scholarly journals Conidial Melanin of the Human-Pathogenic Fungus Aspergillus fumigatus Disrupts Cell Autonomous Defenses in Amoebae

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Iuliia Ferling ◽  
Joe Dan Dunn ◽  
Alexander Ferling ◽  
Thierry Soldati ◽  
Falk Hillmann
Keyword(s):  
Aspergillus Fumigatus ◽  
Dictyostelium Discoideum ◽  
Immune Cells ◽  
Pathogenic Fungus ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Membrane Repair ◽  
Protective Function ◽  
Content Type ◽  
Human Pathogenic Fungus

ABSTRACT The human-pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that causes fatal lung infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrest phagolysosome maturation. How this virulence trait could have been selected for in natural environments is unknown. Here, we found that surface exposure of the green pigment 1,8-dihydroxynaphthalene-(DHN)-melanin can protect conidia from phagocytic uptake and intracellular killing by the fungivorous amoeba Protostelium aurantium and delays its exocytosis from the nonfungivorous species Dictyostelium discoideum. To elucidate the antiphagocytic properties of the surface pigment, we followed the antagonistic interactions of A. fumigatus conidia with the amoebae in real time. For both amoebae, conidia covered with DHN-melanin were internalized at far lower rates than were seen with conidia lacking the pigment, despite high rates of initial attachment to nonkilling D. discoideum. When ingested by D. discoideum, the formation of nascent phagosomes was followed by transient acidification of phagolysosomes, their subsequent neutralization, and, finally, exocytosis of the conidia. While the cycle was completed in less than 1 h for unpigmented conidia, the process was significantly prolonged for conidia covered with DHN-melanin, leading to an extended intracellular residence time. At later stages of this cellular infection, pigmented conidia induced enhanced damage to phagolysosomes and infected amoebae failed to recruit the ESCRT (endosomal sorting complex required for transport) membrane repair machinery or the canonical autophagy pathway to defend against the pathogen, thus promoting prolonged intracellular persistence in the host cell and the establishment of a germination niche in this environmental phagocyte. IMPORTANCE Infections with Aspergillus fumigatus are usually acquired by an inhalation of spores from environmental sources. How spores of a saprophytic fungus have acquired abilities to withstand and escape the phagocytic attacks of innate immune cells is not understood. The fungal surface pigment dihydroxynaphtalene-melanin has been shown to be a crucial factor for the delay in phagosome maturation. Here, we show that this pigment also has a protective function against environmental phagocytes. Pigmented conidia escaped uptake and killing by the fungus-eating amoeba Protostelium aurantium. When ingested by the nonfungivorous phagocyte Dictyostelium discoideum, the pigment attenuated the launch of cell autonomous defenses against the fungal invader, such as membrane repair and autophagy, leading to prolonged intracellular retention. Membrane damage and cytoplasmic leakage may result in an influx of nutrients and thus may further promote intracellular germination of the fungus, indicating that A. fumigatus has acquired some of the basic properties of intracellular pathogens.

scholarly journals Conidial melanin of the human pathogenic fungus Aspergillus fumigatus disrupts cell autonomous defenses in amoebae

2019 ◽  
Author(s):  
Iuliia Ferling ◽  
Joe Dan Dunn ◽  
Alexander Ferling ◽  
Thierry Soldati ◽  
Falk Hillmann
Keyword(s):  
Aspergillus Fumigatus ◽  
Pathogenic Fungus ◽  
Innate Immune ◽  
Natural Environments ◽  
Initial Attachment ◽  
Membrane Repair ◽  
Green Pigment ◽  
Cell Assays ◽  
Human Pathogenic Fungus ◽  
Conidial Surface

AbstractThe human pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that causes fatal infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrest phagolysosome maturation. How such general virulence traits could have been selected for in natural environments is unknown. Here, we used the model amoeba Dictyostelium discoideum to follow the antagonistic interaction of A. fumigatus conidia with environmental phagocytes in real time. We found that conidia covered with the green pigment 1,8-dihydroxynaphthalene-(DHN)-melanin were internalized at far lower rates when compared to those lacking the pigment, despite high rates of initial attachment. Immediately after uptake of the fungal conidia, nascent phagosomes were formed through sequential membrane fusion and fission events. Using single-cell assays supported by a computational model integrating the differential dynamics of internalization and phagolysosome maturation, we could show that acidification of phagolysosomes was transient and was followed by neutralization and, finally, exocytosis of the conidium. For unpigmented conidia, the cycle was completed in less than 1 h, while the process was delayed for conidia covered with DHN-melanin. At later stages of infection, damage to infected phagocytes triggered the ESCRT membrane repair machinery, whose recruitment was also attenuated by DHN-melanin, favoring prolonged persistence and the establishment of an intracellular germination niche in this environmental phagocyte. Increased exposure of DHN-melanin on the conidial surface also improved fungal survival when confronted with the fungivorous predator Protostelium aurantium, demonstrating its universal antiphagocytic properties.

scholarly journals The Putative APSES Transcription Factor RgdA Governs Growth, Development, Toxigenesis, and Virulence in Aspergillus fumigatus

