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The fungusCryptococcus neoformansis a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress abrogated Dragotcytosis frequency, that ROS susceptible mutants ofC. neoformansunderwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH and oxidative stresses and correlated with the macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochasticC. neoformansnon-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.

Phagocytosis is the mechanism of the internalization of large particles, microorganisms and cellular debris. The complement pathway represents one of the first mechanisms of defense against infection and the complement receptor 3 (CR3), which is highly expressed on macrophages, is a major receptor for many pathogens and debris. Key to dissecting the mechanisms by which CR3-mediated phagocytosis occurs, is understanding how the complex actin binding protein machinery and associated regulators interact with actin during phagocytosis, from triggering of receptor, through to phagosome formation and closure. However, how CR3-mediated phagosome completion and closure are orchestrated is not known. Here, we reveal that dynamin-2 is recruited concomitantly with polymerised actin at the site of the nascent phagosomes and accumulates until membrane scission. Inhibition of dynamin activity leads to stalled phagocytic cups and a decrease in the amount of F-actin at the site of phagocytosis. Acute inhibition of dynamin activity in living phagocytosing cells established that dynamin-2 plays a critical role in the effective scission of the CR3-phagosome from the plasma membrane. Thus, dynamin-2 has two distinct roles in CR3-mediated phagocytosis, in the assembly of the F-actin phagocytic cup and during phagosome scission.

Phosphatase and Tensin Homolog on chromosome Ten (PTEN) has emerged as a key protein that governs the response to kidney injury. Notably, renal adaptive repair is important for preventing acute kidney injury (AKI) to chronic kidney disease (CKD) transition. To test the role of PTEN in renal repair after acute injury, we constructed a mouse model that overexpresses PTEN in renal proximal tubular cells (RPTC) by crossing PTENfl-stop-fl mice with Ggt1-Cre mice. Mass spectrometry-based proteomics was performed after subjecting these mice to ischemia/reperfusion (I/R). We found that PTEN was downregulated in renal tubular cells in mice and cultured HK-2 cells subjected to renal maladaptive repair induced by I/R. Renal expression of PTEN negatively correlated with NGAL and fibrotic markers. RPTC-specific PTEN overexpression relieved I/R-induced maladaptive repair, as indicated by alleviative tubular cell damage, apoptosis, and subsequent renal fibrosis. Mass spectrometry analysis revealed that differentially expressed proteins in RPTC-specific PTEN overexpression mice subjected to I/R were significantly enriched in phagosome, PI3K/Akt, and HIF-1 signaling pathway and found significant upregulation of CHMP2A, an autophagy-related protein. PTEN deficiency downregulated CHMP2A and inhibited phagosome closure and autolysosome formation, which aggravated cell injury and apoptosis after I/R. PTEN overexpression had the opposite effect. Notably, the beneficial effect of PTEN overexpression on autophagy flux and cell damage was abolished when CHMP2A was silenced. Collectively, our study suggests that PTEN relieved renal maladaptive repair in terms of cell damage, apoptosis, and renal fibrosis by upregulating CHMP2A-mediated phagosome closure, suggesting that PTEN/CHMP2A may serve as a novel therapeutic target for the AKI to CKD transition.

Legionella pneumophila is the causative agent of Legionnaires’ disease and is capable of replicating inside phagocytic cells, such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication-permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, and RavY does not contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild-type L. pneumophila. Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

For many intracellular pathogens, the phagosome is the site of events and interactions that shape infection outcome. Phagosomal membrane damage, in particular, is proposed to benefit invading pathogens. To define the innate immune consequences of this damage, we profiled macrophage transcriptional responses to wild-typeMycobacterium tuberculosis(Mtb) and mutants that fail to damage the phagosomal membrane. We identified a set of genes with enhanced expression in response to the mutants. These genes represented a late component of the TLR2-dependent transcriptional response to Mtb, distinct from an earlier component that includedTnf. Expression of the later component was inherent to TLR2 activation, dependent upon endosomal uptake, and enhanced by phagosome acidification. Canonical Mtb virulence factors that contribute to phagosomal membrane damage blunted phagosome acidification and undermined the endosome-specific response. Profiling cell survival and bacterial growth in macrophages demonstrated that the attenuation of these mutants is partially dependent upon TLR2. Further, TLR2 contributed to the attenuated phenotype of one of these mutants in a murine model of infection. These results demonstrate two distinct components of the TLR2 response and identify a component dependent upon endosomal uptake as a point where pathogenic bacteria interfere with the generation of effective inflammation. This interference promotes tuberculosis (TB) pathogenesis in both macrophage and murine infection models.

Protein kinase C (PKC)-ε is required for membrane addition during IgG-mediated phagocytosis, but its role in this process is ill defined. Here, we performed high-resolution imaging, which reveals that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-ε colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles were not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implies that Golgi-tethered PKC-ε is a driver of Golgi-to-phagosome trafficking. Finally, we established that the regulatory domain of PKC-ε is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis – its regulatory domain drives Golgi-derived vesicles to the phagosome, whereas catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain.This article has an associated First Person interview with the first author of the paper.

