The Endoplasmic Reticulum-Mitochondrion Tether ERMES Orchestrates Fungal Immune Evasion, Illuminating Inflammasome Responses to Hyphal Signals

ABSTRACT The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies. The pathogenic yeast Candida albicans escapes macrophages by triggering NLRP3 inflammasome-dependent host cell death (pyroptosis). Pyroptosis is inflammatory and must be tightly regulated by host and microbe, but the mechanism is incompletely defined. We characterized the C. albicans endoplasmic reticulum (ER)-mitochondrion tether ERMES and show that the ERMES mmm1 mutant is severely crippled in killing macrophages despite hyphal formation and normal phagocytosis and survival. To understand dynamic inflammasome responses to Candida with high spatiotemporal resolution, we established live-cell imaging for parallel detection of inflammasome activation and pyroptosis at the single-cell level. This showed that the inflammasome response to mmm1 mutant hyphae is delayed by 10 h, after which an exacerbated activation occurs. The NLRP3 inhibitor MCC950 inhibited inflammasome activation and pyroptosis by C. albicans, including exacerbated inflammasome activation by the mmm1 mutant. At the cell biology level, inactivation of ERMES led to a rapid collapse of mitochondrial tubular morphology, slow growth and hyphal elongation at host temperature, and reduced exposed 1,3-β-glucan in hyphal populations. Our data suggest that inflammasome activation by C. albicans requires a signal threshold dependent on hyphal elongation and cell wall remodeling, which could fine-tune the response relative to the level of danger posed by C. albicans. The phenotypes of the ERMES mutant and the lack of conservation in animals suggest that ERMES is a promising antifungal drug target. Our data further indicate that NLRP3 inhibition by MCC950 could modulate C. albicans-induced inflammation. IMPORTANCE The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.

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TolC-Dependent Modulation of Host Cell Death by the Francisella tularensis Live Vaccine Strain

Infection and Immunity ◽

10.1128/iai.00044-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 2068-2078 ◽

Cited By ~ 16

Author(s):

Christopher R. Doyle ◽

Ji-An Pan ◽

Patricio Mena ◽

Wei-Xing Zong ◽

David G. Thanassi

Keyword(s):

Cell Death ◽

Immune Responses ◽

Host Cell ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Live Vaccine ◽

Live Vaccine Strain ◽

Type I ◽

Content Type ◽

Host Cell Death

ABSTRACTFrancisella tularensisis a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of theF. tularensislive vaccine strain (LVS) and demonstrated that a ΔtolCmutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required forF. tularensisto preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the ΔtolCmutant. These findings support a model wherein the immunomodulatory capacity ofF. tularensisrelies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the ΔtolCLVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the ΔtolCmutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection byF. tularensisand highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.

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Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+ Homeostasis To Promote Cell Death

mBio ◽

10.1128/mbio.02250-20 ◽

2020 ◽

Vol 11 (6) ◽

pp. e02250-20

Author(s):

Kathrin Stelzner ◽

Ann-Cathrin Winkler ◽

Chunguang Liang ◽

Aziza Boyny ◽

Carsten P. Ade ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Death ◽

Host Cell ◽

Intracellular Bacterium ◽

Tissue Destruction ◽

Content Type ◽

Host Cell Death ◽

Genome Wide ◽

A Genome ◽

Spread Of Infection

ABSTRACTThe opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.

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Core N-Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death

mBio ◽

10.1128/mbio.00711-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Eun Jung Thak ◽

Su-Bin Lee ◽

Shengjie Xu-Vanpala ◽

Dong-Jik Lee ◽

Seung-Yeon Chung ◽

...

Keyword(s):

Cell Death ◽

Fungal Infection ◽

Host Cell ◽

Host Cells ◽

Glycan Structure ◽

Macrophage Cell ◽

Content Type ◽

Host Cell Death ◽

Intact Core

ABSTRACT Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N-glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N-glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 (alg3Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N-glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro, the alg3Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12, encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N-glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1β (IL-1β) were not reduced in the bone marrow-derived dendritic cells obtained from Asc- and Gsdmd-deficient mice infected with the alg3Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N-glycans. Our results demonstrated N-glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformans. IMPORTANCE We previously reported that the outer mannose chains of N-glycans are dispensable for the virulence of C. neoformans, which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans. Here, we present evidence that an intact core N-glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ, and alg12Δ strains that have defects in lipid-linked N-glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N-glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans, thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N-glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.

