scholarly journals Calcineurin Controls Growth, Morphology, and Pathogenicity in Aspergillus fumigatus

2006 ◽  
Vol 5 (7) ◽  
pp. 1091-1103 ◽  
Author(s):  
William J. Steinbach ◽  
Robert A. Cramer ◽  
B. Zachary Perfect ◽  
Yohannes G. Asfaw ◽  
Theodor C. Sauer ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Clinical Observation ◽  
Immunocompromised Patient ◽  
Fungal Growth ◽  
Pathogenic Fungi ◽  
Wild Type ◽  
Therapeutic Approaches ◽  
Growth Morphology ◽  
Different Types ◽  
Hyphal Morphology

ABSTRACT Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The ΔcnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the ΔcnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the ΔcnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the ΔcnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

scholarly journals Toll-Like Receptor 9 Modulates Immune Responses to Aspergillus fumigatus Conidia in Immunodeficient and Allergic Mice

2008 ◽  
Vol 77 (1) ◽  
pp. 108-119 ◽  
Author(s):  
Hemanth Ramaprakash ◽  
Toshihiro Ito ◽  
Theodore J. Standiford ◽  
Steven L. Kunkel ◽  
Cory M. Hogaboam
Keyword(s):  
Immune Responses ◽  
Aspergillus Fumigatus ◽  
Inflammatory Responses ◽  
Fungal Growth ◽  
Lower Airway ◽  
Interleukin 17 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Immunodeficient Mice

ABSTRACT The role of Toll-like receptor 9 (TLR9) in antifungal responses in the immunodeficient and allergic host is unclear. We investigated the role of TLR9 in murine models of invasive aspergillosis and fungal asthma. Neutrophil-depleted TLR9 wild-type (TLR9+/+) and TLR9-deficient (TLR9−/−) mice were challenged with resting or swollen Aspergillus fumigatus conidia and monitored for survival and lung inflammatory responses. The absence of TLR9 delayed, but did not prevent, mortality in immunodeficient mice challenged with resting or swollen conidia compared to TLR9+/+ mice. In a fungal asthma model, TLR9+/+ and TLR9−/− mice were sensitized to soluble A. fumigatus antigens and challenged with resting or swollen A. fumigatus conidia, and both groups of mice were analyzed prior to and at days 7, 14, and 28 after the conidium challenge. When challenged with resting conidia, TLR9−/− mice exhibited significantly lower airway hyper-responsiveness compared to the TLR9+/+ groups. In contrast, A. fumigatus-sensitized TLR9−/− mice exhibited pulmonary fungal growth at days 14 and 28 after challenge with swollen conidia, a finding never observed in their allergic wild-type counterparts. Increased fungal growth in allergic TLR9−/− mice correlated with markedly decreased dectin-1 expression in whole lung samples and isolated dendritic cell populations. Further, whole lung levels of interleukin-17 were lower in allergic TLR9−/− mice compared to similar TLR9+/+ mice. Together, these data suggest that TLR9 modulates pulmonary antifungal immune responses to swollen conidia, possibly through the regulation of dectin-1 expression.

scholarly journals A Fungus-Specific Ras Homolog Contributes to the Hyphal Growth and Virulence of Aspergillus fumigatus

2005 ◽  
Vol 4 (12) ◽  
pp. 1982-1989 ◽  
Author(s):  
Jarrod R. Fortwendel ◽  
Wei Zhao ◽  
Ruchi Bhabhra ◽  
Steven Park ◽  
David S. Perlin ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Growth Control ◽  
Biomass Accumulation ◽  
Hyphal Growth ◽  
Pathogenic Fungi ◽  
Total Biomass ◽  
Gene Encoding ◽  
Directional Growth ◽  
Solid Media ◽  
Hyphal Morphology

ABSTRACT The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The ΔrasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBΔ113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited ∼65% survival compared to ∼10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.

scholarly journals Phosphoproteomics of Aspergillus fumigatus Exposed to the Antifungal Drug Caspofungin

