Early Intracellular Trafficking ofGranulibacter bethesdensis in HumanMacrophages

Jessica Chu,a* Margery G. Smelkinson,b David W. Dorward,c Kol A. Zarember,a

John I. Gallina

Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health,Bethesda, Maryland, USAa; Biological Imaging Section, Research Technologies Branch, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USAb; Electron MicroscopyUnit, Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and InfectiousDiseases, National Institutes of Health, Hamilton, Montana, USAc

ABSTRACT Granulibacter bethesdensis is a Gram-negative bacterium that infectspatients with chronic granulomatous disease (CGD), a primary immunodeficiencymarked by a defect in NOX2, the phagocyte NADPH oxidase. Previous studies haveshown that NOX2 is essential for killing of G. bethesdensis by neutrophils and mono-cytes and that the bacteriostatic activity of monocyte-derived macrophages (MDM)requires NOX2 and gamma interferon (IFN-�) pretreatment. To determine whether G.bethesdensis evades phagolysosomal killing, a host defense pathway intact in bothnormal and CGD MDM, or whether it occupies a distinct intracellular niche in CGDMDM, we assessed the trafficking patterns of this organism. We observed colocaliza-tion of G. bethesdensis with an early endosome antigen 1 (EEA1)-positive compart-ment, followed by colocalization with lysosome-associated membrane protein 1 (LAMP1)-positive and LysoTracker-positive late phagosomes; these characteristics were similarin both normal and CGD MDM. Despite localization to acidified late phagosomes, vi-able G. bethesdensis cells were recovered from viable MDM in numbers greater thanin the initial input up to 6 days after infection. G. bethesdensis remains, and in somecases appears to divide, within a membrane-bound compartment for the entire6-day time course. These findings indicate that this organism resists both oxygen-dependent and oxygen-independent phagolysosomal antimicrobial systems of hu-man macrophages.

KEYWORDS Gram-negative bacteria, Granulibacter, chronic granulomatous disease,innate immunity, macrophages, microbial pathogenesis, vesicular trafficking

The Gram-negative bacterium Granulibacter bethesdensis infects patients withchronic granulomatous disease (CGD), a primary immunodeficiency caused by

mutations in the phagocyte NADPH oxidase (NOX2) (1). NOX2 activation generates asuperoxide anion, which is transformed into hydrogen peroxide, hypohalous acids, andother oxidants that are required for normal phagocyte bactericidal activity againstcertain bacterial and fungal pathogens, including G. bethesdensis (2, 3). Nine cases of G.bethesdensis infection of CGD patients have been reported (4–6), but this may be anunderestimate, as suggested by anti-G. bethesdensis seropositivity in CGD patients fromwhom bacteria were never isolated (3).

G. bethesdensis is a member of the family Acetobacteraceae but only weakly gener-ates acetic acid from ethanol and can utilize methanol as a sole carbon source, whichclassifies it as a methylotroph (7). Two other methylotrophs, Acidomonas methanolicaand Methylobacterium lusitanum, are also pathogens in CGD patients (4, 8). Additionally,infections in immunocompromised individuals caused by other Acetobacteraceae have

Received 11 October 2016 Returned formodification 20 November 2016 Accepted12 March 2017

Accepted manuscript posted online 20March 2017

Citation Chu J, Smelkinson MG, Dorward DW,Zarember KA, Gallin JI. 2017. Earlyintracellular trafficking of Granulibacterbethesdensis in human macrophages. InfectImmun 85:e00847-16. https://doi.org/10.1128/IAI.00847-16.

Editor Shelley M. Payne, University of Texas atAustin

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to John I. Gallin,jgallin@cc.nih.gov.

* Present address: Jessica Chu, Division ofChemistry and Toxicology Devices, Centerfor Devices and Radiological Health, Foodand Drug Administration, Silver Spring,Maryland, USA.

CELLULAR MICROBIOLOGY:PATHOGEN-HOST CELL MOLECULAR INTERACTIONS

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been reported in recent years (9). Thus, it is becoming increasingly important tounderstand the interactions of these emerging pathogens with the host.