Background: The oleaginous microalga Graesiella emersonii, a potential industrial strain for lipid production, is frequently infected by the endoparasite Amoeboaphelidium protococcarum. It is essential to investigate the microalgae–endoparasite interaction to prevent and control microalgal diseases. However, the underlying mechanisms of microalgal host-pathogen interactions remain largely unknown. In this study, we applied physiological and simultaneous dual transcriptomic analysis to characterize the G. emersonii–A. protococcarum interaction for the first time. Results: Three infection stages were determined according to infection rate and physiological features. Dual RNA-seq results showed that the genes expression of G. emersonii and A. protococcarum were strongly dynamically regulated during the infection. For microalgal hosts, the expression of defense genes involved in the pattern recognition receptors, large heat shock proteins, and reactive oxygen scavenging enzymes (glutathione, ferritin, and catalase) were significantly upregulated during infection. However, some genes encoding resistance proteins (R proteins) with a leucine-rich repeat domain exhibited no significant changes during infection. Furthermore, ubiquitin-mediated proteolysis and endocytosis were strongly affected by A. protococcarum infection. For endoparasite A. protococcarum, genes for carbohydrate-active enzymes, pathogen-host interactions, and putative effectors were significantly upregulated during infection. Furthermore, the genes in cluster II were significantly enriched in pathways associated with the modulation of vacuole transport, including endocytosis, phagosome, ubiquitin-mediated proteolysis, and SNARE interactions in vesicular transport pathways.Conclusions: Our data provide a new promising basis for complete understanding of the algal host defense strategies and parasite pathogenicity. This is beneficial for the screening of resistant microalgal strains and developing control strategies for microalgal diseases.

The membranes of endosomes, phagosomes and macropinosomes can become damaged by the physical properties of internalized cargo, by active pathogenic invasion or by cellular processes, including endocytic maturation. Loss of membrane integrity is often deleterious and is, therefore, prevented by mitigation and repair mechanisms. However, it can occasionally be beneficial and actively induced by cells. Here, we summarize the mechanisms by which cells, in particular phagocytes, try to prevent membrane damage and how, when this fails, they repair or destroy damaged endocytic organelles. We also detail how one type of phagocyte, the dendritic cell, can deliberately trigger localized damage to endocytic organelles to allow for major histocompatibility complex class I presentation of exogenous antigens and initiation of CD8 + T-cell responses to viruses and tumours. Our review highlights mechanisms for the regulation of endocytic organelle membrane integrity at the intersection of cell biology and immunology that could be co-opted for improving vaccination and intracellular drug delivery.

Essential peptidoglycan synthases, like penicillin binding proteins 2 and 3 (PBP2/PBP3) of Escherichia coli, define shape by orchestrating cell elongation and division, respectively. Despite being intensively studied as drug targets, the regulatory rules governing their production remain poorly understood. During infection, the closely related pathogen Salmonella enterica serovar Typhimurium downregulates PBP2/PBP3 production and replace them with alternative peptidoglycan synthases, PBP2SAL/PBP3SAL, absent in E. coli. The bases for such switch in morphogenetic proteins are unknown. Here, we show that the S. Typhimurium regulator OmpR triggers PBP2SAL and PBP3SAL expression responding solely to acid pH and define a shared motif present in upstream regions of the PBP2SAL- and PBP3SAL-coding genes sufficient for such control. The elimination of PBP2/PBP3 in infection conditions is however multifactorial, requiring acidity, high osmolarity and being favoured by OmpR and the Prc protease. Remarkably, we found that E. coli loses the essential PBP3 required for cell division when exposed to both acidity and high osmolarity, the environmental cues encountered by intracellular S. Typhimurium. Therefore, OmpR played a central role in the evolution of this pathogen when co-opting the regulation of PBP2SAL/PBP3SAL and, consequently, promoting a new morphogenetic cycle that made possible increasing progeny inside acidic eukaryotic phagosomes.SignificanceSome enzymes that participate in peptidoglycan metabolism are present exclusively in bacterial pathogens and modify its structure to limit immune recognition. The intracellular pathogen Salmonella enterica serovar Typhimurium is the only example known to date in which a “substitution” of essential peptidoglycan enzymes involved in cell division and elongation takes place during infection. The data presented here support instability of PBP3 in environments with acidity and high osmolarity as a probable selective pressure that promoted the fixation of alternative morphogenetic enzymes. This was possible due to the control that OmpR exerted over these new foreign functions. The acquisition of enzymes like PBP2SAL and PBP3SAL therefore represent a “quantum leap” evolutionary event in S. Typhimurium that made possible the colonization of acidic intracellular niches.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a major cause of morbidity and mortality worldwide. Increasing lines of evidence indicate that certain individuals, which are termed resisters, are naturally resistant to TB infection. The resister phenotype has been linked to host efficient innate immune responses, but the underlying mechanisms and the key immune factors remain unclear. Here, we find that upon Mtb infection, monocyte-derived macrophages (MDMs) from TB resisters exhibited distinctly higher production of TNF-α, IL-1β and IL-6, higher ratio of bacteria in acidic vacuoles, and lower intracellular bacterial loads, as compared to that from the healthy controls, individuals with latent TB infection, and TB patients. Such enhanced anti-Mtb immune capacity of macrophages from resisters largely depends on histone deacetylase 6 (HDAC6), whose expression is specifically maintained in MDMs from TB resisters during Mtb infection. Furthermore, we demonstrate that HDAC6 is required for acidification of Mtb-containing phagosomes in macrophages, thus controlling the intracellular survival of Mtb. Taken together, these findings unravel an indispensable role of HDAC6 in human innate resistance against Mtb infection, suggesting that HDAC6 may serve as a marker for individual TB risk as well as a novel host-directed anti-TB therapeutic target.

