Phagosome Maturation In Macrophages Research Articles

VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages.

Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Vesicle-associated membrane protein 5 (VAMP5), also a plasmalemma SNARE, interacts with SNAP23; however, its precise function in phagocytosis in macrophages remains elusive. To elucidate this aspect, we investigated the characteristics of macrophages in the presence of VAMP5 overexpression or knockdown and found that VAMP5 participates in Fcγ receptor-mediated phagosome formation, although not directly in phagosome maturation. Overexpressed VAMP5 was localized to the early phagosomal membrane but no longer localized to the lysosomal-associated membrane protein 1-positive maturing phagosomal membrane. Analyses using compound-based selective inhibitors demonstrated that VAMP5 dissociation from early phagosomes occurs in a clathrin- and dynamin-dependent manner and is indispensable for SNAP23 function in subsequent membrane fusion during phagosome maturation. Accordingly, to the best of our knowledge, we demonstrate, for the first time, that VAMP5 exerts an immunologically critical function during phagosome formation and maturation via SNARE-based membrane trafficking in macrophages.

Engulf and digest – TRPM7 as key regulator of macrophage phagosome maturation

Localized intracellular calcium fluxes are indispensable for immunologically directed Fc receptor-mediated cellular phagocytosis. A similar dependency on calcium signals has been speculated to occur in efferocytosis, the clearance of non-opsonized apoptotic cell bodies by macrophages. In a recent study published in Nature Communications, Schappe et al. describe the TRPM7 ion channel as mediator of peri‑phagosomal calcium currents, ensuring maturation of the acidifying phagosome. The authors identified a fundamental calcium signaling module provided by TRPM7, which is necessary for clearance of apoptotic cells. This finding updates our current molecular understanding of calcium dynamics, tissue maintenance and immunological clean-up.

Phagosome maturation in macrophages: Eat, digest, adapt, and repeat.

Phagocytosis is a dynamic process that requires an intricate interplay between phagocytic receptors, membrane lipids, and numerous signalling proteins and their effectors, to coordinate the engulfment of a bound particle. These particles are diverse in their physico-chemical properties such as size and shape and include bacteria, fungi, apoptotic cells, living tumour cells, and abiotic particles. Once engulfed, these particles are enclosed within a phagosome, which undergoes a striking transformation referred to as phagosome maturation, which will ultimately lead to the processing and degradation of the enclosed particulate. In this review, we focus on recent advancements in phagosome maturation in macrophages, highlighting new discoveries and emerging themes. Such advancements include identification of new GTPases and their effectors and the intricate spatio-temporal dynamics of phosphoinositides in governing phagosome maturation. We then explore phagosome fission and recycling, the emerging role of membrane contact sites, and delve into mechanisms of phagosome resolution to recycle and reform lysosomes. We further illustrate how phagosome maturation is context-dependent, subject to the type of particle, phagocytic receptors, the phagocytes and their state of activation during phagocytosis. Lastly, we discuss how phagosomes serve as signalling platforms to help phagocytes adapt to their environmental conditions. Overall, this review aims to cover recent findings, identify emerging themes, and highlight current challenges and directions to improve our understanding of phagosome maturation in macrophages.

Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense.

Regulation of cellular metabolism is now recognized as a crucial mechanism for the activation of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. Despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggest that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolysis facilitates glycolytic enzyme aldolase A mediated v-ATPase assembly and the acidification of phagosomes which is critical for lysosomal degradation. Thus, impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in murine macrophages (BMDM). Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense.

Examining the Underappreciated Role of S-Acylated Proteins as Critical Regulators of Phagocytosis and Phagosome Maturation in Macrophages.

Phagocytosis is a receptor-mediated process used by cells to engulf a wide variety of particulates, including microorganisms and apoptotic cells. Many of the proteins involved in this highly orchestrated process are post-translationally modified with lipids as a means of regulating signal transduction, membrane remodeling, phagosome maturation and other immunomodulatory functions of phagocytes. S-acylation, generally referred to as S-palmitoylation, is the post-translational attachment of fatty acids to a cysteine residue exposed topologically to the cytosol. This modification is reversible due to the intrinsically labile thioester bond between the lipid and sulfur atom of cysteine, and thus lends itself to a variety of regulatory scenarios. Here we present an overview of a growing number of S-acylated proteins known to regulate phagocytosis and phagosome biology in macrophages.