Previous studies have shown that the G. bethesdensis type strain, CGDNIH1, isresistant to serum (3). It can be internalized in a serum-dependent manner, and �50%of the initial input is killed by normal neutrophils and normal monocytes after 24 h ata multiplicity of infection (MOI) of 1 (2, 3). Additionally, gamma interferon (IFN-�)-pretreated normal monocyte-derived macrophages (MDM) can exert a bacteriostaticeffect on G. bethesdensis that was not seen in MDM from CGD patients. Neutrophils,monocytes, and MDM from patients with CGD cannot kill G. bethesdensis, indicating theimportance of NOX2 in these cells for activity against this organism. While IFN-�pretreatment significantly increases MDM anti-Granulibacter activities of healthy butnot CGD patient MDM (2), healthy IFN-�-pretreated MDM are less effective at control-ling G. bethesdensis than healthy monocytes and neutrophils. Although the cellularniche(s) in which G. bethesdensis persists in vivo remains to be described, the relativeresistance of this bacterium to MDM in vitro suggests that G. bethesdensis resists hostdefense pathways, such as lysosomal degradation, used by macrophages to control andeliminate other microbes. To explore this possibility, we characterized the early intra-cellular trafficking pathway(s) that G. bethesdensis uses after serum-dependent inter-nalization by normal and CGD MDM.

RESULTSTrafficking of G. bethesdensis through early phagosomes in macrophages.

Microbes are initially internalized into a phagosome that undergoes maturation throughsuccessive fusion with early endosomes, late endosomes, and finally lysosomes (10). Theearly phagosome acquires characteristics of early endosomes, such as the expression ofearly endosome antigen 1 (EEA1) and a mildly acidic (pH 6.1 to 6.5) and weakly hydrolyticlumen. In order to determine whether serum-opsonized G. bethesdensis localizes to earlyphagosomes, we assessed Cy5-labeled G. bethesdensis colocalization with EEA1 in nor-mal and CGD monocyte-derived macrophages (MDM) over a short 2-h time course(Fig. 1A). A peak of colocalization was observed at 15 min for both normal MDM(23.7% � 7.1%, mean � standard deviation [SD]) and CGD MDM (25.4% � 9.9%)(Fig. 1B). There was no statistical difference in the numbers of internalized bacteriaper MDM between normal MDM and CGD MDM (Fig. 1C). Thus, G. bethesdensisinitially traffics to the early phagosome upon internalization, and this localization isthe same in normal and CGD MDM.

Trafficking of G. bethesdensis through late phagosomes in macrophages. Theearly phagosome matures through an intermediate stage to a late-stage phagosomethat acquires lysosome-associated membrane proteins (LAMPs) and an acidic luminalpH (5.5 to 6.0) (10). In the final maturation stage, the late phagosome fuses withlysosomes to generate the LAMP-positive phagolysosome, which decreases further inpH (to �4.5). To determine whether G. bethesdensis traffics to late phagosomes, weassessed the colocalization of Cy5-labeled G. bethesdensis with the acidotrophic dyeLysoTracker for �16 to 18 h using live-cell time-lapse imaging (Fig. 2A; see also MoviesS1 and S2 in the supplemental material). Colocalization between G. bethesdensis andLysoTracker increased until �6 h for normal MDM and until �4 h for CGD MDM beforereaching a maximum and declining slightly (Fig. 2B). There was a trend toward earliercolocalization in CGD MDM than in normal MDM. The complex setup of live-celltime-lapse imaging experiments does not easily allow for prior synchronization ofphagocytosis via centrifugation, as was used for fixed-cell EEA1 (Fig. 1) and LAMP1(Fig. 3, discussed below) microscopy experiments. Instead, image capture beginsimmediately after the addition of bacteria. Thus, the kinetics of bacterial internal-ization are slower for LysoTracker-stained MDM (Fig. 2C) than for EEA1- or LAMP1-stained MDM.

Cy5-labeled G. bethesdensis colocalization with LAMP1 was also measured (Fig. 3Aand B). LAMP1 colocalization peaked around 1 h and slowly decreased thereafter.However, there was no significant difference between normal MDM (54.4% � 14.4%)

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and CGD MDM (40.5% � 22.1%) (Fig. 3C). These results support a trafficking pathwaythat begins with G. bethesdensis internalization into a phagosome and that thenfuses with EEA1-positive early endosomes before subsequent fusion with LAMP1/LysoTracker-positive late endosomes/lysosomes.