The metazoan innate immune system senses bacterial infections by detecting highly conserved bacterial molecules, termed pathogen-associated molecular patterns (PAMPs). PAMPs are detected by a variety of host pattern recognition receptors (PRRs), whose function is to coordinate downstream immune responses. PRR activities are, in part, regulated by their subcellular localizations. Accordingly, professional phagocytes can detect extracellular bacteria and their PAMPs via plasma membrane-oriented PRRs. Conversely, phagocytosed bacteria and their PAMPs are detected by transmembrane PRRs oriented towards the phagosomal lumen. Even though PAMPs are unable to passively diffuse across membranes, phagocytosed bacteria are also detected by PRRs localized within the host cell cytosol. This phenomenon is explained upon phagocytosis of bacteria that specialize in phagosomal escape and cytosolic residence. Contrary to this cytosolic lifestyle, most bacteria studied to date spend their entire intracellular lifestyle contained within phagosomes, yet they also stimulate cytosolic PRRs. Herein, we will review our current understanding for how phagosomal PAMPs become accessible to cytosolic PRRs, as well as highlight knowledge gaps that should inspire future investigations.

We describe the utility of a tandem-tagged autophagy reporter mouse model (CAG-RFP-EGFP-MAP1LC3B) in investigating basal macroautophagic/autophagic flux in the neural retina. Western blot, in situ hybridization, immunohistochemistry, and confocal microscopy showed that CAG promoter-driven expression of RFP-EGFP-MAP1LC3B increased "cytosolic" RFP-EGFP-LC3B-I levels, whereas RFP-EGFP-LC3B-II decorates true phagosomes. We verified that the electroretinographic (ERG) responses of tandem-tagged LC3B mice were comparable to those of age-matched controls. Optimized microscope settings detected lipofuscin autofluorescence in retinas of abca4-/- mice. The majority of retinal phagosomes in the reporter mice exhibited only RFP (not EGFP) fluorescence, suggesting rapid maturation of phagosomes. Only ~1.5% of the total phagosome population was EGFP-labeled; RFP-labeled (mature) phagosomes colocalized with lysosomal markers LAMP2 and CTSD. In the outer retina, phagosome sizes were as follows (in µm2, ave ± SEM): RPE, 0.309 ± 0.015; photoreceptor inner segment-myoid, 0.544 ± 0.031; and outer nuclear layer, 0.429 ± 0.011. Detection of RPE phagosomes by fluorescence microscopy is challenging, due to the presence of melanin. Increased lipofuscin autofluorescence, such as observed in the abca4-/- mouse model of Stargardt disease, is a strong confounding factor when attempting to study autophagy in the RPE. In addition to RPE and photoreceptor cells, phagosomes were detected in inner retinal cell types, microglia, astrocytes, and endothelial cells. We conclude that the tandem-tagged LC3B mouse model serves as a useful system for studying autophagy in the retina. This utility, however, is dependent upon several technical and biological factors, including microscope settings, transgene expression, choice of fluorophores, and lipofuscin autofluorescence.Abbreviations: ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATG: autophagy related; CTSD: cathepsin D; DAPI: (4',6-diamido-2-phenylindole); DIC: differential interference contrast; EGFP: enhanced green fluorescent protein; ELM: external limiting membrane; ERG: electroretinography; GCL: ganglion cell layer; GLUL: glutamine-ammonia ligase (glutamine synthetase); INL: inner nuclear layer; IS-E/M: inner segment - ellipsoid/myoid; ISH: in situ hybridization; LAMP2: lysosomal-associated membrane protein 2; L.I.: laser Intensity; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; O.C.T.: optimal cutting temperature; OS: outer segment; ONL: outer nuclear layer; PE: phosphatidylethanolamine; RFP: red fluorescent protein; R.O.I.: region of interest; RPE: retinal pigment epithelium.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here, we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella enterica serovar Typhimurium. The 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 h and 18 h postinfection and observed widespread changes in metal transport, metal-dependent genes, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 45-fold at 12 h postinfection. Further, manipulation of zinc in the medium alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing the transcriptomic changes to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response. Our results reveal that 129S6 macrophages represent a distinct model system compared to C57BL/6 macrophages. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. The 129S6 macrophages leverage intricate means of manipulating zinc availability and distribution to limit the pathogen’s access to zinc, while simultaneously ensuring sufficient zinc to support the immune response.

Phagocytosis is an important physiological process, which, in higher organisms, is a means of fighting infections and clearing cellular debris. During phagocytosis, detrimental foreign particles (e.g. pathogens and apoptotic cells) are engulfed by phagocytes (e.g. macrophages), enclosed in membrane-bound vesicles called phagosomes, and transported to the lysosome for eventual detoxification. During this well-choreographed process, the nascent phagosome (also called early phagosome, EP) undergoes a series of spatiotemporally regulated changes in its protein and lipid composition and matures into a late phagosome (LP), which subsequently fuses with the lysosomal membrane to form the phagolysosome. While several elegant proteomic studies have identified the role of unique proteins during phagosomal maturation, the corresponding lipidomic studies are sparse. Recently, we reported a comparative lipidomic analysis between EPs and LPs and showed that ceramides are enriched on the LPs. Further, we found that this ceramide accumulation on LPs was orchestrated by ceramide synthase 2, inhibition of which hampers phagosomal maturation. Following up on this study, here, using biochemical assays, we first show that the increased ceramidase activity on EPs also significantly contributes to the accumulation of ceramides on LPs. Next, leveraging lipidomics, we show that de novo ceramide synthesis does not significantly contribute to the ceramide accumulation on LPs, while concomitant to increased ceramides, glucosylceramides are substantially elevated on LPs. We validate this interesting finding using biochemical assays and show that LPs indeed have heightened glucosylceramide synthase activity. Taken together, our studies provide interesting insights and possible new roles of sphingolipid metabolism during phagosomal maturation.

Mycobacterium tuberculosis (Mtb), the main etiology of tuberculosis (TB), is predominantly an intracellular pathogen that has caused infection, disease and death in humans for centuries. Lipid droplets (LDs) are dynamic intracellular organelles that are found across the evolutionary tree of life. This review is an evaluation of the current state of knowledge regarding Mtb-LD formation and associated Mtb transcriptome directly from sputa.Based on the LD content, Mtb in sputum may be classified into three groups: LD positive, LD negative and LD borderline. However, the clinical and evolutionary importance of each state is not well elaborated. Mounting evidence supports the view that the presence of LD positive Mtb bacilli in sputum is a biomarker of slow growth, low energy state, towards lipid degradation, and drug tolerance. In Mtb, LD may serve as a source of chemical energy, scavenger of toxic compounds, prevent destruction of Mtb through autophagy, delay trafficking of lysosomes towards the phagosome, and contribute to Mtb persistence. It is suggest that LD is a key player in the induction of a spectrum of phenotypic and metabolic states of Mtb in the macrophage, granuloma and extracellular sputum microenvironment. Tuberculosis patients with high proportion of LD positive Mtb in pretreatment sputum was associated with higher rate of poor treatment outcome, indicating that LD may have a clinical application in predicting treatment outcome.The propensity for LD formation among Mtb lineages is largely unknown. The role of LD on Mtb transmission and disease phenotype (pulmonary TB vs extra-pulmonary TB) is not well understood. Thus, further studies are needed to understand the relationships between LD positivity and Mtb lineage, Mtb transmission and clinical types.

Staphylococcus aureus is an important human pathogen that can cause a variety of diseases ranging from mild superficial skin infections to life-threatening conditions like necrotizing pneumonia, endocarditis, and septicemia. Polymorphonuclear leukocytes (PMNs; neutrophils in particular herein) are essential for host defense against S. aureus infections, and the microbe is phagocytosed readily. Most ingested bacteria are killed, but some S. aureus strains—such as the epidemic USA300 strain—have an enhanced ability to cause PMN lysis after phagocytosis. Although progress has been made, the mechanism for lysis after phagocytosis of S. aureus remains incompletely determined. Here, we tested the hypothesis that disruption of phagosome integrity and escape of S. aureus from the PMN phagosome into the cytoplasm precedes PMN lysis. We used USA300 wild-type and isogenic deletion strains to evaluate and/or verify the role of selected S. aureus molecules in this cytolytic process. Compared to the wild-type USA300 strain, Δagr, Δhla, ΔlukGH, and Δpsm strains each caused significantly less lysis of human PMNs 3 h and/or 6 h after phagocytosis, consistent with previous studies. Most notably, confocal microscopy coupled with selective permeabilization assays demonstrated that phagosome membrane integrity is largely maintained prior to PMN lysis after S. aureus phagocytosis. We conclude that PMN lysis does not require escape of S. aureus from the phagosome to the cytoplasm and that these are independent phenomena. The findings are consistent with the ability of S. aureus (via selected molecules) to trigger lysis of human PMNs by an undetermined signaling mechanism. IMPORTANCE S. aureus strain USA300 has the ability to cause rapid lysis of human neutrophils after phagocytosis. Although this phenomenon likely contributes to the success of USA300 as a human pathogen, our knowledge of the mechanism remains incomplete. Here, we used a selective permeabilization assay coupled with confocal microscopy to demonstrate that USA300 is contained within human neutrophil phagosomes until the point of host cell lysis. Thus, consistent with a process in macrophages, S. aureus fails to escape into the neutrophil cytoplasm prior to cytolysis.