A novel stress-inducible CmtR-ESX3-Zn2+ regulatory pathway essential for survival of Mycobacterium bovis under oxidative stress

Reactive oxygen species (ROS) are an unavoidable host environmental cue for intracellular pathogens such as Mycobacterium tuberculosis and Mycobacterium bovis; however, the signaling pathway in mycobacteria for sensing and responding to environmental stress remains largely unclear. Here, we characterize a novel CmtR-Zur-ESX3-Zn2+ regulatory pathway in M. bovis that aids mycobacterial survival under oxidative stress. We demonstrate that CmtR functions as a novel redox sensor and that its expression can be significantly induced under H2O2 stress. CmtR can physically interact with the negative regulator Zur and de-represses the expression of the esx-3 operon, which leads to Zn2+ accumulation and promotion of reactive oxygen species detoxication in mycobacterial cells. Zn2+ can also act as an effector molecule of the CmtR regulator, using which the latter can de-repress its own expression for further inducing bacterial antioxidant adaptation. Consistently, CmtR can induce the expression of EsxH, a component of esx-3 operon involved in Zn2+ transportation that has been reported earlier, and inhibit phagosome maturation in macrophages. Lastly, CmtR significantly contributes to bacterial survival in macrophages and in the lungs of infected mice. Our findings reveal the existence of an antioxidant regulatory pathway in mycobacteria and provide novel information on stress-triggered gene regulation and its association with host–pathogen interaction.

Characterization of MORN2 stability and regulatory function in LC3-associated phagocytosis in macrophages.

ABSTRACTMicrotubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a type of non-canonical autophagy that regulates phagosome maturation in macrophages. However, the role and regulatory mechanism of LAP remain largely unknown. Recently, the membrane occupation and recognition nexus repeat-containing-2 (MORN2) was identified as a key component of LAP for the efficient formation of LC3-recruiting phagosomes. To characterize MORN2 and elucidate its function in LAP, we established a MORN2-overexpressing macrophage line. At a steady state, MORN2 was partially cleaved by the ubiquitin-proteasome system. MORN2 overexpression promoted not only LC3-II production but also LAP phagosome (LAPosome) acidification during Escherichia coli uptake. Furthermore, the formation of LAPosomes containing the yeast cell wall component zymosan was enhanced in MORN2-overexpressing cells and depended on reactive oxygen species (ROS). Finally, MORN2-mediated LAP was regulated by plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as SNAP-23 and syntaxin 11. Taken together, these findings demonstrate that MORN2, whose expression is downregulated via proteasomal digestion, is a limiting factor for LAP, and that membrane trafficking by SNARE proteins is involved in MORN2-mediated LAP.

S. Typhimurium Impairs Glycolysis-Mediated Acidification of Phagosomes to Evade Macrophage Defense

Regulation of the cellular metabolism is now recognized as a crucial mechanism for the homeostasis of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. However, despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggested that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolytic enzyme aldolase A is critical for v-ATPase assembly and the acidification of phagosomes upon S. Typhimurium infection, and impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in macrophages. Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense.

Effect of patchouli alcohol on macrophage mediated Helicobacter pylori digestion based on intracellular urease inhibition

BackgroundHelicobacter pylori infects almost half of the world population and is listed as a type I carcinoma factor since 1994. Pogostemon cablin (Blanco) Benth. (Labiatae) has been used to treat gastro-intestinal diseases for thousands of years in many east Asian countries, and the key ingredient, patchouli alcohol (PA), has been observed to exert anti-H. pylori and anti-urease activities. PurposeWe investigated the effect of PA on H. pylori urease and its subsequent influence on macrophage phagosome maturation and function. MethodsIn H. pylori experiment, the berthelot method and pH shock assay were adopted to evaluate the effect of PA on extracellular and intracellular H. pylori urease. And then, Q-PCR and Western blot were carried out to analyze the alterations in the expression of urease-related genes and proteins after PA treatment. In the H. pylori and macrophage cell (RAW264.7) co-culture experiment, the effects of PA on H. pylori-induced phagocytosis and intracellular killing of RAW264.7 were investigated using gentamycin protection assay, and the underlying mechanism was explored by immunofluorescence. ResultsPA at 25 and 50 μM inhibited intracellular H. pylori urease activity but not isolated urease by down-regulating the gene expression levels of ureB, ureE, ureI and nixA and reducing the protein expression level of UreB, thereby inhibiting the acid resistance of H. pylori. PA also recovered the function of macrophage bacterial digestion, and prior treatment with ammonium chloride inhibited the efficacy of PA. ConclusionPA suppressed intracellular H. pylori urease function and maturation, which increased macrophage digestion ability.