G. bethesdensis survives intracellularly despite late phagosome colocalization.Lysosomes contain degradative enzymes capable of killing microorganisms, a defensemechanism employed by phagocytes to control infection. If G. bethesdensis weresensitive to these enzymes, the number of organisms would decrease over time afterfusion with the lysosome. However, as shown in Fig. 3D, median numbers of intracel-lular bacteria for both MDM cell types remained steady from about 1 h until the end ofthe time course. It should also be noted that for later time points, individual MDMexhibited a wide range of intracellular bacterial loads, with some MDM containing morethan 30 bacteria. Thus, decreases in intracellular bacterial numbers were not observedover time. Either G. bethesdensis is resistant to lysosomal antimicrobial factors, or itprevents activation of these factors. It was previously shown that G. bethesdensissurvives in CGD polymorphonuclear leukocytes (PMN), monocytes, and MDM (2, 3),suggesting that it is resistant to the NOX2-independent microbicidal arsenals of thesecell types. In fact, G. bethesdensis resisted human cathelicidin LL-37 (3), an antimicrobialpeptide found in many cell types, including monocytes and neutrophils (11). It is alsopossible that G. bethesdensis survives because it blocks lysosomal fusion with lateendosomes or escapes to another compartment prior to lysosomal fusion, therebypreventing G. bethesdensis contact with microbicidal enzymes.

We sought to confirm the viability of these Cy5-labeled bacteria by enumeratingCFU. CFU in supernatants were quantified to determine numbers of non-cell-associatedbacteria, and MDM were lysed to determine numbers of cell-associated bacteria (i.e.,intracellular bacteria and bacteria bound to the cell surface). This method cannotdifferentiate between intracellular bacteria and adherent bacteria, but the majority arelikely intracellular by 15 to 30 min after phagocytosis synchronization, as observed byconfocal microscopy (Fig. 1). To minimize the contribution of non-cell-associatedbacteria, MDM were infected with Cy5-labeled G. bethesdensis for 1 h, washed threetimes, and incubated with fresh complete medium for up to 6 days. Previous attemptsin our laboratory to use gentamicin to remove non-cell-associated G. bethesdensis hadproven unsuccessful, as gentamicin appeared to kill cell-associated bacteria (data notshown). Internalization of gentamicin by human macrophages and subsequent killingof cell-associated bacteria have been described previously (12, 13).

At 1 h postinfection after being washed, the percentages of remaining non-cell-associated bacteria from the total input bacteria (mean � SD) were 1.1% � 0.5% for

FIG 1 G. bethesdensis colocalizes with EEA1-positive early phagosomes in monocyte-derived macrophages (MDM). MDM fromnormal donors (n � 5) and CGD donors (n � 4) were incubated with Cy5-labeled G. bethesdensis (red) in 10% autologousserum for the indicated time points. MDM were fixed and stained for the early endosome marker EEA1 (green) and nucleus(DAPI; blue). Confocal images were acquired and analyzed with Imaris software as detailed in Materials and Methods. (A)Representative image after 15 min of coincubation of normal MDM. The yellow arrow points to Granulibacter cells colocalizedwith EEA1. Scale bar � 5 �m. (B) Quantitative analysis of percentages of G. bethesdensis cells colocalized with EEA1 inmacrophages from normal and CGD donors (means � SD). (C) Quantitative analysis of numbers of Cy5-labeled G. bethesdensiscells per macrophage for the cells analyzed in panel B.

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normal MDM and 0.9% � 0.3% for CGD MDM (Fig. 4A). By 1 day postinfection, thesepercentages dropped below 0.05% for both normal MDM and CGD MDM, showingthat the majority of the bacteria were cell associated by this time. Numbers ofcell-associated bacteria tended to be higher at 1 day, 3 days, and 6 days postin-fection than at 1 h for both MDM types, though these differences reached statisticalsignificance only for normal MDM (Fig. 4B). A modest decrease in numbers ofcell-associated bacteria was observed between 3 days and 6 days after infection.Although statistical significance was not reached, there was a trend toward greaterbacterial growth in NOX2-deficient CGD MDM. No decrease in MDM viability wasobserved for either normal or CGD MDM (Fig. 5). There was a trend toward increasedmetabolic activity, as measured by a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay, for both normal and CGD MDM in thepresence of bacteria compared to the metabolic activity of MDM alone, which may becontributed by the bacteria themselves. Taken together, these data suggest that G.bethesdensis evades lysosomal killing and survives intracellularly in viable MDM regard-less of a functional NOX2 NADPH oxidase.

G. bethesdensis resides in a membrane-bound compartment. We performedtransmission electron microscopy (TEM) in order to visualize G. bethesdensis insidenormal and CGD MDM (Fig. 6). For the entire 6-day time course, G. bethesdensisremained within a membrane-bound compartment. It was rare to observe a bacteriumlacking clear evidence of an encircling host membrane. At earlier time points (1 h, 1day), the phagosomal membrane appeared to tightly enclose the bacterium, whereasat later time points (3 days, 6 days) both tight phagosomes and enlarged phagosomes

FIG 2 G. bethesdensis colocalizes with acidified late phagosomes in MDM. LysoTracker-labeled MDM(green) from normal donors (n � 3) and CGD donors (n � 4) were incubated with Cy5-labeled G.bethesdensis (red) in 10% autologous serum in a live-cell imaging chamber equilibrated to 37°C in a 5%CO2 atmosphere. Time-lapse images were acquired every 5 min over �16 to 18 h and analyzed usingImaris software as described in Materials and Methods. (A) Representative images at 8 h for normal andCGD MDM. The yellow arrow points toward colocalized signals. Scale bar � 15 �m. (B) Quantitativeanalysis of percentages of Cy5-labeled G. bethesdensis cells colocalized with LysoTracker in macrophagesfrom normal and CGD donors (means � SD). (C) Quantitative analysis of numbers of Cy5-labeled G.bethesdensis cells per macrophage (means � SD) for the cells analyzed in panel B at the 2-h and 16-htime points.

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were observed. In most cases, sections through enlarged phagosomes contained 1 to2 bacteria, but for one CGD donor, multiple bacteria could be found in a singlephagosome at day 6. It is possible that G. bethesdensis replicates within the phagosomeor that it exits and reenters the MDM as a group. However, early time points show asingle bacterium per phagosome, suggesting that G. bethesdensis is initially internalizedas an individual rather than as a group. Moreover, there was evidence of bacterialdivision within the phagosome especially at 3 days and 6 days, when a cleavage furrowcould be seen. These data demonstrate that G. bethesdensis traffics through MDMwithin a phagosomal compartment, where it is capable of replicating, supporting theaforementioned results.

FIG 3 G. bethesdensis colocalizes with LAMP1-positive late phagosomes in MDM. MDM from normaldonors (n � 6) and CGD donors (n � 4) were incubated with Cy5-labeled G. bethesdensis (red) in 10%autologous serum for the indicated time points. MDM were fixed and stained for the lysosomal markerLAMP1 (green) and nucleus (DAPI; blue). Confocal images were acquired and analyzed with Imarissoftware as described in Materials and Methods. (A) Representative micrographs taken at 1 h fromnormal and CGD MDM. Scale bar � 5 �m. (B) Quantitative analysis of percentages of Cy5-labeled G.bethesdensis cells colocalized with LAMP1 in macrophages from normal and CGD donors (means � SD).(C) Quantitative analysis of numbers of Cy5-labeled G. bethesdensis cells per macrophage (means � SD)for the cells analyzed in panel B.

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DISCUSSION

The host macrophage employs innate immune mechanisms to eliminate invadingmicrobes. This includes recognition of microbes via complement, Fc, and Toll-likereceptors, phagocytosis into the endocytic pathway, phagosome maturation, and

FIG 4 Cell-associated G. bethesdensis organisms remain viable. MDM from normal donors (n � 7) and CGDdonors (n � 4) were incubated with Cy5-labeled G. bethesdensis cells in 10% autologous serum for 1 h beforebeing washed to remove non-cell-associated bacteria. Time points were 1 h (immediately after washing) and1, 3, and 6 days (d). (A) CFU present in supernatants after being washed are represented as a percentage ofthe input bacteria (see Materials and Methods). (B) CFU in cellular lysates were enumerated to determinecell-associated bacteria and are expressed per MDM. Statistical testing by the Wilcoxon test comparing valuesat 1 h and those at 1-, 3-, or 6-day time points for normal or CGD MDM (*, P � 0.05).

FIG 5 Infected MDM retain their viability. MDM from normal donors (n � 9) and CGD donors (n � 4) wereincubated with Cy5-labeled G. bethesdensis cells in 10% autologous serum for 1 h before being washedto remove non-cell-associated bacteria. An XTT assay was conducted at 1 h (immediately after washing)and at 1, 3, and 6 days as described in Materials and Methods in order to assess viability. Controlsconsisted of medium only, bacteria only, or noninfected MDM. Statistical testing was by the Wilcoxon testcomparing MDM and MDM with bacteria at each time point (**, P � 0.01).

