Author response: Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Candida albicans hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2 is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3 co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae. https://doi.org/10.7554/eLife.34798.001 eLife digest Billions of microorganisms live on, and in, the human body. Known as the human microbiome, most of these microscopic hitchhikers are harmless. But, for people with a compromised immune system, common species can sometimes cause disease. For example, the yeast Candida albicans, which colonises between 30 and 70% of the population, is normally harmless, but can switch to a disease-causing version that makes branching structures called hyphae. These hyphae grow fast, piercing and damaging the tissues around them. Immune cells called macrophages usually engulf invading microbes. These cells recognise sugars on the outside of C. albicans, and respond by wrapping their membranes around the yeast, drawing the microorganism in, and sealing it into closed structures called phagosomes. Then, the macrophages fill the phagosomes with acid, enzymes and destructive chemicals, which breaks the yeast down. Yet, C. albicans hyphae grow larger than macrophages, making them difficult to control. Maxson et al. have now tracked the immune response revealing how macrophages try to control large hyphae. The immune cells were quick to engulf C. albicans in its normal yeast form, but the response slowed down in the presence of hyphae. Electron microscopy revealed that the large structures were only partly taken in. Rather than form a closed phagosome, the macrophages made a cuff around the middle of the hypha, leaving the rest hanging out. The process starts with a receptor called CR3, which detects sugars on the outside of the hyphae. CR3 is a type of integrin, a molecule that sends signals from the surface to the inside of the immune cell. A network of filaments called actin assemble around the hypha, squeezing the membrane tight. The macrophage then deploys free radicals and other damaging chemicals inside the closed space. The seal is not perfect, and some molecules do leak out, but the effect slows the growth of the yeast. When a phagosome cannot engulf an invading microbe, a state that is referred to as being "frustrated", the leaking of damaging chemicals can harm healthy tissues and lead to inflammation and disease. These findings reveal that macrophages do at least try to form a complete seal before releasing their cocktail of chemicals. Understanding how the immune system handles this situation could open the way for new treatments for C. albicans infections, and possibly similar diseases related to "frustrated engulfment" (such as asbestos exposure, where asbestos fibers are also too large to engulf). However, one next step will be to find out what happens to partly engulfed hyphae, and how this differs from the fate of fully engulfed yeast. https://doi.org/10.7554/eLife.34798.002 Introduction Candida albicans is a commensal fungus that colonizes the epithelial surfaces of 30–70% of healthy individuals (Perlroth et al., 2007). However, in immune-compromised individuals, C. albicans can cause invasive, life-threatening disease. The mortality rate for infected patients is 46–75%, with candidiasis classified as the fourth most common nosocomial bloodstream infection (Brown et al., 2012). Invasive candidiasis is correlated with a switch of C. albicans from its yeast form to a hyphal form, a shift that can be induced in vitro by nutrient deprivation among other cues (reviewed in Sudbery, 2011). In vivo, C. albicans hyphae are capable of invading epithelium and endothelium; in addition C. albicans is capable of forming recalcitrant biofilms and inducing inflammation (Sudbery, 2011). These conditions activate host defense mechanisms for the control and clearance of C. albicans, mounted predominantly by phagocytic cells of the innate immune system. Phagocytes can effectively sense, internalize and kill invasive C. albicans. Accordingly, impairment of the phagocytic response, e.g. by elimination of macrophages and neutrophils, is associated with disseminated candidiasis (reviewed in Netea et al., 2015). Phagocytic cells possess receptors that bind the C. albicans cell wall and trigger uptake of the fungus into a phagosome. The C. albicans cell wall is composed mostly (80–90%) of polysaccharides, containing ≈ 60% β-(1,3) and -(1,6) glucans, and ≈ 40% O- and N-linked mannans (Ruiz-Herrera et al., 2006). As such, the main non-opsonic phagocytic receptors for C. albicans are the