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Eliciane Cevolani Mattos ◽  
Giuseppe Palmisano ◽  
Gustavo H. Goldman
Keyword(s):  
Cell Wall ◽  
Transcription Factors ◽  
Aspergillus Fumigatus ◽  
Protein Kinases ◽  
Pathogenic Fungus ◽  
Fungal Growth ◽  
Wild Type ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
High Concentrations

ABSTRACT Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans. A. fumigatus infections are usually treated with polyenes, azoles, or echinocandins. Echinocandins, such as caspofungin, can inhibit the biosynthesis of the β-1,3-glucan polysaccharide, affecting the integrity of the cell wall and leading to fungal death. In some A. fumigatus strains, caspofungin treatment at high concentrations induces an increase of fungal growth, a phenomenon called the caspofungin paradoxical effect (CPE). Here, we analyze the proteome and phosphoproteome of the A. fumigatus wild-type strain and of mitogen-activated protein kinase (MAPK) mpkA and sakA null mutant strains during CPE (2 μg/ml caspofungin for 1 h). The wild-type proteome showed 75 proteins and 814 phosphopeptides (corresponding to 520 proteins) altered in abundance in response to caspofungin treatment. The ΔmpkA (ΔmpkA caspofungin/wild-type caspofungin) and ΔsakA (ΔsakA caspofungin/wild-type caspofungin) strains displayed 626 proteins and 1,236 phosphopeptides (corresponding to 703 proteins) and 101 proteins and 1,217 phosphopeptides (corresponding to 645 proteins), respectively, altered in abundance. Functional characterization of the phosphopeptides from the wild-type strain exposed to caspofungin showed enrichment for transcription factors, protein kinases, and cytoskeleton proteins. Proteomic analysis of the ΔmpkA and ΔsakA mutants indicated that control of proteins involved in metabolism, such as in production of secondary metabolites, was highly represented in both mutants. Results of functional categorization of phosphopeptides from both mutants were very similar and showed a high number of proteins with decreased phosphorylation of proteins involved in transcriptional control, DNA/RNA binding, cell cycle control, and DNA processing. This report reveals novel transcription factors involved in caspofungin tolerance. IMPORTANCE Aspergillus fumigatus is an opportunistic human-pathogenic fungus causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. Caspofungin is an echinocandin that impacts the construction of the fungal cell wall by inhibiting the biosynthesis of the β-1,3-glucan polysaccharide. Caspofungin is a fungistatic drug and is recommended as a second-line therapy for treatment of aspergillosis. Treatment at high concentrations induces an increase of fungal growth, a phenomenon called the caspofungin paradoxical effect (CPE). Collaboration between the mitogen-activated protein kinases (MAPK) of the cell wall integrity (MapkA) and high-osmolarity glycerol (SakA) pathways is essential for CPE. Here, we investigate the global proteome and phosphoproteome of A. fumigatus wild-type, ΔmpkA, and ΔsakA strains upon CPE. This study showed intense cross talk between the two MAPKs for the CPE and identified novel protein kinases and transcription factors possibly important for CPE. Increased understanding of how the modulation of protein phosphorylation may affect the fungal growth in the presence of caspofungin represents an important step in the development of new strategies and methods to combat the fungus inside the host.

scholarly journals The Strength of Synergistic Interaction between Posaconazole and Caspofungin Depends on the Underlying Azole Resistance Mechanism of Aspergillus fumigatus

2015 ◽  
Vol 59 (3) ◽  
pp. 1738-1744 ◽  
Author(s):  
Eleftheria Mavridou ◽  
Joseph Meletiadis ◽  
Antony Rijs ◽  
Johan W. Mouton ◽  
Paul E. Verweij
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanism ◽  
Fungal Growth ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Synergistic Interactions ◽  
Drug Concentrations