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Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during Legionella Infection

mBio ◽

10.1128/mbio.00839-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 63

Author(s):

Stephen Weber ◽

Maria Wagner ◽

Hubert Hilbi

Keyword(s):

Host Cell ◽

Legionella Pneumophila ◽

Live Cell Imaging ◽

Cell Imaging ◽

Effector Proteins ◽

Host Cells ◽

Wild Type ◽

Host Cell Membrane ◽

Content Type ◽

Temporal And Spatial

ABSTRACTThe causative agent of Legionnaires’ disease,Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, theLegionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different “effector” proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoebaDictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficientL. pneumophila, PtdIns(3,4,5)P3transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)Pwithin 1 min after uptake. Whereas phagosomes containing ΔicmTmutant bacteria remained decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)Ptransiently localized to early phagosomes harboring wild-type or ΔicmT L. pneumophilaand was cleared within minutes after uptake. During the following 2 h, PtdIns(4)Psteadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)Pidentity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboring ΔsidC-sdcAmutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions.IMPORTANCEThe environmental bacteriumLegionella pneumophilais the causative agent of Legionnaires’ pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, theLegionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. UsingDictyosteliumamoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)Pwas slowly cleared from LCVs, thus incapacitating the host cell’s digestive machinery, while PtdIns(4)Pgradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.

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The Als3 Cell Wall Adhesin Plays a Critical Role in Human Serum Amyloid A1-Induced Cell Death and Aggregation in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00024-20 ◽

2020 ◽

Vol 64 (6) ◽

Author(s):

Jiao Gong ◽

Jian Bing ◽

Guobo Guan ◽

Clarissa J. Nobile ◽

Guanghua Huang

Keyword(s):

Cell Death ◽

Cell Wall ◽

Candida Albicans ◽

Critical Role ◽

Membrane Integrity ◽

Cell Aggregation ◽

Serum Amyloid ◽

Mouse Serum ◽

Content Type ◽

Serum Amyloid A1

ABSTRACT Antimicrobial peptides and proteins play critical roles in the host defense against invading pathogens. We recently discovered that recombinantly expressed human and mouse serum amyloid A1 (rhSAA1 and rmSAA1, respectively) proteins have potent antifungal activities against the major human fungal pathogen Candida albicans. At high concentrations, rhSAA1 disrupts C. albicans membrane integrity and induces rapid fungal cell death. In the present study, we find that rhSAA1 promotes cell aggregation and targets the C. albicans cell wall adhesin Als3. Inactivation of ALS3 in C. albicans leads to a striking decrease in cell aggregation and cell death upon rhSAA1 treatment, suggesting that Als3 plays a critical role in SAA1 sensing. We further demonstrate that deletion of the transcriptional regulators controlling the expression of ALS3, such as AHR1, BCR1, and EFG1, in C. albicans results in similar effects to that of the als3/als3 mutant upon rhSAA1 treatment. Global gene expression profiling indicates that rhSAA1 has a discernible impact on the expression of cell wall- and metabolism-related genes, suggesting that rhSAA1 treatment could lead to a nutrient starvation effect on C. albicans cells.

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Real-Time Integration of Cell Death and Proliferation Kinetics at the Single-Cell and Population-Level Using High-Throughput Live-Cell Imaging

SSRN Electronic Journal ◽

10.2139/ssrn.3261823 ◽

2018 ◽

Author(s):

Jesse D. Gelles ◽

Jarvier N. Mohammed ◽

Luis C. Santos ◽

Jerry E. Chipuk

Keyword(s):

Cell Death ◽

Single Cell ◽

Real Time ◽

High Throughput ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Integration ◽

Population Level ◽

Live Cell ◽

Proliferation Kinetics

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Inflammasomes, Autophagy, and Cell Death: The Trinity of Innate Host Defense against Intracellular Bacteria

Mediators of Inflammation ◽

10.1155/2019/2471215 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 20

Author(s):

Teresa Krakauer

Keyword(s):

Cell Death ◽

Host Cell ◽

Host Defense ◽

Intracellular Bacteria ◽

Inflammasome Activation ◽

Type I ◽

Dependent Manner ◽

Cell Death Pathways ◽

Host Cell Death ◽

Molecular Patterns

Inflammasome activation is an innate host defense mechanism initiated upon sensing pathogens or danger in the cytosol. Both autophagy and cell death are cell autonomous processes important in development, as well as in host defense against intracellular bacteria. Inflammasome, autophagy, and cell death pathways can be activated by pathogens, pathogen-associated molecular patterns (PAMPs), cell stress, and host-derived damage-associated molecular patterns (DAMPs). Phagocytosis and toll-like receptor (TLR) signaling induce reactive oxygen species (ROS), type I IFN, NFκB activation of proinflammatory cytokines, and the mitogen-activated protein kinase cascade. ROS and IFNγare also prominent inducers of autophagy. Pathogens, PAMPs, and DAMPs activate TLRs and intracellular inflammasomes, inducing apoptotic and inflammatory caspases in a context-dependent manner to promote various forms of cell death to eliminate pathogens. Common downstream signaling molecules of inflammasomes, autophagy, and cell death pathways interact to initiate appropriate measures against pathogens and determine host survival as well as pathological consequences of infection. The integration of inflammasome activation, autophagy, and cell death is central to pathogen clearance. Various pathogens produce virulence factors to control inflammasomes, subvert autophagy, and modulate host cell death in order to evade host defense. This review highlights the interaction of inflammasomes, autophagy, and host cell death pathways in counteractingBurkholderia pseudomallei, the causative agent of melioidosis. Contrasting evasion strategies used byB.pseudomallei,Mycobacterium tuberculosis, andLegionella pneumophilato avoid and dampen these innate immune responses will be discussed.