C-type lectin family of receptors, including Dectin1, the mannose receptor, and DC-SIGN (reviewed in Hardison and Brown, 2012). The phagosome typically matures rapidly after closure, evolving into an acidic, degradative and microbicidal compartment. Acquisition of antimicrobial properties by this compartment depends on its ability to accumulate and retain toxic compounds, including reactive oxygen species (ROS). Superoxide produced by the NADPH oxidase undergoes dismutation into hydrogen peroxide in the acidic luminal environment generated by the V-ATPase, which additionally favors the catalytic activity of various hydrolases. Transporters such as NRAMP-1, that antagonize microbial growth by depleting the phagosome of nutrients, also depend on phagosomal H+ for the extrusion of metal ions. Unlike most other microbes, C. albicans presents a distinct problem for phagocytes. The hyphal form of C. albicans can grow at a rate of 18.8 μm hr−1 (GOW and Gooday, 1982), quickly exceeding the size of the phagocytes themselves. The challenge is greatest for macrophages, which migrate to infection sites later than the polymorphonuclear cells, and thus encounter growing hyphae (reviewed in Erwig and Gow, 2016). Despite being remarkably plastic, macrophages have difficulty engulfing the much larger C. albicans hyphae, an impasse that no doubt contributes to the pathogenesis of candidiasis. The aim of the current study was to examine the dynamic and complex process of C. albicans phagocytosis by macrophages. We found that attempts to engulf large hyphae result in the formation of incomplete (frustrated) phagosomes, which nevertheless segregate a section of the hypha, preferentially exposing it to microbiostatic products. The mechanism and fungal components underlying the formation of the diffusion barrier established by the phagocyte when generating the frustrated phagosome was analyzed using a combination of imaging, pharmacological and genetic approaches. Results Phagocytosis of C. albicans hyphae To optimize the phagocytosis of C. albicans, which has a cell wall rich in β-glucans (Gow et al., 2011), we used RAW 264.7 macrophages stably expressing the Dectin1 receptor (RAW-Dectin1; Esteban et al., 2011). Yeast or hyphal forms of C. albicans expressing BFP (Candida-BFP; Strijbis et al., 2013) were used as targets to facilitate their visualization. Under the conditions used to generate them, C. albicans hyphae were considerably longer (>15 μm) than the macrophages (8–10 μm in diameter). After 1 hr of co-incubation with the macrophages the yeast form was fully engulfed (Figure 1A), while a significant number of hyphal C. albicans were only partially internalized (68.5% ± 4.5, while 31.5% ± 4.6 were fully internalized; 1019 events from 12 independent experiments), which was verified using fluorescent concanavalin A to label exposed hyphae (Figure 1B). This was similar to the frustrated engulfment of >20 μm C. albicans hyphae reported earlier (Lewis et al., 2012). Transmission electron microscopy confirmed that most hyphae were only partially internalized (Figure 1C) and, in addition, revealed the existence around the neck of the frustrated phagosome of a low-contrast structure seemingly devoid of membrane-bound organelles (Figure 1C, inset), previously interpreted by Strijbis et al., 2013 as accumulated actin. Indeed, this region corresponded to an actin-rich cuff-like structure (Figure 1D); F-actin was so highly accumulated at the cuff that the remainder of the cellular actin could only be visualized when images were overexposed (Figure 1D, inset). Note that the remainder (i.e. the base) of the frustrated phagocytic cup was virtually devoid of F-actin. 3D visualization verified the continuous accumulation of F-actin around the neck of the tubular phagosomes lining individual hyphae and its sharp delineation of the intracellular and extracellular portions of the fungus (Figure 1E,F,G,H and Video 1). This actin cuff was observed for RAW-Dectin1 cells engulfing C. albicans hyphae up to 100 μm in size (data not shown), and occurred in 96.3% ± 1.9 of the partially internalized hyphae (674 events analyzed in 12 independent experiments). These data support published accounts of actin cuff-like structures seen during the phagocytosis of various filamentous targets (García-Rodas et al., 2011; Gerisch et al., 2009; Heinsbroek et al., 2009; Prashar et al., 2013; Strijbis et al., 2013). The occurrence of frustrated phagocytosis with formation of a pronounced actin cuff was not unique to the RAW-Dectin1 cell line; similar features were seen when murine or human primary macrophages were