Significance Lysosomes are organelles that also act as cell-signaling hubs. They regulate functions ranging from antigen presentation to autophagy. Spherical lysosomes can spontaneously elongate into tubules in starving or inflamed immune cells. We describe a DNA-based reagent, denoted Tudor, that tubulates lysosomes in macrophages without triggering either an immune response or autophagy. Chemical imaging revealed that tubular lysosomes differ from vesicular ones in terms of their pH, calcium, and proteolytic activity. Tudor revealed a role for tubular lysosomes in that they enhance MMP9 secretion and phagocytosis in resting macrophages. The ability to tubulate lysosomes in resting immune cells without starving or inflaming them may help reveal new insights into how tubular lysosomes function., Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

The endosomal sorting complex required for transport (ESCRT) is formed by ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins. It conducts vesicular trafficking in eukaryotes through the formation of vesicles and membrane fission and fusion events. The trophozoites of Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents an active membrane movement in basal state that increases during phagocytosis and tissue invasion. ESCRT-III complex has a pivotal role during these events, but ESCRT-0, ESCRT-I and ESCRT-II have been poorly studied. Here, we unveiled the E. histolytica ESCRT-I complex and its implication in vesicular trafficking and phagocytosis, as well as the molecular relationships with other phagocytosis-involved molecules. We found a gene encoding for a putative EhVps23 protein with the ubiquitin-binding and Vps23 core domains. In basal state, it was in the plasma membrane, cytoplasmic vesicles and multivesicular bodies, whereas during phagocytosis it was extensively ubiquitinated and detected in phagosomes and connected vesicles. Docking analysis, immunoprecipitation assays and microscopy studies evidenced its interaction with EhUbiquitin, EhADH, EhVps32 proteins, and the lysobisphosphatidic acid phospholipid. The knocking down of the Ehvps23 gene resulted in lower rates of phagocytosis. Our results disclosed the concert of finely regulated molecules and vesicular structures participating in vesicular trafficking-related events with a pivotal role of EhVps23.

Cystic fibrosis is a hereditary disease that mainly affects secretory organs in humans. It is caused by mutations in the gene encoding CFTR with the most common phenylalanine deletion at position 508. CFTR is an anion channel mainly conducting Cl− across the apical membranes of many different epithelial cells, the impairment of which causes dysregulation of epithelial fluid secretion and thickening of the mucus. This, in turn, leads to the dysfunction of organs such as the lungs, pancreas, kidney and liver. The CFTR protein is mainly localized in the plasma membrane; however, there is a growing body of evidence that it is also present in the intracellular organelles such as the endosomes, lysosomes, phagosomes and mitochondria. Dysfunction of the CFTR protein affects not only the ion transport across the epithelial tissues, but also has an impact on the proper functioning of the intracellular compartments. The review aims to provide a summary of the present state of knowledge regarding CFTR localization and function in intracellular compartments, the physiological role of this localization and the consequences of protein dysfunction at cellular, epithelial and organ levels. An in-depth understanding of intracellular processes involved in CFTR impairment may reveal novel opportunities in pharmacological agents of cystic fibrosis.

The type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels, but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. These data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.

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.

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the antibody-binding fragment (Fab) region of the antibody, the induction of immune-mediated effector functions through the fragment crystallizable (Fc) region is a vital aspect of antibody therapeutics targeting tumor cells. Cross-linking of the Fc gamma receptors (FcγRs) via cell-bound antibodies activate immune effector cells, leading to antibody-dependent cellular cytotoxicity via natural killer (NK) cells. Linking of FcγRs on macrophages triggers the process of antibody-dependent cellular phagocytosis (ADCP), where antibody-opsonized target cells are internalized in phagosomes and degraded through the process of phagosome maturation and acidification. ADCP activity can be challenging to measure accurately due to the difficulty in differentiating target cells that are bound to a macrophage versus those that are internalized within phagosomes. In this chapter, we describe a protocol that measures ADCP activity by labeling target cells with a pH-sensitive dye that fluoresces brightly in mature phagosomes. The ADCP activity of therapeutics is then measured via flow cytometry. This assay is capable of detecting glycosylation differences arising from manufacturing processes and is suitable for evaluation of ADCP activity of monoclonal antibodies (mAb) to support in vitro biological characterization of drug candidates and lead candidate selection for desirable effector functions.

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. Generally, in microbe-challenged macrophages, phagosome-lysosome fusion occurs four to six hours post phagocytic uptake of the microbe. However, live pathogenic mycobacteria can retard its lysosomal delivery up to twenty hours post phagocytic uptake wherein it mitigates the macrophage stress and upregulates proteins for acid tolerance inside lysosomes. The exact sequence of events through which, mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin 1, is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria infected macrophages exhibit an increased level of cAMP and based on receptor stimulus Coronin 1 expressing cells exhibit a higher level of cAMP than non-Coronin 1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Coronin 1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin 1 to depolymerize F actin into G actin at the vicinity of the mycobacteria containing phagosome. Owing to reduced F actin levels the movement of the phagosome towards the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Coronin 1 mediated up-regulation of Cofilin also contributes towards the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Taken together our study provides firsthand information on Coronin1 mediated retardation of phagosome maturation which therefore can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1- and lpg5A-/lpg5B- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania., Author summary Leishmaniasis, the disease caused by parasites of the genus Leishmania, can be divided into cutaneous, muco-cutaneous and visceral leishmaniasis depending on the parasite species and the clinical outcome of the disease. Of the three, cutaneous leishmaniasis is the most common form, which is usually characterized by a localized lesion due to the infection of immune cells, primarily dermal and lymph node-resident macrophages. The time between infection and lesion appearance ranges from weeks to years, while some individuals never develop lesions. The length of this subclinical stage of leishmaniasis depends on a variety of factors: parasite virulence, infectious dose, and the host immune response. Therefore, it remains crucial to develop our understanding of each component of the host-parasite interface and assess the role that each component plays in the clinical outcome. Here, we analyze the interaction between L. major, a cutaneous strain, and the host innate immune factor Trypanosome Lytic Factor (TLF), a sub-class of circulating High-Density Lipoprotein (HDL). TLF provides sterile immunity to most extracellular African Trypanosomes by osmotically lysing the parasites. Lysis is driven by the primate specific protein apolipoprotein L-1 (APOL1), a cation channel-forming protein that is activated by a series of pH-dependent conformational changes. APOL1 inserts into cellular membranes at acidic pH and forms a closed ion channel that subsequently opens when re-exposed to neutral pH, resulting in ion flux. Using transgenic mice producing primate TLF, we show that both human and baboon TLFs ameliorate cutaneous Leishmania major infection and that this reduction in parasite burden correlates with: 1. infectious dose of metacyclic promastigotes 2. the concentration of circulating TLF in plasma and 3. early recruitment of neutrophils at the site of infection. Our results show that the acidification step is essential for TLF-mediated lysis of axenic metacyclic promastigotes of Leishmania in vitro. The susceptibility of metacyclic promastigotes to TLF-mediated lysis is governed by the surface glycoconjugates of Leishmania. We find that surface glycoconjugate-deficient Leishmania are resistant to TLF-mediated killing. Based on these data, we conclude that the shedding of surface glycoconjugates while transitioning from metacyclic promastigotes to amastigotes, results in parasite resistance to TLF-mediated lysis. Whether TLF is effective at killing metacyclic promastigotes of other experimentally tractable Leishmania sp., such as L. infantum and L. donovani, which have different surface glycoconjugate structures is yet to be tested. Our data raise the possibility that TLF may have lytic activity against a broader range of pathogens such as bacteria, viruses, fungi and parasites with surface glycoconjugates that transit through intracellular acidic compartments.

Professional phagocytes represent a critical node in innate immunity and tissue homeostasis through their specialized ability to eat, drink, and digest material from the extracellular milieu. The degradative and microbicidal functions of phagocytes rely on the fusion of lysosomes with endosomal compartments such as phagosomes, resulting in the digestion and recycling of internalized prey and debris. Despite these efforts, several particularly dangerous infections result from a class of tenacious pathogens that resist digestion, often surviving and even proliferating within the confines of the phagosomal membrane. One such example, Candida albicans, is a commensal polymorphic fungus that colonizes ~50% of the population and can cause life-threatening infections in immunocompromised patients. Not only can C. albicans survive within phagosomes, but its ingestion by macropahges triggers a yeast-to-hyphal transition promoting rapid intraphagosomal growth (several microns per hour) while imposing a substantial mechanical burden on the phagosomal membrane surrounding the fungus. Preservation of membrane integrity is essential to maintain the hostile internal environment of the phagosome, a functionality of degradative enzymes and oxidative stress. Yet, biological membranes such as phagosomes have a limited capacity to stretch. Using C. albicans as a model intracellular pathogen, our recent work reveals a mechanism by which phagosomes respond to intraphagosomal growth of pathogens by expanding their surface area, and as a result, maintain the integrity of the phagosomal membrane. We hypothesized that this expansion would be facilitated by the delivery and fusion of membrane from extraneous sources with the phagosome. Consistently, macrophages respond to the yeast-to-hyphal transition through a stretch-induced release of phagosomal calcium, leading to recruitment and insertion of lysosomes that accommodate the expansion of the phagolysosome and preserve its integrity. Below, we discuss this calcium-dependent mechanism of lysosome insertion as a means of avoiding phagosomal rupture. Further, we examine the implications of membrane integrity on the delicate balance between the host and pathogen by focusing on fungal stress responses, nutrient acquisition, inflammasome activation, and cell death.

Mycobacterium tuberculosis can invade different cells with distinct persistence fates because cells are equipped with different host restriction factors. However, the underlying mechanisms remain elusive. Here, we infected THP1 and Raw264.7 macrophages cell lines, A549 epithelial cell line, and hBMEC and bEnd.3 endothelial cell lines with M. tuberculosis and demonstrated that M. tuberculosis significantly inhibited lysosome acidification in THP1, hBMEC, A549, and Raw264.7 cells, while, in bEnd.3 cells, M. tuberculosis was mainly delivered into acidified phagolysosomes and auto-lysosomes. The systematic gene profile analysis of different cells and intracellular M. tuberculosis showed that the phagosome autophagy-pathway-related genes itgb3 and atg3 were highly expressed in bEnd.3 cells. Knockdown of these genes significantly increased the number of viable intracellular M. tuberculosis bacilli by altering phagosomal trafficking in bEnd.3 cells. Treatment with itgb3 agonist significantly decreased M. tuberculosis survival in vivo. These findings could facilitate the identification of anti-M. tuberculosis host genes and guide M. tuberculosis-resistant livestock breeding. IMPORTANCE As an intracellular pathogen, Mycobacterium tuberculosis could avoid host cell immune clearance using multiple strategies for its long-term survival. Understanding these processes could facilitate the development of new approaches to restrict intracellular M. tuberculosis survival. Here, we characterized the detailed molecular events occurring during intracellular trafficking of M. tuberculosis in macrophage, epithelial, and endothelial cell lines and found that ITGB3 facilitates M. tuberculosis clearance in endothelial cells through altering phagosomal trafficking. Meanwhile, the treatment with ITGB3 agonist could reduce bacterial load in vivo. Our results identified new anti-M. tuberculosis restriction factors and illuminated a new anti-M. tuberculosis defense mechanism.