Potassium response and homeostasis in Mycobacterium tuberculosis modulates environmental adaptation and is important for host colonization.

Successful host colonization by bacteria requires sensing and response to the local ionic milieu, and coordination of responses with the maintenance of ionic homeostasis in the face of changing conditions. We previously discovered that Mycobacterium tuberculosis (Mtb) responds synergistically to chloride (Cl-) and pH, as cues to the immune status of its host. This raised the intriguing concept of abundant ions as important environmental signals, and we have now uncovered potassium (K+) as an ion that can significantly impact colonization by Mtb. The bacterium has a unique transcriptional response to changes in environmental K+ levels, with both distinct and shared regulatory mechanisms controlling Mtb response to the ionic signals of K+, Cl-, and pH. We demonstrate that intraphagosomal K+ levels increase during macrophage phagosome maturation, and find using a novel fluorescent K+-responsive reporter Mtb strain that K+ is not limiting during macrophage infection. Disruption of Mtb K+ homeostasis by deletion of the Trk K+ uptake system results in dampening of the bacterial response to pH and Cl-, and attenuation in host colonization, both in primary murine bone marrow-derived macrophages and in vivo in a murine model of Mtb infection. Our study reveals how bacterial ionic homeostasis can impact environmental ionic responses, and highlights the important role that abundant ions can play during host colonization by Mtb.

The Neuropeptides of Ocular Immune Privilege, α-MSH and NPY, Suppress Phagosome Maturation in Macrophages.

The ocular microenvironment has evolutionarily adapted several mechanisms of immunosuppression to minimize the induction of inflammation. Neuropeptides produced by the retinal pigment epithelial cells regulate macrophage activity. Two neuropeptides, α-melanocyte–stimulating hormone (α -MSH) and neuropeptide Y (NPY), are constitutively expressed by the retinal pigment epithelial cells. Together these two neuropeptides induce anti-inflammatory cytokine production in endotoxin-stimulated macrophages and suppress phagocytosis of unopsonized bioparticles. These neuropeptides do not suppress the phagocytosis of opsonized bioparticles; however, they do suppress phagolysosome activation or formation. In this report, we studied the possibility that α-MSH with NPY suppress phagosome maturation within macrophages using opsonized OVA-coated magnetic beads to isolate and analyze the phagosomes. The magnetic bead–containing intercellular vesicles were isolated and assayed for Rab5, Rab7, LAMP1, Iad, and OVA. The macrophages cotreated with α-MSH and NPY were suppressed in Rab7 recruitment to the phagosome with suppression in LAMP1 expression but not in Iad expression. The results demonstrated that the α-MSH/NPY cotreatment suppressed phagosome maturation. In addition, the a-MSH/NPY–cotreated macrophages were suppressed in their ability to Ag stimulate CD4+ T cell proliferation. These results imply a potential mechanism of ocular immune privilege to divert Ag processing to prevent autoreactive effector T cells from binding their target cognate Ag within the ocular microenvironment.

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages.

Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.

The SecA2 pathway of Mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.

SNX10 promotes phagosome maturation in macrophages and protects mice against Listeria monocytogenes infection.

Listeria monocytogenes (L. monocytogenes), which is a facultative intracellular bacterial pathogen that causes listeriosis, is widely used to study the mammalian immune response to infection. After phagocytosis by professional phagocytes, L. monocytogenes is initially contained within phagosomes, which mature into phagolysosomes, where the bacteria are degraded. Although phagocytosis and subsequent phagosome maturation is essential for the clearance of infectious microbial pathogens, the underlying regulatory mechanisms are still unclear. SNX10 (Sorting nexin 10) has the simplest structure of the SNX family and has been reported to regulate endosomal morphology, which might be crucial for macrophage function, including phagocytosis and digestion of pathogens, inflammatory response, and antigen presentation. Our results showed that SNX10 expression was upregulated following L. monocytogenes infection in macrophages. It was also revealed that SNX10 promoted phagosome maturation by recruiting the Mon1-Ccz1 complex to endosomes and phagosomes. As a result, SNX10 deficiency decreased the bacterial killing ability of macrophages, and SNX10-deficient mice showed increased susceptibility to L. monocytogenes infection in vivo. Thus, this study revealed an essential role of SNX10 in controlling bacterial infection.