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eventual degradation in the lysosome (14). The late phagosome undergoes luminalacidification to a pH of �5, required for the activation of lysosomal proteinases (e.g.,cathepsins), which have been shown to lyse bacterial species like Staphylococcus aureusand Acinetobacter (15). In this study, we show that serum-opsonized G. bethesdensistraffics through EEA1-positive early phagosomes within 15 min, followed by subse-quent localization to acidified LAMP1-positive late phagosomes. Despite localizing inacidified late phagosomes, G. bethesdensis survives and replicates until day 6 postin-fection, suggesting that it may evade degradation. These findings were similar for MDMfrom normal donors and CGD donors, who lack a functional NOX2 NADPH oxidasesystem.

Many intracellular bacteria have evolved mechanisms to circumvent lysosomalkilling in order to survive host defenses. In many cases, bacteria escape from latephagosomes/phagolysosomes or completely avoid phagosome-lysosome fusion sothat they can replicate in another intracellular compartment, including the cytosol. Inhuman macrophages, Bordetella pertussis initially localizes to a LAMP-positive phago-some but is thought to later replicate in a compartment that has lost lysosomal markersand gained early endosome markers (16). Avoiding fusion with endosomes and lyso-somes, Legionella pneumophila creates a replicative endoplasmic reticulum (ER)-likevacuole in host cells and later lyses host cells in order to infect neighboring cells (17).Burkholderia cenocepacia escapes from early phagosomes to the cytosol in humanTHP-1 macrophages, where it can replicate and block autophagosome containment(18). Similarly, Francisella tularensis undergoes early phagosomal maturation beforeescape to the cytosol, where it replicates (19). Listeria monocytogenes, Shigella flexneri,Rickettsia species, Mycobacterium marinum, and Burkholderia pseudomallei escape fromthe phagosome to the cytosol, where they take advantage of host actin machinery to

FIG 6 G. bethesdensis remains in a membrane-bound phagosome for 6 days. MDM from normal donors (top [n � 4]) and CGD donors (bottom[n � 4]) were incubated with Cy5-labeled G. bethesdensis cells in 10% autologous serum for 1 h before being washed to remove non-cell-associated bacteria. Samples were collected at 1 h (immediately after washing) and at 1, 3, and 6 days, prepared as described in Materials andMethods, and imaged by transmission electron microscopy. Scale bars are 0.4 �m (A to H, J) and 4 �m (I). Gamma corrections on the images are1.4 (A to H) and 1.1 (I to J). Each time series is from a single representative donor.

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infect neighboring cells (20). Instead of escaping from phagosomes or evading lyso-somal fusion, some bacteria, such as Coxiella burnetii (21) and Mycobacterium tubercu-losis (22), resist and adapt to the acidic environment of the lysosome.

G. bethesdensis localization to early phagosomes reached a peak at 15 min,followed by localization to late phagosomes from 1 to 24 h. Like other members of theAcetobacteraceae family, which includes organisms responsible for vinegar production,G. bethesdensis grows optimally at lower pHs of 5.0 to 6.5 (7), a characteristic that mayenable it to exploit the acidic environment of the late phagosome and phagolysosome.Interestingly, the weak base amantadine caused a dose-dependent decrease in therecovery of viable G. bethesdensis organisms in the presence of normal MDM, while itcaused an increase in the recovery of viable control bacteria in the absence of normalMDM (see Fig. S1 in the supplemental material). Although we have not determined thepH of the MDM phagosome in which G. bethesdensis resided, it is possible that thegrowth of this acid-tolerant microbe is enhanced by phagosomal acidification andreduced in amantadine-induced neutral or alkaline settings.

Though it initially reaches late phagosomes, G. bethesdensis may not stay in thatcompartment for the entire 6-day period. The percentage of colocalization with theLAMP1 marker decreased between 24 and 72 h, which may indicate relocation toanother compartment and/or loss of LAMP1 from the phagosomal membrane. G.bethesdensis appeared to be contained in a membrane-bound phagosome over a 6-dayperiod, as assessed by electron microscopy, suggesting that G. bethesdensis does notescape to the cytosol during that time frame. Of note, although we have occasionallyseen evidence by TEM of rare phagosomes in MDM surrounded by apparently multi-lamellar membranes, numerous efforts in our lab using pharmacologic inhibitors andactivators of autophagy have not detected differences in levels of Granulibacter survivalin monocytes or PMN, suggesting that this pathway may not play a major role inGranulibacter pathogenesis (data not shown). Future studies will explore the latertrafficking pathways, including mechanisms that G. bethesdensis uses to evade lyso-somal killing (e.g., blocking lysosomal fusion, escaping to another compartment prior tolysosomal fusion, resisting killing by lysosomal enzymes) in more depth.