Leprosy reactional episodes are acute inflammatory events that may occur during the clinical course of the disease. Type 1 reaction (T1R) is associated with an increase in neural damage, and the understanding of the molecular pathways related to T1R onset is pivotal for the development of strategies that may effectively control the reaction. Interferon-gamma (IFN-γ) is a key cytokine associated with T1R onset and is also associated with autophagy induction. Here, we evaluated the modulation of the autophagy pathway in Mycobacterium leprae-stimulated cells in the presence or absence of IFN-γ. We observed that IFN-γ treatment promoted autophagy activation and increased the expression of genes related to the formation of phagosomes, autophagy regulation and function, or lysosomal pathways in M. leprae-stimulated cells. IFN-γ increased interleukin (IL)-15 secretion in M. leprae-stimulated THP-1 cells in a process associated with autophagy activation. We also observed higher IL15 gene expression in multibacillary (MB) patients who later developed T1R during clinical follow-up when compared to MB patients who did not develop the episode. By overlapping gene expression patterns, we observed 13 common elements shared between T1R skin lesion cells and THP-1 cells stimulated with both M. leprae and IFN-γ. Among these genes, the autophagy regulator Translocated Promoter Region, Nuclear Basket Protein (TPR) was significantly increased in T1R cells when compared with non-reactional MB cells. Overall, our results indicate that IFN-γ may induce a TPR-mediated autophagy transcriptional program in M. leprae-stimulated cells similar to that observed in skin cells during T1R by a pathway that involves IL-15 production, suggesting the involvement of this cytokine in the pathogenesis of T1R.

Here, a comparative transcriptome investigation was conducted based on high-quality deep sequencing data from the midguts of Apis cerana cerana workers at 7 d post-inoculation (dpi) and 10 dpi with Nosema ceranae and corresponding un-inoculated midguts. PCR identification and microscopic observation of paraffin sections confirmed the effective infection of A. c. cerana worker by N. ceranae. In total, 1127 and 957 N. ceranae-responsive genes were identified in the infected midguts at 7 dpi and 10 dpi, respectively. RT-qPCR results validated the reliability of our transcriptome data. GO categorization indicated the differentially expressed genes (DEGs) were respectively engaged in 34 and 33 functional terms associated with biological processes, cellular components, and molecular functions. Additionally, KEGG pathway enrichment analysis showed that DEGs at 7 dpi and 10 dpi could be enriched in 231 and 226 pathways, respectively. Moreover, DEGs in workers’ midguts at both 7 dpi and 10 dpi were involved in six cellular immune pathways such as autophagy and phagosome and three humoral immune pathways such as the Toll/Imd signaling pathway and Jak-STAT signaling pathway. In addition, one up-regulated gene (XM_017055397.1) was enriched in the NF-κB signaling pathway in the workers’ midgut at 10 dpi. Further investigation suggested the majority of these DEGs were engaged in only one immune pathway, while a small number of DEGs were simultaneously involved in two immune pathways. These results together demonstrated that the overall gene expression profile in host midgut was altered by N. ceranae infection and some of the host immune pathways were induced to activation during fungal infection, whereas some others were suppressed via host–pathogen interaction. Our findings offer a basis for clarification of the mechanism underlying the immune response of A. c. cerana workers to N. ceranae infection, but also provide novel insights into eastern honeybee-microsporodian interaction.

Engagement of LC3-associated phagocytosis (LAP) in response to the uptake of certain particles modulates innate immune responses. Now in Cell Host and Microbe, Akoumianaki et al. (2021) show how a regulatory role of IL-6 on LAP may be at the core of susceptibility to secondary infection during severe sepsis.

Contains fulltext : 238107.pdf (Publisher’s version ) (Closed access) Immune deactivation of phagocytes is a central event in the pathogenesis of sepsis. Herein, we identify a master regulatory role of IL-6 signaling on LC3-associated phagocytosis (LAP) and reveal that uncoupling of these two processes during sepsis induces immunoparalysis in monocytes/macrophages. In particular, we demonstrate that activation of LAP by the human fungal pathogen Aspergillus fumigatus depends on ERK1/2-mediated phosphorylation of p47phox subunit of NADPH oxidase. Physiologically, autocrine IL-6/JAK2/Ninein axis orchestrates microtubule organization and dynamics regulating ERK recruitment to the phagosome and LC3(+) phagosome (LAPosome) formation. In sepsis, loss of IL-6 signaling specifically abrogates microtubule-mediated trafficking of ERK, leading to defective activation of LAP and impaired killing of bacterial and fungal pathogens by monocytes/macrophages, which can be selectively restored by IL-6 supplementation. Our work uncovers a molecular pathway linking IL-6 signaling with LAP and provides insight into the mechanisms underlying immunoparalysis in sepsis.

Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non‐professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis., This study suggests a role of the secreted protein NimB4 in phagosome maturation. Similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.