ABSTRACTThe majority of azole resistance mechanisms inAspergillus fumigatuscorrespond to mutations in thecyp51Agene. As azoles are less effective against infections caused by multiply azole-resistantA. fumigatusisolates, new therapeutic options are warranted for treating these infections. We therefore investigated thein vitrocombination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistantA. fumigatusisolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly (ttest,P< 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates (P< 0.01). FIC-2 values were inversely correlated with POSA MICs (rs= −0.52,P= 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA (rs= 0.76,P< 0.0001) and CAS (rs= 0.52,P= 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) againstA. fumigatusisolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

scholarly journals Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1541-1550 ◽  
Author(s):  
Dev Sriranganadane ◽  
Utz Reichard ◽  
Karine Salamin ◽  
Marina Fratti ◽  
Olivier Jousson ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Fungal Growth ◽  
Low Ph ◽  
Acidic Protein ◽  
Protein Digestion ◽  
Wild Type ◽  
Single Chain ◽  
Encoding Gene ◽  
Complete Protein ◽  
Mature Protease

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

scholarly journals At the metal–metabolite interface in Aspergillus fumigatus: towards untangling the intersecting roles of zinc and gliotoxin

Microbiology ◽  
2021 ◽  
Vol 167 (11) ◽  
Author(s):  
Aimee M. Traynor ◽  
Rebecca A. Owens ◽  
Claudia M. Coughlin ◽  
Maeve C. Holton ◽  
Gary W. Jones ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Drug Targets ◽  
Fungal Growth ◽  
Dependent Manner ◽  
Label Free ◽  
Wild Type ◽  
Transcription Activator ◽  
Zinc Availability ◽  
Metabolic Systems ◽  
Zinc Sensing

Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)−1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc-depleted medium, addition of zinc (1 µM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, triacetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 µM zinc) and ΔgtmA (at >1 µM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 µM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidoreductase- and secondary metabolism-related enzymes was also evident in a zinc- and strain-dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatus.

scholarly journals Azole and Amphotericin B MIC Values against Aspergillus fumigatus: High Agreement between Spectrophotometric and Visual Readings Using the EUCAST EDef 9.3.2 Procedure

2020 ◽  
Vol 65 (1) ◽  
pp. e01693-20 ◽  
Author(s):  
Julia Serrano-Lobo ◽  
Ana Gómez ◽  
Waldo Sánchez-Yebra ◽  
Miguel Fajardo ◽  
Belén Lorenzo ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Amphotericin B ◽  
Fungal Growth ◽  
Sensu Stricto ◽  
Resistance Rate ◽  
Wild Type ◽  
Growth Reduction ◽  
High Agreement ◽  
Content Type

ABSTRACTThe EUCAST EDef 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against Aspergillus spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare visual and spectrophotometric MIC readings of azoles and amphotericin B against Aspergillus fumigatussensu lato isolates. A total of 847 A. fumigatussensu lato isolates (A. fumigatus sensu stricto [n = 828] and cryptic species [n = 19]) were tested against amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole using the EUCAST EDef 9.3.2 procedure. Isolates were classified as susceptible or resistant/non-wild type according to the 2020 updated breakpoints. The area of technical uncertainty for the azoles was defined in the updated breakpoints. Visual and spectrophotometric (fungal growth reduction of >95% compared to the control, read at 540 nm) MICs were compared. Essential (±1 2-fold dilution) and categorical agreements were calculated. Overall, high essential (97.1%) and categorical (99.6%) agreements were found. We obtained 100% categorical agreements for amphotericin B, itraconazole, and posaconazole, and consequently, no errors were found. Categorical agreements were 98.7 and 99.3% for voriconazole and isavuconazole, respectively. Most of the misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or within one 2-fold dilution above the breakpoint. The resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant cyp51A mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against A. fumigatussensu lato isolates may be a convenient alternative to visual endpoint readings.

scholarly journals Novel intermicrobial molecular interaction: Pseudomonas aeruginosa Quinolone Signal (PQS) modulates Aspergillus fumigatus response to iron

Microbiology ◽  
2020 ◽  
Vol 166 (1) ◽  
pp. 44-55 ◽  
Author(s):  
Hasan Nazik ◽  
Gabriele Sass ◽  
Shajia R. Ansari ◽  
Reyhan Ertekin ◽  
Hubertus Haas ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aspergillus Fumigatus ◽  
Biofilm Formation ◽  
Fungal Growth ◽  
Wild Type ◽  
Content Type ◽  
Pseudomonas Quinolone Signal ◽  
Maximal Stimulation ◽  
Fe Homeostasis ◽  
Quorum Sensing Molecule

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af), the commonest bacterium and fungus in compromised host airways, compete for iron (Fe). The Pseudomonas quinolone signal (PQS), a Pa quorum sensing molecule, also chelates Fe, and delivers Fe to the Pa cell membrane using Pa siderophores. In models of Af biofilm formation or preformed biofilms, PQS inhibited Af in a low Fe environment. AfΔsidA (mutant unable to produce siderophores) biofilm was more sensitive to PQS inhibition than wild-type (WT), as was planktonic AfΔsidA growth. PQS decreased WT Af growth on agar. All these inhibitory actions were reversed by Fe. The Pa siderophore pyoverdin, or Af siderophore inhibitor celastrol, act cooperatively with PQS in Af inhibition. These findings all indicate PQS inhibition is owing to Fe chelation. Remarkably, in high Fe environments, PQS enhanced Af biofilm at 1/100 to 1/2000 Fe concentration required for Fe alone to enhance. Planktonic Af growth, and on agar, Af conidiation, were also enhanced by PQS+Fe compared to Fe alone. In contrast, neither AfΔsidA biofilm, nor planktonic AfΔsidA, were enhanced by PQS-Fe compared to Fe. When Af siderophore ferricrocin (FC),+PQS, were added to AfΔsidA, Af was then boosted more than by FC alone. Moreover, FC+PQS+Fe boosted AfΔsidA more than Fe, FC, FC+Fe, PQS+FC or PQS+Fe. Thus PQS-Fe maximal stimulation requires Af siderophores. PQS inhibits Af via chelation under low Fe conditions. In a high Fe environment, PQS paradoxically stimulates Af efficiently, and this involves Af siderophores. PQS production by Pa could stimulate Af in cystic fibrosis airways, where Fe homeostasis is altered and Fe levels increase, supporting fungal growth.

scholarly journals CD137 Signaling Is Critical in Fungal Clearance during Systemic Candida albicans Infection

2021 ◽  
Vol 7 (5) ◽  
pp. 382
Author(s):  
Vuvi G. Tran ◽  
Na N. Z. Nguyen ◽  
Byungsuk Kwon
Keyword(s):  
Candida Albicans ◽  
Defense Mechanisms ◽  
Tumor Necrosis ◽  
Fungal Infections ◽  
Fungal Growth ◽  
Wild Type ◽  
Agonistic Antibody ◽  
Innate Defense ◽  
Surface Receptor ◽  
Candida Albicans Infection

Invasive fungal infections by Candida albicans frequently cause mortality in immunocompromised patients. Neutrophils are particularly important for fungal clearance during systemic C. albican infection, yet little has been known regarding which surface receptor controls neutrophils’ antifungal activities. CD137, which is encoded by Tnfrsf9, belongs to the tumor necrosis receptor superfamily and has been shown to regulate neutrophils in Gram-positive bacterial infection. Here, we used genetic and immunological tools to probe the involvement of neutrophil CD137 signaling in innate defense mechanisms against systemic C. albicans infection. We first found that Tnfrsf9−/− mice were susceptible to C. albicans infection, whereas injection of anti-CD137 agonistic antibody protected the host from infection, suggesting that CD137 signaling is indispensable for innate immunity against C. albicans infection. Priming of isolated neutrophils with anti-CD137 antibody promoted their phagocytic and fungicidal activities through phospholipase C. In addition, injection of anti-CD137 antibody significantly augmented restriction of fungal growth in Tnfrsf9−/− mice that received wild-type (WT) neutrophils. In conclusion, our results demonstrate that CD137 signaling contributes to defense mechanisms against systemic C. albicans infection by promoting rapid fungal clearance.

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