Similar intracellular trafficking patterns for G. bethesdensis were observed in normalMDM and CGD MDM. Additionally, there was no significant difference in numbers ofintracellular G. bethesdensis organisms in normal MDM and CGD MDM, though therewas a trend toward more bacteria and earlier fusion with late endosomes/lysosomes inCGD MDM. Percentages of cell colocalization with LAMP1 in normal MDM and CGDMDM were similar, but increased bacterial numbers in CGD MDM may indicate that G.bethesdensis escapes from LAMP1-positive compartments to a LAMP1-negative repli-cative niche unique to CGD MDM. A comparison of later trafficking pathways in normalMDM and CGD MDM will be a focus of future studies.

One possible limitation of this study is that the high MOI (up to �20 bacteria perMDM), which was required for microscopic visualization of the colocalization betweenbacteria and organelle markers and for TEM, may have affected the ability of normalMDM (with a functional NADPH oxidase) to effectively control bacteria. Previous studiesreported that maximum control of Granulibacter by MDM required 4 host cells perbacterium (an MOI of 0.25), which would have made visualization difficult if notimpossible. Despite these high numbers of G. bethesdensis organisms, MDM remainedviable and normal MDM appeared to exert some control on bacterial outgrowth by day6. In summary, G. bethesdensis persists despite trafficking through acidified late phago-somes, a central host defense mechanism in macrophages.

MATERIALS AND METHODSEthics statement and human cell isolation. Blood samples were taken after we obtained informed

consent from healthy and CGD donors enrolled at the NIH Clinical Center with NIH institutional reviewboard-approved protocols. Monocytes were isolated from peripheral blood collected into Becton Dick-inson (Franklin Lakes, NJ) Vacutainer acid citrate dextrose tubes and differentiated into monocyte-derived macrophages (MDM) in 50 ng/ml macrophage colony-stimulating factor (M-CSF; Invitrogen,

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Carlsbad, CA) for 6 to 7 days as previously described (2). Serum was collected from BD serum separatortubes according to the manufacturer’s instructions, used fresh or snap-frozen, and stored at �80°C.

Bacterial cultures. G. bethesdensis CGDNIH1 was cultured for 2 days and subcultured for 1 day tomid-log phase in YPG medium (5 g yeast extract, 3 g peptone, and 10 g glucose per liter) at 37°C withshaking. After three washes in 150 mM NaCl with vortexing, bacteria were enumerated by determiningtheir optical density at 600 nm (OD600) using a NanoDrop ND-1000 (Grace Scientific, LLC, Clarksburg, MD)and spiral plated (Eddy Jet 2 spiral plater; Neu-Tec Group Inc., Farmingdale, NY) on YPG plates. Plateswere incubated for 3 to 4 days at 37°C, and CFU were enumerated using Neu-Tec Group’s Flash & Growadvanced economical automated colony counter and accompanying Flash & Grow version 1.3 software.

Cy5 labeling of bacteria. One vial of Cy5 mono-reactive dye (GE Healthcare, Pittsburgh, PA) wasdissolved in 40 �l of dimethyl sulfoxide (DMSO). Ten microliters of this Cy5 solution was added to 109

bacteria in 750 �l of 0.1 M NaHCO3 (pH 9.3) and incubated for 1 h at room temperature (RT) in the darkwith vortexing every 15 to 30 min to mix. In order to quench unbound dye, Cy5-labeled bacteria werepelleted at 11,000 � g and incubated for 10 min at RT with 10% normal human serum. Labeled bacteriawere washed 3 times with Hanks balanced salt solution without calcium or magnesium (HBSS�;Invitrogen), pelleted as described above, resuspended in 100 �l HBSS�, enumerated by determining theOD600 using NanoDrop, and plated on YPG plates to obtain colony counts. Bacterial viability wasunaffected by the Cy5 conjugation procedure, and Cy5 signal could be detected on bacteria grown inRPMI 1640 for up to 6 days following labeling. Cy5-labeled bacteria were stored at 4°C and used within24 h of being labeled. Previous studies have shown that G. bethesdensis requires serum for efficientphagocytosis by neutrophils and monocytes (2, 3). Therefore, all assays described below were conductedin the presence of human serum.