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Genetic Screen inChlamydia muridarumReveals Role for an Interferon-Induced Host Cell Death Program in Antimicrobial Inclusion Rupture

mBio ◽

10.1128/mbio.00385-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 4

Author(s):

Amanda M. Giebel ◽

Shuai Hu ◽

Krithika Rajaram ◽

Ryan Finethy ◽

Evelyn Toh ◽

...

Keyword(s):

Cell Death ◽

Immune Responses ◽

Host Cell ◽

Host Defense ◽

Genetic Screen ◽

Content Type ◽

Host Immune Responses ◽

Interferon Stimulated Genes ◽

Host Cell Death ◽

Ifn Γ

ABSTRACTInterferon-regulated immune defenses protect mammals from pathogenically diverse obligate intracellular bacterial pathogens of the genusChlamydia. Interferon gamma (IFN-γ) is especially important in controlling the virulence ofChlamydiaspecies and thus impacts the modeling of human chlamydial infection and disease in mice. How IFN-γ contributes to cell-autonomous defenses againstChlamydiaspecies and how these pathogens evade IFN-γ-mediated immunity in their natural hosts are not well understood. We conducted a genetic screen which identified 31IFN-γ-sensitive (Igs) mutants of the mouse model pathogenChlamydia muridarum. Genetic suppressor analysis and lateral gene transfer were used to map the phenotype of one of these mutants, Igs4, to a missense mutation in a putative chlamydial inclusion membrane protein, TC0574. We observed the lytic destruction of Igs4-occupied inclusions and accompanying host cell death in response to IFN-γ priming or various proapoptotic stimuli. However, Igs4 was insensitive to IFN-γ-regulated cell-autonomous defenses previously implicated in anti-Chlamydia trachomatishost defense in mice. Igs4 inclusion integrity was restored by caspase inhibitors, indicating that the IFN-γ-mediated destruction of Igs4 inclusions is dependent upon the function of caspases or related prodeath cysteine proteases. We further demonstrated that the Igs4 mutant is immune restricted in an IFN-γ-dependent manner in a mouse infection model, thereby implicating IFN-γ-mediated inclusion destruction and host cell death as potentin vivohost defense mechanisms to which wild-typeC. muridarumis resistant. Overall, our results suggest thatC. muridarumevolved resistance mechanisms to counter IFN-γ-elicited programmed cell death and the associated destruction of intravacuolar pathogens.IMPORTANCEMultiple obligatory intracellular bacteria in the genusChlamydiaare important pathogens. In humans, strains ofC. trachomatiscause trachoma, chlamydia, and lymphogranuloma venereum. These diseases are all associated with extended courses of infection and reinfection that likely reflect the ability of chlamydiae to evade various aspects of host immune responses. Interferon-stimulated genes, driven in part by the cytokine interferon gamma, restrict the host range of variousChlamydiaspecies, but how these pathogens evade interferon-stimulated genes in their definitive host is poorly understood. VariousChlamydiaspecies can inhibit death of their host cells and may have evolved this strategy to evade prodeath signals elicited by host immune responses. We present evidence that chlamydia-induced programmed cell death resistance evolved to counter interferon- and immune-mediated killing ofChlamydia-infected cells.

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Importance of PdpC, IglC, IglI, and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis

Infection and Immunity ◽

10.1128/iai.00275-13 ◽

2013 ◽

Vol 81 (6) ◽

pp. 2076-2084 ◽

Cited By ~ 16

Author(s):

Marie Lindgren ◽

Kjell Eneslätt ◽

Jeanette E. Bröms ◽

Anders Sjöstedt

Keyword(s):

Cell Death ◽

Host Cell ◽

Francisella Tularensis ◽

Caspase 3 ◽

Mitochondrial Damage ◽

Content Type ◽

Mitochondrial Superoxide ◽

Cell Death Pathway ◽

Host Cell Death ◽

Nucleosome Formation

ABSTRACTModulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacteriumFrancisella tularensisis highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with theF. tularensislive vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of thepdpC,iglC,iglG, origlIgenes, which encode components of theFrancisellapathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ΔiglIor ΔiglCmutants, although the former strain replicated. The ΔiglGmutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ΔpdpCmutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from theF. tularensisinfection.

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