confronted with C. albicans hyphae (Figure 1—figure supplement 1A and B, respectively). The actin cuff was remarkably stable, lasting for at least 90 min without contracting (Figure 1I). Nevertheless, the actin composing these structures undergoes measurable turnover (treadmilling), since the cuffs underwent gradual disassembly when the cells were treated with latrunculin A, which scavenges actin monomers (last two panels, Figure 1I). These long-lasting yet dynamic cuffs identify the frustrated phagocytic cups generated by macrophages attempting to eliminate C. albicans hyphae. Figure 1 with 1 supplement see all Download asset Open asset Partial phagocytosis of C. albicans hyphae is associated with formation of an actin cuff. Phagocytosis of C. albicans yeast (A) or hypha (B) by RAW-Dectin1 cells. After incubation with Candida-BFP, RAW-Dectin1 cells were fixed and extracellular C. albicans stained using Alexa594-conjugated concanavalin A (red). The fluorescence of the BFP is shown in white here and elsewhere to reveal the location of the Candida-BFP. Inset in (B): overexposure of the concanavalin A signal to show less intense, staining of the macrophage membrane (as in A). Scale bars: 5 μm and 10 μm, respectively. (C) Transmission electron micrograph of a RAW-Dectin1 cell with a partially internalized C. albicans hypha. Area of organelle clearance corresponding to the cuff structure is indicated in inset by arrows. Scale bar: 5 μm. (D) F-actin enrichment at the neck of partial phagosome. RAW-Dectin1 cells were allowed to internalize C. albicans hyphae, fixed and stained with fluorescent phalloidin (green). Actin cuff indicated with a bracket. Inset: overexposure to show the less intense cellular actin. Scale bar: 10 μm. (E–H) 3D rendering of a C. albicans hypha partially internalized by a RAW-Dectin1 cell. After incubation with Candida-BFP (white), RAW-Dectin1 cells were fixed and extracellular portions of the hyphae stained using Alexa647-conjugated concanavalin A (blue). Actin was stained with fluorescent phalloidin (red). Scale bar: 5 μm. (F) 3D rendering sliced near the middle of the tubular phagosome, (G) same as E showing only the hypha (white) and actin (red), and (H) same as E showing only the hypha (white) and concanavalin A (blue). (I) Stability of the actin cuff assessed by live cell imaging. RAW-Dectin1 cells expressing LifeAct-GFP were allowed to internalize C. albicans hyphae and imaged at defined intervals. Where indicated (105 min) 1 µM latrunculin A was added and recording continued. Actin cuff location indicated by bracket. Scale bar: 10 μm. Images are representative of ≥30 fields from ≥3 separate experiments of each type. In this and subsequent figures the outline of the phagocyte (when not readily apparent) is indicated by a dotted grey line. https://doi.org/10.7554/eLife.34798.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 3D rendering of a RAW-Dectin1 cell with a partially internalized Candida-BFP hypha (white), showing the demarcation of concanavalinA (blue) by the actin cuff (red). See Figure 1 for additional information. https://doi.org/10.7554/eLife.34798.005 Dectin1 and cadherins do not localize to the actin cuff We proceeded to probe the receptors whose signaling could potentiate the formation of the actin cuff. Because C-type lectin signaling contributes importantly to C. albicans phagocytosis (de Turris et al., 2015; Tafesse et al., 2015; Xu et al., 2009), we analyzed whether Dectin1 accumulated in the membrane at sites where cuffs were evident. Remarkably, while Dectin1 was clearly concentrated in patches elsewhere along the frustrated phagocytic cup, it was poorly detectable by immunostaining near the actin cuff (ratio cuff: cup 0.60 ± 0.04; n = 30 p<0.0001; Figure 2A and inset). The failure to detect accumulation of Dectin1 at these sites was not attributable to masking of the exofacial epitope, possibly resulting from tight apposition to the hyphae, because similar results were obtained when the receptors were tagged with emerald fluorescent protein and visualized directly in live cells (ratio cuff: cup 0.56 ± 0.04; n = 15, p<0.0001; Figure 2B and inset). Figure 2 with 1 supplement see all Download asset Open asset Assessing the contribution of Dectin1 and cadherin/catenin to the formation of the actin cuff. After incubation with Candida-BFP hyphae, RAW-Dectin1 cells were fixed and monolayers stained and visualized as follows. (A) The distribution of Dectin1-HA was detected by immunostaining (red). Actin was stained using fluorescent phalloidin (green); concanavalin A (blue). Inset: actin cuff shows little colocalization (yellow) with Dectin1-HA. (B) Visualization of Emerald-Dectin1 (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: poor colocalization of actin cuff with Emerald-Dectin1, in yellow. (C) The expression of E-cadherin (top panel) and β-catenin (bottom panel) was assessed by immunoblotting in human macrophages, A431 and RAW-Dectin1 cells; GAPDH was used as loading control. Visualization of: (D) E-cadherin-GFP or (E) β-catenin-GFP transiently transfected into RAW-Dectin1 cells. For both (D) and (E), after phagocytosis and fixation, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). Scale bars: 5 μm. (F) RAW-Dectin1 cells were allowed to internalize C. albicans-hyphae in the presence or absence of 4 mM EDTA. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was 12.7 ± 1.0. For each condition, three independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM. https://doi.org/10.7554/eLife.34798.006 Figure 2—source data 1 Numerical data corresponding to Figure 2F. https://doi.org/10.7554/eLife.34798.008 Download elife-34798-fig2-data1-v2.xlsx In epithelial and endothelial cells, host E- or N-cadherin, respectively, have been reported to contribute to C. albicans internalization (Moreno-Ruiz et al., 2009). This process involved the recruitment of α- and β-catenins and activation of the Arp2/3 pathway for actin nucleation. In agreement with these reports, we observed E-cadherin and β-catenin accumulation at sites of where C. albicans hyphae were being internalized by epithelial A431 cells, with particular accumulation at sites where actin polymerized (Figure 2—figure supplement 1). We considered whether a similar mechanism was responsible for the formation of actin cuffs by macrophages. However, neither E-cadherin nor β-catenin was detectable in RAW-Dectin1 cells or in primary human macrophages by immunoblotting (Figure 2C) or by immunofluorescence (not illustrated). Under comparable conditions, robust signals were obtained when probing A431 cells (Figure 2C). When expressed heterologously in macrophages E-cadherin-GFP was found to line the surface membrane, but was absent from the phagocytic cup (Figure 2D), while β-catenin-GFP was largely soluble and did not accumulate at the cuff (Figure 2E). Thus, E-cadherin and β-catenin are unlikely to mediate phagocytosis of C. albicans in macrophages. Nevertheless, low levels of expression of these proteins (below the level of detection of our assays) or other cadherins may have mediated the internalization. This possibility was assessed by treating the cells with EDTA, which chelates the Ca2+ known to be required for ligand binding by cadherins (reviewed in Brasch et al., 2012). As shown in Figure 2F, omission of Ca2+ had no effect on actin cuff formation in C. albicans-infected RAW-Dectin1 cells. Integrin αM β2 is involved in the formation of the actin cuff Actin can also be tethered to the phagocytic cup via integrins (Freeman et al., 2016). Integrins can be directly or indirectly involved in the phagocytosis of opsonized particles, apoptotic cells and a variety of other targets (reviewed in Dupuy and Caron, 2008) and link with actin filaments via talin and (reviewed in et al., However, activation and ligand binding (reviewed in et al., and be by their with EDTA. Moreover, actin cuffs normally in macrophages (Figure supplement with the that cuff formation was independent of activation of which (reviewed in et al., 2011). one where activation can in the absence of The of the receptor also referred to as is unique in that it a that in a et al., The is separate from the of integrins (reviewed in and, fungal et al., et al., We proceeded to whether CR3, which of αM and β2 is present in the region of the actin cuff. As in Figure both and accumulated in the region of the actin cuff in RAW-Dectin1 cells that had partially internalized C. albicans hyphae cuff: cup ± n = p<0.0001; cuff: cup ± n = p<0.0001; Figure and Moreover, and were also to the cuff (Figure and Figure supplement and inset), as was the of in (Figure and inset). is to actin and branching Figure with 1 supplement see all Download asset Open asset of is for formation of the actin cuff. After incubation with Candida-BFP hyphae, RAW-Dectin1 cells were fixed and extracellular C. albicans stained using Alexa594-conjugated concanavalin A (red). For F-actin was stained using fluorescent phalloidin and actin cuff location indicated with a or bracket. (A) immunostaining (green). Inset: of actin cuff with in yellow. Scale bar: 5 μm. (B) immunostaining (green). Inset: of actin cuff with in yellow. Scale bar: 5 μm. (C) Visualization of transfected Inset: of actin