Mycobacterium tuberculosis (Mtb) causes one of the deadliest infectious diseases worldwide. Upon infection, Mtb is phagocytosed by macrophages and uses its virulence-associated ESX-1 secretion system to modulate the host cell. We showed previously that the ESX-1 secretion system perturbs the Mtb-containing phagosome, and a population (∼30%) of intracellular Mtb is tagged with ubiquitin and targeted to selective autophagy. However, our understanding of how macrophages sense and respond to damaged Mtb-containing phagosomes remains incomplete. Here, we demonstrate that several cytosolic glycan-binding proteins called galectins recognize Mtb-containing phagosomes; in macrophage cell lines and in primary macrophages, galectin-3, -8, and -9 are all recruited to the same Mtb population that colocalizes with selective autophagy markers (ubiquitin, p62, and LC3). To test whether galectins are required for controlling Mtb replication in macrophages, we generated CRISPR/Cas9 knockouts and found that galectin-8−/− and galectin-3/8/9−/− macrophages were similarly defective in targeting Mtb to selective autophagy and controlling replication. This suggests galectin-8 plays a unique role in anti-Mtb autophagy. In investigating galectin-8's role, we identified a novel and specific interaction between galectin-8 and the selective autophagy adapter TAX1BP1 and found that this galectin-8/TAX1BP1 interaction was necessary for macrophages to efficiently target Mtb to selective autophagy. Remarkably, overexpressing galectin-8 increased targeting of Mtb to autophagy and limited Mtb replication. Taken together, these data demonstrate that while several galectins are capable of recognizing damaged Mtb-containing phagosomes, galectin-8 plays a privileged role in recruiting downstream autophagy machinery and may represent a promising target for host-directed tuberculosis therapies. IMPORTANCE Mycobacterium tuberculosis (Mtb) infects one-quarter of the global population and causes one of the deadliest infectious diseases worldwide. Macrophages are the first line of defense against Mtb infection and are typically incredibly efficient at destroying intracellular pathogens, but Mtb has evolved to survive and replicate in this harsh environment. Previous work has found that a portion of intracellular Mtb bacilli damage their phagosomes, leaving them vulnerable to detection by the host and delivery to an antibacterial pathway called selective autophagy. Here, we show that in macrophages, galectin-8 recognizes damaged Mtb-containing phagosomes and targets Mtb to selective autophagy; we found that galectin-8, unlike other highly similar and closely related galectins, is required for targeting and controlling Mtb in macrophages. The specific role for galectin-8 appears to stem from its interaction with TAX1BP1, a selective autophagy adapter protein. Interestingly, overexpressing galectin-8 helps macrophages target and control Mtb, highlighting the importance of galectin-8 in the innate immune response to Mtb.

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3−/− mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target., Interferon (IFN) is an important component of antiviral immunity, but can also be exploited by bacteria for immune evasion. Here the authors show that Listeria monocytogenes (Lm) induces type I IFN to suppress the degradation of Lm virulence proteins, ActA and LLO, and promote Lm infection in an IFITM3-dependent manner, thereby hinting at a potential target for antimicrobial therapy.

SUMMARY Evasion of killing by immune cells is crucial for fungal survival in the host. For the human fungal pathogen Candida albicans, internalization by macrophages induces a transition from yeast to filaments that promotes macrophage death and fungal escape. Nutrient deprivation, alkaline pH, and oxidative stress have been implicated as triggers of intraphagosomal filamentation; however, the impact of other host-derived factors remained unknown. Here, we show that lysates prepared from macrophage-like cell lines and primary macrophages robustly induce C. albicans filamentation. Enzymatic treatment of lysate implicates a phosphorylated protein, and bioactivity-guided fractionation coupled to mass spectrometry identifies the immunomodulatory phosphoprotein PTMA as a candidate trigger of C. albicans filamentation. Immunoneutralization of PTMA within lysate abolishes its activity, strongly supporting PTMA as a filament-inducing component of macrophage lysate. Adding to the known repertoire of physical factors, this work implicates a host protein in the induction of C. albicans filamentation within immune cells., Graphical Abstract, In brief The human fungal pathogen Candida albicans filaments within host macrophages, enabling its escape. Case et al. demonstrate that lysates prepared from macrophage-like cell lines and primary macrophages induce C. albicans filamentation and implicate the immunomodulatory protein prothymosin alpha (PTMA) as a trigger of filamentation produced by host immune cells.

Tick-borne encephalitis virus (TBEV) causes about 5-6 thousand cases annually, while there is still no effective treatment for this virus. To fill this gap, a high-affinity chimeric anti-TBEV antibody ch14D5 has previously been constructed, and high protective activity in a murine TBEV model has been shown for this antibody. However, the mechanism of action of this antibody and the recognized epitope have not been known yet. In this study, it is shown by X-ray crystallography that this antibody recognizes a unique epitope on the lateral ridge of the D3 domain of glycoprotein E, which is readily accessible for binding. The orientation of this antibody relative to the virion surface makes bivalent binding possible, which facilitates the cross-linking of glycoprotein E molecules and thus blocking of surface rearrangements required for infection. Since the antibody tightly binds to this protein even at pH ∼ 5.0, it locks the virion in an acidic environment inside the late endosomes or phagosomes and, therefore, effectively blocks the fusion of the viral and endosomal/phagosomal membranes. We believe that this is why the ch14D5 antibody does not induce an antibody-dependent enhancement of infectionin vivo, which is critical in the development of antibody-based therapeutic agents. In addition, the structure of the antibody-glycoprotein E interface can be used for the rational design of this antibody for enhancing its properties.