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Sang-Cheol Jun ◽  
Yong-Ho Choi ◽  
Min-Woo Lee ◽  
Jae-Hyuk Yu ◽  
Kwang-Soo Shin
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Pathogenic Fungus ◽  
Mrna Levels ◽  
Transcript Levels ◽  
Content Type ◽  
Asexual Sporulation ◽  
Human Pathogenic Fungus ◽  
Growth Development

ABSTRACT The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus. The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA, brlA, and wetA were decreased in the ΔrgdA mutant compared to those in the wild type (WT). Moreover, ΔrgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the ΔrgdA mutant. Comparative transcriptomic analyses revealed that the ΔrgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the ΔrgdA mutant exhibited elevated production of GT, ΔrgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the ΔrgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling. IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus. This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus. Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.

scholarly journals Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4

2004 ◽  
Vol 125 (3) ◽  
pp. 392-399 ◽  
Author(s):  
Sibylla Braedel ◽  
Markus Radsak ◽  
Hermann Einsele ◽  
Jean-Paul Latgé ◽  
Andreas Michan ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Immune Cells ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Toll Like Receptors

scholarly journals A lectin that targets specific bacteria for killing on DNA-based extracellular traps

2020 ◽  
Author(s):  
Timothy Farinholt ◽  
Christopher Dinh ◽  
Adam Kuspa
Keyword(s):  
Dictyostelium Discoideum ◽  
Immune Cells ◽  
Binding Proteins ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Bacterial Killing ◽  
Extracellular Traps ◽  
Bacterial Binding ◽  
Binding Lectin ◽  
Dna Scaffold

AbstractAnimal defenses directed against bacteria include DNA-based extracellular traps (ETs) that are produced by innate immune cells. ET-bound bacteria are prevented from further tissue dissemination and are eventually killed by ET-bound antibacterial proteins. It is unclear how bacteria bind to ETs, though it has been proposed that the negatively-charged DNA scaffold of the ETs is involved. We have found that the bacterial-binding lectin CadA is a component of the ETs produced by the innate immune cells of Dictyostelium discoideum and is required for the binding and killing of two Enterobacteriaceae by ETs, but not other bacteria. Our results suggest that ETs selectively sequester bacteria and that lectins can facilitate bacterial killing by acting as ET-bacteria binding proteins.

scholarly journals Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
Miranda J. Neubert ◽  
Elizabeth A. Dahlmann ◽  
Andrew Ambrose ◽  
Michael D. L. Johnson
Keyword(s):  
Streptococcus Pneumoniae ◽  
Immune Cells ◽  
Pathogenic Bacteria ◽  
Copper Toxicity ◽  
Innate Immune ◽  
Copper Chaperone ◽  
Innate Immune Cells ◽  
Content Type ◽  
Copper State ◽  
Intracellular Copper

ABSTRACT As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria. Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

scholarly journals Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Darel Macdonald ◽  
Darren D. Thomson ◽  
Anna Johns ◽  
Adriana Contreras Valenzuela ◽  
Jane M. Gilsenan ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Cell Fusion ◽  
Pathogenic Fungus ◽  
Brefeldin A ◽  
Culture Conditions ◽  
Competitive Fitness ◽  
Content Type ◽  
Genetic Profiling ◽  
Standard Culture ◽  
Human Pathogenic Fungus

ABSTRACT Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in Aspergillus fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high-throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show that heterozygous cyp51A and arf2 null mutants have reduced fitness in the presence of itraconazole and brefeldin A, respectively, and a heterozygous atp17 null mutant is resistant to brefeldin A.

scholarly journals The Life Cycle Stages of Pneumocystis murina Have Opposing Effects on the Immune Response to This Opportunistic Fungal Pathogen

2016 ◽  
Vol 84 (11) ◽  
pp. 3195-3205 ◽  
Author(s):  
Heather M. Evans ◽  
Grady L. Bryant ◽  
Beth A. Garvy
Keyword(s):  
Cell Wall ◽  
Life Cycle ◽  
Immune Responses ◽  
Immune Cells ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Content Type ◽  
Life Cycle Stages ◽  
Ifn Γ

The cell wall β-glucans of Pneumocystis cysts have been shown to stimulate immune responses in lung epithelial cells, dendritic cells, and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with these innate immune cells. Here we report differences in the responses of both neonatal and adult mice to the trophic and cystic life cycle stages of Pneumocystis murina . The adult and neonatal immune responses to infection with Pneumocystis murina trophic forms were less robust than the responses to infection with a physiologically normal mixture of cysts and trophic forms. Cysts promoted the recruitment of nonresident innate immune cells and T and B cells into the lungs. Cysts, but not trophic forms, stimulated increased concentrations of the cytokine gamma interferon (IFN-γ) in the alveolar spaces and an increase in the percentage of CD4 + T cells that produce IFN-γ. In vitro , bone marrow-derived dendritic cells (BMDCs) stimulated with cysts produced the proinflammatory cytokines interleukin 1β (IL-1β) and IL-6. In contrast, trophic forms suppressed antigen presentation to CD4 + T cells, as well as the β-glucan-, lipoteichoic acid (LTA)-, and lipopolysaccharide (LPS)-induced production of interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) by BMDCs. The negative effects of trophic forms were not due to ligation of mannose receptor. Our results indicate that optimal innate and adaptive immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, trophic forms suppress β-glucan-induced proinflammatory responses in vitro , suggesting that the trophic forms dampen cyst-induced inflammation in vivo .