Infection of monocyte-derived macrophages for fixed-cell imaging. In a tissue culture hood,12-mm, number 1 thickness, round glass coverslips (Electron Microscopy Sciences, Hatfield, PA) weresterilized with 70% ethanol and dried in 24-well tissue culture plates. Monocytes (3 � 105) were addedto each well, which contained RPMI 1640 with 2 mM L-glutamine (Invitrogen) plus 10 mM HEPES (pH 7.2)(Sigma-Aldrich, St. Louis, MO) and 10% autologous human serum, and differentiated into MDM directlyon coverslips. On day 7, spent medium was replaced with 10% autologous-serum-containing mediumand MDM were infected at an estimated multiplicity of infection (MOI) of 5. Plates were centrifuged at362 � g for 8 min at 4°C to synchronize phagocytosis.

Immunofluorescence staining. At the time points indicated in Fig. 1 and Fig. 3, coverslips werewashed once with ice-cold phosphate-buffered saline without calcium or magnesium (PBS�), fixed with4% paraformaldehyde (PFA) for 15 min at RT, washed 3 times with ice-cold PBS�, and stored in PBS�(EEA1 samples) or 5% bovine serum albumin (BSA) in PBS� (LAMP1 samples) at 4°C until all coverslipswere collected. Coverslips were rinsed with PBS� once, permeabilized with 0.1% Triton X-100 in PBS for10 min at RT, washed 3 times with PBS, blocked with 5% BSA in PBS� for 30 min at RT, and incubatedwith primary antibody diluted in 1% BSA in PBST (PBS� containing 0.1% Tween 20) overnight at 4°C. Thenext day, coverslips were washed 3 times with PBS� (5 min per wash), incubated with secondaryantibody in 1% BSA in PBST for 1 h at RT, washed 3 times with PBS� (5 min per wash), counterstainedwith 1 �g DAPI (4=,6-diamidino-2-phenylindole)/ml in PBS� for 5 min, and rinsed with PBS�. Sampleswere kept in the dark for the majority of the procedure, and steps requiring 5-min washes or incubationslonger than 10 min were conducted with gentle agitation. Coverslips were mounted with ProLongDiamond antifade mountant (Invitrogen) overnight at RT in the dark and stored at 4°C prior to imaging.Primary rabbit EEA1 (1:200; catalog no. 3288) and rabbit LAMP1 (1:200; catalog no. 9091) monoclonalantibodies and secondary anti-rabbit IgG(H�L) and F(ab=)2 fragment-Alexa Fluor 488 conjugate (1:1,000;catalog no. 4412) were obtained from Cell Signaling Technology (Danvers, MA).

Fixed-cell microscopy and analysis. Confocal images were acquired with a Leica SP5 confocalmicroscope equipped with a 63�/1.4-numerical-aperture (NA) oil immersion objective, hybrid HyDdetectors, and 488-nm argon and 633-nm HeNe lasers. Two 2 by 2 fields representing two distinct areasof the coverslip were acquired using the tiling feature in the LAS AF software. Imaris (MS Windows 8.0 �64; Bitplane, Concord, MA) was used for image analysis. The spot feature was used to determine the totalnumber of bacteria and the total number of MDM. The number of extracellular bacteria was subtractedfrom the total number of bacteria to determine the number of intracellular bacteria. The average numberof intracellular bacteria per MDM was equal to the total number of intracellular bacteria divided by thetotal number of MDM. Colocalization between Cy5-bacteria (red) and Alexa Fluor 488-stained EEA1 orLAMP1 (green) was determined using the coloc function. The same threshold values for red and greenchannels were applied across all time points for EEA1 or LAMP1 colocalization analysis. Approximately 10to 20 individual cells were analyzed per time point per donor.

Time-lapse confocal microscopy. Lab-Tek number 1.0 borosilicate cover glass chamber slides wereprecoated with 19 �g/ml poly-D-lysine (Corning, Corning, NY), diluted in RPMI 1640 medium for 1 h, andwashed 3 times with RPMI 1640. Two times 105 monocytes per well were differentiated in the chamberslides. On days 6 to 7, spent medium was replaced with fresh medium containing 10% autologous serumand 50 nM LysoTracker Green DND-26 (Invitrogen). Slides were placed in a live-cell imaging chamber(PeCon GmbH, Erbach, Germany) and heated to 37°C with a 5% CO2 atmosphere, and 3 fields per wellwere selected. Once live-cell imaging was initiated, Cy5-labeled bacteria were added at an estimated MOIof 5. Time-lapse images were acquired every 5 min over �16 to 18 h with a Leica SP8 confocalmicroscope equipped with a 63�/1.4-NA oil immersion objective, hybrid HyD detectors, and 488-nmargon and 633-nm HeNe lasers. Colocalization between Cy5 bacteria (red) and LysoTracker (green) wasdetermined using the coloc function in Imaris (Bitplane). The same threshold values for red and greenchannels were applied across all time points.