cuff with in yellow. Scale bar: 10 μm. (D) of (green). Inset: of actin cuff with in yellow. Scale bar: 10 μm. (E) of (green). Scale bar: 10 μm. of Candida-BFP hyphae was allowed to in the presence of the or an control Following phagocytosis, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). for control panel) or Scale bars: 5 μm. Images shown are representative of at least experiments of each (H) The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal Average number of C. albicans per field was ± For each condition, independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM. Figure data 1 Numerical data corresponding to Figure Download The findings support a of by the activation of CR3 directly et al., et al., or in with Dectin1 signaling et al., 2015; et al., 2011), resulting in actin nucleation. This was using the which to between its and et al., and effectively the binding of CR3 to et al., with to show accumulation of CR3 around partially internalized C. albicans hyphae, and their ability to form actin cuffs was (Figure actin cuffs were much less or when CR3 was (Figure The number of fully internalized C. albicans did not between conditions (Figure We that binding of the CR3 to C. albicans was for the of actin cuffs observed during frustrated phagocytosis of the hyphae. of receptor in actin cuff formation Dectin1 and CR3 both bind β-glucans (Brown and et al., et al., et al., and have been reported to during phagocyte to fungal et al., 2015; et al., 2011). Dectin1 has also been reported to with et al., Netea et al., Netea et al., and mannose receptors et al., et al., et al., et al., Netea et al., in the of C. albicans. We to the receptors and involved in actin cuff RAW 264.7 cells levels of Dectin1 (Brown et al., Esteban et al., 2011; et al., a means to the contribution of this receptor to actin cuff As shown in Figure RAW 264.7 cells actin cuffs to RAW-Dectin1 cells, that of the hyphae by Dectin1 was The for Dectin1 in C. albicans phagocytosis et al., 2013; et al., could be when the hyphae were receptors to the with the fungus (Figure Thus, while not in the region of the Dectin1 binding to the hyphae is by its accumulation in the frustrated phagocytic Figure 2A and is required for the subsequent activation of F-actin by Figure 4 with 1 supplement see all Download asset Open asset Assessing the contribution of C. albicans cell wall components to actin cuff (A) RAW or RAW-Dectin1 cells were with Candida-BFP hyphae that had been or Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal Average number of C. albicans per field was ± For each condition, three independent experiments were quantified, with fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM. (B) RAW-Dectin1 cells were allowed to internalize Candida-BFP hyphae in the presence or absence of or For RAW-Dectin1 cells were also allowed to C. albicans min to the addition of as as in Average number of C. albicans per field was ± of C. albicans cell wall for actin cuff were induced to form hyphae in the absence or presence of to and with RAW-Dectin1 cells. Following phagocytosis, monolayers were fixed and C. albicans stained with 10 white (white), extracellular C. albicans stained using concanavalin A (red), and actin stained with phalloidin (green). in is representative of ≥30 fields from ≥3 separate experiments of each type. Scale bar: 5 μm. (D) The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was ± For each condition, three independent experiments were quantified, with fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM. (E) of C. albicans in actin cuff The was and induced to form hyphae in the presence or absence of 5 and with RAW-Dectin1 cells for Following phagocytosis, cells were fixed and C. albicans stained with 10 white (white), extracellular C. albicans stained using fluorescent concanavalin A (red), and actin stained with fluorescent phalloidin (green). is representative of ≥30 fields from ≥3 separate Scale bar: 5 μm. (F) The effect of on actin cuff were as in (E), with of as and staining as in as in Average number of C. albicans per field was ± (G) Actin cuffs are observed during phagocytosis of hyphae. After incubation with hyphae, monolayers were fixed and stained with 10 white Actin stained with phalloidin (green). representative of ≥30 fields from separate Scale bar: 5 μm. Figure data 1 Numerical data corresponding to Figure Download Figure data 2 Numerical data corresponding to Figure Download Figure data Numerical data corresponding to Figure Download Figure data 4 Numerical data corresponding to Figure

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