscopy was used to determine uptake of transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) following pharmacological inhibition of endocytosis pathways. NHBE cells were pretreated with pharmacological inhibitors prior to a 4-h incubation with TR or CE NTHI strain 86-028NP(pGM1.1). NTHI was visualized by GFP fluorescence (green), and epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red). The far-right panel depicts infected cells with no inhibitor (Untreated) for comparison. Each experiment was performed in three biological replicates, and representative images are shown. Bar, 10 µm. (C) Viable intracellular bacteria were enumerated following gentamicin treatment of NHBE cells that were incubated with transiently restricted (TR) or continuously exposed (CE) NTHI for 1 h in the presence of pharmacological inhibitors compared to untreated controls. Statistical significance was determined by analysis of variance with means from duplicate wells from three independent biological replicates, and error bars represent standard errors of the means (**, P  < 0.01; ****, P  < 0.0001). The invasion index was calculated as the number of viable intracellular bacteria divided by total associated bacteria.
FIG 6
FIG 6
Inhibition of macropinocytosis redirects transiently restricted NTHI to the endolysosomal pathway and decreases intracellular survival. (A to D) NHBE cells were pretreated with 60 µM EIPA prior to coculture with transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) and visualized for colocalization of NTHI with EEA1 by fluorescence microscopy. NTHI was visualized by GFP fluorescence (green), EEA1 was labeled with rabbit antibody to EEA1 and visualized with donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (red), and epithelial cell membranes were visualized with wheat germ agglutinin conjugated to Alexa Fluor 350 (blue). Representative images depict colocalization of TR (A) or CE (C) NTHI with EEA1 as observed by yellow fluorescence or red EEA1 labeling closely surrounding clusters of green NTHI bacteria. Bar, 10 µm. (B) EIPA pretreatment of NHBE cells significantly increases the number of colocalization events of TR NTHI with EEA1 in infected cells compared to infected cells that were not treated with EIPA. (D) Colocalization of CE NTHI with EEA1 does not significantly change in the presence or absence of EIPA. Statistical significance was determined by Mann-Whitney U test of colocalization events from a total of 200 independent cells from three biological assays. (E) Viable intracellular bacteria were enumerated following gentamicin treatment of NHBE cells infected with TR or CE NTHI in the presence and absence of EIPA. Statistical significance was determined by two-tailed Student’s t test of the means for duplicate wells from three independent biological replicates, and error bars represent standard errors of the means. (F to H) Intracellular bacterial communities were enumerated in chinchilla middle ear epithelial cells incubated with TR NTHI in the presence or absence of EIPA. (F and G) Bacteria were visualized by GFP fluorescence (green), epithelial cell members were stained with wheat germ agglutinin conjugated to Alex Fluor 594 (red), and host and bacterial DNA were labeled with Hoechst stain (blue). Bar, 10 µm. (H) Statistical significance was determined by two-tailed Student’s t test of the mean from duplicate wells from each of three independent biological replicates, and error bars represent standard errors of the means.
FIG 7
FIG 7
Proposed model for differential trafficking of TR NTHI through macropinocytosis resulting in IBC formation. In the absence of EIPA (top), both transiently restricted (TR, left) and continuously exposed (CE, right) NTHI enter the cells through endolysosomal pathways: clathrin-mediated endocytosis (blue circles), lipid raft/caveola-mediated endocytosis (orange rectangles), and macropinocytosis (membrane ruffling by actin polymerization, red). The ability to enter the cell through these various pathways appears to be independent of prior heme-iron status. Once internalized, CE NTHI (yellow bacteria) traffics to the early endosomes (red circles) and finally to the lysosomes (purple circles), where the bacteria are degraded. TR NTHI (green bacteria) also enters the cells through endolysosomal pathways and traffics to the early endosomes and lysosomes. In contrast, the subpopulation of transiently restricted NTHI that enters through macropinocytosis either completely evades or escapes this pathway (indicated by “?”) to form intracellular bacterial communities in the cell cytoplasm. In the presence of the macropinocytosis inhibitor EIPA (bottom), trafficking of continuously exposed NTHI through the endolysosomal pathway remains unchanged. Transiently restricted NTHI, entering the cell through clathrin- or lipid raft/caveola-mediated endocytosis, now localizes to the early endosomes. This shift in trafficking targets TR NTHI for degradation by the endolysosomal pathway and significantly decreases intracellular survival of this population.

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