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Transmission electron microscopy. Thirteen-millimeter plastic Thermanox coverslips (ElectronMicroscopy Sciences) were sterilized in 24-well tissue culture plates as described above and 4 � 105

monocytes differentiated into MDM directly on coverslips. On days 6 to 7, spent medium was replacedwith 10% autologous-serum-containing medium and MDM were infected at an estimated MOI of 5 withCy5-labeled G. bethesdensis. Plates were centrifuged at 362 � g for 8 min at 4°C to synchronizephagocytosis. After 1 h of infection, MDM were washed 3 times with RPMI 1640 medium before freshmedium containing 10% autologous serum was added. Coverslips were collected immediately after cellswere washed (1 h) or 1, 3, or 6 days after they were washed. Coverslips were washed with PBS� andincubated in 2.5% glutaraldehyde in Sorenson’s phosphate buffer (Electron Microscopy Sciences) for 1 to2 h before removal to fresh fixative. Following primary fixation, the samples were postfixed, contrasted,dehydrated, and embedded in Araldite resin (Structure Probe, Inc., West Chester, PA) essentially aspreviously described (23), except that dehydration steps included 3-min cycles, once in 70% ethanol andthree times in 100% ethanol. Araldite infiltration was extended to one 10-min cycle in 50% resin, one20-min cycle in 75% resin, and one 40-min cycle for each of two changes in 100% resin.

XTT viability assay and CFU assay. Ninety-six-well tissue culture plates were precoated with 19�g/ml poly-D-lysine as described above, and 5 � 104 monocytes per well were differentiated on theplates. On days 6 to 7, spent medium was replaced with fresh medium containing 10% autologousserum, and MDM were infected with Cy5-labeled G. bethesdensis at an estimated MOI of 5. Controlsconsisted of bacteria in the absence of MDM (XTT assay, CFU assay), MDM in the absence of bacteria (XTTassay), and medium lacking both bacteria and MDM (XTT assay). Plates were centrifuged at 362 � g for8 min at 4°C to synchronize phagocytosis. After 1 h of infection, all wells containing MDM were washed3 times with RPMI 1640 medium before fresh medium containing 10% autologous serum was added.Samples were processed immediately after washing (1 h) or at 1, 3, or 6 days after washing. The XTT assaywas carried out as previously described (2). For the CFU assay, supernatants were collected from wellscontaining MDM and the volume was replaced with an equivalent volume of RPMI 1640 containing 10%fetal bovine serum (FBS). Supernatants were centrifuged at 400 � g for 3 min at 4°C to pelletnonadherent cells and transferred to new tubes. Wells containing infected MDM or bacteria only wereincubated with saponin and subjected to shearing with a needle, as previously described (2). Lysates andcleared supernatants were diluted and spiral plated, and CFU were enumerated as described above. TheCFU in supernatants were considered non-cell-associated bacteria, while the CFU in lysates wereconsidered cell-associated bacteria (i.e., intracellular or attached to the cell surface). Non-cell-associatedCFU remaining at each time point are represented as a percentage of the total input bacteria recoveredafter 1 h of incubation in the absence of MDM. The average number of cell-associated bacteria per MDMwas equal to the total number of cell-associated bacteria in lysates divided by the total number of MDMplated.

Graphical and statistical analysis. Data were graphed and analyzed statistically using GraphPadPrism (version 6; GraphPad Software, La Jolla, CA).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00847-16.

SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.SUPPLEMENTAL FILE 2, AVI file, 2.3 MB.SUPPLEMENTAL FILE 3, AVI file, 4.3 MB.

ACKNOWLEDGMENTSWe acknowledge Harry L. Malech, Suk See DeRavin, and Steven M. Holland for

generously providing us with CGD patient samples and thank the CGD patients whocontributed to this study. We also thank Juraj Kabat for confocal microscopy imageanalysis advice and Chris Combs and Daniella Malide of the NIH-NHLBI imaging corefacility for use of their stimulated emission depletion (STED) microscope and usefuldiscussions.

This work was supported by the National Institutes of Health Intramural ResearchProgram. The views expressed here are those of the authors and not necessarily thoseof the U.S. Government.

J.C., M.G.S., D.W.D., and K.A.Z. designed and performed the experiments. J.C., M.G.S.,D.W.D., K.A.Z., and J.I.G. analyzed and interpreted the results. J.C., K.A.Z., and J.I.G.prepared the manuscript.

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