scholarly journals The Fungal Quorum-Sensing Molecule Farnesol Activates Innate Immune Cells but Suppresses Cellular Adaptive Immunity

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Ines Leonhardt ◽  
Steffi Spielberg ◽  
Michael Weber ◽  
Daniela Albrecht-Eckardt ◽  
Markus Bläss ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Immune Cells ◽  
Innate Immune ◽  
Interleukin 12 ◽  
Innate Immune Cells ◽  
Content Type ◽  
Gm Csf ◽  
Immature Dendritic Cells ◽  
Macrophage Colony ◽  
Quorum Sensing Molecule

ABSTRACTFarnesol, produced by the polymorphic fungusCandida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and macrophage inflammatory protein 1 alpha [MIP-1α]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance.IMPORTANCEFarnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeastCandida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor.

scholarly journals Yersinia pestis Subverts the Dermal Neutrophil Response in a Mouse Model of Bubonic Plague

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Jeffrey G. Shannon ◽  
Aaron M. Hasenkrug ◽  
David W. Dorward ◽  
Vinod Nair ◽  
Aaron B. Carmody ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Immune Cells ◽  
Innate Immune ◽  
Health Concern ◽  
Virulence Plasmid ◽  
Innate Immune Cells ◽  
Bubonic Plague ◽  
Content Type ◽  
Large Numbers ◽  
Neutrophil Response

ABSTRACTThe majority of humanYersinia pestisinfections result from introduction of bacteria into the skin by the bite of an infected flea. Once in the dermis,Y. pestiscan evade the host’s innate immune response and subsequently disseminate to the draining lymph node (dLN). There, the pathogen replicates to large numbers, causing the pathognomonic bubo of bubonic plague. In this study, several cytometric and microscopic techniques were used to characterize the early host response to intradermal (i.d.)Y. pestisinfection. Mice were infected i.d. with fully virulent or attenuated strains of dsRed-expressingY. pestis, and tissues were analyzed by flow cytometry. By 4 h postinfection, there were large numbers of neutrophils in the infected dermis and the majority of cell-associated bacteria were associated with neutrophils. We observed a significant effect of the virulence plasmid (pCD1) on bacterial survival and neutrophil activation in the dermis. Intravital microscopy of i.d.Y. pestisinfection revealed dynamic interactions between recruited neutrophils and bacteria. In contrast, very few bacteria interacted with dendritic cells (DCs), indicating that this cell type may not play a major role early inY. pestisinfection. Experiments using neutrophil depletion and a CCR7 knockout mouse suggest that dissemination ofY. pestisfrom the dermis to the dLN is not dependent on neutrophils or DCs. Taken together, the results of this study show a very rapid, robust neutrophil response toY. pestisin the dermis and that the virulence plasmid pCD1 is important for the evasion of this response.IMPORTANCEYersinia pestisremains a public health concern today because of sporadic plague outbreaks that occur throughout the world and the potential for its illegitimate use as a bioterrorism weapon. Since bubonic plague pathogenesis is initiated by the introduction ofY. pestisinto the skin, we sought to characterize the response of the host’s innate immune cells to bacteria early after intradermal infection. We found that neutrophils, innate immune cells that engulf and destroy microbes, are rapidly recruited to the injection site, irrespective of strain virulence, indicating thatY. pestisis unable to subvert neutrophil recruitment to the site of infection. However, we saw a decreased activation of neutrophils that were associated withY. pestisstrains harboring the pCD1 plasmid, which is essential for virulence. These findings indicate a role for pCD1-encoded factors in suppressing the activation/stimulation of these cellsin vivo.

scholarly journals Microbiome management in the social amoeba Dictyostelium discoideum compared to humans

2019 ◽  
Vol 63 (8-9-10) ◽  
pp. 447-450 ◽  
Author(s):  
Timothy Farinholt ◽  
Christopher Dinh ◽  
Adam Kuspa
Keyword(s):  
Recent Work ◽  
Dictyostelium Discoideum ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Social Amoeba ◽  
The Social ◽  
Physical Barriers ◽  
Social Amoebae

Social amoebae and humans use common strategies to orchestrate their interactions with the bacteria in their respective environments and within their bodies. These strategies include the elimination of bacteria by phagocytosis, the establishment of mutualistic interactions, the elaboration of physical barriers, and the deployment of innate immune cells. Many of the molecular mechanisms that humans and social amoebae employ differ, but there are striking similarities that may inform studies in each organism. In this topical review we highlight the similarities and consider what we might learn by comparing these highly divergent species. We focus on recent work in Dictyostelium discoideum with hopes of stimulating work in this area and with the expectation that new mechanistic details uncovered in social amoebae-bacteria interactions will inform microbiome management in humans.

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