Quantitative analysis of phagosome formation and maturation using an Escherichia coli probe expressing a tandem fluorescent protein.

Phagosome formation and maturation are essential innate immune mechanisms to engulf and digest foreign particles. To analyze these processes quantitatively, we established a specific Escherichia coli probe expressing a tandem fluorescent protein, comprising glutathione S-transferase fused with monomeric Cherry (mCherry) and monomeric Venus (mVenus). We demonstrated that mVenus was more susceptible to bleaching in an acidic environment than mCherry, and that the mVenus:mCherry fluorescence intensity ratio can be used to monitor phagosomal pH changes during maturation. Using this probe, we revealed that synaptosomal-associated protein of 23 kDa, a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, actively regulated phagocytosis of E. coli and subsequent phagosome maturation in macrophages. Our results indicated that this probe has the potential to be a powerful tool in understanding the molecular mechanisms of phagosome formation and maturation.

Species dependent impact of helminth-derived antigens on human macrophages infected with Mycobacterium tuberculosis: Direct effect on the innate anti-mycobacterial response.

BackgroundIn countries with a high prevalence of tuberculosis there is high coincident of helminth infections that might worsen disease outcome. While Mycobacterium tuberculosis (Mtb) gives rise to a pro-inflammatory Th1 response, a Th2 response is typical of helminth infections. A strong Th2 response has been associated with decreased protection against tuberculosis.Principal findingsWe investigated the direct effect of helminth-derived antigens on human macrophages, hypothesizing that helminths would render macrophages less capable of controlling Mtb. Measuring cytokine output, macrophage surface markers with flow cytometry, and assessing bacterial replication and phagosomal maturation revealed that antigens from different species of helminth directly affect macrophage responses to Mtb. Antigens from the tapeworm Hymenolepis diminuta and the nematode Trichuris muris caused an anti-inflammatory response with M2-type polarization, reduced macrophage phagosome maturation and ability to activate T cells, along with increased Mtb burden, especially in T. muris exposed cells which also induced the highest IL-10 production upon co-infection. However, antigens from the trematode Schistosoma mansoni had the opposite effect causing a decrease in IL-10 production, M1-type polarization and increased control of Mtb.ConclusionWe conclude that, independent of any adaptive immune response, infection with helminth parasites, in a species-specific manner can influence the outcome of tuberculosis by either enhancing or diminishing the bactericidal function of macrophages.

Quantitative Immunofluorescence to Study Phagosome Maturation.

Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes, and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation. These changes can be assessed at the single-phagosome level by using immunofluorescence methods to study phagosome maturation. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Most commonly, phagosome maturation in macrophages can be determined by staining the cells for Lysosomal-Associated Membrane Protein I (LAMPI) and measuring the fluorescence intensity of LAMPI around each phagosome by microscopy or flow cytometry.

Topical Simvastatin as Host-Directed Therapy against Severity of Cutaneous Leishmaniasis in Mice.

We recently demonstrated that statins mediate protection against intracellular pathogens, Mycobacterium tuberculosis and Listeria monocytogenes in mice. Here, we investigated the immunomodulatory potential of simvastatin as a topical or systemic host-directed drug therapy in controlling inflammatory responses in an experimental mouse model of cutaneous leishmaniasis caused by Leishmania major (LV39). In an ear infection model, topical application of simvastatin directly on established lesions significantly reduced severity of the disease reflected by ear lesion size and ulceration. The host protective effect was further accompanied by decreased parasite burden in the ear and draining lymph nodes in both BALB/c and C57BL/6 mice. Pre-treatment of these mice on a low-fat cholesterol diet and systemic simvastatin also reduced footpad swelling, as well as parasite burdens and ulceration/necrosis in the more robust footpad infection model, demonstrating the prophylactic potential of simvastatin for cutaneous leishmaniasis. Mechanistically, following L. major infection, simvastatin-treated primary macrophages responded with significantly reduced cholesterol levels and increased production of hydrogen peroxide. Furthermore, simvastatin-treated macrophages displayed enhanced phagosome maturation, as revealed by increased LAMP-3 expression in fluorescent microscopy and Western blot analysis. These findings demonstrate that simvastatin treatment enhances host protection against L. major by increasing macrophage phagosome maturation and killing effector functions.

Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis.

More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis.

Novel cell-based in vitro screen to identify small-molecule inhibitors against intracellular replication of Cryptococcus neoformans in macrophages

The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of C. neoformans. The Prestwick Chemical Library® of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca2+ channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease.